KR800000298B1 - Process for the production of antibiotic a-30912 - Google Patents

Abstract

Antibiotic A-30912(I), useful as fungicide, was prepd. by submerged aerobic fermn. of Aspergillus regulosis NRRL 8113. Thus, 100 L medium contg. glucose 25, starch 10, peptone, molasses 5, casein hydrolyzate 4, CaCO3 2, MgSO47H2 (0.5g/L,anc Czapek's mineral stock 2m/L was inoculated with A.rugulosus and incubated at 25≰C with stirring and aeration at 0.5 vol/vol/min for 4 days. I was extd. with MeOH, re-extd. with CHC13 and pptd. with Et2O to yield 20g I. I factors comprised A,B,C and D.

Description

Preparation of Antibiotic A-30912

1 is the I. R. spectrum of antibiotic A-30912 factor A,

2 is the I. R. spectrum of antibiotic A-30912 factor D,

3 is the I. R. spectrum of antibiotic A-30912 factor B,

4 shows l. Of antibiotic A-30912 factor C. R. Spectrum.

The present invention relates to a method for preparing a new antibiotic A-30912 (at least 7 factors represented by A, B, C, D, E, F, G factors) effective as an antifungal agent. The antibiotic A-30912 mixture is prepared by culturing a new strain, Aspergillus rugu1osus NRRL 8113.

As used herein, `` antibiotic mixture '' refers to the state of the mixture of each antibiotic factor produced together in the culture. As is well known to those skilled in the manufacture of antibiotics by fermentation, the ratio of each individual of the resulting antibiotic mixture may vary depending on the fermentation conditions used.

Each antibiotic factor of the antibiotic according to the present invention is represented by antibiotics A-30912 factors A, B, C, D, E, F and G.

The present invention relates to a preparation of a novel antibiotic A-30912 mixture and antibiotic A-30912 factors B, C, D, E, F and G.

The present invention also provides a method for preparing and separating an A-30912 mixture comprising A, B, C, D, E, F and G factors, and a method for separating A, B, C, D, E, F and G factors. It is about.

The present invention relates to a novel preparation for preparing an antibiotic A-30912 mixture comprising the following factors: A, B, C, D, E, F and G.

a) Incubate Aspergillus Rugurossus NRRL 8113 in a medium containing an assimilation source of carbohydrates, nitrogen, and inorganic salts in-depth until a substantial amount of antibiotic is produced that can exhibit antimicrobial activity:

b) optionally separating the antibiotic A-30912 mixture from the medium:

c) optionally separate antibiotics A-30912 factors A, B, C, D, E, F and G from the antibiotic A-30912 mixture.

Antibiotic A-30912 mixture is extracted from fermentation medium with polar organic solvent.

A known compound, staligmatocytostine, is also prepared by Aspergillus lugurossus NRRL 8113. Steligmatocystine is extracted either separately with a non-polar organic solvent or with a mixture of antibiotics A-30912 with a polar organic solvent. In the latter case, the antibiotic A-30912 mixture is concentrated to sterigmatocystin by concentrating the extractant and adding the concentrate to an excess of a non-polar organic solvent such as diethyl ethyl to separate the antibiotic A-30912 mixture as a precipitate. Remove from Steligmatocystine is separated into the filtrate. The antibiotic A-30912 mixture is further purified by column chromatography.

Antibiotic A-30912 mixture and its respective A-30912 factor are used as antifungal agents.

As described above, the figures are shown on the I. R. spectrum for the A-30912 factor using potassium bromide disk.

Antibiotic A-30912 Factor A

Factor A-30912 is also similar to antibiotic Ekenocandin B, a polypeptide recently published in the references. [F. Benz et al., Helv. Chim. Acta 57, 2, 459-2, 477 (1974)

Antibiotic A-30912 Factor A is a white amorphous solid. The result of elemental analysis of factor A can be expressed as the following composition percentage:

Carbon: 56.52% Hydrogen: 7.29%

Nitrogen: 8.68% Oxygen: 27.09%

The approximate _{formula for} antibiotic A-30912 Factor A is C _51-53 H _79-83 N ₇ O _17-19 . The elemental analysis of factor A-30912 within this range is particularly in agreement with the formula of C ₅₂ H ₈₁ N ₇ O ₁₈ H ₂ O.

(Calculated: C, 56.24; H, 7.54; N, 8.84; O, 27.39:

The molecular weight of the antibiotic A-30912 factor by mass spectrometry and titration is 1,100 evil.

The I. R. spectrum of Antibiotic A-30912 Factor A measured in potassium bromide disk is shown in FIG. The following relates to the observed maximum absorption peaks:

2.97 (strong), 3.39 (medium), 3.47 (weak), 5.99 (medium), 6.10 (strong), 6.49 (medium), 6.56 (medium), 6.90 (medium), 9.13 (weak) and 11.77 (weak) microns . (`` Strong '' is a strong peak, '' Medium '' is a medium peak, and '' Weak '' means a weak peak.)

The U. V. absorption spectra of antibiotic A-30912 factor A in neutral and acidic methanol show maximum absorption at 225 nm (ε 18,000), 275 nm (ε 3,000) and 284 nm (solder ε 2,800). The U. V. absorption spectrum of factor A in basic methanol shows maximum absorption at 245 nm (ε 16,000) and 290 nm (ε 3,000), and also shows terminal absorption.

C ¹³ nuclear magnetic resonance spectra of antibiotic A-30912 factor A have the following characteristic peaks in deuterated methanol:

δ 176.1, 174.3, 173.4, 172.7, 172.4, 169.8, 158.4, 132.8, 130.9, 129.6, 129.0, 116.2, 77.0, 75.7, 74.4, 71.3, 70.9, 69.6, 68.3, 62.4, 58.7, 56.9, 56.1, 52.9, 39.0, 38.5, 36.8, 35.2, 33.9, 32.9, 32.6, 30.7, 30.4, 30.2, 28.2, 27.0, 26.5, 23.6, 20.1, 19.6, 14.4 and 11.3 ppm

Antibiotic A-30912 Factor A has the following non-luminescence.

-44° (C=0.5, 메탄올중에서)

-44 ° (C = 0.5 in methanol)

-156°(C =0.5, 메탄올중에서)

-156 ° (C = 0.5 in methanol)

The electrical titration of antibiotic A-30912 Factor A in 66% dimethylformamide aqueous solution shows that there is a titration group with pKa = 12.8 (initial pH 6.9).

Amino acid analysis of antibiotic A-30912 factor A after hydrolysis suggests the presence of threonine, hydroxyproline and other unidentified amino acids in the world.

Antibiotic A-30912 Factor A is soluble in various organic solvents such as methanol, ethanol, dimethylformamide, dimethylsulfoxide and ethyl acetate, and insoluble in nonpolar solvents such as diethyl ether and petroleum ether. Antibiotic A-30912 Factor A is also soluble in aqueous solutions and especially well in aqueous solutions with pH above 7.0.

A-30912 factor D

Antibiotic A-30912 Factor D is a white amorphous solid. The result of an elemental analysis of factor D of A-30912 has the following composition percentage:

C, 56.37% H, 8.17% N, 8.54% O, 26.92%

Antibiotic A-30912 Factor D has a molecular weight of 1,100, and the amino acid analysis shows a structure similar to that of antibiotic A-30912 Factor A.

I. R. spectra with potassium bromide disk of factor D-30912 are shown in FIG.

The following are the characteristic peak absorption peaks in the I. R. spectrum:

2.98 (strong), 3.31 (weak), 3.36 (solder), 3.40 (medium), 3.48 (weak), 5.76 (weak), 6.01 (strong), 6.10 (solder), 6.49 (medium), 6.57 (medium), 6.90 (medium), 7.81 (about), 8.07 (about) and 9.16 μ (about).

The U. V. absorption spectrum of the antibiotic A-30912 factor D in neutral and acidic methanol has a maximum absorption at 225 nm (ε 18,000) and 275 nm (ε 2,500).

The U. V. absorption spectrum of A-30912 factor D in basic methanol has a maximum absorption at 240 nm (ε 11,000) and 290 nm (ε 3,000).

=50°(C=0.34, 메탄올중에서)Antibiotic A-30912 Factor D has the following non-luminescence:

= 50 ° (C = 0.34 in methanol)

Amino acid analysis of antibiotic A-30912 factor D after hydrolysis suggests that there are threonine, hydroxypropine, histidine and three other unidentified amino acids. One of the unidentified antibiotic A-30912-factor-Damino acids is identical to one of the antibiotic A-30912-factor-Aamino acids not yet identified.

Antibiotic A-30912 Factor D is soluble in various organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide and ethyl acetate, but insoluble in non-polar organic solvents such as diethyl ether and petroleum ether. Antibiotic A-30912 Factor D is well soluble in aqueous solution. It is especially soluble in aqueous solutions with a pH greater than 7.0.

A-30912 factor B

Antibiotic A-30912 Factor B is a white amorphous solid. A-30912 Factor B has the following composition percentages, as determined by elemental analysis.

C, 57.36; H, 5.92; N, 8.75; O, 26.19

I. R. spectra of antibiotic A-30912 Factor B in potassium bromide disks are shown in FIG. The following are the characteristic peak absorption peaks in the I. R. spectrum:

2.99, 3.41, 3.49, 6.06, 6.15, 6.54, 6.61, 6.94, 7.62, 8.07, 9.26 and 9.39 microns.

The U. V. absorption spectrum of A-30912 factor B in neutral and acidic methanol has a maximum absorption at 223 nm (solder, ε 16,000) and 278 nm (ε 2,400). The U. V. absorption spectrum of antibiotic A-30912 in basic methanol has a maximum absorption at 242 nm (ε 13,000) and 292 nm (ε 2,800).

Antibiotic A-30912 Factor B has approximately the following non-oprecitivity:

-47°(C =0.5, 메탄올중에서)

-47 ° (C = 0.5 in methanol)

-170°(C =0.5, 메탄올중에서)

-170 ° (C = 0.5 in methanol)

The electrophoresis of A-30912 factor B in a 66% dimethylformamide aqueous solution showed that the pKa had an appropriate group with a bad 13.0 (initial pH of 7.91).

Amino acid analysis of A-30912 Factor B after standard acid hydrolysis suggests the presence of threonine, hydroxyproteins and some unidentified amino acids.

Factor A-30912 is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethylsulfoxide and ethyl acetate, but insoluble in nonpolar organic solvents such as diethyl ether and petroleum ether. Factor A-30912 is also soluble in aqueous solutions, especially in aqueous solutions with a pH above 7.0.

A-30912 factor C

Antibiotic A-30912 Factor C is a white amorphous solid. A-30912 factor C has the following rough composition percentage as a result of elemental analysis:

C, 56.76%: H, 7.88%: N, 10.61%: O, 25.09%

Results of the I. R. spectrum with antibiotic A-30912 factor C as potassium bromide disk are shown in FIG.

The following shows the maximum absorption peaks in the I. R. spectrum:

2.58, 3.39, 3.43, 3.51, 6.01, 6.12, 6.47, 6.90, 7.04, 7.22, 7.38, 8.00, 3.30 and 9.13 microns.

The spectra of A-30912 factor C in neutral and acidic methanol have maximum absorption at 223 nm (shoulder ε 7,300) and 275 nm (ε 1,350). The U. V. spectrum of antibiotic A-30912 in basic methanol has maximum absorption at 240 nm (ε 12,400) and 290 nm (ε 5,200).

Factor A-30912 has the following approximate non-luminescence:

-33°(c=0.5메탄올중에서)

-33 ° (in c = 0.5 methanol)

-119°(C =0.5메탄올중에서)

-119 ° (in C = 0.5 methanol)

Electric titration of A-30912 factor C in 66% dimethylformamide aqueous solution suggests that the titration group has a pKa of 13.08 (initial pH is 7.93).

The amino acid analysis of A-30912 factor C after standard acid hydrolysis suggests that there are threonine, hydroxyproline and a few unidentified amino acids.

Factor C is soluble in various organic solvents such as methanol ethanol, dimethylformamide, dimethyl sulfoxide and ethyl acetate, but insoluble in non-polar organic solvents such as diethyl ether and petroleum ether.

A-30912 Factor C is also soluble in aqueous solutions, especially in aqueous solutions with pH above 7.0.

Seven individual factors of the antibiotic A-30912 mixture can be isolated and isolated by insecticide chromatography (TLC). Silica gel is the preferred adsorbent and benzene: methanol (7: 3, V: V) is the preferred solvent system.

Antibiotic A-30912 factor by bioautography by TLC using silica gel (Merck, Darmshtar) and benzene: methanol (7: 3) solvent system, and by Candida albicans The Rf value of AG is equal to the heart mark (I).

TABLE I

The Rf values of the antibiotic A-30912 Factor A using various paper-chromatography and Candida albicans as detection microorganisms are shown in the following table (II).

TABLE II

The microorganisms useful for the preparation of the antibiotic A-30912 mixture were isolated from soil recovered from the Pompeii ruins in Italy. The microorganism that produces A-30912 is classified as Aspergillus Lugurosus Tom and Leifé, belonging to the Aspergillus nidulans form group.

This taxonomy is according to the method described in the references.

[See K.B. Raper and D. I. Fennel, `` The Genus Aspergillus '', 1965 edition from The Williams and Wilkins compony in Balibore]

The color name is according to I SCC-NBS method by reference.

(See K. L. Kelly and D. B. Judd, `` The ISCC-NBS Method of Designatind Co1or and a Dictionary of Color Names '' U. S. Dept. of Commerce, Cire 553, Washington, D. C., 1955]

The color schemes of the mertz and the foul are as described in the reference.

[See: A. Maerz and M. R. Paul, `` Dicitionaryl of Color, '' 1950 edition from MeGrow-Hill Bood Company in New York, NY.

Unless otherwise indicated, the cells were cultured at 25 ° C.

Culture Characteristics of Aspergillus Rogurossus NRRL 8113

Incubation is made slowly and after 15 days at 25 ℃ will be 1.5 to 2.0cm in diameter.

The colony surface is convex, velvety, corrugated near the center over time, and a central projection is formed. Mycelium terminal is two-wide, completely buried colorless mycelium, wave-like. Pink-brown output is formed at the end of aerial hyphae. After 7 to 14 days, a light purple soluble material is produced in the agar around the colonies.

This material diffuses into colonies over 15 hours.

After 5 days, the surface of the colony is white to pale yellow, and the inside of the colony is brownish-orange in the center and brownish-purple in the incidence. After 10 days the colony becomes medium yellowish-pink. After 14 days, the toloni becomes pale-grey-red (29 with ISCC-NBS and 11-A-7 days for Mertz and Paulo). The margins are congenital, resulting in wart-shaped projections and dark yellow (84 in ISCC-NBS and 10-L-5 in Mertz and Paulo). Dull yellow folds of capsular cells scattered around and appear randomly on the surface and edges of the colonies. Over time, dark yellow areas and edges become yellowish green. After three weeks an orange-purple tint can be found in the new parasitic part of the edge.

At first, the interior of the colony is slightly concave. As they mature, the colonies become flattened against the surface of the KEPCO and the interior of the colonies becomes slightly wrinkled. After 10 days, the interior becomes light brown (57 in ISCC-NBS and 5-A-10 in Mertz and Paulo). After 15 days it becomes gray-red (19 with ISCC-NBS and Bertz and Paulo 6-J-3).

Conidia are rare and conidia are sparsely scattered on the surface, sometimes in clusters or even in worm bands. The conidia head is initially loose radioactive and progresses to a more dense state, circular or short, over time.

The round head is 70μ-80μ in diameter and usually 50μ. The tubular head is 140μ long and 70μ wide.

Conidia are round or semi-circular, with many wrinkles and generally greenish-golden.

Diameters range from 2.8 microns to 3.9 microns with an average of 3.2 microns.

Basal spines cog-shaped on both sides and colorless. The main basal process is 4.7μ to 11.0μ long and averages 7.9μ. The widest part is 2.4μ and the thin part is 1.6μ. Side ridges appear one or a pair from main ridges and are shaped like flasks. The widest part is 3.0μ and the upper part of the fine thin part is 0.4μ and tubular. The tube top is 1.2μ long. The overall length is 5.5 microns to 12.6 microns and the average is usually 9.2 microns.

The vesicles are round to medium or semicircular, and the top is flat and gradually browns over time. It has a diameter of 7.4 μm to 11.2 μm and usually averages 9.4 μm.

Conidia are smooth, relatively thick membranes, initially transparent, but brightly cinnamon-brown. The vesicles are slightly wider, and they are a little thin near the foot cells. The average width is 5.9μ. Conidia have a length of 47.7μ to 166.6μ and an average of 106μ.

It is produced from submerged hyphae or outwardly from parasitic mycelial parts.

Asystole status does not appear until 20 days have passed. Small clusters of yellow, capsular cells appearing on the surface are found in the mycelium anywhere.

It has been one or more closed cleistothecia.

Capsular cells round to semi-circular or ovoid to long oval, with thick walls and transparent. Circular capsular cells 18-24 microns in diameter, usually 21.8 microns.

Closed membranes are round to semicircular, surrounded by thick walls, relatively rough and fibrous. It is relatively colorless at first, but gradually changes from reddish-purple to darker over time.

Malt Extract Agar Badge

Colonies grow at 25 ° C. and quickly expand to 4-5 cm after 10-12 days. At first it was ash-white. After 4 days, the colonies become olive and green (90 with ISCC-NBS and 23-E-6 days with Mitz and Paulo).

The terminal part, which is wave-shaped or slightly sulphate, has a dense, short and white parasitic mycelium. Small yellow clusters of capsular cells are found anywhere in the mycelium.

It consists of capsular cells covered with one or more closed membranes. After 20 days, the capsular cells cover much of the conidia spore head. Colonies turn grayish-yellow (ISCC-NBS 90 and Mertz and Paulo 11-B-1).

Asymptomatic condition occurs in 15 days. Tiny yellow clusters of capsular cells appearing on the surface are found in some conditions in mycelia. It consists of capsular cells propagated into one or more closed membranes. Capsular cells cluster on mycelial head. Capsular cells round to semicircular or ovoid to long oval, surrounded by thick membrane and transparent. The diameter of circular capsular cells is 18μ to 24μ and usually 21.8μ.

Closed membrane is round to semicircular and dark red light brown. It is 389μ to 700μ in diameter and usually 506μ.

Asystole is fragile, transparent, round or ovoid. Semicircular follicles range from 8.7μ to 11.9μ in diameter with an average of 10.3μ. The size of the ovoid vesicles ranges from 10.3 μm to 14.2 μ × 8.7 μm to 10.3 μm with an average of 12.2 μ × 9.1 μm.

Asystole reddish-orange, lugurose, two parallel, middle wrinkly fine, 0.8 wide, with unbroken head. Asystole is lenticular through the long axis.

When the head is around, the follicular spores are circular. The diameter in the case of a circular shape is 4.9μ to 6.3μ and usually 5.4μ.

The two characteristics of Aspergillus rogurossus, the strain that produces antibiotic A-30912, differ from those of A. rogurossus described in the reference. The strain that produces A-30912 is larger in the conidia spore head and follicular spores. Aspergillus rugurosus, useful for preparing antibiotics A-30912 mixtures, is stored and by reference media. This is known and known as NRRL 8113.

Reference: Northern Resional Research Laboratory (Agricultural Research Institute of the United States Department of Agriculture in Peoria, Illinois, USA 61604, expressed as N. R. R. L.)

One of the many media can be used for the culture of Aspergillus tugurosus NRRL 8113. However, in view of manufacturing economics, that is, which medium is preferred depending on the optimum yield and the ease of product separation.

Thus, for example, the preferred carbohydrate source for large-scale fermentation is glucose, although molasses, starch, lactose, sucrose, maltose, glycerol and the like can also be used. Preferred nitrogen sources are enzyme-hydrolyzed casein and those such as meat peptone and wheat glutamate.

Nutrient inorganic acids may be mixed into the medium. These include conventional soluble salts capable of producing ions such as sodium, magnesium, calcium, chlorides, carbonates, sulfates, nitrates and the like.

Trace elements, which are essential for the growth and development of microorganisms, must also be included in the medium.

These trace elements are usually present as impurities in other media components in amounts sufficient to grow the microorganisms.

It may be necessary to add a small amount (eg 0.2 ml / l) of polypropylene glycol as a defoaming agent to solve the foaming problem in large scale fermentation.

In order to produce a full mass production of the antibiotic A-30912 mixture, it is preferable to carry out aerobic deep culture in a tank.

A small amount of antibiotic A-30912 mixture can be obtained by shaking in a flask.

Normally, inoculating microorganisms in large tanks in the form of spores in the production of antibiotics delays the time, so it is preferable to inoculate the strains of nutritional strains. Caps or mycelium parts of microorganisms can be inoculated in small amounts of media to produce and prepare influenced strains of fresh and actively growing microorganisms. Nutritional strains are transferred to larger tanks. The medium used for the cultivation of nutritional strains may be the same as that used for large scale fermentation, but other mediums may also be used. Microorganisms producing the antibiotic A-30912 are cultured at temperatures between 20 ° and 43 ° C., and microorganisms are well grown between 25 ° and 30 ° C. The optimum temperature for preparing the antibiotic A-30912 mixture is 25 ° C.

As in the conventional aerobic deep culture process, sterile air should be injected into the medium. The amount of air used to prepare an effective amount of antibiotic is preferably at least 0.4 volume of air (V / V / M) per medium volume per minute.

During fermentation for the production of an antibiotic A-30912 mixture, a test sample of the alcohol extract of the whole medium solution can be taken and tested for antimicrobial activity against microorganisms known to be sensitive to antibiotic A-30912. A potent microorganism useful for testing for antibiotic A-30912 is Candida albicans. As a biological test, a paper-disk test is conveniently performed on the agar plate inoculated with the strain.

In general, the antimicrobial activity is detected the next day after the start of fermentation. Usually the maximum production of antimicrobial products is between 3 and 6 days.

Antibiotic A-30912 is an antifungal agent. An example of the antifungal action of antibiotic A-30912 is a biopsy with antibiotics A-30912 factors A and D. This test is summarized in the following table (II).

Antifungal action is measured by conventional disc diffusion methods (6 mm pads in a solution containing the test substance and then placed on agar plate inoculated with the test microorganisms). The result is a test compound that inhibits the test microorganisms. The minimum inhibitory concentration (MIC) per disc containing

TABLE III

Factor A-30912 is the most active in the disk-diffusion test, which is a biometric test for skin filamentous fungi. The results of this test are summarized in Table (IV).

Table IV

Antifungal action of antibiotic A-30912 is better demonstrated in vivo. In vivo testing is carried out in the following manner.

Three test doses are administered to Candida albicans-infected rats in three stages immediately, 4 hours and 24 hours after infection. Inhibitors represented by the test animals are measured at an ED of ₅₀ . 50% inhibitory effective amount expressed in mg / kg in rats:

See W. Wick et al., J. Bacteriol. 81, 233-235 (1961) The ED ₅₀ values of the antibiotic A-30912 factor A against Candida albicans in rats are 30 mg / kg (intraperitoneal injection) and 50 mg / kg (subcutaneous injection). The ED ₅₀ value of the antibiotic A-30912 Factor D against Candida albicans in rats is 33 mg / kg. (Subcutaneous injection)

There was no sign of acute toxicity when antibiotic A-30912 factor A was injected intraperitoneally or subcutaneously into rats twice daily for 100 mg / kg (600 mg / kg total). In addition, intraperitoneal injection of antibiotic A-30912 factor A three times a day at 200 mg / kg (total 600 mg / kg) shows no signs of acute toxicity.

Subcutaneous injection of antibiotic A-30912, Factor D, with 14 mg / kg (42 mg / kg total) three times a day shows no signs of acute toxicity.

When used as an antifungal agent, antibiotic A-30912 is administered parenterally, usually in admixture with a pharmaceutical nontoxic carrier or diluent. The dose of antibiotic A-30912 depends on various conditions, such as the nature of the infected organism and the extent of infection.

In order to more fully obscure the present invention, the following examples are given.

Example 1

A. Shake Fermentation in Flasks

Aspergillus Lugurosus NRRL 8113 medium is maintained in 18 × 150 ml agar medium with the following composition:

* N-Z-Amine A

It is a product of Sheffield Chemical Co., Norwich, NY.

Aspergillus Lugurosus NRRL 8113 is inoculated on the slope, and the inoculated slope is incubated at 25 ° C. for about 7 days. Cover the mature slope culture with bovine serum and inoculate half of the resulting suspension with dense loopospores in 50 ml of nutrient-type medium having the following composition:

* N-Z-CaSe is a product of Sheffield Chemicals, Norwich, NY.

Inoculated nutrient medium is incubated in a 250 ml spouted Erlenmeyer flask on a shaker rotating at 250 RPM through a 2 inch diameter arc for 24 hours at 25 ° C.

This inoculated nutritional medium can be used for inoculation of the nutritional medium in the second stage soon. Alternatively and preferably the medium may be kept in the gaseous state of liquid nitrogen for storage for subsequent use.

The medium is filled for storage in several small vials as follows: In each vial, 2 ml of inoculated nutrient-type media and 2 ml of glycerol-lactose solution with the following composition:

The prepared suspension is stored in the gaseous state of liquid nitrogen.

The thus prepared storage suspension (1 ml) is inoculated into 50 ml of the first stage nutritional medium having the same composition as described in the nutritional medium described above.

The inoculated first stage nutrient medium is incubated in a 250 ml spouted Elenmeyer flask for 22 hours at 25 ° C. on a shaker rotating at 250 RPM through a 2 m diameter arc.

B. Fermentation in Tank

In order to increase the dose of the inoculum, the above-mentioned 10 ml of the inoculum nutrient medium for the first inoculation mentioned above was used to inoculate 400 ml of the nutrient medium for the second phase with the same composition. The second stage medium is incubated for 25 hours at 25 ° C. in a 2 l wide wide conical flask on a shaker rotating at 250 RPM through a 2 inch arc.

The second stage nutrient-type medium (800 ml) cultured as described above is used to inoculate 100 liters of dense medium having the following composition:

* W. P. No. 159: Inolex Biomedical Corp. Product.

** N-Z Amine A is a product of Sheffield Chemicals, Norwich, NY.

*** The composition of Zabeck's mineral stock is as follows:

At a pressure between 16 and 181b, the pH of the medium after sterilization in autoclave at 121 ° C. for 30 minutes is 6.8. The inoculated medium is left to ferment in 165 L fermentation tank at a temperature of 25 ° C. for 4 days. Fermentation broth is infused with sterile air at a rate of 0.5 V / V / M. Fermentation medium is stirred at a speed of 300 RPM with a conventional stirrer.

Example 2

Isolation of Mixture of Antibiotic A-30912

The whole fermentation broth obtained by the method described in Example 1 (200 L) was stirred with methanol (200 L) for one hour and filtered through a filter (Hyflo superset: diatomaceous earth manufactured by Manville Product Corp.): pH of the filtrate Adjust to 4.0 by adding 5N hydrochloric acid. The acidified filtrate is extracted with two equal portions of chloroform. The chloroform extracts were combined and concentrated to 4 l in vacuo. This concentrate is added to about 60 liters of diethyl ethyl to precipitate the A-30912 mixture. The precipitate is separated by filtration and dried to obtain 38 g of a gray powdered antibiotic A-30912 mixture. This oily substance is dissolved in methanol (500 ml). Methanol solution is added to diethyl ethyl (7.5 L) and precipitated to give a further antibiotic A-30912 mixture. This precipitate is also filtered, separated and dried to yield a further 3.5 g of antibiotic A-30912 mixture.

Example 3

A-30912 Isolation of Factor A

The antibiotic A-30912 mixture (20 g) obtained as described in Example 2 is placed in a silica gel column (4-x10 < 7 > -cm film) in a mixture of acetonitrile and water (95: 5). Acetonitrile: water (95: 5) is flowed into this column at a rate of 1 to 2 ml per minute to elute to obtain a fraction having a volume of about 20 ml. Each fraction is acetonitrile: water (95: 5) as a solvent system, and the thin layer chromatography (using silica gel), which is subjected to biological self-test with Candida albicans, is observed for the presence of the product.

Combine the fractions 74 to 125 and concentrate. The concentrated solution is left to crystallize to additionally yield 124 mg of staligmatocysteine. Combine fractions 212-273 and concentrate in vacuo to give an oily material. This oily substance is dissolved in a small amount of methanol. Methanol solution is added to diethyl ether (15 times volume). The resulting precipitate is filtered and dried to obtain 23 mg of antibiotic A-30912 Factor D. Fractions 274 to 4,337 contain the antibiotics A-30912 factors A, B, C and D. Fractions 482 to 900 contain the antibiotics A-30912 ≠ factor A, E, F and G. Combine fractions 438-481 and concentrate in vacuo to yield an oily substance. This oily substance is dissolved in a small amount of methanol and this methanol solution is added to diethyl ether (15 times capacity). The resulting precipitate is isolated and dried to obtain 2.17 g of antibiotic A-30912A factor.

Example 4

Isolation of Antibiotic A-30912 Factor D

Some purified antibiotic A-30912 mixture containing antibiotics A-30912 Factors B, C and D are obtained by the method of Example 3 from fractions 274 to 437. The foreign material (750 mg) was chromatographed with a silica gel column (2.2 × 51 cm, film silica gel), and fractions having a volume of about 15 ml were collected using the following solvent as the eluent.

The column fractions are quenched by thin layer chromatography using silica acetonitrile: water (95: 5) and benzene: methanol (7: 3) as solvent solvents and subjected to biological self-test with Candida albicans. 535-685 fractions containing antibiotic A-30912 factor D are combined and concentrated in vacuo to give an oily substance. This oily substance is dissolved in a small amount of methanol and the methanol solution is added to diethyl ether (15 times capacity). The resulting precipitate is filtered off and dried to yield 69 mg of antibiotic A-30912 factor D.

Example 5

Isolation of Antibiotic A-30912 Factors B and C

Antibiotic A-30912 Some purified A-30912 antibiotic complexes containing Factors A, B, C, and D are obtained according to the method described in Example 3 from fractions 212-437. The foreign material (18 g) is dissolved in a minimum amount of acetonitrile: water (4: 1) and chromatographed on an alumina column (3.8 x 56 cm film) to obtain and collect fractions containing about 20 ml each. The column is eluted with the following solvent.

The column fractions are checked by silica gel thin layer chromatography according to the method described in Example 4. Based on this result, the fractions are collected and concentrated to obtain an oily substance. The oily substance is dissolved in a small amount of methanol, and the methanol solution is added to 10 to 15 times the volume of diethyl ether to precipitate. The contents of each factor of the fraction combined with the weight of the material obtained according to the method of collecting fractions are summarized as follows.

^* Insoluble material obtained prior to ether precipitation

To obtain purified A-30912 Factor C, fractions 6-28 (2 g) were dissolved in methanol, adsorbed on a sufficient amount of silica gel (grade 62), dried and filled with acetonitrile (1.9 x 80 cm, grade 62). On top of each other).

The column is eluted by flowing acetonitrile at a rate of 2 ml / min and fractions of about 10 ml are collected. Change the solvent to acetonitrile: water (99: 1) in fraction 117. Check the column fractions again by thin layer chromatography.

Based on the results, the fractions are combined and concentrated to obtain an oily residue that is dissolved in a small amount of methanol and the methanol solution is precipitated with 10 to 15 times the volume of diethyl ether. The summary of each factor content and weight of each fraction is as follows:

Claims (1)

Aspergillus lugurose (NRRL 8113) was incubated under aerobic in-depth culture conditions until a substance having a substantial amount of antimicrobial activity was produced in a medium containing carbohydrate, nitrogen and an assimilation source of inorganic salts. Process for the preparation of antibiotic A-30912, characterized by separating factors A, B, C and D.

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