JPH0142274B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing certain previously unknown tetraene antifungal antibiotics by a harsh process using a novel microorganism belonging to the genus Streptomyces. The present invention also relates to novel tetraene antibiotics. Description of the Prior Art Tetraene antibiotics are substances whose basic properties include a structure containing a conjugated double bond which gives them very interesting biological properties. These compounds include, for example, amphotericin B (US Pat. Nos. 2,908,611 and 2,908,612), phi-
lippine complex) (UK Patent No. 783,486), nystatin (UK Patent No. 866,600), pimaricin, also known as tenesetine or natamycin (UK Patent No. 852,883 and German Patent No. 1024,206) , Candicidin (US Pat. No. 2,992,162), and the like. These substances are UV and
They can be distinguished from each other by IR spectra and by chromatography (paper, thin layer or high pressure). Tetraene antibiotics are primarily produced by microorganisms of the order Actinomycetales, in particular of the genus Streptomyces and Streptoverticillinum. Usually, biosynthesis of these compounds does not take place, but the appearance of several tetraenes in one and the same medium is observed. The mechanism of action in eukaryotic cells is that of membrane sterols,
Cell collapse occurs when the selective permeability conferred by these sterols changes. Tetraenes are widely used in the pharmaceutical field, and although the toxicity of these tetraenes is due to intravenous administration, tetraenes are mainly used topically or orally because they are not particularly toxic. However, the recent creation of tetraene as an interesting substance has led to the discovery that there are some differences between its effects on membrane permeability in tumor cells and normal cells. It is. Also, other interesting facts have been established that blood cholesterol levels are reduced when these compounds (candicidin) are administered orally. Additionally, certain types of tetraene are known to act on prostate enlargement. This has been confirmed by studies of the microformations that give rise to these substances. SUMMARY OF THE INVENTION The present invention describes the production of several types of tetraene substances with particular antifungal activity;
The most notable of these is a novel substance named Ab400 by the applicant, whose main biological property is that it inhibits the growth of fungi and yeast; Accordingly, these tetraene substances can be used to treat infections caused by pathogenic microorganisms (belonging to the above-mentioned classes) in animals and plants. Ab400 is Streptomyces sp, strain SF-1
It is produced in a culture of aerobic bacteria related to the genus Streptomyces (ASA). Another property of this microorganism is that it produces other tetraene substances, the most prominent of which is the already known tetraene called pimaricin. In addition, the present invention will explain the extraction and confirmation of the above-mentioned substances, as well as the antifungal spectrum of Ab400. Characteristics of microorganisms The microorganisms used were isolated from soil collected near Madrid, and were Streptomyces sp, strain SF.
-1 (ASA) in the collection belonging to the Antibioticos Società Anonyme of León (Spain) and deposited with the National Collection of Industrial Bacteria (NCIB), Tray, Research Station, Aberden, Scotland, under No. NCIB 11738. Sykes and Skinner
(1973), The Actinomycetales, published by Academic Press.
The guidelines set out in ``Actinomycetales'' include:
Confirmation and conclusion of the toxicity of the microorganism revealed that the microorganism is neither overcoiled nor sporangial, but has an aerial mycelium with sporophores containing many spores in a chain. It is reported that it belongs to “International Streptomyces
Project” -ISP- (“Int.J.Sist.Bacterio-
The method described by Shirling and Gottlieb in 16:313-40, 1966) describes the characteristics of species. Under phase contrast microscopy, fine particles SF-1
(ASA) has 1 to 6 somewhat dense spirals in ISP-3 and ISP-4 media, and 10
It was observed to have spiral sporophytes with chains of more than 0.8 x 0.5μ oval spores. In ISP-2 and ISP-5 media, these sporophytes do not show true spirals, but occasionally dense hooks were observed when one spiral was photographed closely. The spores showed a warty surface under an electron microscope. The characteristics of the cultures in different media are shown in the table. All cultures were performed at 28°C. There are two ways to color,
namely Ed.P.Lechevalier by Mr. E.Seguy
(Paris) (1936), “Code Universal de
Couleurs' sheet method and the regular names for the colors, and by the method of assigning these subdivision names. The assimilation spectrum of the nitrogen (N) source is shown in the table. 1% glucose and 1.5% agar (Lyudemann GM
(Luedemann GM), "Int.J.Sist.Bacteriol."
21:240-47 (1971) was used as the base medium. E. Schilling assimilation spectra of carbon (C) sources.
B. and Gotstrieb D. method (“Int.J.
Bacteriol.'' 16:313-340 (1966). The results are shown in the table. Physiological properties are shown in the table. In summary, the strains in the study had the following phylogenetic characteristics:

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[Table] 1. British Patent No. 846933 (1960) 2. Schilling EB and Gotrieb D. "Int.
J.Sist.Bacteriol.'' 18:69-189 (1968) 3 Schilling EB and Gotrieb D. ``Int.
J.Sist.Bacteriol.'' 22:265-394 (1972) 4 Results obtained by this laboratory (a) Belongs to the spiral part. (b) It has spores with a warty surface. (c) Does not produce melanoid pigments. (d) The color of sporulated aerial mycelium is violet or red depending on the medium used. (e) Produces a soluble red (reddish brown) dye that does not act on the PH indicator. (f) The color of the substrate mycelium changes from chestnut with red to violet chestnut depending on the medium. (g) Assimilates the following materials as carbon sources: - glucose - glycerol - inositol - starch fructose - cellobiose mannitol - trehalose galactose mannose It is not clear whether suchrose and roughnose are assimilated. (h) Other physiological properties: 1. Causes significant hydrolysis of gelatin. 2 Reduce nitrate to nitrite. 3 Do not hydrolyze milk casein. 4. Withstands up to 4% NaCl. 5 It is mesophylic. (i) Produces several types of tetraene antifungal antibiotics. Bergey's Handbook of Quantitative Bacteriology
Manual of Determinative Bacteriology)
(1974, 8th edition, The Williams and
Wilkins Company) and Cooperative Description of the Standard Strain of Streptomyces (Wilkins Company);
description of Type strains of
The paper ``International Streptomyces Project'' was published by Schilling and Gotrieb in ``Lnt.J.Sist.
Bacteriol.'' 18:69-189, 1968; Ibid. 18:279
392, 1968; Ibid. 19:391-512, 1969; and Ibid. 22:265-394, 1972) and the phylogenetic characteristics of Streptomyces species in these documents published from 1974 to the present. Hey, SF-
No microorganisms similar to ASA 1 (ASA) could be found. In particular, pimaricin, S. natalensis (S. nata
-lensis), S. chattano
−ogensis) and S. gilbosporeus (S.
gilbospo-reus). The results of this study are tabulated and confirm that Streptomyces sp. SF-1 (ASA) is different from other pimaricin products. The microorganisms described here are wild strains, but naturally occurring mutants and mutants induced by mutagenic agents also give rise, together or separately, to the group of tetraene antibiotics. be able to. For this purpose, wild-type strains and their mutant descendants can be used in the process described below. Production by Fermentation The tetraenes described herein can be produced in solid or liquid media; liquid media are highly suitable for producing large quantities of the compound. In this liquid medium, antibiotics can be produced by surface culture and submerged culture. In particular, submerged culture is ideal for producing large amounts of product. The medium used contains at least the following materials: (a) assimilable carbon sources such as glycose, fructose, mannitol, glycerol, starch,
Dextrin, cellobiose, trehalose,
Mannose and its mixtures. (b) assimilable carbon sources, such as vegetable cake meal (soybeans, cotton, ground nuts, etc.), corn, stave liquor, vegetable or animal peptones;
Yeast, urea, ammonia salts, etc. and mixtures thereof. (c) Inorganic salts that produce a medium with pH buffering capacity or that supply inorganic elements. (d) Vegetable or animal oils used as foam suppressants or as carbon sources. silicon. The pH fermentation range that can be used in the method of the present invention is between 5.1 and 8.4, and the optimal temperature is between 25 and 30℃.
is within the range of The tetraenes of the present invention can be made by fermentation using a regular antibiotic fermentation process. That is, strain sporophytes are inoculated with lyophilised spores in slanted tubes containing medium, incubated at an optimal temperature, and the spores and mycelia from such tubes are initiated into the growth phase in submerged culture. These stages can be longer or shorter depending on the process volume to be fermented. If large fermentation tanks are used, the number of stages reaches 5. When fermenting in a flask, the flask is shaken thoroughly on a suitable vibrating table in a thermostatically controlled chamber; when fermenting in a tank, the tank is energized, stirred, and stirred to achieve optimal levels of production. PH control, dissolved _O2 control, temperature control and nutrients, alkali and acid addition are required. All processes must be performed under completely sterile conditions to avoid contamination with foreign microorganisms.
The medium can be sterilized in the container or placed in an already sterilized container if the container has been previously sterilized. Once production begins, there will be enough mycelium in the medium.
The production gradually increased over time until day 5 or 6 of fermentation, then the increase stopped. In order to study the amount of antibiotic in the prosthesis as a standard against Ab400, we showed a single active substance on paper and high pressure liquid chromatography and a minimum inhibition of 0.5 μg/ml against Sacoharomyces cerevisiae ATCC 9767. A purified sample with a certain concentration was used. As a bimarishing standard, Koninklijke NedeRlandsche Gist-&
A 900 U/mg sample (Batchi 9117A) made by Spiritusfabriek NV) was used. Microbial broth that has already been patented
Although the existence of Ab400 has not been described, we conducted a comparative study on Streptomyces sp. SF-1 (ASA), St. natalensis NRRL-2651, and St. It was confirmed whether or not it was produced during fermentation in a medium. The results are shown in the table. From this table, Streptomyces sp.SF−
1 (ASA) produces large amounts of Ab400 and bimaricin, indicating that the strain of the present invention is clearly different from the well-known pimaricin-producing strain that has already been patented. It can be seen that Ab400 of the former of the two strains mentioned above was not produced to a detectable extent in all the media tested. Furthermore, a different characteristic of the SF-1 (ASA) strain is that it produces a strong apple odor in the fermentation process.

[Table] The medium known as MCH-1 is described in British Patent No. 852883 and contains 2% glycerol, 0.5% Phytone, 0.5% peptone, 0.3% yeast extract, 0.3% meat extract and 0.25% _CaCO3. and has a pH of 7.6. Also, MFNa-1
The medium known as German Patent No. 1024206
5% soy flour, 1% glycose, 0.5% (NH ₄ ) ₂ SO ₄ , 0.02% KH ₂ PO ₄ , 1%
Contains CaCO ₃ , 0.5% soybean oil and 0.1% corn stable liquor, and has a PH of 6.3. Oil Release and Separation The minimum inhibitory concentration (MIC) of the fermented and filtered process was generally 1/2000 against Saccharomyces cerevisiae. Filter the whole broth through a Hyflo-Supercell with a pH in the range of 5-6;
The filtered broth was adjusted to pH 8.0 with sodium hydroxide and then extracted with a water-immiscible aliphatic alcohol such as butanol. The organic extract was concentrated under vacuum to reduce the volume to 1/20 of the original volume, thereby precipitating a white powder, which was referred to as "raw antifungal material".
It is called "antifungal". The precipitate was filtered and dried under vacuum at room temperature. The obtained product is a very pale yellow powder, and its MIC is about 1/1500000 (0.67 mcg/
ml). The yield of this product was 0.7 g/filtered broth. Such "crude antifungals" are relatively water soluble and, when examined by paper chromatography, have determined the presence of at least two major antibiotics that are active against S. cerevisiae. When a "crude antifungal substance" is dissolved in water at a rate of approximately 4 mg/ml, this solution is adjusted to pH 10.2, and left at +1°C (for 24 hours), the crude antifungal substance becomes Ab400. Equivalent to 1/10000000 (0.1mcg/ml)
A white substance was precipitated with a MIC of . This Ab400
was obtained with a yield of 6.6% of the initial "crude antifungal". Ab400 is 290, 304 and
It shows maximum absorption in the ultraviolet spectrum at 318 nm, and this property is similar to that of polyene tetraene.
enes). (Saoshi Omura, “Macrolide Antibiotics” page 353). The UV spectrum of 'Ab400' in methanol shows that this substance is a tetraene,
The infrared spectrum of potassium bromide was also shown to be similar to the tetraene antifungal pimaricin, although not identical. Pimaricin was added in a butanol + ethanol + water (5:4:1, V) chromatographic system.
Has a lower R _f than Ab400. The above crude antifungal substance was added to a LiCrosorb rp-18-column (5μ), 0.06M
When processed by high pressure liquid chromatography (HPLC) with a mobile phase of ammonium citrate, PH 5.0 + acetonitrile (3:1:v/v), the crude antifungal substance consists of a mixture of seven chemicals. I made sure of that. One of these chemicals is pimacillin, the other is Ab400 in the present invention,
The other five chemicals had antibiotic activity against Satucharomyces cerevisiae and a characteristic ultraviolet spectrum of tetraene materials. Ab400 has the following physicochemical properties: 1 Elemental analysis: C = 54.5%, H = 7.1%, N = 2.9%, O
(diff.) = 35.5% Empirical formula: C ₂₂ H ₃₅ O ₁₁ N 2 Molecular weight: 489.522 3 In the ultraviolet spectrum, in methanol,
290, 304 and 318nm at 4mg/ml concentration of Ab400
shows the maximum absorption of 4 Infrared spectrum (in potassium bromide):
It has an infrared spectrum shown in FIG. 5 Solubility in solvents: Very soluble in dimethyl sulfoxide (DMSO), slightly soluble in _H2O and N-butyl alcohol. It is also insoluble in benzene ether, ethyl acetate and methylene chloride. 6 Distinction between chlorine, acid and neutral: Chlorinated compounds 7 Color of substance: White powder 8 Melting point: 220-225°C (with decomposition) Antibacterial activity Ab 400 was tested for antibacterial activity. Tests showed no activity against bacteria and no positive activity against yeast: Candida albicans Candida anomala Candida dolonii Candida desusei Candida melinii Candida pelliculosa Candida pulcherrima Candida tropica tropicalis Cryptococcus albidus Cryptococcus difluens Cryptococcus neoformans Debaryonyces hansenii Debaryonyces klockenii Geotricium versiforme hansenii La (Hansenula) pseudopelculosa (Hansenula) suaveolens (Hansenula) Pichia (Pichia) pseudopolymorpha (pseudopolimorfa) Pichia (membranaefaciens) The present invention will now be described by way of example. explain. Example 1 A double base was made from the following composition: Cottonseed flour...20g Corn Steep liquor...10g Dextrin...10g Glycose...50g (NH ₄ ) ₂ SO ₄ ...5g NaNO ₃ ...2.5g K ₂ HPO ₄ …1g FeSO ₄・7H ₂ O …0.05g MgSO ₄・7H ₂ O …0.05g MnSO ₄・H ₂ O …0.05g ZnSO ₄・7H ₂ O …0.1g Distilled water ad … 1000ml PH was adjusted to 6.2 with KOH. 200 ml of each medium was placed in each 1000 ml Ellenmeyer flask. 1 g of CaCO ₃ was placed in each flask. Each flask was sterilized at 121°C for 30 minutes. Streptomyces
sp. strain SF-1 (ASA) inoculated with a spore suspension.
This suspension was made from slant tube cultures on any solid medium that sporulates microorganisms. After inoculation, the medium was incubated for 5 days in a thermostatically controlled chamber at 28° C. with constant vibration on an orbital vibration table with a 5 cm stroke and a speed of 240 rpm. The innoculum thus prepared was used to inoculate a fermentation medium with the following composition: Soybean flour...40g Glucose... _60g ( _NH4 ) _2SO4 ... _{5g K2HPO4} ... ...13g CaCO ₃ ...10g distilled water ad ...1000ml PH is 6.2 with dilute sulfuric acid before adding carbonate.
It was adjusted to Each flask contained 200 ml of sodium. 1 ml of soybean oil was added to each of these flasks. Each flask was sterilized at 121°C for 30 minutes. After sterilization, the medium was inoculated with a 5% inoculum. This was incubated in a chamber for 4 days at 28°C on a shaking table with the same characteristics as described for the inoculum. The analysis of the broth at the end of the fermentation is as follows: PH 5.5-6.5; sugar 2%; ammonium N: 100 mg/; total soluble N: 950 mg/; and mycelial volume: 32. It has a minimum inhibitory concentration of 1/4000 to 1/5000 against S. cerevisiae ATCC 9767; and in HPLC (900 U/
mg pimaricin) 300-500mcg/ml
It showed Ab400 activity and pimaricin activity of 1000-12000 mcg/ml. Example 2 A double base was prepared from the same composition as the inoculum in Example 1, and 200
1 ml into an Ellenmeyer flask,
The medium was sterilized at 121°C for 30 minutes. The medium thus prepared was inoculated with a spore suspension of the proprietary microorganism. Spread this medium in 5cm steps and
Cultures were grown for 5 days at 28°C in a thermostatically controlled chamber with constant vibration on an orbital vibration table with a vibration rate of 240 rpm. Using the inoculum thus prepared, a medium 1 having the same composition as the fermentation medium of Example 1 at a proportion of 0.5% is prepared.
inoculated in an 8 volume fermentation tank containing .
The following fermentation conditions were used: temperature 28°C, aeration 0.3v/
v/m, stirring 400 r.pm, and fermentation time 90 hours.
At this time, the fermentation medium had pimaricin activity of 500-1000 mcg/ml and Ab activity of 300-600 mcg/ml. The same type of standard was used as in Example 1. Example 3 3540 ml of total broth at PH 5.9 was filtered under vacuum on a High Flow Supercell to obtain 2150 ml of red clear filtered broth with MIC=1/2222. Adjust the pH to 8.0 with 25% sodium hydroxide,
The fluffy precipitate was filtered off in the same manner as in the above example. In the first extraction, the organic phase was centrifuged to 700 ml.
A filtration broth with 50% butanal was obtained. The aqueous phase was extracted twice with 350 ml of butanal as described above. The total clear yellow butanal extract was concentrated under vacuum to 1/20 of the initial volume resulting in the precipitation of a white powder, washed with butanal after filtration, dried under vacuum at room temperature, and 1.52g (i.e.
0.71 g/ml) of "crude antifungal substance" was obtained, which showed MIC=1/1450000 (0.69 mcg/ml). The product was dissolved in 380 ml of water and the pH of the solution was adjusted to 10.2 with sodium hydroxide. After 24 hours at +1°C, the fluffy precipitate was filtered, washed with water and dried under vacuum at room temperature to give 101 mg of Ab400 (6.7
%) was obtained. The MIC of this Ab400 is 1/10000000
(0.1 mcg/ml). Example 4 3800 ml of total broth at PH 5.6 was filtered under vacuum on a high flow super cell to obtain 2500 ml of filtered broth with MIC=1/2000. The PH was adjusted to 8.0 with the addition of sodium hydroxide and the broth was refiltered in the same way. This broth was extracted with butanol as described in the example above and the butanol extract was concentrated under vacuum to 1/20 of its original volume. The precipitated "crude antifungal substance" was filtered, washed with butanol, and dried under vacuum at room temperature.
A product with MIC=1/1666000 (0.60mcg/ml) was obtained. Dissolve the above crude antifungal substance in 500ml and make a solution of
Adjust the pH to 10.2 with sodium hydroxide and bring the solution to +
Stored at 1°C for 24 hours. After drying, the precipitated Ab400
131mg (6.6%) and its MIC is 1/10000000
(0.1 mcg/ml). Example 5 3750 ml of total broth at PH 5.9 was filtered on a High Flow Supercel to obtain a filtered broth with MIC=2500. The broth was adjusted to PH 8.0 as described in the example above, filtered and extracted with butanal. After concentrating the organic extract, MIC=1/
Crude antifungal substance with 2222000 (0.45mcg/ml)
1.9g was obtained. This crude antifungal substance was dissolved in 475 ml of water (PH 10.2) and after 24 hours at 1°C,
Ab400 146mg with 10000000 (0.1mcg/ml)
(7.7%).

[Brief explanation of drawings]

FIG. 1 is a curve diagram showing the infrared absorption spectrum of Ab 400 antifungal tetraene antibiotic obtained in the present invention.

Claims (1)

[Claims] 1 The following physicochemical properties: (1) Elemental analysis: C = 54.5%, H = 7.1%, N = 2.9%, O
(diff.) = 35.5% Empirical formula: C ₂₂ H ₃₅ O ₁₁ N (2) Molecular weight: 489.522 (3) In the ultraviolet spectrum, in methanol,
290, 304 and 318nm at 4mg/ml concentration of Ab400
shows the maximum absorption of (4) Infrared spectrum (in potassium bromide):
It has an infrared spectrum shown in FIG. (5) Solubility in solvents: Very soluble in dimethyl sulfoxide (DMSO), slightly soluble in H ₂ O and N-butyl alcohol. It is also insoluble in benzene ether, ethyl acetate and methylene chloride. (6) Distinction between base, acid and neutral: Chlorinated compound (7) Color of substance: White powder (8) Melting point: 220-225℃ (with decomposition)
A tetraene antifungal antibiotic named Ab400. 2 Streptomyces sp, strain SF-1 (ASA)
It is characterized by fermenting the medium by a microorganism called Ab400 and extracting a tetraene antibiotic from the mycelium and/or from the medium.
A method for producing a tetraene antifungal antibiotic named Ab400. 3. Aerating at a temperature of 25 to 30°C during the fermentation,
3. A method according to claim 2, wherein the base medium contains assimilable carbon and nitrogen sources and inorganic salts. 4 Using glycose, fructose, mannitol, glycerol, starch, dextrin, cellobiose, trehalose, mannose and mixtures thereof as carbon sources, and vegetable cake meal, corn, staple liquor, as storage sources.
4. The method according to claim 3, wherein plant or animal peptones, yeast, ammonium salts or mixtures thereof are used. 5 filtering the resulting fermentation broth, adjusting the pH to about 8.0, extracting water and concentrating under vacuum, thereby precipitating the antibiotic complex or crude antifungal substance;
This precipitate was dissolved in water at a rate of about 4 mg/ml, the pH of the solution was adjusted to about 10.2, and then by precipitation.
A method according to claim 2, 3 or 4 for obtaining a tetraene antibiotic named Ab400.