JPS5867189A - Polyene anti-bacterial antibiotic and production thereof - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the production of a polyene antifungal antibiotic, which has been previously unknown, by the enzymatic process of a novel organism belonging to the genus Streptomyces.
The present invention also relates to a novel tetraene antibiotic.

Description of the Prior Art Polyene antibiotics are substances whose basic properties include a structure containing a valvular double bond which gives them very interesting biological properties.

These compounds include, for example, amphotericin B (U.S. Pat. No. 2,908,611 and 2.908.6).
No. 12 Mei M), Philippine Complex (ph
ilippine complex) (UK Patent No. 788,486), Nyscutin (UK Patent No. 866,600), Pimaricin (also known as Tenesetine or Natamycin) (UK Patent No. 852)
, 888 and German Patent No. 1,024,20
Candicidin (U.S. Pat. No. 2,999)
2,162 specification), etc. These substances can be distinguished from each other by UV and IR spectra and by chromatography (paper, thin layer or high pressure).

Polyene antibiotics are ActinO
-mycetales), especially the genus Streptomyces and Streptovertisium
ticillinm) microorganisms.

Usually, biosynthesis of these compounds does not take place, but the appearance of several polyenes in one and the same medium is observed.

The mechanism of action in eukaryotic cells is one that operates on membrane sterols, resulting in cell collapse when the selective permeability conferred by these sterols is altered.

Polyenes are widely used in the pharmaceutical field, and although the toxicity of these polyenes is due to intravenous administration, since polyenes do not have any particular toxicity, they are mainly used topically or orally.

However, the recent creation of polyenes as interesting substances has led to the discovery that there are some differences in their effects on membrane permeability in tumor cells and normal cells. It is. Another interesting fact has also been established that blood cholesterol levels are reduced when these compounds (candicidin) are administered orally. Additionally, certain polyenes are known to act on enlargement of the prostate gland. This has been confirmed by studies of the microorganisms that produce these substances.

SUMMARY OF THE INVENTION In the present invention, we describe the production of several polyene substances with particular antifungal activity, of which the applicant has named Ab 4 (+.0) and whose main biological properties have been described. have the property of inhibiting the growth of fungi and yeasts, and this property makes these polyene substances suitable for use in the treatment of infections caused by pathogenic microorganisms (belonging to the above-mentioned classes) in animals and plants. I can do it.

Ab 400 was produced in a culture medium of aerobic bacteria belonging to the genus Streptomyces, and the product was transformed into a culture of aerobic bacteria belonging to the genus Streptomyces.
p, and the strain was named 5F-1 [MuSA]. Another property of this microorganism is that it produces other polyene substances, the most prominent of which is the already known tetraene called pimaricin.

In addition, in the present invention, extraction of the above-mentioned substance and mg
, and the antifungal spectrum of Ab 400.

Characteristics of the microorganisms The microorganisms used were collected from the vicinity of Madrid, in a collection belonging to the Antibiotics Societa Anonyme, and in the National Collection of Industrial Bacteria, Trey Research Station, Aberden, Scotland, in ANCIBII, F88.
NOIB).

Shike 7. (5ykes) and Skinner (5kin
rler) (1978), Academic Press, ``The Act.
The guidelines shown in the inomycetales include:
As a result of the confirmation and conclusion of the toxicity of the microorganism, the organism is not spiral-shaped or sporangium-shaped, but has an aerial mycelium with chain-shaped sporophytes that contain many spores. It is reported that it belongs to

“International Streptomyces Project J-ISP-(Int, J, 5ist,
Baote-riology16: ala ~4
Shirling at 0,1966
The method described by O and Gottlieb describes the characteristics of the species. Under phase contrast microscopy, microorganism 5F-1 (ASA) is 15P-a
and 1 to 6 somewhat dense spirals in l5P-4 medium and 10 or more 0.8
Spiral sporophyte with chains of ovoid spores? In the 0ISP-2 and 15P-Is media which were observed to have these sporophytes do not show true spirals, but sometimes when one spiral is photographed up close! & Dense hooks observed 0
The spores showed a warty surface under electron microscopy.

The characteristics of the cultures in different media are shown in Table 1.

All cultures were performed at 28°C. Color can be determined in two ways: Ea by Mr. E. Seguy, P. Leche
valiez (Paris) (1986)
, r (7ode Universal de
couleurs J (7) C) and the regular names for colors, and the method of naming these subjects.

Nitrogen (N) i (7) assimilation subek) st (assim
Ilat soil spectrum) are shown in Table H. 1
% glucose and 1.5% agar (Ludemann,
Mr. M. (LuedemannG, M.), r In
t, J, 5ist, Baoteriol, 21
:240-47 (1971) J) was used as the base medium.

Schilling E assimilation spectra of carbon (C) sources.

B, and Gottlieb D. (Int.

J, 5ist, Bacteriol, 16:
31 B-340 (1966)). The results are shown in Table ■.

Physiological properties are shown in Table ■.

In summary, the strains in Kenshin had the following phylogenetic characteristics.

Table ■ Elephant assimilation control-1------------NH4No8-
-----------N-Z amine A -------
−−−+Yeast extract −−−−−−−−−−++
÷+asparagine =−−−−−−−+
＋＋＋Glutanoic acid −−−−−−−−
±Qin Table for 14 days at 28°C Table carbon source Growth Qin control −−−−−−−−−−−−−−−−−−−−−D-Glucose −−−−−−−−−−− −＋＋Sucroseーーーーーーーーーーーーーーーーーーーーーー１ ±Inositol −−−−−−−−−−−−−−
±D-7 Lactose −−−−−−−−−−−＋＋Raffinose −−−−−−−−−−−−−−
±L-arabinose ------------D-xy4-su-------D-mannitol
−−−−−−−−−−−＋＋L−Rhamnose −1−−
−−−−−−−−D-galactose −1−−−−−−
--- ±D-arabinose ---
−−−−−−−α-Melibiose −−−−−−−−−
−−−Glycerol −1−−−−−−−−−−Hao
++β-lactose −−−−−−−−−−
−−−Starch −−−−−−−−−−−−−−−−
+Table 1 (Continued) D-Ribose −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−10trehalose−−−−−−−−−−−−−−−−−−−−−−−−−− ”””−−”−−−−−−＋＋Sorbose −−−−−−−−−−−−−−−−−1−Sorbitol−−−−−111−−−− −−Mannose −−=−−”−””−”−”−−−−＋＋Dulcitol −−−−−−−−−−−−−−−−−−−Melezitose −−−−−−−−− ------------1≧ Shu 1 British Patent No. 846,938 (1960) 2 Schilling E, B, and Buto 91100 "Eng. nt.

J, 5ist, Bacteriol, Jl 8
: 69-189 (1968) 8, Schilling E, B, and Tetraene IL Mr.
Int.

J* 5ist, Bacterol, JAS! :
265-394 (1972) Arc The results obtained by this laboratory a) belong to the spiral part.

b) It has spores with a warty surface.

C) Does not produce melanoid pigments.

d) The color of the sporulated aerial mycelium is either violet or red depending on the medium used. 08) It produces a soluble red (reddish brown) pigment that does not act on pH indicators.

f) The color of the substrate mycelium changes from reddish chestnut to violet chestnut depending on the medium.

g) Neglucose, which assimilates the following materials as a carbon source:
-Glycerol-inositol
- Starch - 7-lactose - Cellobiose - Mannitol - Trehalose-galactose
-mannose sucrose (5uchr, ose
) and 7/-s assimilated is not clear.

h) Other physiological properties: L<results in significant hydrolysis of latin.

1. Reduce nitrate to nitrite.

& Does not hydrolyze milk casein.

Withstands up to 44% Na0j.

L) lV filtrate (mesophyl 0).

i) Generates several tetraene antifungal antibiotics.

``Pergay's Handbook of Mycology (Be3rl)
lf9fB 1annualor D6termina
1=-ye BaCt8riOIOgy ) J
(1974, 8th edition, The Williams and Wilkins Company), and “Cooperative Description of Streptomyces spp.
Description of Type 5trai
ns of StreptomyCes) The paper ``International Streptomyces Project'' published by Schilling and Gotliep in J.
Int, J, 5ift, Bacteriol Co 18:69-189.1968: Ibid. 18 + 2
! 79-892.1968: Ibid. 19:891-51
2.1969: and the same document 22:265-894.1
972) and the phylogenetic characteristics of Streptomyces species in these documents published from 1974 to the present, no microorganisms similar to 5F-IAsA) could be found.

compared. The results of this study are shown in Table V and confirm that Streptomyces sp. SF-1 (ASA) is different from other pimaricin products.

The microorganisms described here are wild strains, but also naturally occurring mutants and mutagens.
mutants derived from kT-, together or separately, can give rise to a group of antibiotics. For this purpose, the wild-type strain and its 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 amounts of the compound. In this liquid medium, antibiotics can be produced by surface culture and submerged culture. In particular, in-liquid i@culture is ideal for producing large amounts of product.

The medium used contains at least the following materials: a) Assimilable carbon sources such as glucose, 7-lactose, mannitol, guar, wal, starch, textrin, cellobiose, trehalose, mannose and mixtures thereof.

b) Assimilable carbon sources, e.g. vegetable cake meal (soybean,
(cotton, crushed nuts, etc.), corn stay-7' IJ car, vegetable or animal peptones, yeast, urea, ammonia algae, etc., and mixtures thereof.

C) Inorganic IJ that produces a medium with pH buffering capacity
Inorganic salts that supply type i or inorganic elements.

d) Vegetable or animal oils used as foam suppressants or as carbon sources. silicon.

The I)H fermentation range that can be used in the method of the present invention is 6
．． ranges from 1 to 8.4, and the optimal temperature is 25 to aO℃
is within the range of

The tetraenes of the present invention can be made by fermentation using a regular antibiotic fermentation process.

That is, the strain sporophyte was lyophilized to 5 pores (1yOphilised 5pores) in a tilted tube containing the medium.
After inoculation and culturing at the optimum temperature, the growth phase in submerged culture is initiated with spores and mycelia from such tubes.

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 flasks, the flasks are thoroughly shaken on a suitable shaking table in a thermostatically controlled chamber; when fermenting in tanks, the tanks are vented, stirred, and stirred to achieve optimal levels of production. pH control, dissolution 0. It is necessary to control, temperature control and add nutrients, alkalis and acids. 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 started, sufficient mycelium was generated in the medium for 48 hours and the production gradually increased until day 5 or 6 of fermentation, then the increase stopped.

In order to study the antibiotic spear in the pros as a product factor for Ab 400, a single active substance was shown on paper and high pressure liquid chromatography, and Sacca! Mrs. Cereviche - 0.5μg/for ATOO9767
A test shoulder with a minimum inhibitory concentration of - was used. Koninklisike, Delft, Netherlands, as a pimaricin standard.
Koninklijke Nede Schist and Spirits 7 App Tour
Rlandsche G15t-acSpiri
900 made with tusfabriek N, V, )
A sample of U/wI9 (batch 9117A) was used.

We conducted a comparative study on the presence of Ab400 in a patented microbial process, which was not described, to confirm whether this substance was produced during fermentation in a conventional culture medium. .

The results are shown in Table ■. From Table 1, it can be seen that the strain of the present invention is clearly different from the well-known pimaricin-producing strain that has already been patented, since Streptomyces sp. 5F-1 (ASA) produces large amounts of Ab 400 and pimaricin. It can be seen that in all the media tested, Ab 400, the former of the two strains mentioned above, was not produced as quickly as it could be detected. Furthermore, such 5F-1 (A
SA) A different patent of the strain is that it produces a strong apple odor in the fermentation process.

Table II SFMF-13 medium contains 4% soybean flour, 6% glucose, 0.5% (NH, N, aSO4, 0.18%,
Contains HPO4,gH,O, 1% caco, and 0.5% soybean oil, and has a pH of 6.2.

The medium known as MOH-1 has a British patent @ssz
, SSA specification, 2% glycerol, 0.
Contains 5% phytone, 0#5% peptone, 0.8% yeast extract, 0.3% meat extract and 0.25@caco, and has a pH of 7.6. Also, MFNa-1
The medium known as
06 specification, 5% soybean flour, 1% glucose, 0.5% (NH, 5,, 'o, 0.02% KH, P
04.1% 0aOOB, 0.5% soybean oil and 0.1%
Contains corn stave liquor and has a pH of 6.8.

Extraction and separation After one fermentation, the minimum inhibitory concentration (
MIC) was generally 172,000 for Satsukalomi Shichisu Shichishi Vishie.

H3/flo-8uparcall all the liquids with a pH range of 5 to 6.
The filtered prosthesis 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 l/l of the initial volume.
20, thereby precipitating a white powder, which is referred to as "raw antifungal material". The precipitate was filtered and dried under vacuum at room temperature. The substance is an extremely pale yellow powder, and this MIO is about 1/1.5 that of the yeast mentioned above.
fl O,000 (0,67mcg7'az). The yield of this product is 1! Hit 0.
It was 7g.

Such "crude antifungal substances" are relatively water soluble and have at least 20% active against Saccharomyces cerevisiae when prepared by paper toweling.
It was confirmed that the main antibiotics of various types existed. 〇 "Crude antifungal substance" was dissolved in water at a ratio of about 4 to 1/-.
Adjust this solution to pH 10.2 and release to +1"C.
(over 24 hours), the crude antifungal substance was known as “antifungal 1 J”/10,000,000 (0,1H
Ab 400 with a MIO of
A white substance corresponding to . This antifungal substance l or Ab 400 is 6.6 of the initial “crude antifungal substance”.
The UV spectrum of "Antifungal" in methanol, obtained in excellent yield, shows that the material is a tetraene, and the infrared spectrum in potassium bromide, although not identical, shows that the tetraene antifungal pimaricin. Pimaricin was shown to be similar to butanol + ethanol + water (5:471
, V) has a lower Ry in the chromatographic thread than "Antifungal 1".

Li0rosorp rp-18-column (Li0rosor
brp-18-column) (5 μn, 0.0
6 M <ammonium citrate mobile phase, PH5,0+
"crude antifungal" using acetonitrile (8:1 v/v) and high pressure liquid chromatography (HPLC) at 800 nm, class 74 substances with antibiotic activity against Satucharomyces cerevisiae and tetraene UV spectrum It was confirmed that

Elemental centesimal composition of "Antifungal Substance 1" (elem
entry centisimal composition
) is C54.5%, H7.1%, N2.9%
and O(dir,)85.5% C□H,40□,N
gave the minimum empirical formula of , and the minimum molecular layer was 488.522.

Antibacterial Activity Ab 400 was tested for antibacterial activity. Test results showed activity against bacteria and yeast.

There was no siteip activity: Candida albicans (trop
icalis) Next, the invention will be explained by way of example.

Example 1 A medium was made from the following composition: Cottonseed flour.
20g Corn Steep Liquor ・・・・−・
10 g dextrin・・・・・・・・・・・・
・・・10g glucose・・・・・・・・・・・・・
...N 50g (NH4)2SO4...
・・・・・・・・・ 5gNaN0 ・・・・
・・・・・・・・・・・・・・・ 8.5g, H
PO4・・・・・・・・・・・・・・・・・・ 1
gF8S0. 7HO・・・・・・・・・・・・・・・
0.05 gm MgSO4,7H,0・・・・・・・・・・・・・・・
0.05 gMnSo4. H, O-・-------・
−・・−・0.05 gZnSo, 7HO・・・・
・・・・・・・◆・・ 0.1gm Distilled water ad ・・・・・・・・・・・・・・・・・・
1ooov pH was adjusted to 6.2 with KOH.

Each 200-liter medium was placed in each 1,000-liter Erlenmeyer flask.

19 of OaC01 was added to each flask.

Each flask was sterilized at 121°C for 80 minutes.

The medium thus prepared was inoculated with a spore suspension of Streptomys sp., strain 5F-1 (ASA). This suspension was made from slant tube cultures on any solid medium that sporulates microorganisms.

11+ diameter, this medium was subjected to 5 C11 steps and 24 O
The cells were cultured for 5 days in a chamber controlled by a thermostat at 8-8° C. while being constantly vibrated on an orbital vibration table with a speed of rpm. The innoculum thus prepared was used to inoculate a fermentation medium consisting of the following composition: Soybean flour °°°°°°°°0°""' 40
g glucose...11■...60
g(NH4n, S04...
5 gK, HPO4・・・・・・・・・
...... 18 g CaOO8....... 10 g distilled water ad mouth...
...---10001141) H was adjusted to 6.2 with dilute sulfuric acid before adding charcoal #1M.

200ml of sodium each! I put it in a flask. 1 - of soybean oil was added to each of these flasks. Each flask was sterilized briefly at 121°C for 80 minutes.

After sterilization, the medium was inoculated with a 5% inoculum.

This was incubated for 4 days at 28°C on a vibrating table with the same characteristics as described for the inoculum in a chamber. The analysis of the broth at the end of the fermentation is as follows: M5.5-6.5; Sugar 2%; Ammonium N: 100 @ 9/l: Total soluble N: 950
"9/ '; and bacterial volume (mycelial v
olume) II 32゜Has a minimum inhibitory concentration of 1/4000 to 115000 against S. cerevisiae-ATOO9767; and 900 U/■ as standard in HPL (E)
300-500 mcg/m of Ab using pimaricin
400 activity and pimaricin activity of 1000-12000 mCg/-.

Example 2 A medium was prepared from the same composition as the inoculum of Example 1, with a ratio of 200-1! The medium was sterilized in Ellenmeyer flasks at 21°C for 80 minutes.

The medium thus prepared was inoculated with a spore suspension of the proprietary microorganism. This medium was added for 5 (it steps and 24
Cultures were incubated for 5 days at 28° C. in a thermostatically controlled chamber with constant vibration on an orbital vibrating table with a lifting speed of Orpm.

The inoculum thus prepared was used to inoculate an 81 g fermentation tank containing medium 11 having the same composition as the fermentation medium of Example 1 at a rate of 0.5%.

The following fermentation conditions were used: temperature 28°C, aeration 0.8v/v
/m, stirring 40 Or, p, m, and fermentation time 9
0 hours. At this time, the fermentation medium is 500-1100 Ona
Pimaricin activity of /- and 800-600 mcg /
- had lb activity. The same type of standard was used as in Example 1.

Example 8 pHIS, 9T8540- whole proso
Passed under vacuum on Purcel, MIO-1/2,2
Reddish transparent p-transparent with 22,150
I got -. Adjust pH to 8.0 with 25% sodium hydroxide.
The fluff-like precipitate was separated from house to house in the same manner as in the above example.

In the first extraction, the organic phase was centrifuged to obtain p-perpros with 700-butanal. The aqueous phase was extracted twice with 350-butanal as described above. The total clear yellow I/) butanal extract was concentrated under vacuum to an initial volume of 1720 resulting in the precipitation of a white powder, washed with butanal after filtration and dried under vacuum at room temperature, 1.52 g
(i.e., 0.719 per f perpros 11), and this substance is MIO-1/1,450
°000 (0.69 mcg/m).

The product was dissolved in 880-m 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 yield 101 ■ of antifungal substance 1; 6.7% of the starting "crude antifungal substance". The MIO of this antifungal substance is 1/1
It was 0,000,000 (o, 1mcg/vd).

Example 4 A total broth of 8,800 at pH 5,6 was passed under vacuum over a Via 0-Supercell and MMC-1/2!0
A strained broth of 2,5QQmj with 0.00 was obtained.

The pH was adjusted to 8.0 by the addition of sodium hydroxide,
The broth was refiltered in the same way. This gloss 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 material" was filtered, washed with butanol and dried under vacuum at room temperature to give MIO-1/1,6
A product with a concentration of 66,000 (0,60 mcg//Lt) was obtained.

Dissolve the above crude antifungal substance in 500@j and adjust the pH of the solution.
was adjusted to 10.2 with sodium hydroxide and the solution was heated to +1°C.
It was stored for 24 hours. Dry, precipitate antifungal substance 1 is 1
81m119 (6.6%), its MIC is 1/
It was 10,000,000 (0,1' conductive Og/m).

ヱ1L pH 5,9, 3+750WLt whole proso via tf
Filtration over f-Supercel yielded p-filtrate with MIO-2,500. The broth was adjusted to pH 8.0 as described in the example above, filtered and extracted with butanal. Concentrated organic extract, MIO-1/2,
1.9 g of crude antifungal substance having a concentration of 222,000 C0.45 mcg/v4) was obtained. This crude antifungal substance
After dissolving in water (pH 10,2) at 24°C at 1°C.
1/10,000,000 (0.1mog
/ ml) of antifungal substance 1 146 mg (7
, 7%).

Patent applicant Antipioticus. Sochete Anonymous Page 1 continued 0 Inventor Jose María Fernández Saucerjuaro Villanueva de U Cañada (Madrid), Spain 66

Claims (1)

[Claims] 1 Streftomyces sp, strain SF-1 (ASA)
1. A method for producing a polyene antifungal antibiotic, which comprises fermenting a medium using a microorganism known as a microorganism, and extracting the antibiotic from the mycelium and/or the medium. & In the fermentation, aeration is carried out at a temperature of 25 to 80°C,
2. The method for producing a polyene antifungal antibiotic according to claim 1, wherein the medium contains an assimilable carbon and nitrogen source and an inorganic salt. & Using glucose, lactose, mannitol, glycerol, 'll1mC textrin, cellobiose, trehalose, mannose and mixtures thereof as carbon sources and vegetable cake meal, cornstap liquor, vegetable or animal peptones as nitrogen sources ,
3. The method for producing a polyene antifungal antibiotic according to claim 2, using yeast, urea, ammonium salt, or a mixture thereof. Table Patent for filtering the resulting fermentation process, adjusting the pH to about 8.0, extracting the water and concentrating under vacuum, thereby precipitating the antibiotic complex or crude antifungal substance and separating pimaricin from it. A method for producing a polyene antifungal antibiotic according to claim 1, 2 or 8. 5. Filter the resulting fermentation broth, adjust the pH to about 8.0, extract the water and concentrate under vacuum, thereby precipitating the antibiotic complex or crude antifungal substance, and pouring this precipitate into water. Dissolve at a ratio of about 4s9/- and adjust the pH of the solution to about 1O02
A process for producing a polyene antifungal antibiotic according to claim 1.2 or 8, wherein the tetraene antibiotic designated as Ab 400 is obtained by precipitation. & Filter the resulting fermentation process, adjust the pH to about 8.0, extract the water and concentrate under vacuum, thereby precipitating the antibiotic complex or crude antifungal substance, and pouring this precipitate into water. Dissolve at a rate of approximately 4s9/gLt, and adjust the pH of the solution to approximately 10.
．． 2 and then obtained by precipitation 0 54.5%,
A with an elemental centesimal composition of H7.1%, N2.9% and O35.5% and a minimum inhibitory concentration of 1/10,000,000 against Sacca 4.
A tetraene antifungal antibiotic named b400.