FR2511034A1 - PROCESS FOR THE PREPARATION OF POLYENIC ANTIFUNGAL ANTIBIOTIC AND NOVEL TETRAENIC ANTIBIOTIC - Google Patents

Abstract

THE INVENTION CONCERNS A PROCESS FOR OBTAINING POLYENIC ANTIFUNGAL ANTIBIOTICS, CHARACTERIZED IN THAT A FERMENTATION OF A CULTURE MEDIUM IS CARRIED OUT BY MEANS OF A MICROORGANISM CALLED STREPTOMYCES SP. CEPA SF-1 (ASA), BY EXTRACTING THE SAID ANTIBIOTICS FROM MYCELIUM AND / OR THE CULTURE MEDIUM.

Description

The present invention relates to a process for obtaining

  polyenic antifungal antibiotics, some of which

  unknown until this date, by means of a process of iron-

  ment of a new microorganism belonging to the genus Streptomyces.

  The invention also relates to a new anti-

tetraene biotic.

  Polyene antibiotics are substances whose fundamental characteristic is to have a structure with conjugated double bonds which give them

  some very interesting biological properties.

  Examples of these compounds include amphotericin B (US Pat. Nos. 2,908,611 and 2,908,612), the philipine complex (English Patent No. 783,486), nystatin (English Patent No. 866,600), pymaricine , also called tennecetin and natamycin (English patent no. 852,883 and German patent no. 1,024,206), candicidin (American patent no. 2,992,162), etc. These substances can be differentiated from each other by their UV and IR spectra and by Chromatography (paper,

thin or high pressure).

  Polyene antibiotics are the main product

  palement by microorganisms of the order of Actinomycetales in particular by Streptomyces and Streptoverticillium The biosynthesis of these compounds is not usually carried out unequivocally, but we observe the appearance of various

  polyenes in the same culture medium.

  The mechanism of action on eukaryotic cells consists in acting on the sterols of the membranes, causing the lysis of the cell until altering the selective permeability

that these sterols give it.

  The use of polyenes in the field of medicine is important, although given their toxicity by the intravenous route, their main application was for local use, or by oral route since then the

toxicity is practically zero.

  What makes polyenes substances of current interest is the discovery that there is a difference in action on the permeability of the membrane of tumor cells and normal cells Another interesting fact is that they cause a decrease blood cholesterol levels when some of these

  compounds (candicidins) are administered orally.

  We also know that certain polyenes have an action against an enlarged prostate. This is why we undertook

  to look for microorganisms producing these substances.

  Concretely, the present invention describes the production of various polyenic substances having an antifungal activity among which stands out a new one, called by the applicant Ab 400, whose characteristic

  main biological is to inhibit the growth of

  gnons and yeasts so that it can have a

  useful in the treatment of infections due to micro-

  pathogenic organisms (belonging to the classes mentioned)

  both in animals and plants.

  The substance Ab 400 is produced in the culture medium by an aerobic germ belonging to the genus Streptomyces whose denomination is Streptoinyces sp cepa

  SF-1 (ASA) In addition, a characteristic of this microorganism

  me is the production of other polyenes among

  which stands out for its quantity, the tetraene already

  nude named pimarycine (see notes above).

  It is also described the extraction and the ident-

  tification of these substances, as well as the anti

of the substance Ab 400.

  The microorganism used was isolated from a soil sample from the vicinity of Madrid, and deposited in the collection of ANTIBIOTICOS SA in Leon, under the name of Streptomyces sp cepa SF-1 (ASA) and in the collection " NATIONAL COLLECTION OF INDUSTRIAL BACTERIA "(NCIB), Torry Research Station, Aberdeen, Scotland, under n NCIB

11,738.

  For its taxonomic characterization we follow the indi-

  cations which appear in "The Actinomycetales" of SYKES

  and SKINNER (1973) Academic Press, arriving at the conclusion

  that this microorganism belongs to the genus Streptomes, since it presents an aerial mycelium with sporophores

  with multiple spores in a chain, which are neither verti-

  blinked or sporangiated No fragmentation was observed

mycelium.

  For the characterization of the species we followed the

  processes described by SHIRLING and GOTTLIEB in the "Internatio-

  nal Streptomyces Project "-ISP (Int J Sist Bacteriology 16: 313-40, 1966) Under a phase-construct microscope, it is observed that the microorganism SF1 (ASA) in the ISP-3 and ISP-4 media presents the sporophores in the form spiral with from 1 to 6 more closed loops, with cats of more than 10 oval spores of 0.8 x 0.5 p

  ISP-2 and ISP-5 these sporophores do not present truth

  spirals, but we can clearly observe more or less closed hooks which on certain occasions manage to close in a turn The spores observed under the electron microscope

  have a warty surface.

  The characteristics of the cultures in the various environments are presented in Table I. All the cultures are done with 28 C The designation of the colors is done in two ways, the first according to the plates of the "Universal code of colors", of E SEGUY (1936 ) éd P LECHEVALIER (Paris) and the second by giving them names of subjective forms, using the usual names

for these colors.

  The assimilation spectrum of nitrogen sources (N) is given in Table II; as the basal medium, 1% glucose and 1.5% agar are used (LUEDEMANN GM Int. J Sist Bacteriol 21: 240-47 (1971). The assimilation spectrum of carbon sources is made according to the technique by SHIRLING EB and GOTTLIEB D. (Int J Sist Bacteriol 16: 313-340 (1966) The results

are given in Table III.

  The physiological characteristics are given in Table IV. In summary, the strain studied has the following taxonomic characteristics:

TABLE 1

MORPHOLOGICAL CHARACTERS

  AND PHYSIOLOGICALS OF STREPTOMYCE CULTURES Sp SF-1 (ASA) Ref Medium Duration Growth Aerial mycelium Mycelium substrate Pigment Observations (days) soluble 1 ISP-2, 21 +++ orange 180 red 116 strong, growth agar to purple brown blackish brown velvety brown , whitish malt no sporulation abundant yeast indicator of p H ISP-3, 21 ++ violet 675 violet 687 very growth agar with violet gray light chestnut light velvety dark flour good madder oat sporulation

_ _______ __ _______-

  1 ISP-M, 21 ++ violet 675 violet 687 little growth agar of violet gray brown abundant velvety dark salts madder sporulation of regular starch 1 ISP-5 21 violet 675 yellow 312 strong growth agar purple gray black black velvety glycerol dark greenish no of abundant asparagine indicator sporulation ______ _____________ _______ of p H Jn 'Ln W TABLE 1 (continued) MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF STREPTOMYCAL CULTURES Sp SF-1 (ASA) Medium Duration Growth Mycelium Mycelium Pigment Observations (Days) soluble substrate brown red red growth 707 no velvety dicator few spores Agar of 14 ++ black purple purple 641 of p H If one Bennet clear black of adds CO Ca,

with CO Ca bougie le nb de âpo-

  (0.1%) 3 res increases Agar 14 + __ _ Czapek growth very little (sucrose) abundant Agar 14 ++ cream negative cream Emerson growth grainy with CO 3 Ca

(0.1%)

  Agar 14 ++ red 41, purple 641 positive sporulation

  glucose brown black of red little abundant

  asparagine reddish dante candle beef extract Agar 14 +++ violet 606 violet 641 strong lcroissance glucose gray violet black of Pas in veloutée extract of yeast indicator candle color with red phosphate Ref. r%) Ln -.1 ui TABLE 1 (end) MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF STREPTOMYCE CULTURES Sp SF-1 (ASA) Medium Growth r i t t Duration (Days) Agar at 14

the tyro-

  sine granular growth N 172 14 ++ little little ATCC abundant sporulation Agar 14 +++ red 87 red 87 positive not dark gray hydrolysis milk 1 SHIRLING, EB & GOTTLIEB, D Int J Sist Bacteriol 16: 313-40 ( 1966) 3 WAKSMAN, SA "The Actinomycetes", Vol 2 (1961)

  4 GORDON, R E & SMITH, M M J Bacteriol 69: 147-50 (1955).

  Catalog of the "American Type Culture Collection", (ATCC), p 518 (1980)

  6 LUEDEMANN, G M, Int J Sist Bacteriol 21: 240-7 (1971).

  Lt CD o Ref. Aerial mycelium s Yu c se tl ri ua mt Soluble pigment Observations

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TABLE II

ASSIMILATION OF N SOURCES

  BY STREPTOMYCES sp SF-1 (ASA) Witness

NO 3 NH 4

  N-Z amine A Yeast extract Asparagine Glutamic acid Growth + ++ + + Soluble pigment produced ++ + 14 days at 28 C

TABLE II

ASSIMILATION OF SOURCES OF

  C BY STRIEPTOMYCES sp SF-1 (ASA) Source of C Control Growth a * * * Dglucos e ++ Sucrose + Inositol * + D-Fructose * * + Raf finose a + LArabinose * D-Xylos e ** Dl-Man nitol *** ++

11-Rhamnose -

  D-Galactose * + D-.Arabinose GL-Melibios e Glycerol * * + 0-Lactos eo kmidon o * e * + D-Ribose ** Cel lobiose * ++ Trehalose * ++ Sorbos e * * Sorbitol TABLE III (' end)

ASSIMILATION OF C SOURCES

  BY STREPTOMYCES sp SF-1 (ASA) Source of C Mannose Dulcitol Melecitosis

21 days at 28 C.

Growth ++

TABLE IV

  PHYSIOLOGICAL CHARACTERISTICS OF STREPTOMYCES sp SF-1 (ASA) Production of melanin pigments a) ISP-6 b) ISP-7 c) ISP-1 Hydrolysis of gelatin

Starch hydrolysis.

  Skimmed milk Reduction of nitrates Resistance to Na Cl Growth at various temperatures Response Reference negative negative negative strong slight hydrolysis negative positive 4% is the mesophilic limit At 14 Days a strong brown pigmentation occurs

dark purple.

  1 SHIRLING, E B & GOTTLIEB, D Int J Sist Bacteriol.

16: 313-40 (1966).

  2 WAKSMAN, S A "The Actinomycetes" Vol 2 (1961) The Williams

and Wilkins Company.

  3 GORDON, R E & SMITH, M M J Bacteriol 69: 147-50 (1955) 4 LUEDEMANN, G M Int J Sist Bacteriol 21: 240-7 (1971)

TABLE V

  TAXONOMIC DIFFERENCES BETWEEN PIMARYCIN PRODUCING STREPTOMYCES

  Phenotypic character St gilbosporeus St chattanoogensis St natalensis Streptomyces sp.

ATC_ 1335 (2) NRRL 2 6143

  ATCC-13 326 (1) ATCC-13358 NRRL 2 651 SF-I (ASA) (4

  Melanic pigment S negative negative negative Color of the aerial mycelium red? yellow or white gray or red red or purple Color of the mycelium substrate chestnut yellow not studied chestnut yellow chestnut yellow + red Soluble pigment, negative positive yellow negative positive red

Form of sporo-

  phores spiral spiral spiral spiral Spore wall shape spiny thorny warty spiny Nitrate reduction positive positive not studied positive Resistance to Na Cl no growth no growth

at 3% to 3%

  Use of carbon sources a) xylose ++ b) sucrose ++ + c) inositol + ++ + r ', Ln u

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1 English patent no 846 933 (1960)

  2 SHIRLING, E B and GOTTLIEB, D Int J Sist Bacteriol.

18: 69-189 (1968)

  3 SHIRLING, E B and GOTTLIEB, D Int J Sist Bacteriol.

22: 265-394 (1972).

4 Laboratory results.

  a) Belongs to the Spirals section.

  b) Has spores with a warty surface.

  c) Does not produce melanoid pigment.

  d) The color of the sporulated aerial mycelium belongs to the series

  purple or red, depending on the medium used.

  e) Produces a soluble red (reddish brown) pigment which is not indicative of p H. f) The mycelium of the substrate has a color which varies

  from reddish chestnut to purple chestnut, depending on the environment.

  g) Assimilates the following bodies as the sole source of carbon: glucose glycerol inositol starch fructose cellobiose mannitol trehalose galactose mannose The assimilation of sucrose and raffinose is doubtful. h) Has other physiological characteristics: 1 Strongly hydrolyzes gelatin 2 Reduces nitrates to nitrites 3 Does not hydrolyze milk casein

4 Resists up to 4% Na Cl.

Is mesophilic.

  i) Produces various tetraenic antifungal antibiotics.

  By observing the taxonomic characteristics of the species

  de Streptomyces present in "Bergey's Manual of Determi-

  E native Bacteriology "(8 edition 1974 The Williams and Wilkins Company), and studying the International Streptomyces Project" published by SHIRLING and GOTTLIEB in "Cooperative

  description of Type strains of Streptomyces "(Int J Sist.

  Bacteriol 18: 69-189, 1968; ibid 18: 279-392, 1968; ibid. 19: 391-512, 1969, and ibid 22: 265-394, 1972), as well as publications from the year 1974 to this date,

  encountered no microorganism resembling SF-1 (ASA).

  We compare in particular with Streptomyces which also produce piramycin, that is to say S natalensis, S Chattanoogensis and S gilbosporeus In Table V are exposed the results of this study, from which it is concluded that Streptomyces sp SF-1 (ASA) is

  distinct from other pymaricin producers.

  The microorganism described in this invention is

  the sylvan strain, but its mutants, obtained sometimes spontaneously

  in particular as much as 8 t induced by mutagens,

  can also produce the group of poly- antibiotics

  enics in conjunction or separately, which means that in the process described below, we can use

  both the silvestre strain and its descendants by muta-

  tion. The production of the tetraenes described in the invention can be carried out in solid or liquid media, although the latter are more suitable for obtaining large quantities of the compounds. In liquid media, antibiotics can be obtained both by surface and submerged culture, last means being the best for obtaining

a large amount of product.

  The production medium must be composed of at least: a) An assimilable source of carbon such as glucose, fructose, mannitol, glycerol, starch, dextrin, cellobiose, trehalose, mannose, alone or mixed

11034

  b) An assimilable source of nitrogen such as flour from vegetable doughs (soybeans, cotton, peanuts, etc.), corn liqueur macerated, vegetable or animal peptones, yeast,

  urea, ammonia salts, etc., alone or mixed.

  c) Mineral salts to confer a damping power of

  p H in the middle or to supply it with mineral elements.

  d) Vegetable or animal oils to be used as

  antifoam as well as carbon source Silicones.

  The interval of p H of fermentation with which we

  obtains a large production, from 5.1 to 8.4 The temperature

  optimum ture is between 25 and 300 C. To obtain the tetraenes by fermentation, which is the object of the invention, the usual processes are followed in the fermentation for antibiotics, that is to say, from lyophilized spores, inclined tubes filled with a medium in which the strain sporulates well, we incubate at the optimal temperature and with the spores and the mycelium coming from these tubes we begin the growth phase in submerged liquid culture These The phases are more or less large and more or less numerous according to 'The volume of broth that we want to ferment In the case where we use fermentation tanks of large volume,

  these phases could go up to 5.

  For fermentation in a flask it is enough to agi-

  on an appropriate agitator plate and in a climatic chamber

  infused; to proceed in tank, this one must include systems of aeration, agitation, regulation of p H, of

  regulation of dissolved oxygen, regulation of temperature

  rature and the addition of nutritive agents, bases and acids, so that the production is optimal All these processes must be carried out under conditions of absolute sterility to avoid contamination with foreign microorganisms The media can be sterilized in the containers, or they can be introduced beforehand sterilized,

  as long as the container is already.

  The production begins once there is a sufficient quantity of mycelium in the culture medium, which occurs from 48 h; this production increases slowly until the 5 th or 6 th day of fermentation, the increase

tion then stopping.

  To study the amount of existing antibiotic

  in the broth, we use a sample of Ab 400

  purified tillon which by paper chromatography and high pressure liquid (HPLC) indicates a single active substance and which, compared to Saccharomyces cerevisiae ATCC 9767 has a minimum inhibitory concentration of 0.5 pg / ml As a model of pimarycine we use a sample supplied by KONINKLIJKE NEDERLANDSCHE GIST & SPIRITUSFABRIEK NV from

  Delft, Holland, (Batch 9117 A) 900 U / mg.

  Although the existence of the substance Ab 400 has not been described in the broths of already patented microorganisms, a comparative study has been made between Streptomyces

  sp SF-1 (ASA), St natalensis NRRL-2651 and St chattanoo-

  gensis ATCC 13 358 to find out whether in fermentation in the media described by the patents and in one of those used in this patent, the said substance is produced The results are set out in Table VI In this one we can observe the clear differences between the strains already patented, which produce pimarycin, and that which is the subject of the invention, since while Streptomyces sp SF-1 (ASA) produces in all the media tested, in addition to pimarycin, a high quantity of Ab 400, in 'The two strains mentioned there is no production

  detectable of the latter substance Another characteristic

  differential tick of the strain SF-1 (ASA) is to produce

  in the fermentation broths a strong apple smell.

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BOARD

  VI PRODUCTION OF Ab 400 AND PIMARYCIN BY ST NATALENSIS, ST CHATTANOOGENSIS & ST SP SF-i (ASA) Medium Fermentation microorganism mcg / ml Pi mcg / mi marycin Ab 400 S sp SF-i (ASA) S natalensis

NRRL 2651

S chattanoogen-

at ATCC 13,358

(SFMF-3 N

MCH 1 x M Na 1 me MF Na-1

MCI {1

  (MF Na-1 i K The medium SFMF-3 contains 4% of soy flour, 6% of glucose, 0.5% of 504 NH) 2; O 13) of 00 HK 2; 1% CO Ca & 0.95%

soybean oil p H 6.2.

  The medium called MCH-1 is described in English patent NO 852 883 and contains 2% glycerol, 0.5% phytone, 0.5% peptone, 0.3% yeast extract, 0.3% extract

  meat and 0.25% CO 3 Ca p H 7.6.

  o o o O The medium called MF Na-1 is described in German Patent No. 1,024,206 under the name of D and consists of 5% soy flour; 1% glucose; 0.5% 504 (NH 4) 2; 0.02% P 04 H 2 K; 1% C 03 Ca; 0.5% So Ja oil and 0.1%

4 2 3

  maux liqueur macerated p H 6.3.

  When fermentation is considered complete

  born, the minimum inhibitory concentration (MIC) of the filtered broth is normally 1/2000, compared to

Saccharomyces cerevisiae.

  The total broth, the p H of which fluctuates between 5 and 6, is filtered through Hyflo-Supercell and the broth filtered

  is adjusted to p H = 8.0 using sodium hydroxide, in addition

  continuously with an immiscible aliphatic alcohol

with water like butanol.

  The organic extract is concentrated under vacuum until its volume is reduced to 1/20 th of the initial volume, whereby a white powder is precipitated, which is called "antifungal

  "We filter this and dry it under vacuum at room temperature.

  rature ambient The MIC of this powder, very slightly yellow, is of the order of 1 / 1,500,000 (0.67 mcg / ml) relative to the yeast mentioned The yield of this product is 0.7 g

per liter of filtered broth.

  The "crude antifungal" is relatively soluble in water and contains, at least, two main antibiotics, as found by chromatography on

  paper, active towards Saccharomyces cerevisiae.

  If the "crude antifungal" is dissolved in water at a concentration of about 4 mg / ml and if this solution is adjusted to p H = 10.2, leaving it to stand at + 1 for 24 hours, a white substance preciptiates which has a MIC of 1 / 10,000,000 (0.1 mcg / ml) which is called

  "antifungal 1" and which corresponds to Ab 400 indicated This anti-

  fungal 1 or Ab 400 is obtained with a yield of 6.6%

  if we refer to the crude antifungal "starting.

  The ultraviolet spectrum in methanol of the "anti

  fungal 1 "indicates that it is a tetraene and its infrared spectrum in potassium bromide is similar well

  that not identical to that of the tetraene antifungal pima-

  rycin. Pimarycin has an Rf lower than that of "antifungal 1" in a butanol + chromatographic system

ethanol + water (5: 4: 1, v).

  By high pressure liquid chromatography (HPLC) with a column of Li Crosorb RP-18 (5 u), mobile phase of 0.06 M ammonium citrate, p H = 5.0 + acetonitrile (3: 1, v) and detection at 300 nm, we identify in the "crude antifungal", 7 substances with antibiotic activity vis-à-vis

  Saccharomyces cerevisae and with ultraviolet spectra of tetraenes.

  We find that the elemental centesimal composition of the 1 "antifungal is C 54.5%; H 7.1%; N 2.9%; O (dif), 5%; which leads to the minimum empirical formula C 22 H 34011 N and therefore at a minimum molecular weight

488,522.

ANTIMICROBIAL ACTIVITY

  The antimicrobial activity of Ab 400 is studied, by observing the absence of activity with respect to bacteria and the positive activity with regard to yeasts: Candida nt and n C ry-pto cocecus n Dlebaryonyces I Geotricunm Hans enula I Pichia I albicans anomala dolonji desusei Melinji pelliculosa pulcherrimna tropi calis albidus difluen s neoformaris hansenii kiaockeriii versiformne Ps eudopelliculosa suaveol ens ps eudopoimio rfa mienbranaefaciens

EXAMPLE 1

  A medium is prepared having the following composition: Cottonseed flour 20 g Macerated mals liquor 10 "Dext; rine * * * 10 n Glucose 50"

4 (4) 2 5

N 0.Na 2.5 g

4 4 2

J

PO 4 HE 2

  504 Fe 7 H 20 0.05 g 504 Mg7 H 20 0.05

504 MN H 20 0.05

  SO 4 Zn 7 H 20 o X 0.1 "Distilled water q sop 1000 ml

The p H is adjusted with KOH to 6.2.

  The medium is distributed in 1000 ml Erlenmeyer flasks

200 ml each.

  1 g of CO 3 Ca is added to each Erlenmeyer flask.

  Sterilized at 121 ° C for 30 minutes.

  The medium thus prepared is inoculated with a suspension.

  Zion of spores of Streptomyces sp Cepa SF-I (ASA) This suspension is prepared from a culture in an inclined tube in any solid medium in which one can spore

the microorganism.

  Once this medium has been sown, it is incubated for

  for 5 days in an air-conditioned room at 28 ° C., and stirring continuously on an orbital shaker plate with an eccentricity of 5 cm and a stirring speed of 240 rpm The inoculum thus prepared is used to seed the fermentation medium, which has the following composition: Soy flour 40 g Glucose 60 "

SO 4 (NH 4) 2 5

  PO 4 HK 2 a a 13 3 3 CO 3 Ca ,, 10 n Distilled water q s p 1000 ml Adjust the p H before adding the carbonate, to

  6.2 with dilute sulfuric acid.

  We distribute in 1 liter flasks

200 ml of medium per flask.

  1 ml of soybean oil is added to each balloon.

  Sterilized at 121 ° C for 30 minutes.

  the medium, once sterile, is seeded at 5% with the inoculum. We incubate at 28 C for 4 days, in a

  chamber and on an agitator tray having the same characteristics

  as described for the inoculum.

  Analysis of the broth at the end of fermentation indicates: p H 5.5 6.5; 2% sugar; N ammoniacal: 100 mg / l; Total N

* soluble: 950 mg / l and volume of mycelium: 32.

  Opposite Sachh cerevisiae ATCC 9767 it has a minimum inhibitory concentration of 1/4000 to 1/5000; by HPLC it exhibits an Ab 400 activity of 300-500 mcg / ml and of pimarycin from 1,000 to 1,200 mcg / ml, using, as a model, pimarycin having a power of 900 U / mg /

1,1034

EXAMPLE 2

  A medium is prepared having the same composition as the inoculum of Example 1, and which is distributed in 1 liter Erlenmeyer flasks, at the rate of 200 ml of medium. It is sterilized at 121 ° C. for 30 minutes. The medium is seeded thus prepared with a suspension of spores of the microorganism which is the subject of the invention. Incubate for 5 days at 28 ° C. in an air-conditioned chamber and with continuous shaking on an orbital shaker plate with an eccentricity of 5 cm and

a stirring speed of 240 rpm.

  The inoculum thus prepared is used to seed 0.5% of 8-liter fermentation tanks with 4 liters of medium of the same composition as the fermentation medium.

  from example 1 The fermentation conditions are as follows

  temperature: 28 C, ventilation 0.3 v / v / m, agitation

  400 rpm, fermentation time 90 hours The middle of the farm-

  At this time, pimarycin activity of 500-1000 mcg / ml and Ab 400 activity of 300-600 mcg / ml are used.

  the same type of models as in Example 1.

EXAMPLE 3

  3540 ml of total broth of p H 5.9 is filtered through a pre-layer of Hyflo-Supercell, obtaining 2 150 ml of transparent filtered broth of reddish color and MIC = 1/2 222 It is adjusted to p H 8.0 with sodium hydroxide and the Kde flake precipitate is filtered in the same way, and is discarded. A first extraction of this filtered broth is carried out with 700 ml of butanol, centrifuging the organic phase. The aqueous phase is subjected to two other extractions with 350 ml of butanol each time, in the same manner as above h concentrates under vacuum the total butanolic extract, transparent yellow, until reducing its volume to 1/20 of the initial volume, whereby a white powder is precipitated which, once filtered, washed with butanol and dried under vacuum at room temperature weighs

  1.52 g; or 0.71 g per liter of filtered broth This "anti

  crude fungal "has an MIC = 1/1 450,000 (0.69 mcg / ml).

  The product is dissolved in 380 ml of water and

  adjust this solution to p H 10.2 with sodium hydroxide.

  After 24 h at + 1 ° C., the flaky precipitate is filtered,

  washed with water and dried under vacuum at room temperature

  ambient ture, obtaining 101 mg of antifungal 1; 6.7% by

  compared to the starting "crude antifungal" The IJC of this anti-

  fungal 1 was 1 / 10,000,000 (0.1 mcg / ml).

EXAMPLE 4 3800 ml of total broth of p H 5.6 is filtered under vacuum through

  Hyflo-Supercell, and 2500 ml of filtered broth of MIC = 1/2000 are obtained. Adjust to p H 8.0 with sodium hydroxide and start again to filter in an analogous manner. Extraction with butanol as in the previous example and we concentrate the extract

  butanolic under vacuum up to 1/20 th of its starting volume.

  The precipitated crude antifungal is filtered, washed with butanol and dried under vacuum at room temperature,

  and 2.0 g of MIC = 1/1666,000 (0.60 mcg / ml) are obtained.

  The above crude antifungal is dissolved in 500 ml and the solution is adjusted to p H 10.2 with hydroxide

  of sodium, and it is stored at + 1 C for 24 hours.

  The precipitated antifungal 1 weighs once dry 131 mg;

  6.6% The MIC is then 1 / 10,000,000 (0.1 mcg / ml).

EXAMPLE 5

  From 3750 ml of total broth at p H 5.9

  2300 ml of broth filtered through Hyflo- are obtained

  Supercell, with an MIC = 2,500. Adjust to p H 8.0, filter and extract with butanol in the same manner as above. After concentrating the organic extract, 1.9 g are obtained.

  crude MIC antifungal = 1/2 222,000 (0.45 mcg / ml).

  The crude antifungal is dissolved in 475 ml of water and at p H

  , 2 we produce after 24 h at + 1 C 146 mg of antifun-

  gic 1, 7.7% MIC = 1 / 10,000,000 (0.1 mcg / ml).

Claims (6)

  1 Process for obtaining polyfunctional antifungal antibiotics   nics, characterized in that a fermentation of a culture medium is carried out by means of the microorganism called Streptomyces sp cepa SF-1 (ASA), by extracting said   mycelium and / or culture medium antibiotics.   2 Process for obtaining poly- antifungal antibiotics   eniques according to claim 1, characterized in that said fermentation is aeropobic, preferably at a temperature between 250 and 300 C and in that the culture medium contains assimilable sources of carbon and nitrogen and mineral salts.   3 Process for obtaining poly- antifungal antibiotics   eniques according to claim 2, characterized in that the carbon sources used are glucose, fructose, mannitol, glycerol, starch, dextrin, cellobiose, trehalose, mannose, alone or mixed, and as sources of nitrogen, we use flour from vegetable doughs, liqueur from my macerated Is, vegetable or animal peptones,   yeast, urea, ammonia salts, alone or mixed.   4 Method for obtaining poly- antifungal antibiotics   eniques according to one of claims 1 to 3, characterized   in that the broth resulting from the fermentation is filtered, in that it is adjusted to p H approximately equal to 8, in that the moisture is extracted and in that it is concentrated under vacuum, in   thus precipitating a complex of antibiotics or   crude fungal from which pimarycin is isolated.   Method for obtaining poly- antifungal antibiotics   eniques according to one of claims 1 to 3, characterized 1 1,034   in that the broth resulting from the fermentation is filtered, in that it is adjusted to p H approximately equal to 8, in that the moisture is extracted and that it is concentrated under vacuum, thus precipitating a complex of antibiotics or a crude antifungal, which is dissolved in water at a concentration of approximately 4 mg / ml, by adjusting the solution to p H approximately equal to 10.2 and then what we get   by precipitation a tetraene antibiotic called Ab 400.   6 Tetraenic antifungal antibiotic obtainable   according to the method of claim 5, of an elastic composition   centesimal mental C 54.5%; H 7.1%; N 2.9%; O -   , 5% and minimum inhibitory concentration vis-à-vis   1: 10,000,000 Saccharomyces cerevisiae.