BE557372A - - Google Patents

Description

       

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   The present invention relates to a new antibiotic agent as well as to its method of preparation.



   The new antibiotic agent discovered by the Applicant will, for the sake of simplicity, be designated during the present description by the arbitrary name of "marcomycin".



   The present invention relates more particularly to a new antibiotic substance, "marcomycin", exhibiting the following properties: it has a molecular weight of

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 about 436, calculated from analytical results; pK '# values of about 7.20 and 8.95, determined with a 66% solution in dimethylformamide; a melting point, with decomposition, in the range of about 1600 to 180 ° C; it is relatively insoluble in solvents such as ether, chloroform, ethylene chloride, hexane and benzene, it is soluble in water and is relatively soluble in organic solvents such as ethanol, methanol, dimethylformamide and glacial acetic acid;

   is weakly basic and likely to form salts with acids; it is stable in aqueous solutions over a pH range of from about 1 to about 10; its composition in% is the following: carbon = 46.40%, hydrogen = 7.48%, nitrogen = 6.82% and (by difference) oxygen = 39.30%; in aqueous solution, it does not exhibit an absorption spectrum in the ultraviolet; finally, when mixed with mineral oil, it shows the following sharp bands of infrared absorption spectrum: 3.03, 6.01, 6.12, 6.24, 6.34 , 8.10, 8.72, 9.30, 9.63 and 11.25 microns.



   The invention also relates to a process for obtaining an antibiotic agent, which consists in cultivating under aerobic conditions a strain producing marcomycin of Streptomyces hygroscopicopicus in a culture medium containing sources which can be assimilated by carbohydrate molds. , nitrogen and mineral salts, until effective antibiotic activity is produced by that organism in the above culture medium.



   At the base, marcomycin is a non-crystalline, colorless solid substance which is very soluble in water and relatively soluble in solvents such as ethanol, methanol, dimethyl la / formamide, acid. glacial acetic and analogues.



  It is relatively insoluble in solvents such as ether, chloroform, ethylene chloride, hexane, benzene, and the like.

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  It melts with decomposition in an indeterminate range between approximately 160-180 C.



   Marcomycin is weakly basic and exhibits pK '# values of approximately 7.20 and 8.95, determined with a 66% solution in dimethylformamide. Basicity appears to arise from the presence of one or more amino groups in the molecule. Marcomycin forms salts with ordinary acids. For example, the hydrochloride, sulfate, phosphate, maleate and the like can be easily prepared. Such salts are, in general, very soluble in water. Marcomycin also forms salts or salt-like complexes with high molecular weight acids, for example with p (p-hydroxyphenylazo) benzene aulfonic acid, methyl orange and the like. These salts or complexes are relatively insoluble in water.



   It is observed that the antibiotic is stable in aqueous solution at a pH of between 1 and 10 approximately.



   The average of several elemental analyzes shows that marcomycin has approximately the following composition: 46.40% carbon, 7.48% hydrigen, 6.62% nitrogen and 39.30% oxygen, this last content being calculated by difference. The above values indicate that * marcomycin has the empirical formula C15H30O9N2. The baseline molecular weight of marcomycin empirically derived from analytical results is approximately 436.



   The absorption spectrum in the ultraviolet of an aqueous solution of maroomycin does not show absorption maxima.



   The infrared absorption spectrum of a sample of marcomycin mixed with mineral oil shows the following fairly broad net absorption maxima. $, 03, 6.01, 6, la, 6,24 , 6.34, 8.10, 8.72, 9.30, 9.63 and 11.25 microns. The

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 The infrared absorption curve of this mixture is shown in the accompanying drawing, on which the wavelengths in microns have been plotted on the abscissa and the percentage of transmission on the ordinate.



   Marcomycin at baseline shows antibiotic activity against Gram-positive and Gram-negative microorganisms including the following: Staphylococus aureus, Mycrobacterium tuberculosis (H 37 RV), Staphylococus aureus, ( resistant to streptomycin,) Bacillus subtilis, Mycrobacterium phlei, Mycrobacterium tuberculosis (607). Mycrobacterium avium, Salmonella gallinarium, Escherichia coli, Aerobacter aerogenes and Klebsiella pneumoniae.

   Administered orally in the form of capsules, tablets or as a constituent of the diet, marcomycin is effective against parasitic animals, such as amoeba, flagellaria, trichinae, pulmonary parasites, strongyles and other strongyloid parasites.



   The Applicant has discovered that marco-mycin can be produced by culturing certain strains of actinomycetes under aerobic conditions in a culture medium containing assimilable sources of carbohydrates, nitrogen and mineral salts. , until this culture medium exhibits significant antibiotic activity, and then subjecting the medium to various methods serving to isolate marcomycin.



   ¯ Due to the imprecision of taxonomic studies of Aatinomyces groups, there is always some doubt as to the classification of any newly discovered organism. However, the organisms which have been shown to be able to produce marcomycin appear to resemble most closely an actinomycete, which is Streptomyces hygroscopicus, an organism first described by de% en in the periodical

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 Proc. Linn. New South-Xales, 56: 345-370 (1931).

   The three organism strains which the Applicant used to produce marcomycin are deposited prematurely at the Culture Strain Collection of the "Northern Regional Research Laboratories" in Peoria (State of Illinois) and are desired by the strain numbers NRRL 2387, NRRL 2388 and NRRL 2389.

   These three strains were isolated respectively from soil samples collected from the following regions: Marion County (Indiana), Saunders County (Nebraska) and Vigo County (Indiana). @ The isolation process consists of placing a highly diluted sample of this soil on nutrient agar on a plate, incubating this plate until the growth of the organism is complete, then collect separate colonies of this organism using a loop of sterilized platinum wire.



   The three above-mentioned strains look different when viewed superficially and are characterized by their ability to produce marcomycin under suitable culture conditions and by certain particular characteristics which show that they are all relevant to the species. hygroscopicus.



  In the description which follows, detailed taxonomic studies of the aforementioned strains of actinomycetes which produce marcomycin will be indicated.



   In these studies, the methods used are those commonly used in the taxonomy of actinomycetes. The carbon and nitrogen utilization tests are carried out according to the methods of Pridham and Cottlieb, J. Bact., 56 107-114 (1948) The publications of Waksman, Soil Sci., 8: 71-215 (1919 ), Jensen, Soil Sci., 30: 59-77 (1930), and "Conn. NY Agr. Exp. Sta. Tech.



  Bull. 60, 1-25 (1917) "provide the basis for further testing and characterization. To denote most of the stains

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 The system indicated by Ridgway, Color Standards and Color Nomenclature (1912) was used.



   TABLE I
 EMI6.1
 
<tb> NRRL <SEP> 2387 <SEP> NRRL <SEP> 2388 <SEP> NRRL <SEP> 2389
<tb>
<tb>
<tb>
<tb>
<tb> This <SEP> culture <SEP> forms <SEP> Agar-agar <SEP> synth-
<tb>
<tb>
<tb>
<tb>
<tb> a <SEP> mycelium <SEP> vegeta- <SEP> tick <SEP> - <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> tif <SEP> typical <SEP> coated <SEP> and <SEP> identical <SEP> to <SEP> Identical <SEP> to
<tb>
<tb>
<tb>
<tb>
<tb> strongly <SEP> branched <SEP> NRRL <SEP> 2387 <SEP> NRRL <SEP> 2387
<tb>
 producing an upright branched aerial mycelium. The aerial spores originate from short branching conidiophores forming thick, close spirals. As the culture matures, dense clusters of spores mixed with aerial hyphae cover the surface of the culture.



  When viewed under a microscope, the individual spores have a typical geometric shape, ie free. or in the form of spiral chains.



   They have an ap- proximately square or rectangular profile, but the surface of the cell

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 NRRL NRRL 2388 NRRL 2389 - which is turned or has been turned towards the outer side of the spiral chain is longer than the inward facing surface of the spiral, giving it the appearance of a wrench vault. This characteristic form persists in the detached spores. After about 2 weeks of incubation, refractive spherical corpuscles appear in abundance in the aero mycelium either at the extremity or along the body. individual hyphae.

   These corpuscles vary widely in size, but are usually several times the diameter of an individual hypha. These structures do not appear to be cellular but rather formed by globules of an exudate. liquid.

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   Other backgrounds
NRRL 2387 NRRL 2388 NRRL 2389
It is also formed Analogous to NRRL. It forms spirals of the characteristic spirals 2387, but we, as for the NRRL 2387. characteristics on a mete- do not observe the corpuscles globular glucose-aspa- globules on the larvae which one finds, on rabbit, on starch cultures of 32 glucose-asparagine and Emerson and over days on starch-agar-agar ne se-asparagine agar media,. are not observed on containing calcium malate calcium malate calcium. or agar-agar of.E- or agar-agar
The glo- merson corpuscles.

   No Es- from Emerson. bulars, which were found with agarve on glucose-starch agar. asparagine and calcium malate were not observed on starch and Emerson's agar in cultures incubated for 32 days.



   TABLE II
NRRL 2387 NRRL 2388 NRRL 2389
Synthetic ggar-agar
A moderate amount Like NRRL 2387, except Like NRRL 2387 except of vegeta- ble mycelium that the pigmentation - that the a- tive colorless mycelium is in- aerial mycelium nothing is all a- bracketed in the mid- is uniformly gri- bord light brown, then place, at the same time is not stained and brown, then finally a small amount becomes almost black almost black. It consists of mycelium a- charcoal-like in the presence of nothing speckled gray 2 to 4 weeks.

   In a trace of a pig- and white, additionally colored, greenish growth ment solu-

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 NRRL 2387 NRRL 2388 NRRL 2389. mouse gray pale at the is a little more level to gray and the mouse cavities frank at the top of the even surface of the slice; more widely there are some distributed. black areas at the edge of the edge. There is a layer, producing spores, of pulverulent spores growing flat and very abundant when viewed under the microscope.

   Pitting occurs in the surface of the spore crust near the growth margin, especially at dark colored locations; the production of cavities coincides with the disappearance of droplets of exudate of similar size which had been produced by excretion during growth. No soluble pigment.

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   NRRL 2387 NRRL 2388 NRRL 2389
Glucose-asparagine agar Vegetative mycelium Like NRRL 2387, but Like NRRL 2387, but buried, colorless, the aerial mycelium the underside of the culture - present in quantity is more copious, the re takes a significant color. unifor- buff from the air, rather light, becoming black with a trace of a pigment; coloring like charcoal and not soluble. The my- blanche below moist in 2 4 aerial celium is with a topcoat. weeks sparse, white, with the lower middle color of the mouse-gray slice which is thicker and darker to dark brown along the edges. almost black.

   Myce- The color ranges from light aerial lium- white (in the center) until, initially stained with that to mouse-gray to the exsuperior droplets dt becomes dat where black (soot-colored) appear more late and wet at the end of the cavities; the surface 2 to 4 weeks. becomes dull and damp.



   There is a trace of a soluble brownish pigment.



   Calcium Malate Agar
Moderate amount of Like NRRL 2387, but Like NRRL 2387, but vegetative growth - the aerial mycelium there is a ve pigment, buff reverse - develops td, soluble pinkish in color. Amount of fashion- becomes pale brown and light cinnamon pink, reed of an eventually moist mycelium and and a white aerial mycelium that black. At the base of the nothing flat-and white.

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   NRRL 2387 NRRL 2388 NRRL 2389 takes at the. mature band, .11 exists, a It occurs late- a spotty appearance growth under- form on top with. small colony shoots not brown growth. mouse-gray and spore-forming and over- clear which becomes' black with a little high growth. re and wet, after session black with soot. 2 to 4 weeks of in- uneven surface with cubation. There are colonies, sporu- lar growths in weak col- lation to monoliths in the form of derivation especially at the non-sporulated buds but. The medium at the base of the strip does not contain insoluble salts as for NRRL 2387. Presence of a soluble buff pigment.



   Emerson's agar Significant growth Like NRRL 2387, but Like NRRL 2387; but from a vegetative mycelium the aerial mycelium the underside of the growing, Isabel- light brown color becomes sance. 'Vegetative is the to upside down. Myce- black and moist in 2 red-orange which aerial fon- lium abundant at 4 weeks, except 'this to take a and white becoming at the end of the band sien- gray-gray and brown where the growth is not scorched .Black mycelium with transparent tuf-. The fresh aerial is fes of a blanket growth is pro-white, becomes a tiny white, thoroughly wrinkled and dark brown, and finally colo- fissured. dull black.

   The cro- nies.Pigment solu- tion is wrinkled and pale light brown. cracked with a soluble mahogany-red pigment, takes on a ter @@ burnt sienna after 28 days.

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   NRRL 2387 NRRL 2388 NRRL 2389
Nutrient Agar Moderate amount of Same as NRRL Same as NRRL with colorless vegetative growth and white aerial mycelium. The medium generally darkens slightly, but the pigment is too pale to have a characteristic color.



   Glucose Nutrient Agar Growth is so- The growth is Growth of the milàire type to that of like that of the Emerson type, but the myce- Emerson's agar, Emerson, but there aerial lium is wrinkled, but a little less a- has a sparse and leaping white mycelium. The mycelium nothing more. (almost transparent) airy powdery abundant.The surface- a slight white and mouse-gray coloration that entire becomes brown appearing to becomes uniformly finally black; of the tip of gray-mouse except in very little cross- slice.

   Pigment the marginal areas superimposed sance soluble white earth color and one cons-white. Pigment so- sienna burnt after a light pale brown luble or 28 days. colo- growth non-existent. nies superimposed also white. Light brown soluble pigment.

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  NRRL 2387 NRRL 2388 NRRL 2389, ..



   Starch agar Moderate amount of Same as NRRL Looks like NRRL 2387 vegetative growth 2387, but not stained for ve with growth; pigmentation u- growth and the important pig- ment of myceniform mycelium mentation, but it aerial lium; aerial at all there is a small lightly colored phases, but passing amount of speckled white pigment and through the same series of soluble orange-pink shadows of color color changes- at the top of the tran- gray at the beginning, then their (from white at cultivation.The color darker gray-brown and at growth is more as incu- black).

   Vigorous growth and more continuous labor; eventually becomes uniform as that of completely black gray placements. NRRL 2387 and her- Gradually turning- Starch is hydrated eventually as to black. Petilysed (width of the completely black. The almites droplets of hydrolysis zone don is hydrolyzed (zo-exudate occurring 28 mm). Does not hydrolysis earlier. The width only 20 mm): face of the growth shows microscopic cavities at maturity. Traces of a soluble greenish pigment. The starch is hydrolyzed (the hydrolysis zone is 21 mm wide).

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   NRRL 2387 NRRL 2388 NRRL 2389
Potato pad culture
Variable growth Like NRRL 2387, but Like NRRL 2387, but in double tubes the growth and the aerial mycelium is wheat. Vegetable growth is gray, and the underside of the developed branch less vigorous; is black raised character, presence of a myc- in places adhering rough and fissured, colorless aerial lium to the glass.
Growth abundant or white. under the pad is so- a water-readable aerial mycelium. The with white patches of dark cinnamon and gray, the pig- ture to wine red. gray with a light brown coloration. The aerial mycelium is raised, powdery and has some cavities. The pad is darker.



   Gelatin (25) Growth is mea- As NRRL 2387, but Like NRRL 2387, but diocre, amorphous and in-liquefaction is liquefaction is colored. Slightly less complete liquefaction in 28 days. strongly delayed, complete in 28 beginning after in- days. around 20 days, but almost complete after 28 days. No soluble pigment.

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   NRRL 2387 NRRL 2388 NRRL 2389
Sunflower milk (30 C) Growth ring As NRRL 2387, but As NRRL 2387; with a min crust- the crust ring- final pH 7.6. this healthy mycelium is more massive. nothing white. Hydrolysis final pH 7.6. without coagulation, almost complete in 6 days, and complete in 9 days, alkaline reaction (final pH 7.5).



   Sunflower Milk (3700) Ring of a growth- Like NRRL 2387, but Like NRRL 2387. The color of the ring of growth- bluish purple with this has a delicate color of the aerial mycelium si- gly mahogany. white.Previous rapid hydrolysis, in some cases the formation of soft lumps. Alkaline reaction. nutrient medium Poor growth Like NRRL 2387, Like NRRL 2387. submerged, flakes neat and adherent growth. even less is No vigorous soluble pigment.

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   NRRL 2387 NRRL 2388 NRRL 23 89
Glucose nutrient medium n Developed growth, Like NRRL 2387, but Like NRRL 2387, mainly sub-growth is submerged, soft and a- somewhat poor. inherent; very few supernatant colonies. No soluble pigment.



   Tyrosine medium Some colonies Like NRRL 2387 Like NRRL 2387 white supernatants with some growth a-
 EMI16.1
 morph, cloudy and flaky. No soluble pigment.



   Cellulose band Cellulose is par- Like NRRL 2387, Like NRRL 2387, so much consumed, but growth but growth but growth is important. is important. is not abundant.



   In Tables III, IV and V below, the results of use tests carried out with the strains of organisms are indicated. In these tables, the symbols are used for simplicity; + = growth and product use = no growth, no use # = limited growth, probably poor use S = exceptionally good sporulation f low

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 BSP = brown pigment, soluble OSP = orange pigment, soluble BUP = brown-orange pigment C. significant growth
TABLE III
Use of carbon sources for crops
 EMI17.1
 
<tb> Substratum <SEP> NRRL <SEP> 2387 <SEP> NRRI <SEP> 2388 <SEP> NRRL <SEP> 2389
<tb>
<tb>
<tb>
<tb>
<tb> L <SEP> (+) <SEP> arabinose <SEP> + <SEP> + <SEP> (S)

   <SEP> +
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (+) <SEP> xylose <SEP> + <SEP> + <SEP> +
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (+) <SEP> rhamnose <SEP> '+ <SEP> + <SEP> +
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (-) <SEP> fructose <SEP> + <SEP> (S, f <SEP> BSP) <SEP> + <SEP> (S, f <SEP> BSP) <SEP> + <SEP> (S, <SEP> BOP)
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (+) <SEP> galactose <SEP> + <SEP> +
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (+) <SEP> glucose <SEP> + (S, f <SEP> BSP) <SEP> + <SEP> (S) <SEP> + <SEP> (S)
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (+) <SEP> mannose <SEP> + <SEP> (S, f <SEP> BSP) <SEP> + (se ± <SEP> BSP) <SEP> + <SEP> ( S, <SEP> f <SEP> BSP)
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> cellobinose <SEP> + (S, f <SEP> BSP) <SEP> + <SEP> (S, f <SEP> BSP) <SEP> + <SEP> (S, <SEP> OSP)

  '<SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (+) <SEP> lactose <SEP> + <SEP> + <SEP> +
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (+) <SEP> maltose <SEP> + <SEP> (S) <SEP> + <SEP> (S) <SEP> + <SEP> (S)
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> sucrose <SEP> =
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (+) <SEP> raffinose <SEP> '<SEP> - <SEP> +
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> inulin- <SEP> -
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> mannitol <SEP> + <SEP> (S) <SEP> + <SEP> (S) <SEP> + <SEP> (S)
<tb>
<tb>
<tb>
<tb>,
<tb>
<tb>
<tb>
<tb> dulcitol- <SEP> - <SEP> -
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> (-) <SEP> sorbitol <SEP> - <SEP> - <SEP> -
<tb>
 
 EMI17.2
 inositol.

   (BOP) (BOP) + (BOP) salicilin BOP BOP dextrin0 + (S, f BSP) + (S, f'SP) + (S, BSP) starch + (S,! BSP) + (S,! BSP) + (S, BSP)
 EMI17.3
 
<tb> Sodium <SEP> acetate <SEP> <SEP> + <SEP> + <SEP> +
<tb>
<tb>
<tb> <SEP> sodium citrate <SEP> <SEP> - <SEP> + <SEP> +
<tb>
<tb>
<tb> Sodium <SEP> formate <SEP> <SEP> - <SEP>
<tb>
<tb>
<tb> Malate <SEP> of <SEP> sodium <SEP> + <SEP> + <SEP> +
<tb>
<tb>
<tb>
<tb> Salicylate <SEP> from
<tb>
<tb> sodium <SEP> - <SEP> - <SEP> -
<tb>
<tb> Succinate <SEP> of
<tb>
<tb> sodium <SEP> + <SEP> +
<tb>
<tb>
<tb>,

  <SEP> sodium tartrate <SEP> - <SEP> - <SEP> -
<tb>
<tb>
<tb> asparagine <SEP> + <SEP> + <SEP> +
<tb>
 

 <Desc / Clms Page number 18>

 
TABLE IV Use of nitrogen sources for crops
 EMI18.1
 
<tb> Substratum <SEP> NRRL <SEP> 2387 <SEP> NRRL <SEP> 2388 <SEP> NRRL <SEP> 2389
<tb>
<tb>
<tb>
<tb>
<tb> Sulfate
<tb>
<tb>
<tb>
Ammonium <tb> <SEP> + <SEP> (S, <SEP> f <SEP> BSP) <SEP> + <SEP> (S, <SEP> f <SEP> BSP) <SEP> + <SEP > (S, <SEP> BOP)
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> asparagine <SEP> + <SEP> (S, <SEP> f <SEP> BSP) <SEP> + <SEP> (S, <SEP> f <SEP> BSP) <SEP> + <SEP> ( S, <SEP> f <SEP> BSP)
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> nitrate <SEP> + <SEP> (f <SEP> BSP).

   <SEP> + <SEP> +
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> TABLE <SEP> V
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Reduction <SEP> of <SEP> nitrates <SEP> to <SEP> nitrites
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> NRRL <SEP> 2387 <SEP> NRRL <SEP> 2388 <SEP> NRRL <SEP> 2389
<tb>
<tb>
<tb>
<tb>
<tb> Source <SEP> from <SEP> Crois- <SEP> Test <SEP> at <SEP> Growth- <SEP> Test <SEP> at.Crois- <SEP> Test <SEP> at
<tb>
<tb>
<tb>
<tb> carbon <SEP> session <SEP> nitrite <SEP> session <SEP> nitrite <SEP> session <SEP> nitrite '
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Glycerin <SEP> C <SEP> + <SEP> C <SEP> + <SEP> C <SEP> +
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Glucose <SEP> C <SEP> - <SEP> C <SEP> trace <SEP> C <SEP> +,

  
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Sucrose <SEP> C <SEP> + <SEP> C <SEP> + <SEP> C <SEP> +
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Starch <SEP> C <SEP> - <SEP> C <SEP> - <SEP> C <SEP> -
<tb>
 
As noted above, the strains can be grown
NRRL 2387, NRRL 2388 and NRRL 2389 in the culture medium to obtain effective antibiotic agents. The culture medium can be any one of many, since, as shown in the above usage tests, organisms are capable of using many sources of energy. However, in order to achieve economical production, maximum yield and easy isolation of antibiotics, certain culture media are preferable. Thus, the presently preferred source of the carbohydrate in the culture medium is glucose.

   Other sources which can be incorporated are starch, sucrose, dextrin, molasses and the like. Preferred sources of nitrogen are corn liquor, soybean flour and soluble distillation liquors, but other sources can also be used, including casein, mixtures of amino acids, peptones (also of meat - than soy) and similar products. We can use sources

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      nitrogen from the mineral kingdom, such as nitrates or ammonium salts.



   The mineral nutrient salts to be incorporated into the medium include the usual salts capable of liberating sodium, potassium, calcium ions and phosphate, chloride, sulphate, etc. ions.



   As with the growth and development of other microorganisms, it is necessary to incorporate traces of essential elements into the culture medium to produce the growth of the actinomycete used in accordance with the invention. Such traces of elements are usually introduced as occasional impurities due to the addition of the other constituents of the medium. ,
The strains of the organisms used to produce the marcomycin according to the invention can easily be cultivated, and their growth conditions are much less critical than for many other, actinomycetes. Thus, for example, this organism can grow in many media with markedly different pH.

   However, it is preferable to adjust the pH of the culture medium before inoculation of the organism to a value between 6.0 and 7.5 and, preferably, equal to about 6.5. As has been observed for other actinomycetes, the medium gradually becomes alkaline throughout the growth period of the organism used for the production of the antibiotic, and this alkalinity can reach a pH between 7.2 and 8 , Approximately 0 or more, the final pH being a function, at least in part, of the. Initial pH of the medium, the buffers present in that medium, and the period of time that the organism is allowed to grow.



   As preferred for the production of other antibiotics in bulk quantities, the cultivation conditions, submerged and aerobic, are those preferred for the production of large quantities of marcomycin. To prepare quantities

 <Desc / Clms Page number 20>

 limited antibiotic, shake bottles and surface cultures in bottles can be used. For the preparation of large quantities of product, it is desirable to use the submerged aerobic culture in tanks.

   It is well known that when producing in large special tanks it is preferable to use the vegetative form of the organisms for inoculation of the production tanks in order to avoid a pronounced loss in the production of the antibiotic and therefore inefficient use of the facility.



  Accordingly, it is desirable to first produce a vegetative / getative inoculum of the organisms by inoculating a relatively small amount of the culture medium with the spore form of the organisms and, when a vegetative, fresh and active inoculum has been obtained. , to transfer this vegetative inoculum aseptically into the large tanks. The medium in which the vegetative inoculum is produced may be the same as that used for the production of the antibiotics or may be different.



   Good growth of organisms can be obtained at temperatures between about 25 and about 32 ° C. Optimum production of the antibiotic appears to occur when the culture medium is maintained at about 26-30 G.



   As is customary to produce antibiotics using submerged culture methods, sterile air is blown into the culture medium. For efficient growth of the organism and for the production of the antibiotic, the volume of air used in the production of marcomycin in trays preferably exceeds 0.1 volume of air per minute per volume of medium. culture. More efficient growth and better production of the antibiotic are obtained when the volume of air used amounts to at least one volume per minute per volume of culture broth.

 <Desc / Clms Page number 21>

 



   The rate of production of marcomycin and the concentration of the active antibiotic in the culture medium can be easily followed during the period of growth of the microorganism by testing samples of the culture medium for use. de) to determine their antibiotic activity vis-à-vis organisms known to be sensitive to the antibiotic, for example Bacillus subtilis. The bioassay can be performed using the standard haze measurement methods, or the cup plate methods, or the paper disc test on an agar plate.



   In general, maximum production of antibiotics is obtained after inoculation of the culture medium within about two to five days, when using a submerged aerobic culture or a culture in a shaker flask, and in a approximately five to ten days when using surface culture.



   Marcomycin can be recovered from the culture medium and separated from other substances that may be present, using extraction and adsorption techniques.



  Due to the very high solubility of marcomycin in aqueous solution and the resulting unfavorable distribution ratio for the antibiotic in mixtures of organic solvents and water, it is necessary to use relatively large volumes of solvents. organic extraction.



  Accordingly, it is desirable to saturate, prior to extraction, the filtered culture broth with a highly soluble inorganic salt, for example ammonium sulfate, to achieve a more favorable coefficient distribution for the solvent for use. extraction.



   Antibiotic substances that are extracted from the

 <Desc / Clms Page number 22>

 organic solvent, which is preferably a lower alcohol, such as propanol, butanol, etc., can be collected in crude form by vacuum evaporation of the solvent. The relatively crude product thus obtained is then purified by precipitation from suitable solvents, by chromatography or by countercurrent extraction.



   It is presently preferred to use adsorption methods for the recovery of marcomycin because such methods eliminate the use of the relatively large volumes of solvents which are required when using extractive techniques. Currently, carbon is the preferred adsorbent for the separation of antibiotics from filtered culture broth. The antibiotic substance attached to the adsorbent is recovered by the usual elution methods, using an aqueous organic solvent.

   To recover the marcomycin, ion exchange resins of acidic character are used, for example a cation exchanger consisting of a copolymer of methyl acrylate and of divinyl benzene comprising functional hydroxyl groups, such as the product. known under the trademark "IRC-50". The separation of marcomycin from other antibiotics can be easily achieved, using ion exchange resins and taking advantage of the basic nature of marcomycin.

   Thus, solutions containing antibiotics, including marcomycin, can be subjected to successive treatment with acidic and basic ion exchangers, followed by elution of marcomycin from the acidic resins.
Those skilled in the art will readily understand that various combinations of the above methods and modifications can be used to isolate and purify the novel antibiotic according to the invention.

 <Desc / Clms Page number 23>

 



   Marcomycin can be recovered from the culture media used for its preparation, mixed with hygromycin in a more or less purified form, and such a mixture can be used to add it to animal feed. or for similar uses and to get the interesting ones. effects / of both antibiotics.



   The implementation of the invention will be better understood with the aid of the non-limiting examples below: EXAMPLE 1
A sporulated culture of Streptomyces h ros-copicus, strain NRRL 2387, is produced by growing this organism on a nutrient agar medium having the following composition -.-
 EMI23.1
 
<tb> Starch <SEP> - <SEP> 20
<tb>
<tb> Asparagine
<tb>
<tb> Extract <SEP> from <SEP> meat <SEP> from <SEP> beef <SEP> 3 <SEP> gr
<tb>
<tb> Agar-agar <SEP> 20 <SEP> gr
<tb>
<tb> Water, <SEP> sufficient <SEP> quantity
<tb> for <SEP> 1 <SEP> liter
<tb>
 .The
The medium is inoculated with spores, and incubated for about five days at about 26 C.

   The spore culture is covered with a small amount of water and the support is gently scratched to loosen the spores and to obtain an aqueous suspension of these spores.



   The suspension thus obtained of the spores from the strain NRRL 2387 is used to inoculate 1500 cm3 of a sterile vegetative culture medium having the following composition
 EMI23.2
 
<tb> Glucose <SEP> 15 <SEP> gr
<tb>
<tb> <SEP> soy flour <SEP> <SEP> 15 <SEP> gr
<tb>
<tb> Solid <SEP> materials <SEP> of <SEP> the
<tb>. <SEP> liquor <SEP> from <SEP> but <SEP> 5 <SEP> gr
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> 5 <SEP> gr
<tb>
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 2,5 <SEP> gr
<tb>
<tb> Water, <SEP> sufficient <SEP> quantity
<tb> for <SEP> 1 <SEP> liter
<tb>
 

 <Desc / Clms Page number 24>

 
The medium thus inoculated, contained in a 6 liter flask, is grown by shaking it constantly for 48 hours at a temperature of 2800 in order to obtain the vegetative form of the organism.

   The vegetative inoculum is then used to inoculate a sterile productive culture medium having the following composition (the percentages indicated are weight-volume percentages):
 EMI24.1
 
<tb> Olucoe <SEP> 3.0%
<tb>
<tb> Soy <SEP> <SEP> flour <SEP> 2.5%
<tb>
<tb> Solid materials <SEP> <SEP> of <SEP> liquor <SEP> of <SEP> but <SEP> 0.5%
<tb>
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 0.2%
<tb>
<tb> Chloride <SEP> of <SEP> sodium <SEP> 0.5%
<tb>
<tb> Water, <SEP> sufficient <SEP> quantity
<tb> for <SEP> 666 <SEP> liters
<tb>
 
The inoculated culture broth, which is contained in a 946 liter fermentation apparatus, is kept at a temperature of about 28 ° C and stirred during the fermentation period,

   then aeration is carried out with sterile air taken in. An amount of about one volume per volume of broth per minute. Fermentation is allowed to proceed for about 100 hours, during which time the pH of the culture broth gradually varies between about 6.2 and 7.0. At the end of the fermentation period, the antibiotic activity of this medium can be determined in the usual way. A fermentation broth thus obtained contains about 200 g of maromycin (content which can be determined by the paper disc test using B. subtilis as a control organism).

   The filtered culture broth is passed through a column with a dimension of 102 x 864 mm, which contains a cation exchange resin (previously washed with caustic soda to give it a sodium state) formed by a copolymer. of methyl acrylate and of divinyl benzene, and having hydroxy groups

 <Desc / Clms Page number 25>

 functional, the passage taking place at a flow rate of approximately 100 cm3 per minute. An example of a suitable cation exchange resin of this type is that which is commercially available under the designation "IRC-50" and which is sold by "Röhm and Haas Company". After passing all of the broth through the column, the column is washed with deionized water until the effluent is substantially colorless.

   The column was then eluted with about 55 liters of 0.1 N hydrochloric acid, at which time the effluent was colorless and the pH was about 1.5. The pH of the acid elution is adjusted to a level of 6.9 using 10% aqueous caustic soda solution, and concentrated to low volume. The concentrated elution product is then frozen and dried while still frozen. The residue is taken up in 29 liters of deionized water and filtered. The precipitate obtained is removed. The pH of the filtrate is adjusted to 10.5, about 2.9 kg of activated carbon (brand "Norite SG") are added and the mixture is stirred for about one hour at room temperature.



  The charcoal is then removed by filtration and the filter cake washed by resuspending it in about 14.5 liters of deionized water while stirring for about 30 minutes. The suspension is filtered and the suspension wash is repeated once more. The filtrates are rejected. The filter gSteau, consisting of the activated carbon which has adsorbed the marcomycin, is eluted by stirring it for about one hour at room temperature in about 14.5 liters of a mixture containing 1.45 liters. of concentrated ammonia, 4.35 liters of water and 8.7 liters of acetone. One removes the eluting liquid by filtration and this elution is repeated using the same mixture of ammonia, acetone and water.



  The eluting liquors are combined and concentrated by evaporation.

 <Desc / Clms Page number 26>

 tion under vacuum to a volume of approximately 550 cm3. 3.5 liters of acetone are added to the concentrated residue while stirring. A gummy product precipitate is obtained, containing marcomycin, which adheres to the walls and to the bottom of the container. The supernatant liquid is decanted from this gum and the latter is dissolved in a minimum quantity of absolute methanol. The methanolic solution is then poured slowly into 9 liters of ether, while stirring, which operation produces the precipitation of the marcomycin.

   The supernatant methanol-ether mixture is decanted from the precipitate, then the latter is washed three times with ether, decanting the ether from the precipitate after each washing. The precipitate is dried by vacuum evaporation of the adhering ether. Approximately 144 g of marcomycin are thus obtained, having the properties mentioned in the present description.



   Marcomycin irritates the skin and protective measures should be taken when handling it as a dry powder.



   The acid addition salts of marcomycin can be prepared by reacting equivalent amounts of marcomycin and the selected acid in solution in a suitable inert solvent. Precipitation of the salt followed by filtration and drying gives the desired salt in solid form.



     A solution of about 250 mg of marcomycin in about 3 cm3 of methanol is made with 0.3 cm3 of concentrated sulfuric acid. A white amorphous precipitate immediately forms, which is separated by filtration and washed with a small amount of ether. The bitter solid product is the sulfuric acid addition salt of marcoimycin.



   Marcomycin sulfate is a white, amorphous substance. When dissolved in aqueous acidic or alkaline solutions, and examining the absorption of light in

 <Desc / Clms Page number 27>

 in the ultraviolet region of the spectrum, there is only general absorption of low intensity. The electrometric counting of marcomycin sulfate in 66% solution in aqueous dimethylformamide indicates pK # values of 7.2 and 8.9, the initial pH value being 2.4. The molecular weight calculated from the results of the draw is approximately 532.



   For the preparation of marcomycin dihydrochloride, 0.5 g of marcomycin is dissolved in 20 cm3 of methanol and 0.3 cm3 of concentrated hydrochloric acid is added to this solution.



  The mixture is heated until it is homogeneous, then ether is added until precipitation begins. On subsequent cooling, the marcomycin dihydrochloride separates out as a white, amorphous precipitate. This precipitate is collected by filtration, washed with ether and dried. Further purification can be accomplished by redissolving the salt in a minimum amount of hot metahnol and precipitating the product with ether.



  Marcomycin dihydrochloride is a white, amorphous substance which is soluble in water and melts with decomposition at approximately 170-175 C.



   To a solution containing 4 g of marcomycin in 50 cm3 of water, a saturated aqueous solution of p- (p'hydroxyphenylazo) benzene sulfonic acid is added dropwise until precipitation is complete. The resulting crystalline precipitate is collected by filtration. After recrystallization of the product from an alcohol-water solution, the salt obtained melts with decomposition at about 220-230 C. The p- (p'-hydroxyphenylazo) benzene sulfonate of marcomycin thus prepared contains 2 moles of p- (p ' -hydroxyphenylazo) benzene sulfonic acid per mole of marcomycin.



   When treating aqueous solutions of marcomycin with solutions of phosphotungstic acid, with methyl orange

 <Desc / Clms Page number 28>

 or by Reinicke's salt, precipitates form which can be used to recover marcomycin from this solution. The antibiotic can also be recovered from the precipitates of the above kind by treating them with an excess quantity of aqueous alkali in order to break up the complex salt or the adduct, then applying extraction and recovery processes similar to those indicated above.



  EXAMPLE 2
The pH of 37.85 liters of a fermentation broth obtained by the method of Example 1 was adjusted to 4.0 and then subjected to centrifugation to remove the mycelium. The pH of the supernatant liquid is then adjusted to 8.0 and this liquid is passed through a 102 mm x 457 mm column, which contains activated charcoal (trade mark "Norite SG"). The antibiotic substances present in the fermentation broth are. thus absorbed on the charcoal. After passing all of the fermentation broth through the column, an equal part mixture of acetone and 0.02 N hydrochloric acid was used to remove the antibiotics from the column.

   When the active antibiotic is substantially removed, which results from the microbiological testing of the elution product, the last evaporated to dryness in vacuo, which provides a residue consisting of marcomycin and together with a certain amount of other antibiotic substances such as organisms of the Streptomyces family. This mixture is suitable for incorporation in animal feed, in order to obtain anthelmintic effects.

** ATTENTION ** end of DESC field can contain start of CLMS **.


    

Claims (1)

CLAIMS ' 1. A process for producing an antibiotic agent, which comprises culturing under aerobic conditions a marcomycin-producing strain of Streptomyces hygroscopicus in a culture medium containing sources, assimilable by mold, of carbohydrate, nitrogen, and salts <Desc / Clms Page number 29> minerals, until this organism has a notable antibiotic activity in the above-mentioned culture medium. 2. The process of claim 1 for the production of marcomycin which comprises culturing the strain under aerobic conditions and in a submerged state in a culture medium containing carbohydrate, nitrogen and water. mineral salts which can be assimilated by molds, then recovering the marcommycin from this culture medium. 3. The process of claim 1 for producing a mixture containing marcomycin which comprises culturing the strain under aerobic conditions and in a submerged state in a culture medium containing hydrate. carbon, nitrogen and mineral salts which can be assimilated by molds and then recover from this culture medium a solid product containing the antibiotic. 4.The process of claims 1, 2 or 3, wherein the strains used for the production of marcomycin of the variety. EMI29.1 Streptomyces hygroscopicus are called Streptomyces hvgroscopicus NRRL 2387, HRRL 2388 or NRRL 2389. 5. The process for the production of the antibiotic marcocin, as described with reference to any of the examples given. 6. An antibiotic substance having the following properties: a molecular weight of about 436, calculated from the test results; pK '# values of about 7.2 and 8.95, determined with 66% aqueous dimethylformamide solution; a melting point, with decomposition, in the range of about 160 to 180 C; relative insolubility in solvents, such as ether, chloroform, ethylene chloride, hexane and, benzene, solubility in water and relative solubility in organic solvents, such as as ethanol, methanol, dimethylformamide and glacial acetic acid, <Desc / Clms Page number 30> low basicity and ability to form salts with acids; stability in aqueous solutions over a pH range of from about 1 to about 10; following composition in percent: carbon 46.40%; hydrogen = 7.48%; nitrogen = 6.82%; and by difference, oxygen = 39.3%; no absorption in the ultraviolet spectrum in aqueous solution; and, when mixed into mineral oil, this substance shows the following sharp bands of infrared absorption spectrum 3.03; 6.01; 6.12; 6.24; 6.34; 8.10; 8.72; 9.30; 9.63 and 11.25 microns. 7. The antibiotic marcomycin, when prepared by the method of any one of claims 1 to 5.