BE511318A - - Google Patents

Description

       

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  ANTIBIOTICS 'AND PROCESS FOR THEIR PREPARATION.



   The invention relates to novel chemical compounds useful as therapeutic agents because of their antibiotic properties. more particularly, the invention relates to two new antibiotic products to which the names of Antibiotic C and Antibiotic D have been arbitrarily assigned.
Antibiotic C is a crystalline basic compound that melts at
 EMI1.1
 about 182 at 1840C and which contains only the elements carbon, hydrogen, oxygen and nitrogen. It has an optical rotation (o () 26 of about + 180.9 in methanol, a molecular weight of about 552 in D solution: in the chamber, and a probable empirical formula C26H37o8X5 based on analytical data t 56.85% carbon, 6.96% hydrogen and 12.82% nitrogen.

   It is further characterized in that it exhibits absorption maxima in the ultraviolet at 256 and 311.5 millimicrons with an El% of respectively 358 and 291 in 0.1 N hydrochloric acid, at 250 and 320.5 millimi-
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 crcns with an E1% of 270 and 536 respectively in a phosphate buffer at pH 7, p and 1 cm 330 millimicrons with an E% of 626 in sodium hydroxide <0.1 N. The absorption bands more intense in the infrared absorption spectrum of antibiotic C are those located at or near 5.98, 6.09? 6o2L ,, 9 7.995 9.16s 9.50 and 9.62. microns.



  Another group of characteristic moderate intensity absorption bands lie in the region of 9.8 to 12.7 microns. As the free base, Antibiotic C is relatively sparingly soluble in cold water and readily soluble in hot water, which it separates on cooling. it can be extracted from aqueous solutions with n-butanol, since it is readily soluble in this alcohol as well as in other lower aliphatic alcohols, but it is insoluble in ethyl ether.



     Antibiotic C forms a hydrochloride readily soluble in water and methanol. it is precipitated from the latter solvent by adding ether
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 ethyl. At a concentration of 3.12 to 15.6 micrograms cm3 the crystalline C antibiotic completely inhibits the growth of Mvcobacteri tuber-

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 culosis var. hominis H37RV.



   Antibiotic C gives negative results in Molisch, biuret, Sakaguchi., Ninhydrin, ferric chloride and Fehling tests. It gives a positive result to the identification of the arylamino group with c
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 Ehrlich's reagent and a positive result in the Bratton Mars'ta7l test for the same group. The Rf index, using the ascension technique, in ethyl acetate (3), water (3) and acetic acid (1) for anti-
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 biotic C is p, 16, The Rf index in n-butanol (Ia), acetic acid (1) and water (4) is 0.28.



   Antibiotic C is precipitated from its aqueous solution by a large number of the usual base precipitators, for example a-
 EMI2.4
 p-hydroxyazobenzene-p = sia3.fonic acid, picric acid, orange II, flavianic acid, p-nitrobenzeneazochromotropic acid, Congo Red, Millins Yellow 3G. The precipitates formed are solids or gums insoluble in water, soluble for the most part in lower alcohols or in methyl cellosolve.



   Antibiotic D is an amorphous basic solid, pale tan in color, containing only the elements carbon, hydrogen, oxygen and nitrogen. It is very soluble in water and methanol, is soluble in hot ethanol and separates from the latter solvent on cooling. It is insoluble in acetone, ethyl ether, ethyl acetate and benzene. It is further characterized in that it exhibits absorption maxima in the ul-
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 traviolet at 37, millimicrons with E1% 123 in 0.1 N hydrochloric acid, at 302 millimicrons with a 1 cm EliÎo 'of 125 in phosphate buffer at pH 7.0 and at 321.5 millimicrons with an EI% 143 in sodium hydroxide Oel N, At a concentration of 12.5 cm micrograms / cm3, antibiotic D completely inhibits the growth of Xycobacterium tuberculosis var. hominis E37 Rv.

   Antibiotic D has an Rf number of 0.0 in ethyl acetate (3), water (3) and acetic acid (1) and an Rf number of 0.19 in
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 n-butanol (10). acetic acid (1) and water (4). Antibiotic D also gave negative results in tests with Ehrlich's reagent and Bratton-Marshall reagents. it can be precipitated from aqueous solutions by numerous usual precipitating agents for bases, for example
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 p-hydroxyazobenzene-p-sulfonic acid, picric acid, orange II, flavianic acid, p-nitrobenzeneazochromotropic acid, Congo Red, Millins Yellow 3G. The precipitates which form are solids or gums which are soluble for the most part in lower alcohols and in methyl cellosolve.



   Antibiotics C and D are very active against certain acid-resistant bacteria, which are agents of tuberculosis. The activities of antibiotics C and D against Mycobacterium tuberculosis var. hominis H37Rv are approximately equal to those of several other antibiotics.
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 known cues such as viomycin, streptothricire, and neomycin, and superior to that of aureomycin. These new antibiotics are also highly bacteriostatic active against gram-positive bacteria as well as against certain gram-negative bacteria.

   Some examples of gram-positive bacteria that generate
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 diseases are Di "plococcus pneumoniae. Streptococcus hemolyticus and Micrococcus pyogeners var. aureus. Among the gram-negative bacteria against which these products show their bacteriostatic action are pathogenic bacteria such as Hemophilus pertussis and Escherichia Colin Le Table I more vividly represents the remarkable antibiotic spectra of antibiotics C and D compared to the known antibiotic spectra produced by actinomycetes.

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  1 A v L àà A U le
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 Antibacterial activity of C and D ar-tibiotics and antibiotics + clinically tested and produced by Actinomycetesc
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<tb> Anti- <SEP> Anti- <SEP> Auréo- <SEP> chlor- <SEP> Néo- <SEP> strep- <SEP> Strep- <SEP> Terra- <SEP> VioTypes <SEP> de <SEP > bacteria <SEP> bioti- <SEP> bioti- <SEP> myci- <SEP> amphé-- <SEP> myci- <SEP> tomy- <SEP> tothri-
<tb>
 
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 cu.e C que D ne nicol ne cine cine mycine - glycine GRAM-POSITIVE TYPES Diplococcus pneumoniaen 1.0 10 0.05 z0 100 25 2.5 1.0 50
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<tb> Micrococcus <SEP> pyrogenes <SEP> var.

   <SEP> aureus <SEP> 25 <SEP> 5PO <SEP> 0.1 <SEP> 5.0 <SEP> 0.75 <SEP> 2.5 <SEP> 5.0- <SEP> 0.75 < SEP> Plus <SEP> of
<tb> 10 <SEP> 100
<tb>
 
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 streptococcus hemolyticus 5.0 2.5 0.1 1.0 50 50 50 to over 0.75 Over
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<tb> of <SEP> 100 <SEP> 100
<tb> TYPES <SEP> RESISTANT <SEP> TO ACID <SEP>.
<tb>
 
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  Mycobacterilm tuberculosis var. hominis 3.1 12, 25- 6.25- 6.25- 6.25 1z, 5 25 6.25H37RVa 15.6 100 12.5 as, 5 50 12 5 M.ycobacterium s¯ ATCC 607 Parent culture 25 12 , 5 12.5 50 0.39 0.39 3.12 0.78 1.56 Aureomycin resistant 25 12.5 200 0.39 Os39 6.25 25 6.25
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<tb> Resistant <SEP> to <SEP> the <SEP> neomycin <SEP> 25 <SEP> 12.5 <SEP> 12.5 <SEP> plus <SEP> of <SEP> 0.39 <SEP> 3 , 12 <SEP> 1.56 <SEP> 1.56
<tb> 12.5
<tb> Resistant <SEP> to <SEP> the <SEP> streptomycin <SEP> 50 <SEP> 25 <SEP> 25 <SEP> 0.19 <SEP> plus <SEP> of <SEP> 6.25 <SEP > 0.78 <SEP> 0.78
<tb>
 
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 Streptothricin resistant 25 25 6.25 39 100 2. 25 1 6 ï2'- .........

   bzz25 0, 6, 5 5 1.56 3, Resistant to Terramycin 50 12.5 200 0.39 0.39 6.25 50 z25 Resistant to Viomycï.ze 25 12.5 25 0.19 3.12 6 , 25 3.12 12.5 GRAM-NEGATIVE TYPES Aerobacter aeroenes> loo 25 0.1 0.5 0.5 2.5 0.75- 1.0 25 Lscherichia coli zs 5 Escherichia coli i 100 3 loo 0.75 z95, 5 10 5.0 5.0 i 100 Hemophilus pertussis 5.0 1.0 0.2 0.2 z, 0 z, 0 7.5 093 50 Klebsiella pneumoniae> 100> 100 0.5 1.0 0 , 25 0.5 0.5 2.5 10 µµbriōgomma loo 5Q 0.1 l, 0 10 z5 10- 1.0 100 ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ 25 + Those who are not listed on this table lack significant anti-tuberculosis activity,

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In accordance with the present invention, antibiotics C and D are produced by aerobic cultivation of cultures of an actinomycete of the genus Streptomyces,

   designated by laboratory number C1730. This microorganism exists among others in the soil. Cultures can be obtained by mixing cultures of specific bacteria inhibited by antibiotics C and D with aqueous agar and adding soil containing Streptomyces C1730. After incubation of the mixture for one to ten days, colonies appear. of the desired actinomycete and other antagonists. the streptomyces C1730 colonies are selected, transferred to fresh culture medium and subsequently separated in the form of a pure culture by the usual methods.

   A live culture of this microorganism has been deposited with the Culture Office of the PAREE DAVIS & COMPANY Company in Detroit, Michigan under number 04918.



   Under the microscope, the organism is an aerobic actinomycete which forms a fragile branched aerial mycelium, rarely or not at all partitioned; it gives rise to unicellular conidia chains, and is therefore a member of the genus Streptomyces. When grown on glucose-tryptone-agar medium, the young, moist primary mycelium appears colorless to yellowish, later turning gray. The secondary aerial mycelium is initially white and later turns gray, little or no pigment appears in the agar. Surface colonies are circular, erect, folded, or radial-veined with full or wavy margins.



  The primary moist mycelium is glassy and profuse branched. The secondary aerial mycelium is also abundantly branched. Primary, secondary, and sometimes tertiary branches appear and may be alternate, opposite, or occasionally in whorls. The aerial mycelium is wavy, and frequently forms terminal loops or loose spirals. The extreme parts of these aerial branches subdivide into conidial chains 15 to 55 microns in length. Conidia are glassy, spheroid to ovoid, averaging 0.85 microns in diameter (range 0.5 to 1.2) and 1.3 microns in length (range 0.8 to 1.8).

   Table II shows the characteristic differences between streptomyces C1730 and other antibiotic-producing actinomycetes in terms of appearance and actinophagous susceptibility.

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  Appearance, on giucose-% ryp% one-agar and actinophagous susceptibility of streptomyces C1730 and other antibiotic varieties of Streptomyces.
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  Antibiotic Varieties 9 E L i u m Substrate Susceptibility Streptomyces produces Color Form Color Color for- au 2, griseub aerial and mesa mycelium in aerophag.



  ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ Aerial aerial and upside down 1-'Agar SaC1730 Antibiotics wavy, White to Colorless to None 0
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<tb> C <SEP> & <SEP> D <SEP> loops <SEP> and <SEP> gray <SEP> Yellowish <SEP> to
<tb> spirals <SEP> Gray
<tb>
 
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 a.autibiotieus Actinomycin Right White to Black Black 0 Gray a.aureofaciens Aureomycin Right White to yellow to None 0
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<tb> Gray <SEP> Tan
<tb> S.floridae <SEP> Viomycin <SEP> Right <SEP> to <SEP> l- <SEP> White <SEP> to <SEP> purple <SEP> None <SEP> +
<tb> kindly <SEP> Lavender <SEP> at
<tb> wavy <SEP> Green <SEP> grayish
<tb>
 
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 Sfradiae Neomycin Right,

   bou- White to ro- Colorless None 0
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<tb> Fradicine <SEP> keys <SEP> and <SEP> spi- <SEP> se <SEP> coquilla- <SEP> to <SEP> Tan
<tb> lines <SEP> occa- <SEP> ge
<tb> sional
<tb>
 
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 e.griseus Streptomycin Right to the - White to pink Colorless to None +
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<tb> Actidione <SEP> slightly <SEP> on- <SEP> light <SEP> to <SEP> green <SEP> Brown <SEP> light <SEP> to
<tb> dulé <SEP> grayish <SEP> light <SEP> Lavender
<tb> S.griseus <SEP> Griseine <SEP> Right <SEP> to <SEP> l- <SEP> White <SEP> to <SEP> Pink <SEP> Incolo- <SEP> None <SEP> 0
<tb> slightly <SEP> on- <SEP> clear <SEP> to <SEP> Green <SEP> re <SEP> to <SEP> Tan
<tb> dulé <SEP> grayish <SEP> clear
<tb> S.lavendulae <SEP> Streptothrici- <SEP> Right, <SEP> bou- <SEP> White <SEP> to <SEP> Black <SEP> Black <SEP> 0
<tb>
 
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 ne, Lavendulhie,

   keys and spi- Rose to
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<tb> Streptoline <SEP> stripes <SEP> occa- <SEP> Lavender
<tb> sional
<tb> S.rimosus <SEP> Terramycin <SEP> Spirals <SEP> White <SEP> yellow <SEP> None <SEP> 0
<tb>
 
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 .g.SP ,, - A105 Actinorubin Spirals White to Pink to None 0
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<tb> Pink <SEP> to <SEP> Red
<tb> Gray
<tb>
 
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 S. venezuelae Chloramphenicol Right White to Black Black 0
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<tb> ¯¯¯¯ <SEP> ¯¯¯ <SEP> Gray
<tb>
 
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 'SELMAN A, Gd.AKMAN,; r, .Bact.k.6 "300,1943 + = susceptible 0 = not susceptible.

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   The body liquefies the gelatin slowly without forming a pigment, and peptonizes the sunflower milk, usually with a basic reaction. In Gottlieb's synthetic medium, the body uses many carbon sources, including 1-arabinose, dextrose, d-fructose, d-galactose, i-inositol, lactose, maltose, d- mannitol, rhamnose, starch, and d-xylose; less well inulin, raffinose, d-sorbitol, and sucrose, and it does not use dulcitol. The body uses inorganic and organic sources of nitrogen.



   Table III provides a comparison of the ability of Streptomyces 01730 and the various known antibiotic actinomycetes to use different carbon sources when growing on a medium composed of agar, inorganic salts and the carbon source tested.

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  U'M-Llsa'Mon a1nyaraes ae caroone par -Le Trej2T, 2Xces u-utiu and a-awres -vai-.LU (, V5 Luit,. Ptomyces biiotics raised on synthetic agar 2.o = no growth ô + = weak growth; z notable growth a +++ = good growth: ++++ = very intense growth.
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<tb>



  SOURCE <SEP> OF <SEP> CARBON.
<tb>
 
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  Varieties Antibioti- ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ ¯¯¯¯¯¯¯¯.¯¯¯¯¯¯¯¯¯¯ of Strep- ques pro> 1-ara- Dul- d- d-ga- i-ino- Inu- Lac- Mal- d-man - Raf- Rham- d-wsor- Su- d-xy7 '
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<tb> tomyces <SEP> duits <SEP> bino- <SEP> ci- <SEP> fruc- <SEP> lac- <SEP> sitol <SEP> line <SEP> tose <SEP> tose <SEP> nitol <SEP > fi- <SEP> nose <SEP> bitol <SEP>. <SEP> cro- <SEP> lose <SEP> you <SEP> tose <SEP> tose <SEP> nose <SEP> se <SEP> se
<tb>
 
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 S.C.

   Antibiotics ++++ 0 ++++ ++++ +++ pu to, ++ ++++ +++ 0 ++++ 0 to 0 +++
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<tb> 1730 <SEP> C <SEP> & <SEP> D <SEP> to <SEP> lèg. <SEP> to <SEP> to <SEP> to <SEP> leg. <SEP> to <SEP> to
<tb> ++++ <SEP> + <SEP> ++++ <SEP> ++++ <SEP> ++ <SEP> + <SEP> ++ <SEP> ++++
<tb> S.antibio- <SEP> Actinomycin <SEP> + <SEP> 0 <SEP> +++ <SEP> +++ <SEP>. ++++ <SEP> 0 <SEP> ++++ <SEP> ++++ <SEP> ++++ <SEP> 0 <SEP> +++ <SEP> 0 <SEP> 0 <SEP> ++
<tb> ticus
<tb> S.aureofa- <SEP> Aureomycin <SEP> ++++ <SEP> 0 <SEP> ++++ <SEP> ++++ <SEP> 0 <SEP> 0 <SEP> ++ < SEP> +++ <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> ++++ <SEP> ++
<tb> ciens
<tb>
 
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 S. floridae viomycin 0 to 0 ++++ ++++ 0 0 to + â '++++ ++++ 0 0 0 to 0 to ++++
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<tb> ++ <SEP> + <SEP> ++ <SEP> + <SEP> +
<tb>
 
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 S. fradiae Neomycin, ++++ 0 0 to. ++++ 0 0 â, leg.

   ++ 0 0 0 0 a 0 +++
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<tb> Fradicine <SEP> + <SEP> + <SEP> + <SEP> to <SEP> to <SEP> leg,
<tb> + <SEP> +++ <SEP> +
<tb>
 
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 sgriseus streptomycin, 0 a 0 ++++ ++++ 0 0 a, 0 ++++ ++++ 0 0 the 0 to the 0 to ++++
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<tb> Actidione <SEP> leg, <SEP> leg. <SEP> to <SEP> leg. <SEP> leg.
<tb>



  + <SEP> + <SEP> +++ <SEP> + <SEP> +
<tb>
 
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 S., - riseus Griseine ++++ 0 ++++ +++ 0. 0 to 0 ++++ ++++ 0 ++++ 0 to o to ++++
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<tb> leg, <SEP> to <SEP> leg. <SEP> leg.
<tb>



  + <SEP> +++ <SEP> + <SEP> +
<tb>
 
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 S.lavendu- Streptothri- 0 0 0 to leg. 0 0 to 0 ++++ 0 0 0 to 0 0 0 to
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<tb> lae <SEP> cine, Lavenduline <SEP> to <SEP> ++++ <SEP>. <SEP> + <SEP> to <SEP> leg, <SEP> to <SEP> + <SEP> ++++
<tb>
 
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 Streptolin ++++ ++++ + +++ S. rimo su-a Terramycin ++++ 0 ++++ ++++ ++++ 0 ++++ ++++ ++++ + 0 ++++ 0 leg, +
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<tb> S.sp. <SEP> Actinorubin <SEP> ++++ <SEP> 0 <SEP> ++++ <SEP> ++++ <SEP> +++ <SEP> ++++ <SEP> ++++ < SEP> ++++ <SEP> ++++ <SEP> ++++ <SEP> ++++ <SEP> leg. <SEP> ++++ <SEP> ++++
<tb> A <SEP> 105 <SEP> +
<tb>
 
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 S.venezue- Chloramphenicol ++++ 0 ++++ ++++ 0 0 + ++++ 0 0 ++++ 0 0 ++++
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<tb> lae
<tb>
 
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 . 2) JcGo PRIDHAM & Dai GOTTLIEB, J. Bacta569 08'1.

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   As mentioned above, antibiotics C and D are produced by aerobic cultivation of a culture of Streptomyces C1730. This is usually accomplished by inoculation into an appropriate culture medium with Streptomycosis C1730 and incubation of the mixture under aerobic conditions at about 20 to 40 ° C for about two to fifteen days. In the case of an aqueous nutrient medium, the solid material existing in the culture mixture is separated and the desired antibiotics are separated from the culture liquid by the methods described below.

   When using a solid nutrient medium, the culture medium is suitably washed with an antibiotic solvent such as water or methanol and the desired antibiotics are separated from the extract during the incubation phase. , the temperature is preferably maintained in the vicinity of 22 to 29 C. When using aqueous nutrient media, the initial pH should be between 6 and 8.2 and preferably on the alkaline side between approximately pH 7.3 and pH 7 , 7. for inoculation of the nutrient medium, one can use spore suspensions, subcultures or culture medium of Streptomyces C1730. the microorganism can be cultivated in the aqueous nutrient medium in various ways.

   For example, the microorganism can be cultivated under aerobic conditions on the surface of the medium, or it can be cultivated below the surface of the medium, i.e. in the submerged state, if it is introduced at the same time. oxygen. The preferred method of producing antibiotics C and D on a large scale involves the use of submerged or deep cultures of Streptomyces C1730. according to this embodiment of the invention, a sterile aqueous culture medium is inoculated with Streptomyces C1730 and is subjected to incubation with stirring and aeration at a temperature between 20 and 40 C, preferably around 25 to 35 C , for about one to four days.

   Under these conditions, the organism develops in the form of numerous more or less separated particles dispersed throughout the mass of the medium, contrasting with the more or less continuous film present on the surface of the medium in the surface culture process. Thanks to this distribution of the organism in the mass of the medium, it is possible to cultivate at one time large volumes of the inoculated nutrient medium in the large reservoirs and vats usually used in the fermentation industry, it has been found that Fixed tank fermenters, fitted with suitable stirring and aeration apparatus, as well as horizontal rotating drum fermenters are particularly interesting from this point of view.

   However, for the preparation of smaller quantities of antibiotics or cultures of the microorganisms, this submerged culture can be carried out in small shaken or shaken flasks by means of suitable mechanical devices.



   Stirring and aeration of the culture medium can be carried out in different ways, stirring can be achieved by means of a propeller or similar mechanical stirring apparatus, by rotating or shaking the fermenter itself, by means of various pumping devices, or by passing air or other gases containing oxygen through the medium, aeration can be effected by injecting air, or other gases containing oxygen, in the fermentation mixture through open tubes, perforated tubes, porous diffusion media such as carbon rods, carborundum, sintered glass, etc.

   or it can be produced by spraying, spraying or pouring the wort into or through an oxygen-containing atmosphere. The oxygen-containing gas must be free of spores, bacteria and other contaminants before it is introduced into the culture medium. This can very advantageously be achieved by filtration through a bed of activated charcoal. The degree of aeration resulting in optimum production of antibiotics C and D depends somewhat on the kind of tank employed, the speed and type of agitation, the pH, temperature and other conditions of the medium.

   In any case, the oxygen-containing gas must be supplied at a sufficient flow rate to maintain a slight positive pressure in the fermenter and thus eliminate the possibility of contamination of the culture by the outside air, it has been found that in devices close

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 30 liter bowl type agitators, a flow rate of ten to fifteen liters of air per minute gives good results when used with a turbine-type mechanical agitator with six blades 10 cm (four inches) in diameter, rotating At 100-200 rpm, against four 25 by 400 mm (1 inch by 16 inch) peripheral vertical baffles, a large number of culture media can be used in the growth phase.

   These media must contain a source of assimilable carbon, a proteinaceous material, mineral salts and traces of various foods, vitamins and other growth stimulants which usually exist as impurities in the other constituents of the medium, for the optimum production of 'antibiotic C and D, the nutrient medium must also contain a source of fat, by the term source' of assimilable carbon is meant here also polyhydric alcohols and mono-di- and polysaccharides, while the term protein material includes a protein unmodified or degradation products of proteins, particularly products such as those derived from the hydrolysis of proteins, these degradation products of proteins include proteases, peptones, polypeptides, peptides and amino acids.



   As sources of assimilable carbon, arabinose, fructose, galactose, lactose, maltose, rhamnose, xylose, glycerin, mannitol, rhamnitol etc. can be used. it is possible to use materials based on complex carbohydrates such as starches, dextrins, fermentation and distillation residues, solid compounds of corn maceration water, corn maceration liquor, dry fermentation residues, etc. .. @ among the protein materials that can be used in the different media, we can cite beef extract, acid-hydrolyzed casein, beef peptone, soybean oil flour, dried fermentation residues. , defatted and partially hydrolyzed soybean flour,

   mixtures of amino acids from natural or synthetic sources, urea, purines and the like. The use of soybean oil meal or soy protein derivatives as a component of the medium is desirable, because such component provides a small amount of fat for use by the microorganism.



   The main mineral requirements of the medium can be satisfied by adding 0.1 to 0.5% of one or more inorganic salts such as sodium chloride, calcium carbonate, bipotassium phosphate, monopotassium phosphate, etc. The other constituents of nutrient medium, ie vitamins, growth stimulating agents, etc ... are generally present in sufficient quantity in more impure assimilable sources of carbon and / or proteinaceous materials.



    Separation of antibiotics C and D from the crude culture can be accomplished by a number of different means. When a solid culture medium is used in the growth phase, the mycelium is extracted with a solvent for antibiotics C and D, such as water; and separating the products from the extract as described later.

   When using a liquid culture medium, in the growth phase the mixture can be filtered to remove the mycelium or the pH of the mixture can be adjusted with acid to about 2 to 4 and then the mycelium separated by filtration. to obtain the antibiotics from the above extracts, one can apply solvent extraction, using as solvents lower aliphatic alcohols immiscible with water such as n-butanol, or by adsorption processes and elution, using as adsorbents respectively activated carbon or ion exchangers, and as eluent acidified methanol or aqueous ammonia.



   The above processes give a crude concentrate which can be further purified by fractionation with organic solvents, for example by dissolution in methanol and fractional precipitation with ethyl ether, or by precipitation with arylsulfonic acid followed by regeneration. from the dye salt thus formed.

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   The separation of antibiotics Q and D from the purified crude concentrate can be effected in a number of ways, for example, antibiotic C can be obtained as a crystallized solid by recrystallization of the concentration residue from the aqueous effluent. . or else, an aqueous solution of the crude concentrate containing both antibiotics C and D can be treated with ammonia to precipitate antibiotic C by fractionation. Finally, antibiotic C can be obtained from a solution of the two antibiotics by adding potassium acetate to effect precipitation by pH adjustment and desalting. Mother liquors from: any of the above separation processes are effectively used to obtain the antibiotic D.



  Chromatography on cellulose provides an advantageous separation of the antibiotic D. The latter produced by cellulose is adsorbed from its solution in a mixture of acetic acid, ethyl acetate and water. Elution with water gives an effluent liquor from which antibiotic D can easily be obtained by concentration.



   The invention is illustrated by the following examples: EXAMPLE 1 - a mixture which consists of 180 gr is introduced. powdered corn starch, 180 gr. of acid hydrolyzed casein, 90 gr. of oilseed flour of soya beans, 90 gr. of sodium chloride and a sufficient quantity of water to form a volume of 18 liters, in a 30 liter fermenter of the fixed glass tank type with a stainless steel cover and a turbine impeller. The fermenter is glass lined and carries four vertical 25 by 400 mm (1 inch by 16 inch) interior baffles. The fermenter is also fitted with devices for introducing three currents of air in the wake of the impeller.



   The pH of the mixture in the fermenter is adjusted to 7.5 using a 10 N solution of sodium hydroxide. 72 gr. Are added to the mixture in the fermentation apparatus. of a mixture of raw lard and mineral oils containing mono- and biglycerides and 18 gr. of calcium carbonate. the fermenter and the medium are sterilized under a water vapor pressure of 1.4 kg / cm2 (20 pounds) for two hours. the fermenter and its contents are cooled and then removed from the autoclave, the medium is inoculated aseptically with 10 cm3 of a suspension of streptomyces C1730 spores prepared by shaking a culture medium of Streptomyces C1730 on seven day old glucosetryptone-agar with 10 cm3 of 0.01% aqueous solution of Marseille soap.



   After inoculation, the culture mixture is incubated at 24-26 C for 64 hours. During incubation, sterile air is passed through the annular diffuser at a rate of 0.9 to 1.16 liters of air per liter of medium per minute, and the agitator is rotated at a rate of. speed of about 200 revolutions per minute.



   The course of antibiotic production is followed during incubation by taking samples from time to time and testing them against Escherichia coli. After 64 hours, each milliliter of culture is equivalent to 920 micrograms of standard when tested against Escheridia coli. The standard is a concentrate of antibiotics C and D having a potency such that a solution of 2.5 mierograms of standard per cm3 produces a 50% inhibition of Escheridia coli.



   We combine the "beers" from several fermentations carried out as described above, at the end of the incubation, the pH of the medium is adjusted to 2.47 using sulfuric acid, the mycelium is removed by filtration, and it is washed completely with water.

   The combined filtrate and washings (volume 41.1 liters) are adjusted to pH 7, 1a and extracted with six 10 liter portions of n-butanol. the combined extracts of n-butanol are concentrated in a flash evaporator at a temperature below 29 ° C. to 10.6 liters. an essentially inert pale yellow precipitate which forms during concentration is filtered off, extracted

 <Desc / Clms Page number 11>

 the filtrate with 8 portions of 1 1/2 liters of 0.01 N aqueous hydrochloric acid. the combined acid extracts are treated with a sufficient quantity of resinous ion exchanger (basic stage) to remove the excess acid and the solution is concentrated in a flash evaporator at 30 ° C to 1.5 liters.

   This volume of solution is dried in the frozen state and the residue, which weighs 20.08 gr. gives a test of 452 micrograms of standard per mgr. against Escherichia coli. to further purify the crude concentrate, 5.0 gr is added. to 25 ml of absolute methanol. about 1.4 gr are separated. of an insoluble white solid and washed with four 5 ml portions. of absolute methanol. The combined supernatant liquids are diluted to 50 ml with absolute ethanol. 100 ml are added. ethyl ether, the yellowish-brown precipitate which forms is separated by centrifugation, washed with ethyl ether and dried in vacuo.

   The production is 2.4 gr. and the product gives a test of 900 micrograms of standard per mgr. against Escheridia coli. The purified crude product consists of a mixture of the hydrochloride salts of the two antibiotics and is very soluble in water. to separate the antibiotic C from the purified crude concentrate, 500 mgr. of the latter, dissolved in 65 ml of water (pH 4.9) through a column which contains a resinous ion exchanger at the basic or hydroxyl stage. the column is then washed with water until the liquid leaving no longer reacts with Ehrlich's reagent. the liquid exiting is concentrated by drying in the frozen state to obtain a product soluble in 25 ml of hot water.

   Cooling the solution to 0-1 C gives a gelatinous precipitate which is recrystallized from hot water to produce a crystalline mass of antibiotic C in the form of needles which melt at 180-182 C. The spectrum d Ultraviolet absorption gives the following measurements:
 EMI11.1
 
<tb>
<tb> 0.1 <SEP> N <SEP> HCl <SEP> Lambda <SEP> = <SEP> 257 <SEP> millimicrons <SEP> E1 <SEP> cm1% <SEP> = <SEP> 368
<tb> id. <SEP> = <SEP> 311.5 <SEP> id, <SEP> id. <SEP> = <SEP> 292
<tb> <SEP> buffer of <SEP> phosphate <SEP> id. <SEP> = <SEP> 249 <SEP> id. <SEP> id. <SEP> = <SEP> 266
<tb> at <SEP> pH <SEP> 7.0 <SEP> id. <SEP> = <SEP> 321 <SEP>. <SEP> id, <SEP> id. <SEP> = <SEP> 535
<tb> 0.1 <SEP> N <SEP> NaOH <SEP> id. <SEP> = <SEP> 329 <SEP> id, <SEP> id.

   <SEP> = <SEP> 639
<tb>
 
The Rf index (ascension technique) for the antibiotic C in ethyl acetate (3), water (3), acetic acid (1) is 0.16; in n-butanol (la), acetic acid (1), water (4), it is 0.28.



   Antibiotic C gives a positive Bratton-Marshall test and a positive Ehrlich reagent test. It gives a test of 814 micrograms / mgr. standard against Escheridia coli. to separate the antibiotic D, 150 mgr. crude concentrate purified in a mixture of three parts of ethyl acetate, 3 parts of water and 1 part of acetic acid. The solution is introduced at a flow rate of 3-4 ml per hour into a column containing 35 g. of a cellulose called "Solka Floc". The column was then developed with fresh solvent and 3 ml fractions collected at hourly intervals.

   When the outgoing fractions become negative for Ehrlich's reagent, it is eluted. column per 100 ml. of solvent and then with water. The eluate is dried (approximately 100 ml) in the frozen state to produce a brown powder. Dissolving this product in methanol and precipitating it with ethyl ether gives a light tan powder which gives 1070 micrograms / mg of standard against Escherièhia coli. The Rf index in ethyl acetate (3), water (3), acetic acid (1) is 0.0 while the Rf index in n-butanol (la) , acetic acid (1), water (4) is 0.19. The solid antibiotic D exhibits marked activity when tested against Mycobacterium tuberculosis var. hominis H37Rv.

   The absorption spectra of antibiotic D for different pH values show only one maximum; 'at 314 (millimicrons., 1' Et% is 123 in 0.1 N hydrochloric acid, at 302 millimicrons, the E1 cm1% is 125 in phosphate buffer at pH 7.0, and at 321 , 5 millimicrons, the E1 cm1% is

 <Desc / Clms Page number 12>

 143 in 0.1N sodium hydroxide.



    F.XF - MPT.E 2 n- is introduced into each element of a number of 1 liter Erlenmeyer flasks a mixture consisting of 1.5 gr, of cerelose, 1.5 gr. of glycerin, 0.9 gr. of partially hydrolyzed casein, 0.75 gr. of peptone, 0.3 gr. brewer's yeast, 0.75 gr. of solid residues of maceration of corn, 0.75 gr. of soybean oil flour, 1.5 gr. of butanol-acetone fermentation residue, 1.5 gr. of sodium chloride, 0.3 gr.

   of calcium carbonate and sufficient water to bring the volume to 300 ml. the vials are covered with cotton gauze and sterilized under a steam pressure of 1.265 Kg / cm2 (18 pounds) for twenty-five minutes, then each vial is aseptically inoculated with 1 cm3 of spore suspension of Streptomyces 01730 prepared from a culture of Streptomyces C1730 on glucose-tryptone-agar, 30 days old. The vials are shaken on a rotary-type shaking machine, rotating at 150 rpm for four days, for During the incubation period, the temperature is maintained at 22-24 C. At the end of the incubation period, the cultured "beer" has a pH of about 6.7.



   A test against Staphylococcus aureus shows that the cultured beer contains a sufficient amount of antibiotic C and antibiotic D to produce 63% inhibition of the growth of the test organism at a dilution of 1%. the mycelium is filtered off from the broth contained in the above flasks and the pH of the filtrate is adjusted to 8.6 to 7.17 with 3 N sulfuric acid. The antibiotic is separated by 100 ml. of filtrate by four extractions in portions of 25 ml. of n-butanol. the combined alcohol extracts are evaporated to dryness at less than 45 ° C. to obtain the antibiotics C and D in the form of a crude concentrate.



   1.0 gr is dissolved. of crude concentrate in 20 cm3 of water. a solution of 550 mg of p-hydroxyazobenzene-p-sulfonic acid in 11 cm3 is added, which causes the immediate formation of a gummy precipitate. the mixture is centrifuged and the supernatant liquid decanted, the gummy residue washed with water, and recrystallized from 6 ml. of absolute ethanol and 9 ml. The precipitate which forms on cooling is separated by centrifugation, washed well with water and dried in vacuo. It weighs 570 mgr. the dye complex is then dissolved in 10 ml. of methanol and precipitated as a yellow-orange powder by adding 30 ml. of ethyl ether, It weighs 475 mgr.



   450 mg of this dye complex are dissolved in 20 ml. 50% methanol and an aqueous solution of barium acetate is added until the precipitation of barium p-hydroxyazobenzene-p-sulfonate is complete, the precipitate is separated by centrifugation and the supernatant is added to the liquid. 1 ml. excess 2N sulfuric acid after having separated all of the barium ions in the form of barium sulfate, the latter is separated by centrifugation. the supernatant light yellow liquid is passed through a synthetic resin ion exchanger column (basic stage). The light yellow liquid exiting has a pH of 7.6.

   the column is washed with water until the washings no longer give results with Ehrlich's reagent. The exiting liquid and the washings give, on drying in the frozen state, a light yellow powder, weighing 173 mg, which gives the test 1150 micrograms of standard per mg. against Escherichia coli. to 49 mg. of purified crude concentrate containing the two antibiotics C and D, 1 ml. water and enough 1N ammonium hydroxide to bring the pH to 9.0. A white precipitate forms which is separated by centrifugation.

   The solid was washed with cold water and recrystallized from aqueous methanol to obtain light yellow colored needles, which melt at 180-182 ° C, identical to the antibiotic C obtained in Example 1.

 <Desc / Clms Page number 13>

 



   Antibiotic D can be separated from the aqueous ammoniacal liquor which supersedes by the cellulose chromatography method described in Example 1.



    EXAMPLE 3 a mixture which consists of 180 gr. of glycerin, 90 gr. of soybean oil flour, 180 gr. of hydrolyzed casein -. by an acid, 90 gr. of sodium chloride and a quantity of water sufficient to bring the total volume to 18 liters, in a fermentation apparatus. 30 liter glass fixed tank type with stainless steel cover and turbine-like impeller. The fermentation apparatus is lined with glass and carries four interior vertical baffles of 25 x 400 mm, (1 x 16 inches). The fermentation apparatus is fitted with a device for introducing three streams of air in the wake of the impeller.



   The pH of the medium in the fermentation apparatus is adjusted to 7.5 by means of a 10 N sodium hydroxide solution. 180 g are added to the mixture in the fermentation apparatus. of lard as an anti-foaming agent and 18 gr. of calcium carbonate, the fermentation apparatus and the medium are sterilized under a water vapor pressure of 1.4 kg / cm2 (20 pounds) for two hours. the fermentation apparatus and its contents are cooled and removed from the autoclave. the medium is aseptically inoculated with 10 ml of a streptomyces C1730 spore suspension prepared by stirring a seven day old glucose-tryptone-agar streptomyces C1730 culture medium with 10 ml. from an aqueous solution to.

   0.01% Marseille soap.



   After inoculation, the culture medium is incubated at 25 ° C. for eighty-eight hours. During incubation, sterilized air is passed through the annular diffuser at a rate of 0.3 to 1.0 liters of air per liter of medium per minute and the agitator is rotated at a speed of about 200 revolutions per minute. The course of antibiotic production is monitored during incubation by taking samples from time to time and testing them against Escherichia coli. At the end of forty hours, each milliliter of culture is equivalent to 188 micrograms of standard when tested against Escheridia coli.

   After sixty-four hours, each milliliter is equivalent to 410 microgr .; and after eighty-eight hours, each milliliter is equivalent to 636 microgr. standard.



   The acidified crude culture is filtered to remove the mycelium and the filtrate is adjusted to pH 7.0 with dilute sodium hydroxide, added to a column containing 600 cm3 of a cation exchange resin in the hydrogen cycle. 7.5 liters of slurry filtrate at pH 7.0. After washing the column with water, the antibiotics are quantitatively separated from the column by elution with 5 N ammonium hydroxide. The antibiotics can be obtained from the eluate in the form of a crude concentrate which can be purified by the methods described in Examples 1 and 2.



   Antibiotic C can be separated from this purified crude concentrate by the following process: added at 500 mgr. of purified crude concentrate in 7 ml. of water, a concentrated solution of potassium acetate containing 50 gr. in 25 ml. of water, until the pH of the total solution is 8.1. A gummy precipitate forms which solidifies after standing for a short time.



  The solid is separated by filtration and washed thoroughly by trituration under water. The solid washed and dried in a vacuum weighs 160 mgr. and, after recrystallization from aqueous methanol it gives a needle mass, melting at 178-179 C, identical to the antibiotic C.



   Antibiotic D is obtained from the crude concentrate purified by the method described in Example 1.



    EXAMPLE @
A mixture consisting of 180 gr. of glycerin, 90 gr.

 <Desc / Clms Page number 14>

 of soybean oil flour, 180 gr, acid hydrolyzed casein, 90 gr. sodium chloride and a quantity of water sufficient to bring the total volume to 18 liters, in a 30 liter fermentation apparatus of the fixed glass tank type with a stainless steel cover and a turbine-like impeller . The fermentation apparatus is glass lined and carries four vertical interior baffles measuring 25 x 400 mm (1 x 16 inches). The fermentation apparatus is fitted with a device allowing the introduction of 3 air streams in the wake of the impeller. the pH of the medium is adjusted to 7.5 in the fermentation apparatus by means of a 10 N sodium hydroxide solution.

   180 gr is added to the mixture in the fermentation apparatus. of lard as an anti-foaming agent and 18 gr. of calcium carbonate, the fermentation apparatus and the medium are sterilized under a pressure of 1.4 kg / cm2 (20 pounds) of water vapor for two hours. The fermentation apparatus and its contents are cooled and removed from the autoclave, the medium is inoculated aseptically 10 ml. of a suspension of streptomyces C1730 spores prepared by stirring a culture medium of Streptomyces C1730 on glucos.e-tryptone-agar, seven days old with 10 ml of a 0.01% aqueous solution of Marseille soap .

   After inoculation into the culture medium, the latter is incubated at 25 ° C. for eighty-eight hours. During the incubation, sterilized air is passed through the annular diffuser at a flow rate of 0.3 to 1.0 liters of air per liter of medium per minute and the agitator is rotated at a speed of about 200 revolutions per minute. the course of antibiotic production is monitored during incubation by taking samples from time to time and testing them against Escheridia coli.

   After forty hours, each milliliter of culture is equivalent to 188 microgr. standard when tested against Escheridia coli: after sixty hours, each milliliter equals 410 microgr, and after eighty-eight hours, each milliliter is equivalent to 636 microgr. standard. the pH of 100 cm 3 of filtrate is adjusted to 7.5 by means of an aqueous solution of sodium hydroxide and the mixture is stirred for 20 minutes with 300 mgr. activated carbon. The suspension is filtered and the carbon cake is stirred with 25 cm3 of acidified methanol containing 0.05 cm3 of 5N hydrochloric acid per 100 cm3 of methanol. The mixture is filtered and the elution is repeated.

   The methanol extracts are combined, concentrated in vacuo and the resulting concentrate contains about 50% of the activity of the initial broth.



   Antibiotics C and D can be separated from the culture mixture by any of the methods described in the previous examples.



   EXAMPLE 5.



   Into each cell of a number of 500 ml Erlenmeyer flasks is introduced a mixture which consists of 1.0 g. of glycerin, 0.5 gr. of soybean oil flour, 0.5 gr. of commercial peptone, 0.5 gr. of sodium chloride and sufficient water to bring the volume to 100 ml. The pH of the contents of the flasks is adjusted to 7.5 using 10 N potassium hydroxide solution and 0.1 g is added. of calcium carbonate. The vials are covered with cotton gas and sterilized under a pressure of 1.05 kg / cm2 (15 pounds) of water vapor for twenty minutes, then each vial is inoculated with 1 ml. a suspension of spores from a seven day old glucose-tryptone-agar culture medium.

   The vials are stirred in a rotary type stirrer rotating at 160 revolutions per minute for six days. the temperature is maintained at 24-26 C during the incubation period. At the end of the incubation period, the culture broth has a pH of 8.3. In a test against Escheridia coli, one milliliter of broth is equivalent to 800 microgr, of standard.



   Antibiotics C and D can be separated from the culture broth by any of the methods described in Examples 1, 2 and 3.

 <Desc / Clms Page number 15>

 



   EXAMPLE 6 The pH of a mixture which consists of 1.0 g of glycerin, 0.5 g of oil flour is adjusted to 7.5 using 10 N potassium hydroxide solution. of soybeans, 0.5 gr. brewer's yeast from which bitterness has been removed, 0.5 gr. of sodium chloride and a sufficient quantity of water to bring the volume to 100 ml. - and 0.1 gr. of calcium carbonate the mixture is introduced into each component of a number of 500 ml Erlenmeyer flasks. the vials are covered with cotton gauze and sterilized under a steam pressure of 1.05 kgr / cm2 (15 pounds) then inoculated into each vial 1 ml.

   of a suspension of Streptomyces C1730 spores from a thirty-five day old culture medium on glucosetryptone-agar, the vials are stirred on a rotary shaker rotating at 160 revolutions per minute for five days. during the incubation period, the temperature is maintained at 24-26 C.



  At the end of the incubation period, the culture broth has a pH of 8.2.



  Tested against Escheridia coli, one milliliter of this broth is equivalent to 710 mgr. standard. Antibiotics C and D can be separated from the culture broth by any of the methods described in Examples 1, 2 and 3.



   EXAMPLES A nutrient medium which consists of 1.25 g is made alkaline at a pH of 7.3 by means of a 10 N solution of sodium hydroxide. of glycerin, 0.75 gr. of casein hydrolyzed by acids, 1.25 gr. of solid residues from maceration of corn, 1.25 gr. of sodium chloride and sufficient water to bring the volume to 250 ml. After addition of 0.25 gr. of calcium carbonate, the medium is introduced into a Fernbach flask fitted with a cotton stopper and sterilized by steam at a pressure of 1.265 Kgr / cm2 (18 inches) for twenty five minutes. The medium is inoculated with 2 ml. of a suspension of Streptomyces C1730 spores originating from a culture medium on glucose-tryptone-agar seven days old, the resulting mixture is subjected to incubation at 29 ° C. for eleven days.

   At the end of this time, the culture broth has an activity equivalent to 273 micrograms of standard per milliliter of broth tested against Escheridia coli. Antibiotics C and D can be separated from the culture broth by any of the methods described in Examples 1, 2 and 3.



   CLAIMS.


    

Claims (1)

1. - Process for the production of Antibiotic C and Antibiotic D, characterized in that a nutrient medium is inoculated with Streptomyces C 1730. The inoculated nutrient medium is cultivated under aerobic conditions at a temperature of about 20 to 40 C for about two to. fifteen days and the antibiotic C and the antibiotic D thus produced are separated from the culture medium, 2. - Process according to claim 1, characterized in that the culture is carried out in the submerged state in an aqueous nutrient medium having a pH of between 6 and 9, containing a protein material, and an assimilable carbon source is used. . 3. - Antibiotic C, basic chemical compound, characterized by the following properties: it is crystalline when it is pure and melts at approximately 182 to 184 C; it contains only the elements carbon, hydrogen, oxygen and nitrogen; it has the probable empirical formula C26H37O8N5, a molecular weight of about 552 in solution in camphor, an optical rotation ([alpha]) D56 in methanol of about + 180.9; it contains an arylamino group; it gives a negative test to carbohydrates, proteins, polypeptides, phenols, enoles, monosubstituted guanidines, ketones, aldehydes and alpha-amino acids; its absorption spectrum of ultraviolet rays has the following maxima: in dilute hydrochloric acid, to 256 and 311.5 millimicrons with values of E11 cm1% of respectively 358 and 291; in a phosphate buffer <Desc / Clms Page number 16> te at pH 7.0 at 250 and 320.5 millimicrons with Et% values of 270 and 536, respectively; in sodium hydroxide diluted to 330-1 cm millimicrons with an E1 cm1% value of 626; it has high intensity absorption maxima in 1 cm 1 -infra-red at or around 5.98, 6.09, 6.24, 7.99.9,16, 9.50 and 9, 62 microns and moderate intensity in the infrared in the region of 9.8 to 12.7 microns; it is soluble in silue acid, in lower aliphatic alcohols, moderately soluble in al, insoluble in ethyl ether; it precipitates from an aqueous solution during the formation of dye salts by means of the usual base precipitators such as p-hydroxyazobenzene-p-sulfonic acid, picric acid and orange II; it has an Rf index (technique by climbing) of 0.16 in ethyl acetate (@ 3), water (3) and acetic acid (3) and an Rf index of 0.28 in n-butanol (10), acetic acid (1), water (4) and it has antibiotic activity against Mycobacterium tuberculosis var, hominis H37 Rv. 4. - Antibiotic D, basic chemical compound, characterized by the following properties: it contains only the elements carbon, hydrogen, oxygen and nitrogen; its absorption spectrum of ultraviolet rays has the following maxima: in hydrochloric acid diluted to 314 millimicrons with a value of E1 cm1% of -123; in pH 7.0 phosphate buffer at 302 mm with 1 at an Et% value of 125, and in sodium hydroxide diluted to 321.5 mm with an Et% value of 143; it is very soluble in water and methanol, soluble in hot ethanol and insoluble in ethyl ether; it precipitates from an aqueous solution during the formation of dye salts by means of the usual base precipitation agents, such as p-hydroxyazobenzene-p-sulfonic acid, picric acid and orange II ; it has an Rf index (climbing technique) of 0.0 in ethyl acetate (3), water (3), acetic acid (1) and an Rf index of 0.19 in n-butanol (10), acetic acid (1) water (4); and it has antibiotic activity against Mycobacterium tuberculosis var. hominis H37Rv.