BE881509A - ANTIBIOTICS C-19393 S2 AND C-19393 H2 AND THEIR SALTS, THEIR PREPARATION PROCESS FROM MICROORGANISMS AND THEIR USE - Google Patents

Description

       

  The present invention relates to antibiotics

  
  <EMI ID = 1.1>

  
soil and research antibiotics produced by microorganisms in order to explore and research new antibiotics. As a result of this research, the authors of the present invention have discovered that certain microorganisms can produce new antibiotics, that these microorganisms belong to the genus Streptomyces,

  
that the culture of these microorganisms in an appropriate culture medium leads to an accumulation of antibiotics in the culture broth and that said antibiotics have an antimicrobial activity with respect to Grampositive and Gram-negative bacteria The authors of the present invention have isolated the above antibiotics which / are confirmed to be new based on their chemical and physical properties and which have the following formula (I)

  

  <EMI ID = 2.1>


  
  <EMI ID = 3.1>

  
The present invention therefore relates to:

  
(1) the antibiotic C-19393S2 or C-19393H2 represented by the formula
  <EMI ID = 4.1>
(where R is -SO3H or hydrogen) ,,, and

  
  <EMI ID = 5.1>

  
  <EMI ID = 6.1>

  
In the context of the present invention the anti

  
  <EMI ID = 7.1>

  
producing these antibiotics and belonging to the genus Streptomyces is used. A typical example of a microorganism

  
  <EMI ID = 8.1>

  
rant a suspension of a soil sample, collected in Sweden, in sterilized water, on a culture medium

  
  <EMI ID = 9.1>

  
magnesium sulfate, 5 ug / ml bleomycin and 2% agar, having a pH of 7 and purifying repeatedly

  
  <EMI ID = 10.1>

  
The microbilogic characteristics of the strain C-19393 of Streptomyces sp. are as follows: a) morphological characteristics:

  
The aerial mycelium having a width of about

  
  <EMI ID = 11.1>

  
is branched monopodically from the main axis to form side chains. Straight or slightly flexible spore chains, that is to say what is called "rectus flexibilis" (hereinafter abbreviated as "RF") are observed at the end of the side chain. Each spore has a cylindrical formula (0.35 to

  
  <EMI ID = 12.1>

  
neither specific organs such as spherical sporangia and sclerotia nor spores likely to move.

  
b) Culture characteristics:

  
The cultivation characteristics of the layer on
-Various backgrounds are shown in Table 1 below. Unless otherwise specified, specifications are observed. <EMI ID = 13.1>

  
Table 1
  <EMI ID = 14.1>
   <EMI ID = 15.1>

  
grayish yellow are abundantly formed. No soluble pigments are produced.

  
c) Physiological characteristics
(1) 7one temperature for growth:

  

  <EMI ID = 16.1>


  
(2) Liquefaction of gelatin: positive

  
(3) Starch hydrolysis: positive

  
(4) Peptonization of skim milk: positive

  
coagulation of skimmed milk ': negative

  
(5) Production of melanoid pigments

  
tyrosine-agar: negative

  
peptone-yeast-iron-agar extract: negative (6) Assimilation of carbon sources (PridhamGottlieb agar):

  
is shown in Table 2 below Table 2

  

  <EMI ID = 17.1>


  
Note: -: zero growth

  
4 Uncertain growth

  
+: growth

  
According to the various characteristics above,

  
  <EMI ID = 18.1>

  
myces. The authors of the. present Invention have sought the taxonomic position of the strain C-19393 based on the above observations namely: the strain develops the

  
  <EMI ID = 19.1>

  
the surface of the spores is smooth, the melanoid pigments

  
and the soluble pigments are not produced in the medium, no defined opposite color is observed and the layer does not use mannitol, referring to the following bibliography:

  
Reference 1. S.A. Walcsman, "The Actinomycetes",

  
Flight. 2, 1961

  
  <EMI ID = 20.1>

  
Bacteriology, 8th Edition, 1974

  

  <EMI ID = 21.1>


  
However, the authors of the present invention have not found any strain in references 1 to 3 which has all of the above characteristics of the strain.

  
  <EMI ID = 22.1>

  
but Streptomyces rallier !, which does not use mannitol, 'differs from strain C-19393 in that the former is not

  
  <EMI ID = 23.1>

  
followed by the name "Czapek") and, also because the types

  
  <EMI ID = 24.1>

  
are often different. Among the strains indicated in the

  
  <EMI ID = 25.1>

  
strain C-19393 in the use of carbon sources other than mannitol, but 11 differs from strain 1-19393 in that it * produces soluble red to pink pigments.

  
Among the 41 strains indicated in the table

  
  <EMI ID = 26.1>

  
Streptomyces canescens does not use mannitol but <EMI ID = 27.1>

  
only develops slightly on the Czapek and, moreover, does not use xylose and rhamnose. Strains similar to strain c-19393 in the use of carbon sources other than mannitol are described below, but each

  
  <EMI ID = 28.1>

  
clues in parentheses. The color of the aerial mycelium and the production of soluble pigments are directly compared between the scuche C-19393 and the strains typical of the respective species. Streptomyces chrysomallus (coloration of the aerial mycelium and production of soluble pigments), Streptomyces citreofluorescens (color of the aerial mycelium and production

  
  <EMI ID = 29.1>

  
on the Czapek), Streptomyces globisporus (large spore diameter, low use of starch), Streptomyces globisporus subsp. vulgaris (weak growth on Czapek), Streptomyces griseinus (weak growth on Czapek and coagulation of skimmed milk), Streptomyces by vus
(production of soluble pigments and large diameter of spores!

  
  <EMI ID = 30.1>

  
or

  
Project "(ISP) described in reference 3, / there are in all
34 strains characterized by a white to yellow aerial mycelium,

  
  <EMI ID = 31.1>

  
defined opposite, not producing soluble pigment and having RF spore chains and a smooth spore surface, are directly compared and examined against references 1, 2 and 3 and if necessary against the originals of these references., by cultivating the strain on a "T medium" on which the strain C-19393 shows culture properties

  
  <EMI ID = 32.1>

  
a characteristic property distinct from the fact that it cannot assimilate mannitol which can be assimilated by most of the species belonging to the genus Streptomyces which have a white to yellow aerial mycellium and, consequently, <EMI ID = 33.1>
-species and designates it by the name 'streptomyces sp. C-19393. This strain was deposited at the "Fermentation Research Institute, Agency of Industrial Science and Technology, Tsukuba, Japan" with the deposit number: FERM-P n [deg.] 4774; at <EMI ID = 34.1>

  
we know that various properties of Streptomyces are not defined and are susceptible to spontaneous or artificial mutations. So the strain which can be used in the present invention includes any strain which belongs to the genus Streptomyces and can produce the antibiotics C-19393S2 and / or C-19393H2.

  
The culture according to the method used for the production of the antibiotic of the present invention can be carried out by growing the above strain in a culture medium containing nutritive products which can be assimilated by microorganisms. The sources of carbon as a component of the medium can be for example: glucose, starch, glycerin, dextrin, sucrose, millet jelly, molasses, etc. Examples of nitrogen sources that may be mentioned include: meat extract, dried yeast, yeast extract, soy flour, maceration liquor for mats, wheat germ, wheat flour cotton seeds, ammonium sulfate, ammonium nitrate, etc.

  
If necessary mineral salts such as carbonate. calcium, sodium chloride, potassium chloride, salt of phosphoric acid, etc. as well as organic and mineral substances facilitating the growth of the micro-organism and fa-

  
  <EMI ID = 35.1>

  
prayed ..

  
Also, heavy metal salts, such as ferrous sulfate, copper sulfate, etc. and vitamins <EMI ID = 36.1>

  
in the middle if necessary, In addition, anti-foaming products and surfactants such as silicone oil, polyalkylene glycol ether, etc. can be added in the middle. Other organic substances. and minerals that facilitate growth

  
  <EMI ID = 37.1>

  
appropriate.

  
The culture can be carried out by the conventional methods generally used for the production of anti-

  
  <EMI ID = 38.1>

  
culture in liquid medium. The culture in liquid medium can be carried out in stationary culture, agitated culture, culture. shaken or aerobic culture, but it is preferably carried out in culture. agitated under aeration. Culture is carried out

  
  <EMI ID = 39.1>

  
at a pH of about 4 to about 8, for about 8 hours to about 168 hours, and preferably for 24 to 144 hours.

  
  <EMI ID = 40.1>

  
mainly extracellularly in the fermentation broth and therefore it is advantageous to separate the resulting culture into microbial cells and a supernatant by centrifugation or filtration then separate the desired antibiotic from the supernatant. However, the desired antibiotic can also be obtained directly from the fermentation bollon.

  
Tests to determine the activity of the product thus obtained can be carried out against the Comamonas terrigena IPO 13299, as a test organism, and in use.

  
  <EMI ID = 41.1>

  
performed by the process generally used to isolate the. metabolites produced by microorganisms. for example <EMI ID = 42.1> water-soluble, mostly extracellularly produced, a process comprising removing

  
  <EMI ID = 43.1>

  
separation, purification and harvesting of the active substance from the filtrate is generally used to isolate the

  
  <EMI ID = 44.1>

  
the advantage of the difference in behavior and

  
the degree of solubility in various solvents, the difference in the behavior or speed of precipitation, and the difference in the adsorption affinity, as well as ion exchange chromatography, molecular sieve chromatography, concentration under pressure reduced and lyophilization, etc. can be used, alone or in appropriate combinations, in any order or

  
repeatedly. Examples of adsorbents that can be used. are activated carbon, adsorbent resins, anion exchange resins, cellulose, powder, silica gel, etc. or.vehicles having molecular sieve properties. Examples of elutlon solvents which can be used are aqueous solutions of water-soluble organic solvents such as acetone, methanol, ethanol, propanol, butanol, isopropanol, isobutanol and the like, or aqueous solutions of acids or alkalis, or a buffer

  
or aqueous solutions of mineral salts or organic salts although the solvents which can be used differ depending on the type of vehicle.

  
  <EMI ID = 45.1>

  
the fermentation broth obtained once the culture is completed is filtered using a filtration aid to eliminate microbial cells. The resulting filtrate is passed through a column of activated carbon in

  
  <EMI ID = 46.1>. Adsorbed is eluted with a system of hydrophilic solvents. Most of the antibacterial activity is found. in the aqueous eluate. Since the antibiotic substance of the present invention is acidic in nature, the echananionic resins

  
  <EMI ID = 47.1>

  
402 and 410 (Rohm & Haas Co., U.S.A.), "Dowex" -1 (Dow and Chemical Co ,, U, S.A.), "Dialon SA" -2lA and C (Mitsubishi Chemical Industries, Japan) may be advantageously

  
  <EMI ID = 48.1>

  
biotic is further eluted with an aqueous solution of sodium chloride or a buffer solution. Elimination of the salts of the eluate can be carried out by rendering the eluate weakly acidic and by still subjecting this eluate to chromatography on activated carbon and then to an elution with

  
  <EMI ID = 49.1>

  
active is concentrated under reduced pressure at low temperature and methanol or ethanol is added to the concentrate. The precipitate formed is removed by. filtration and the resulting hydro-alcoholic solution is concentrated again under reduced pressure. To the concentrated residue acetone or the like is added and the precipitate is separated by filtration. The powder thus obtained can then be. advantageously purified by column chromatography using

  
  <EMI ID = 50.1>

  
(Pharmacia Co., Sweden) and an "XAD" adsorbent resin (Rohm

  
and Haas Co. U.S.A.) or a high porosity type resin "Dialon HP-20" (Mitsubishi Chemical Industries, Japan hereinafter sometimes referred to as "Dialon HP-20"). For this, an aqueous solution of the powder obtained above is passed and adsorbed on "DEAE Sephadex" 25 (form Cl-) and after washing with water, the column is eluted with a solution

  
  <EMI ID = 51.1>

  
killed. using a mixture of isobutanol and water, but the best results can be obtained by eluting under neutral or weakly basic conditions adjusted by adding dilute ammonia or similar products. The substance

  
  <EMI ID = 52.1>

  
adsorbed is eluted in fractions with water. The active fractions are collected and concentrated and the concentrate is subjected to column chromatography using QAE

  
  <EMI ID = 53.1> <EMI ID = 54.1>

  
the eluate by chromatography on activated carbon in the same way as described above. The eluate is concentrated and the concentrate is subjected to column chromatography on "XAD-II" then eluting and fractionating with water.

  
These fractions are found to include fractions showing a single peak on a liquid chromatogram as described below. The active fractions are collected and concentrated to dryness under. reduced pressure at low temperature, and acetone or a similar product is added to the

  
  <EMI ID = 55.1>

  
  <EMI ID = 56.1>

  
specifically, the fermentation broth obtained once

  
the completed culture is filtered using the filter aid to remove. ' microbial cells. The resulting filtrate is passed through a column of activated carbon under neutral or weakly acidic conditions and

  
  <EMI ID = 57.1>

  
hydrophilic. Since the antibiotic substance of the present invention is acidic in nature, exchange resins

  
  <EMI ID = 58.1>

  
can be used advantageously for further purification. The active substance is further eluted with the aqueous sodium chloride solution or a buffer solution. The elimination of the salts of the eluate can be carried out by making the eluate neutral or weakly acid and by subjecting it again to chromatography on activated carbon, then eluting with a hydro-alcoholic mixture, etc. The eluate containing the active substance is concentrated under reduced pressure and at low temperature and methanol or ethanol is added to the concentrate. The precipitate formed is eliminated. by filtration and the resulting hydro-alcoholic solution is again concentrated under reduced pressure, For further purification of the resulting concentrate, the <EMI ID = 59.1> of elution with water to remove the buffer.

   The active eluted fractions are concentrated under reduced pressure at low temperature and the concentrate is lyophilized to obtain C-19393H2 in the form of a white powder.

  
The compounds of the present invention can respectively form alkali metal salts or ammonium salts. As an example of metallic salts of each

  
  <EMI ID = 60.1>

  
lithium, etc.

  
The chemical and physical properties of the disodium salt of C-19393S2 obtained in Example 1 described below are as follows

  
(1) appearance: white powder

  
  <EMI ID = 61.1>

  
(c = 0.5 in water)

  
  <EMI ID = 62.1>

  
The samples are dried over pentoxide

  
  <EMI ID = 63.1>

  

  <EMI ID = 64.1>


  
(the oxygen content is the rest calculated by subtracting the contents of the other elements)

  
(4) Molecular weight: (calculated as containing 2 atoms of

  
  <EMI ID = 65.1>

  
(6) Absorption spectrum in the ultraviolet:

  
The spectrum measured in water is shown in Figure 1 and the maximum value is as follows:

  

  <EMI ID = 66.1>
 

  
(7) Infrared absorption spectrum <EMI ID = 67.1> a potassium bromide tablet is shown in the figure, and the main peaks (number of waves) are as follows:

  

  <EMI ID = 68.1>


  
(8) Thin layer chromatography ("Cellulose f" (Tokyo

  
Kasei Co., Ltd. Japan);

  

  <EMI ID = 69.1>


  
(9) Liquid chromatography under

  
  <EMI ID = 70.1>

  
(10) solubility:

  
Insoluble in chloroform, ethyl acetate, acetone; sparingly soluble in ethanol, butanol,

  
  <EMI ID = 71.1>

  
acetic acid; very soluble in water.

  
(11) Color reactions

  
Positive: reaction of Ehrlich and permanganate

  
potassium

  
Negative: reactions to Ninhydrin, Greig-Leaback,

  
Dragendorff, ferric chloride and. Sakaguchi.

  
The physical and chemical properties of salt

  
  <EMI ID = 72.1>

  
further are:

  
(1) appearance: white powder

  
(2) elementary analysis {$) ^ determined on the sample <EMI ID = 73.1>

  

  <EMI ID = 74.1>


  
(3) Molecular weight (calculated as containing 1 atom of

  
  <EMI ID = 75.1>

  
(4) Molecular formula

  
  <EMI ID = 76.1>

  
(5) Specific rotary power:

  

  <EMI ID = 77.1>


  
(6) Ultraviolet absorption spectrum

  
The spectrum measured in water is shown in Figure 3 and the maximum values are as follows:

  

  <EMI ID = 78.1>


  
(7) Infrared absorption spectrum

  
The measured spectrum of the product dispersed in a potassium bromide pellet is shown in Figure 4 and the main peaks (wave number) are as follows:

  

  <EMI ID = 79.1>


  
  <EMI ID = 80.1>

  
(9) Thin layer chromatography / using "Cellulose f" (Tokyo Kasei Co. Ltd., Japan))
  <EMI ID = 81.1>
(10) High pressure liquid chromatography

  
(Waters Associates Ine. U.S.A.) <EMI ID = 82.1>

  
(11) Colored reactions

  
Positive: Ehrlich reactions to permanganate

  
potassium.

  
Negative: Reactions to. ninhydrin, Greig-

  
Leaback, Dràgendorff ', ferric chloride and Sakaguchi

  
(12) Solubility:

  
Insoluble in chloroform and ethyl acetate:
sparingly soluble in acetone, ethanol and butanol; soluble in methanol and water. According to the properties described above, the

  
  <EMI ID = 83.1>

  
antibiotics of the type (i-lactam and based on each of the elementary analyzes and the estimated molecular weights, the estimated molecular formulas are

  
  <EMI ID = 84.1>

  
  <EMI ID = 85.1>

  
  <EMI ID = 86.1>

  
max

  
and 287 + 4 nm on the ultraviolet absorption spectrum,

  
it is believed that the antibiotics of the present invention have the same chromophore group as in MM-4550 among the known antibiotics of the p-lactam type.

  
Table 3 shows the comparative results of the

  
  <EMI ID = 87.1>

  
  <EMI ID = 88.1>
  <EMI ID = 89.1>
   <EMI ID = 90.1>

  
vis, Gram-positive and Gram-negative bacteria.

  

  <EMI ID = 91.1>


  
Note) Medium: Agar broth

  
  <EMI ID = 92.1>

  
strong beta-lactamase inhibitor activity and, by

  
  <EMI ID = 93.1>

  
various bacteria resistant to penicillin derivatives

  
  <EMI ID = 94.1>

  
penicillin derivatives and / or cephaloaporin derivatives. This increase in antimicrobial activity <EMI ID = 95.1>

  
in table 5.

  
Table 5 Effect of strengthening of the antimtcro- activity

  
  <EMI ID = 96.1>

  
C-19393S2

  

  <EMI ID = 97.1>


  
  <EMI ID = 98.1>

  
to produce beta-lactamase.

  
Medium: coagar infusion

  
(Elken Chemical Co. Japan).

  
As can be seen from the anti spectrum

  
  <EMI ID = 99.1>

  
The invention shows antimicrobial activity against Grain-positive and Gram-negative bacteria. Therefore

  
  <EMI ID = 100.1>

  
bacterial infections in mammals (eg mice, rats, dogs, humans), different species of birds (eg hens, ducks).

  
  <EMI ID = 101.1>

  
dissolves this antibiotic in physiological saline solution to prepare an injectable solution which can be administered parenterally, for example by subcutaneous or intramuscular route, at a dose of 2 to 200 mg / kg / day,

  
  <EMI ID = 102.1> <EMI ID = 103.1>

  
Invention can be used as a disinfectant. For example,

  
  <EMI ID = 104.1>

  
  <EMI ID = 105.1>

  
0.1 to 1% (weight / volume) or an ointment containing 2 to

  
  <EMI ID = 106.1>

  
white petroleum jelly or lanolin as a base, can be used as a bactericide or disinfectant for hands, limbs, eyes, ears, etc. of the above animals.

  
As can be seen from the results of the

  
  <EMI ID = 107.1>

  
Inhibitor of beta-lactamase and therefore remarkably increases the sensitivity of bacteria resistant to

  
  <EMI ID = 108.1>

  
beta-lactamase. Therefore C-19393S2 can be used for the treatment of infections in mammals
(for example mice, rats, dogs, human beings) and volatile species (for example chickens; ducks etc.) in particular in bacterial infections due to bacteria resistant to beta-lactam antibiotics, in particular

  
  <EMI ID = 109.1>

  
porine.

  
  <EMI ID = 110.1>

  
other beta-lactam agents for the treatment of Infections, for example by antibiotic-resistant E. coli

  
  <EMI ID = 111.1>

  
ampicillin are dissolved in physiological saline to prepare a solution for injection which can be administered parenterally, for example by subcutaneous injection

  
  <EMI ID = 112.1>

  
administered orally at a dose of 1 to 00 mg / kg / day, preferably 5 to 100 mg / kg / day in the form of capsules <EMI ID = 113.1>

  
cephalexin.

  
When C-19393S2 is used as a disinfectant, a liquid preparation, for example an aqueous solution

  
  <EMI ID = 114.1>

  
  <EMI ID = 115.1>

  
per gram of white petrolatum or lanolin, as a base, can be used as bactericides or disinfectants for the hands, limbs, eyes, ears, etc. of the above animals.

  
The antibiotic C-19393S2 is also intended to be used as an intermediate product for the synthesis of new types of products. pharmaceutical. The antibiotic of the present invention is stable in aqueous solution in the region of neutral pH.

  
The physiological properties of C-19393H2 are as follows.

  
  <EMI ID = 116.1>

  
C-19393H2 vis-à-vis the various microorganisms is shown in Table 6; and according to these results we believe that

  
  <EMI ID = 117.1>

  
not towards Gram-positive and Gram-negative bacteria.

  
  <EMI ID = 118.1>

  

  <EMI ID = 119.1>


  
Note) Medium: Agar broth As shown in Table 6 above, the antibiotic

  
  <EMI ID = 120.1>

  
and Gram-negative and, therefore, can be used for the treatment of bacterial infections in mammals for example: mice, rats, dogs, humans and the like and in poultry species, for example hens, ducks and the like.

  
  <EMI ID = 121.1>

  
is dissolved in physiological saline solution to prepare an injectable solution which can be administered by parenteral route, for example subcutaneously or

  
  <EMI ID = 122.1> rence 0.5 to 20 mg / kg / day. Also, for oral administration, the antibiotic C-19393H2 is mixed with lactose and encapsulated to make a preparation in capsules which can be administered at a dose of 1 to 100 mg / kg / day, preferably 5 to 50 mg / kg / day.

  
In addition, C-19393H2 obtained according to the present invention can be used as a disinfectant. For example,

  
  <EMI ID = 123.1>

  
  <EMI ID = 124.1>

  
(weight / volume) or an ointment containing 0.2 to 20 mg of

  
  <EMI ID = 125.1>

  
white or lanolin as a base, can be used as bactericides or disinfectants for hands, limbs, eyes, ears, etc. of the above animals.

  
The antibiotic C-19393H2 is also intended to be used as an intermediate product for the synthesis of new types of pharmaceutical products. The antibiotic of the present invention is stable in aqueous solution in the region of neutral pH.

  
As described above, the MM-4550 can be taken as an example as an antibiotic relatively similar to the antibiotics of the present invention C-19393S2 and

  
  <EMI ID = 126.1>

  
  <EMI ID = 127.1>

  
  <EMI ID = 128.1>

  
770-772, 1977) which is the same substance as MM-4550 is very Unstable (Umezawa et al., The journal of Anti-

  
  <EMI ID = 129.1>

  
The present invention is illustrated by the following descriptive and nonlimiting examples in which, unless otherwise indicated, all the percentages are expressed by weight / volume.

Example 1

  
A culture of Streptomyces sp. strain C-19393 (IFO 13886, ATCC 31486) on 200 ml of T medium loaded into a one liter Erlenmeyer flask to obtain spores. The resulting spores are then suspended in sterilized water at a concentration of 1.2 x 108 live cells / ml. The spore suspension is diluted with sterilized water to a volume

  
equal to ten times the initial volume, and 1 ml of the diluted suspension is used to inoculate 40 ml of a medium for

  
  <EMI ID = 130.1>

  
inoculated germ is then grown on a rotary shaker at 28 [deg.] C for two days. The resulting culture is used to inoculate 500 ml of inoculated culture medium loaded into a Sakaguchi shaker flask and the inoculated inoculated broth is cultured on a

  
  <EMI ID = 131.1>

  
The culture of sprouts thus obtained is transferred to a 50 liter fermentation tank made of stainless steel containing 30 liters of a seeded medium containing 15 ml of "Actocol" (Takeda Chemical Industries, Ltd., Japan) and

  
  <EMI ID = 132.1>

  
  <EMI ID = 133.1>

  
culture broth is transferred to a container of

  
  <EMI ID = 134.1>

  
main and. cultivated at 30 [deg.] C for 5 days, with aeration of 840 l / minute and stirring of 180 revolutions / minute. The seeded medium used above is composed

  
  <EMI ID = 135.1>

  
raw soy, 10 g mats maceration liquor,

  
  <EMI ID = 136.1>

  
3 g of sodium chloride and 5 g of calcium carbonate precipitated per liter of broth which has been adjusted to a pH of 7 before being sterilized, and the main culture medium used above is composed of 30 g of glucose ,

  
30 g soluble starch, 15 g defatted soy flour,
15 g cotton seed flour, 0.25 g monopotassium phosphate, 0.6 g dipotassium phosphate, 0.002 g

  
  <EMI ID = 137.1>

  
  <EMI ID = 138.1>

  
media used above are steam sterilized to
120 [deg.] C for? 0 minutes.

  
The fermentation broth thus obtained is filtered

  
  <EMI ID = 139.1>

  
obtain 1230 1 of a filtrate which is then adjusted to pH 6.3 and passed through a column filled with 100 liters of charcoal

  
  <EMI ID = 140.1>

  
  <EMI ID = 141.1>

  
of water, respectively. The eluate containing C-19393H2 is

  
  <EMI ID = 142.1>

  
  <EMI ID = 143.1>

  
eluted with 3? liters of an aqueous solution of chloride

  
  <EMI ID = 144.1>

  
column filled with 4 liters of activated carbon. After washing

  
  <EMI ID = 145.1>

  
  <EMI ID = 146.1>

  
diaper Isobutanol; ammonia N / 20 (8:92) and the eluate is concentrated to a volume of 150 ml under reduced pressure. 1350 ml of methanol are added to the concentrate and the precipitate thus formed is removed by filtration. The filtrate is concentrated

  
  <EMI ID = 147.1>

  
  <EMI ID = 148.1>

  
is washed successively with aqueous solutions of

  
  <EMI ID = 149.1>

  
desired antibiotic substance is eluted with 1500 ml of an aqueous solution of sodium chloride O, 4M. The eluate is adjusted to pH 5 and passed through a column filled with
500 ml of activated carbon. After washing with 1.5 l of water, the

  
  <EMI ID = 150.1>

  
ammonia N / 20 (8:92) and the eluate is concentrated to dryness;

  
acetone is then added to the residue to obtain 2.4 g of a pale yellow powder. After dissolving the powder obtained in a small amount of water, the solution is passed through

  
  <EMI ID = 151.1>

  
  <EMI ID = 152.1>

  
The fractions that show antibiotic activity are

  
  <EMI ID = 153.1>

  
through a column filled with 200 ml of "QAE-Sephadex

  
  <EMI ID = 154.1> <EMI ID = 155.1>

  
filled with 600 ml of activated carbon. After washing the column with 1.8 l of water, the column is eluted with 3 liters

  
  <EMI ID = 156.1>

  
concentrated to dryness and acetone is added to the residue to obtain 1.07 g of a powder. A 620 mg portion of the powder thus obtained is dissolved in a small amount of water and the solution is passed through a column filled with

  
  <EMI ID = 157.1>

  
is then eluted in fractions with water and each of the fractions which shows antibiotic activity is subjected to analysis by liquid chromatography as described above. The fractions which show a single peak are collected. wheat and concentrated to dryness and acetone is added to

  
  <EMI ID = 158.1>

  
C-19393S2 in the form of a white powder.

Example 2

  
  <EMI ID = 159.1>

  
  <EMI ID = 160.1>

  
12 1) and the column is washed with 6 liters of water eluted with

  
  <EMI ID = 161.1>

  
The eluate is passed through a column filled with 25 ml of activated carbon. After washing with 75 liters of water, the anti-

  
  <EMI ID = 162.1>

  
under reduced pressure. 15 liters of methanol are added to the concentrate and the precipitate thus formed is removed by filtration. The filtrate is concentrated to a volume of 2 liters and passed through a filled column, with 5 liters "Diaion

  
  <EMI ID = 163.1>

  
fractionated with 5 liters of water and 10 liters of a methanol: water mixture (1: 9). The active fractions are combined and concentrated, and the concentrate is passed through a column filled with 3 liters of "DEAE-Sephadex A-25"

  
  <EMI ID = 164.1>

  
0.02M sodium chloride aqueous solution and the desired antibiotic is eluted and fractionated with 12 liters of 0.05M aqueous sodium chloride solution. The active fractions are passed through one. column filled with 2 liters of "Dialon HP-20"

  
  <EMI ID = 165.1>

  
watery

  
your / sodium chloride., and, after lifting with 10 liters of a 5% aqueous sodium chloride solution, the active principle is eluted and fractionated with 10 liters of a methanol mixture: aqueous chloride solution 5% sodium (5:95) and 10 liters of a methanol mixture: aqueous solution of

  
  <EMI ID = 166.1>

  
passed through a column filled with 500 ml of activated carbon and, after washing with 1.5 l of water, are eluted with

  
  <EMI ID = 167.1>

  
and the concentrate is passed through a filled column

  
  <EMI ID = 168.1>

  
eluted and fractionated with water. The fractions with antibiotic activity are collected and concentrated and the concentrate is passed through a column filled with

  
  <EMI ID = 169.1>

  
with 400 ml of an aqueous solution of sodium chloride

  
  <EMI ID = 170.1>

  
through a column filled with 200 ml of activated carbon and, after washing with 0.6 liters of water, are eluted with 1

  
  <EMI ID = 171.1>

  
propanol is added to the concentrate to prepare a solution

  
  <EMI ID = 172.1>

  
  <EMI ID = 173.1>

  
the column is eluted with water. The active fractions are concentrated and the concentrate is subjected to chromatography.

  
  <EMI ID = 174.1>

  
sant "RP-18" (E.Merck & Co. Germany West) as vehicle and eluted with a 0.02M phosphate buffer mixture:

  
  <EMI ID = 175.1> <EMI ID = 176.1>

  
(149 µm to 74 µm mesh) and eluted in fractions with water. Each of the fractions which reveals the antimicrobial activity is subjected to analysis by liquid chromatography as described above. Fractions that show only one.

  
  <EMI ID = 177.1>

  
  <EMI ID = 178.1>

Example 3

  
1150 liters of a culture broth filtrate prepared by a process similar to that described in the example

  
  <EMI ID = 179.1>

  
with 100 liters of activated carbon. After washing with 300 liters of water, the column is eluted with 350 liters of a mixture

  
  <EMI ID = 180.1>

  
  <EMI ID = 181.1>. Is eluted with 100 liters of an aqueous chloride solution <EMI ID = 182.1>

  
  <EMI ID = 183.1>

  
been treated with 40 liters of a 5% aqueous sodium chloride solution, and the column is washed with 20 liters of a 5% aqueous sodium chloride solution. Then C-19393S2 and C-I9393H2 are eluted from the column with 40 liters of water and 60 liters of a methanol mixture: 5% aqueous sodium chloride solution (5:95) respectively. The eluate

  
  <EMI ID = 184.1>

  
a column filled with 2 liters of activated carbon. After washing with 6 liters of water, the column is eluted with 10 liters of an 8% isobutanol-ammonia N / 20 mixture and the eluate is concentrated. The concentrate is passed through a filled column

  
  <EMI ID = 185.1>

  
with 1 liter of 0.2M aqueous sodium chloride solution, the column is eluted with 1 liter of 0.4M aqueous sodium chloride solution. Then, the eluate is desalted by chromatography on activated carbon and is subjected to column chromatography with "Amberlite XAD-II" and we continue to work in an identical manner to that of Example 1

  
to obtain 100 mg of the disodium salt of the antibiotic C-19393S2 in the form of a white powder.

Example 4

  
  <EMI ID = 186.1>

  
2 liters of activated carbon. After washing with 6 liters of water, the column is eluted with 10 liters of an isobutanol mixture:
ammonia N / 20 (8:92) and the eluate is concentrated. The concentrate is passed through a column filled with 200 ml of

  
  <EMI ID = 187.1>

  
of an aqueous solution of 0.02M sodium chloride, the column is eluted with 1 liter of an aqueous solution of 0.04M sodium chloride. Once the eluate has been desalted by chromatography on activated carbon, it is subjected to chroma'-

  
  <EMI ID = 188.1>

  
  <EMI ID = 189.1>

  
some water. The fractions which show a single peak by liquid chromatography are combined and concentrated and the concentrate is lyophilized to obtain 48 mg of the sodium salt

  
  <EMI ID = 190.1>

CLAIMS '

  
  <EMI ID = 191.1>

  
formula

  

  <EMI ID = 192.1>


  
in which 8 represents a group -SO HH or hydrogen, and the salts of these antibiotics.

  
2. Process for the preparation of antibiotics C-19393S2 and / or C-19393H2 characterized by the fact that it comprises the culture of a microorganism belonging to the

  
  <EMI ID = 193.1>

  
from assimilable carbon sources and digestible nitrogen sources until antibiotics C-19393S2

  
  <EMI ID = 194.1>

  
middle, and the recovery of these antibiotics.


    

Claims (1)

<EMI ID = 195.1> by the fact that the microorganism is Streptomyces sp. C-19393 (IFO 13886). 4. Essentially pure culture of the microorganism belonging to the genus Streptomyces having the characteristics identifiable with those of IFO 13886, characterized in that said culture is capable of producing in a culture medium containing assimilable carbon sources and digestible sources of nitrogen, a quantity recovered <EMI ID = 196.1> 5. Medicament characterized by / that it contains an antibiotic as defined in claim 1. 6. Medicament according to claim 5 characterized in that it additionally contains one or more antibiotics from the penicillin and / or cephalosporin series.