CH513974A - Process for the preparation of a mixture of the antibiotics quintomycin A, B, C and D. - Google Patents

Description

  

  
 



  Process for the preparation of a mixture of the antibiotics quintomycin A,
B, C and D
The present invention relates to a method for producing a mixture of the antibiotics quintomycin A, B, C and D; this method is characterized in that Streptomyces lividus or its mutants are cultured under aerobic conditions in a medium containing a carbon source and a nitrogen source and the antibiotics are then obtained from the culture broth.



   The above-mentioned new fungus was obtained under the name No. 2230-N1 strain in the Fermentation Laboratory of Technical Institute, 2, 5, 4-chome, Inage-Higashi, Chiba-shi, Japan with the deposit number of No. 50 FLTI deposited. The same strain was later deposited under the name Streptomyces lividus in the American Type Culture Collection with the deposit number ATCC 21178.



   The above-mentioned antibiotic substance produced according to the invention was first named as No. 2230 substance, and only later was named quintomycin. It contains a new antibiotic substance, the quintomycit-A, expressed by the molecular formula C29H55N5O19, the pentahydrochloride with a specific rotation of [a] n22 of +59; a new antibiotic substance quintomycin-B expressed by the molecular formula C29H55N5O18, whose pentahydrochloride has a specific rotation of la] D22 of +50;

   and a new antibiotic substance quintomycin-D expressed by the molecular formula C23H4sNsOI3 whose pentahydrochloride has a specific rotation of [ca, "of +36, and also quintomycin-C, which is a synonym for a known antibiotic substance paromomycin produced by Streptomyces rimosus forma paromomycinus.



  The above three antibiotic substances are new substances.



   The main components of the antibiotic substance quintomycin produced according to the invention are thus new antibiotic substances, namely quintomycin-B and quintomycin-A. The quintomycin produced according to the invention, which contains these new substances, in particular quintomycin-B, has a high biological effectiveness against Pseudomonas aeruginosa and moreover has an extremely low toxicity against warm-blooded animals. Known antibiotic substances have a high biological effectiveness against the abovementioned bacterium, but are not satisfactory with regard to their toxic behavior. The Streptomyces lividus used according to the invention has the following microbiological properties:
1.

  morphology
Streptomyces lividus n.s.p- appears on a synthetic medium in the form of a light gray growth that gradually turns dark blue to black. No soluble pigment is formed, or on a very rare substance, a soluble pigment of a light pink or raspberry color occurs. Aerial hyphae are short and irregularly branched in a single shape. The sporophores formed are generally rectilinear, and in rare cases curved loops (threads or spirals are not formed). Three to ten spores appear at the end of a sporophore.



  The structure of the spore surface was observed through an electron microscope, and it was found to be oval or ellipsoidal (0.2 x 0.3-0.5µ).



     II. Bacterial culture characteristics of the various
Media: (synthetic media) W = growth, M = aerial mycelium, L = soluble pigment Glycerine Czapek's agar W: moderate, light gray to dark blue or black
M: moderate, velvety, light gray to dark blue or black
L: none, rarely light pink or strawberry colored glucose-asparagine-agar W: moderate, cream-colored to dark blue or charcoal gray
M: moderate, velvety, light gray to dark blue
L: no calcium malate agar W: moderate, white to dark blue
M: very poor, white or light blue
L: none (organic medium) nutrient agar W: good, wrinkled, mostly yellow, brown or black
M: poor, light gray
L: no starch agar W: good, light gray to dark blue or black M: velvety, white or light gray
L: no potato glucose agar W:

   moderate, dark blue or dark gray, easily penetrating the medium
M: poor, light gray
L: none, slightly light pink peptone glucose agar W: good, dark blue or dark gray
M: poor, white to gray
L: brick red tyrosine agar W: moderate, light brown to dark gray
M: weak white
L: none or light brown gelatin perforated W: good, white or light gray
M: none
L: no litmus milk W: moderate, cream-colored on the surface
M: none
L: no change
111. Biochemical properties: reaction
Chromogen formation negative
Tyrosinase negative
Nitrate reduction positive
Cellulose decomposition negative
Milk coagulation negative
Milk peptonization positive
Starch hydrolysis positive (strong)
Gelatin liquefaction positive (considerable)
IV.

  Use of carbon sources:
Carbon source use
D-xylose
L-arabinose
D-glucose ++
D-galactose ++
D-fructose +
D-lactose
D-maltose
Sucrose
D-raffinose +
Trehalose ++
Inositol ++
Sorbitol '++
D-mannitol ++
Dextrin +
Strength +
Rhamnose
Inulin
Dulcit
Carbon source use
Glycerin ++
Sodium citrate +
Sodium acetate +
Calcium malate + By comparison with the classification given in Bergey's Manual of Determinative Bacteriology, 7th edition, it was found that the new fungus used according to the invention is similar to S. gedanensis (S. ged.) In the following relationship: psychrophilic to mesophilic growth ; no soluble pigment is produced in the organic medium; strong proteolytic effect; Growth on synthetic media dark to black to mostly blue-black; and a series mycelium white to gray. S.



  ged. on the other hand, shows cream-colored to brownish growth in a potato medium and does not produce aerial mycelium and soluble pigment, while S. lividus (S. liv.) shows dark gray or grayish-black growth with poor aerial mycelium, and usually produces soluble pigment although in In some rare cases it is light pink in color. On a starch medium, S. ged. to a yellow to cream-colored growth, and S. liv. has a light gray to blackish blue or black growth. S. ged. behaves negatively in milk peptonization and nitrate reduction, while S. liv. shows positive behavior in both cases.



   As a strain with psychrophilic to mesophilic growth and which does not produce any soluble pigment in the organic medium, S. griseolus (S. gris.) Can be named; this shows a mouse-gray growth on synthetic medium, produces a white to gray aerial mycelium and just forms sporophores. S. faciculus, which is broom-shaped sporophores, also belongs to this strain. A comparison of these two strains with the S. liv used according to the invention. has resulted in the following.



  In a nutrient agar, S. gris. a brownish growth with a soft surface, produces a deep dull gray aerial mycelium and does not produce any soluble pigment, while S. liv. shows wrinkled moist yellow-brown growth, and produces no soluble pigment with almost no aerial mycelium. In a potato medium, S.



  gris. a yellowish white to black growth, forms an aerial mycelium of a white-gray color and produces a brown to black soluble pigment, while S.



  liv. has a dark gray or grayish black growth, only forms a poor aerial mycelium and does not produce any soluble pigment, although at times it is light pink in color. In gelatin there is S. gris. a yellowish flaky membrane and a sediment and forms a white aerial mycelium and a pale brown medium, while S. liv. produces a white or light gray growth, however, does not form aerial mycelium and soluble pigment. In milk, S. shows gris. a strong red membrane growth and leads to slow coagulation, while S. Iiv. shows a yellowish white growth on the surface and does not coagulate.

 

   S. fasciculus (S. fasc.) Shows good lichenoid growth and forms an aerial mycelium that is colorless and covered with a dark gray, powdery or velvety coating, while S. liv. shows light gray to dark blue or black growth and forms white or white gray aerial mycelium that is short and not as abundant. In a milk medium, S.



  fasc. positive to both coagulation and peptonization and shows good growth on cellulose, while S. liv. is only positive towards peptonization in the milk medium and shows no growth on a cellulose medium.



   When comparing characteristic properties of different series of Streptomyces in the manual of S.A. Waksman The Actinomycetes, Volume 2, nothing was found that appears on S. liv. indicates. S. intermedius, S. parvullus, S. craterifer, (S. crat.) And S. cellulose belong to the series of Cinereus, but can be regarded as similar to S. liv. insofar as spiraling is + or -, melanin is - and an aerial mycelium is white to gray.



   In a nutrient agar, S. crat. shows colorless growth, and in a starch agar, it shows culture outgrowth, thin and colorless growth does not form aerial mycelium. In a potato medium, S. crat. shows a yellowish white growth and forms a white to mouse-gray aerial mycelium. S. liv. on the other hand, shows good wrinkled moist yellow-brown growth in nutrient agar, or good growth from light gray to dark blue or black, and forms velvety white or light gray aerial mycelium in starch agar, and dark gray growth in potato medium.



   S. Cellulose grows well on cellulose and quickly coagulates milk into cellulose and shows yellow or lemon yellow growth in a synthetic medium.



  These characteristics distinguish it from S. liv.



   S. parvullus differs from S. liv. in that it forms sporophore twists in long, closed spirals, it shows yellow growth in synthetic medium, produces yellow soluble pigment in gelatin, produces brown soluble pigment and abundant gray aerial mycelium in milk, and results in very slow peptonization .



   The S. liv. differs from S. intermedius in that the latter shows good growth of olive green in cellulose, folded growth from brown to green brown in a potato medium shows green brown growth of olive green in cellulose, folded growth from brown to green brown in potato medium and produces a greenish brown soluble pigment and shows slow liquefaction in gelatin and produces a greenish brown soluble pigment.



   With the exception of S. griseolus, S. cellulose and S. parvullus, the above-mentioned known strains do not produce any antibiotic substance.



   Information is now given on fungi of the genus Streptomyces which produce antibiotic substances belonging to the aminocyclitol group. According to Waksman, S. fradiae shows a thin, soft, colorless, sometimes orange-yellow substrate growth on a synthetic medium and forms an aerial mycelium which is pale red, sea mussel red or salmon red in color.



   This distinguishes the above Streptomyces from the S. liv used according to the invention. Waksman's classification also indicates that S. albogriseolus shows red to purplish-red growth on potato medium, and the sporophores produce spirals. As a result, it differs from the S. liv used according to the invention. S. rimosus, f. paromomyceticus, S. christomyceticus, S. kanamyceticus and S. pulveraceus all show a yellow or yellowish white to brown growth and have no dark gray to dark blue or bluish black growth like the S. Oliv used according to the invention. does.



   From the above it follows that the above-mentioned strain producing the antibiotic quintomycin has been identified as a new strain belonging to the genus Streptomyces.



   For the sake of completeness, the details of the microbiological properties of strains belonging to the genus Streptomyces and which genus produce antibiotic substances which in turn belong to the aminocyclitol group are given in paragraphs I to VI below. The microbiological properties of strains belonging to other genera and which antibiotic substances belonging to the aminocyclitol group produce are also given in paragraphs VII and VIII.



      1.



  Strain Streptomyces rimosus forma paromomycinus Antagonistic properties Paromomycin properties Morphology Sporophores, forms dense
Tufts of spirals; the aerial mycelium is short and straight, but seldom spiral-shaped glycerine-asparagine-W: yellowish white in color, agar turns brown to orange-brown through aging.



   M: white
L: no calcium malate agar W: yellowish brown
M: white
L: no nutrient agar W: light yellowish white, colored to yellowish brown
M: none
L: no starch agar W: yellowish white colored, with darker brown centers
M: absent or white
L: no potato agar W: lichenoid, colored yellowish white to reddish brown
M: white to gray to dark brown
L: yellowish brown gelatin W: yellowish white colored to brownish
M: white
L: no milk W: heavy surface membrane, yellowish white colored to yellowish
M: gray-white nitrate reduction positive cellulose decomposition negative milk coagulation negative milk peptonization positive hydrolysis of starch positive (limited) gelatin liquefaction positive (slow) II.



  Strain Streptomyces kanamyceticus Antagonistic properties Kanamycin properties Morphology The aerial mycelium develops from the submerged mycelium on a little medium and its
Branching is not abundant and it has the sporophores at its end. Spirals and turns were generally not observed.



  Glycerin-Czapek's-Agar W: colorless, later changing to lemon yellow
M: white to yellow and occasionally greenish or slightly reddish in color
L: occasionally pale brown glucose asparagine agar W: colorless to yellow with a pale reddish white
M: poor, white, slightly reddish white, greenish yellow or yellow
L: occasionally pale brown calcium malate agar W: yellow
W: orange
M: yellowish white nutrient agar W: yellowish white
M: absent or white
L: none potato agar W: wrinkled, pale yellowish brown to yellow
M: poor, white
L: none Nitrate reduction positive Milk coagulation doubtful Milk peptonization doubtful Starch hydrolysis positive Gelatin liquefaction positive
111.



  Strain Streptomyces fradiae Antagonistic properties Neomycin properties Morphology Monopodially branched sporophores straight or flexible, but not true spirals. Spirals are formed in certain media.



  Glucose-Asparagine-AgarW: limited, shiny, leather yellow colored, lichenoiderk
edge
M: late, sea shell red Malate-Glycerin-Agar W: orange
M: sea shell red nutrient agar W: limited, yellowish, orange to leathery yellow
M: none
L: no starch medium W: common, colorless
M: sea shell-red potato agar W: limited, orange-colored white to pink or red
L: absent or brown-colored gelatin W: dense, yellowish white to brownish
M: white
L: none litmus milk W: yellowish white ring
L: becomes alkaline Nitrate reduction negative Cellulose decomposition negative Milk coagulation positive Milk peptonization positive (fast) Starch hydrolysis positive
IV.



  Strain Streptomyces albogriseolus Antagonistic properties Neomycin properties Morphology Monopodially branched Sporophc ren, produce short, compact
Spirals, on average 4-6
Twists. Spherical or oval spores, covered with numerous long, fine hairs Nutrient agar M: white to ash gray starch agar M: white to dark gray milk W: orange-colored ring nitrate reduction positive milk peptonization positive starch hydrolysis positive gelatin liquefaction positive
V.

 

  Strain Streptomyces pulveraceus antagonistic property zygomycin (identical to paromomycin)
Aerial mycelium generally develops well. The sporophore forms spirals and the spores are spherical to ellipsoidal.



   The spiral adhering state.



  Glycerin-Czapek's-Agar W: colorless, later colored brown
M: powdery, light gray brown to light gray olive
L: none glucose asparagine agar W: orange to xanthine orange
M: powdery, light gray olive
L: none or becomes leathery yellow calcium malate agar W: yellow, penetrates the
Medium one
M: small or skimpy, smoky gray
L: no nutrient agar W: colorless, folded
M: none
L: none starch agar W: colorless to yellow-ocher, penetrates deep into the medium
M: poor, light gray olive
L: none tyrosine agar W: colorless to brown in color
M: light gray olive
L: no milk W: colorless, surface growth
L: brown Nitrate reduction positive (strong) Cellulose decomposition negative Milk coagulation negative Milk peptonization positive Starch hydrolysis positive Gelatin liquefaction positive VI.



  Strain Streptomyces chrestomyceticus Antagonistic intrinsic aminosidin (identical to paromomycin) Morphology Air mycelia are generally strong, but rarely have hooks or spirals Glucose-Asparagine-AgarW: yellow
M: absent potato glucose agar W: colorless
M: poor white peptone-glucose agar W: good, white or yellowish white
M: excessively white tyrosinase positive milk coagulation positive milk peptonization negative (neutral) starch hydrolysis positive gelatin liquefaction positive VII.



  Strain Micromonospora purpurea Antagonistic properties Gentamicin properties Morphology No aerial mycelium, increased colony, sinuous abundant
Growth, waxy, no diffusible pigment. Surface: terra cotta. The back: red brown. Mycelium long, branched, regular, not separated by any partition, 0.5 g in diameter. Sporophores simple, Spo; ren spherical to ellipsoidal,
1.0 R in diameter glucose-asparagine-agarW: clean, peach-colored peptone-glucose-agar W: good, burgundy red nitrate reduction positive gelatin liquefaction positive (weak) VIII.



   Strain Micromonospora echinospora Antagonistic properties Gentamicin properties Morphology No aerial mycelium, increased colony, noticeable-convoluted good
Growth, waxy, pale amber, diffusible
Pigment. Surface: deep red brown. Mycelium long, branched, not separated by a partition. No
Spore glucose asparagine agar W: weak peptone glucose agar W: good, burgundy red nitrate reduction variable gelatin liquefaction positive
The use of carbon sources for the above-mentioned fungi, with the exception of fungus IV, are listed in the following table together with similar information for the S. lividus used according to the invention.



   Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Micromono- Micromono lividus kanamyceticus fradiae pulveraceus rimosus f. chrestomyce- spora spora paromomycinus ticus purpurea chinospora L-arabinose - + + + + + + rhamnose - - - ++ - + + D-xyrose - - + ++ - + + D-glucose ++ + ++ + + + D -Galactose ++ + + ++ ++ + + + D-fructose + + - + ++ + D-lactose - - - ++ ++ + + + D-maltose ++ + + ++ ++ + sucrose - + - - + + Trehalose ++ <++ + + + D-raffinose + + - ++ + - Dextrin + + ++ ++ + Inulin - - - - - + Starch + + ++ + + Dulcit - - Glycerin ++ + + ++ + - L-inositol ++ - ++ D-mannitol ++ + - - ++ + - sorbitol ++ + - + ++ +

   -
According to the present inventive method, a new and useful antibiotic substance is thus produced; this method according to the invention is characterized in that Streptomyces lividus or its mutants are cultured under aerobic conditions in a medium containing a carbon source and a nitrogen source, and the antibiotics are obtained from the broth. Said nutrient medium can also be an inorganic substance d. H. contain a mineral, in addition to the carbon and nitrogen source.



   Various substances known for this purpose can be used as carbon and nitrogen sources for cultivation according to the method according to the invention above. As examples of the carbon source, there can be mentioned starch, glucose, glycerin, maltose, inositol, fructose, dextrin, sucrose and lactose.



  As the nitrogen source, there can be mentioned a yeast extract, polypeptone, dry yeast, meat extract, corn steep water, soybean meal, inorganic nitrate and ammonium salt.



   Examples of the above-mentioned inorganic substances or minerals are sodium chloride, dipotassium phosphate, magnesium sulfate, iron sulfate, calcium chloride, calcium carbonate, calcium hydroxide, iron chloride, zinc sulfate and cobalt chloride.



   The cultivation can be carried out under aerobic conditions, either in a solid nutrient medium or a liquid medium. It is preferred to conduct the cultivation in a liquid medium under aerobic conditions. When growing in a liquid medium under aerobic conditions, known anti-foam agents such. B. liquid paraffins, fatty oils and silicone oils can be used.



      The cultivation temperature is about 20-39 "C, advantageously about 25-37" C. it is advisable to cultivate in a cultivation medium at a pH of 6.6-7.5, preferably at 7.0 to 0.3. The cultivation period is usually at least 2 days.



  For example, a period of two days to two weeks, or two days to a week, is perfectly adequate. If desired, a longer cultivation period is possible, but not necessary. If the production of quintomycin in the broth has reached a sufficient amount, advantageously if the amount has reached a maximum, the cultivation is stopped and the quintomycin contained in the broth is recovered. It can be obtained either by allowing the broth to flow through an adsorbent and then eluting it with the aid of a suitable eluting liquid or by treating the broth with a suitable solvent for the purpose of extraction; In general, however, the first method mentioned above is preferred.

  If the above adsorbing and eluting method is used, a suitable adsorbent is added to the culture broth to adsorb the desired product with sufficient stirring; after removing the culture broth with the mycelium, the adsorbent can then be subjected to the elution operation. It is also possible to add an adsorbent to the culture broth, from which broth the mycelium has previously been removed by suitable means such as filtration and centrifugation; then the above-described process can be continued, or the broth, which has been purified from the mycelium, can be passed through a column filled with a suitable adsorbent and subsequently subjected to the above-mentioned process.

 

   Any solid adsorbent which adsorbs the desired products and from which the desired product can then be eluted by a suitable eluting liquid can be used as the adsorbent for the above-mentioned purpose. Cation exchange resins are preferred as the adsorbent in the recovery process described above. In particular, weakly acidic cation exchange resins are used with advantage. As specific examples of these cation exchange resins, there may be mentioned Amberlite IRC-50, Amberlite IRC-84, Amberlite CG-50 and carboxymethyl cellulose known under the trade name. In addition to these, other solid adsorbents such as. B.



  Cellulose powder, activated charcoal, alumina gel, silica gel and alumina / silica gel can be used.



   As the eluting liquid, an aqueous solution of acids such as inorganic and organic acids and aqueous solutions of alkaline materials such as caustic alkalis, ammonia and ammonium salts can be used.



   These acids mentioned include mineral acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and lower aliphatic acids such as acetic acid and formic acid. It is advisable to use these acids in the form of aqueous solutions with a concentration of 0.1 to 1.0 N. As examples of the above-mentioned alkaline materials, there can be mentioned sodium hydroxide, potassium hydroxide, ammonia and ammonium formate. It is also expedient that these alkaline materials in concentrations of about 0.1 to 3.0 N are used. Of all the above materials, the use of mineral acids or ammonia water is recommended.



   The pH of the eluate obtained is, if desired, adjusted to a value close to the neutral point and by removing the liquid at the lowest possible temperature, the desired quintomycin can be obtained in the form of a solid powder. Removal of the liquid can be accomplished in any of a number of known ways. In this case, however, drying by freezing out or by atomizing the liquid is preferred. If this operation is carried out at as low a temperature as possible, heating under reduced pressure can be carried out.



  But it is also possible to concentrate the liquid by drying in vacuo under such conditions that a stable salt, such as. B. forms the hydrochloride or the sodium salt. The quintomycin in the eluate can also be converted into a solid powder in such a way that the eluate is extracted with a suitable extraction solvent and then the solvent is removed at the lowest possible temperature.



   If activated carbon is used as a solid adsorbent, it is eluted with an aqueous acid solution. In this case, acetone or a lower aliphatic alcohol such as methanol or ethanol, which is adjusted to a pH of less than 5, advantageously less than 4, is used in addition to the acids listed above.



   If quintomycin is obtained from the cultivation broth by solvent extraction, higher fatty acids such as lauric acid and stearic acid are used as auxiliaries. Water-immiscible aliphatic alcohols are used as solvents. With advantage such alcohols such. B. nor iso-butanol and amil alcohol are used. An alcohol layer is known by decanting or other
Separating agent is separated into two layers and the alcohol is evaporated at the lowest possible temperature. For example, this is used for evaporation
Atomization drying, in addition to heating under reduced pressure, is possible.



   It is preferred, as stated above, the quintomycin produced according to the invention by an adsorption or elution process or by a solvent
Win extraction. However, as long as the quintomycin in the culture broth is not substantially destroyed, any separating agent for a solid dissolved in a liquid broth can be used.



   The new antibiotic produced according to the invention
Quintomycin is a white to white-gray solid substance, and can be used as an antibiotic substance in a wide range, or if desired, after appropriate purification by repeated application of the above-mentioned extraction processes. If desired, the quintomycin produced according to the invention can be separated into four antibiotic substances.



  As already mentioned above, these four antibiotic substances are named Quintomycin-A, Quintomycin-B, Quintomycin-C and Quintomycin-D. Quintomycin-A, Quintomycin-B and Quintomycin-D are new substances. The quintomycin produced according to the invention consists mainly of quintomycin-B.



   The following describes the means for separating the quintomycin obtained according to the invention into these four substances mentioned above.



   If the quintomycin obtained from the culture broth is redissolved in water, or is eluted with an aqueous liquid, an acid or an alkaline substance, as mentioned above, it can be chromatographed while maintaining the pH of the eluting liquid at 6.2 to 12 with or without adjustment, into which quintomycin-A, quintomycin-B, quintomycin-C, and quintomycin-D are broken down.



   Both cation and anion exchange resins can be used. When using a column filled with a cation exchange resin, weakly acidic cation exchange resins such as resins of the trademarks Amberlite IRC-50, Amberlite IRC-84, Amberlite CG-50, furthermore carboxymethyl cellulose and the resin of the trademark CM-Sephadex are advantageously used ; an aqueous solution of quintomycin adjusted to pH 6.2 to 12 is adsorbed by the column, after which the column is developed. Using ammonia water or an aqueous ammonium formate solution as the eluting liquid, the quintomycin is separated off by a gradient or stepwise process.



   According to the gradient process, the quintomycin-A is eluted in sections 45-50 under the working conditions listed below in Example 4; the quintomycin-B in sections 53-58, the quintomycin-C in sections 88-90, and then the quintomycin-D is eluted. According to the stepwise method, the quintomycin-A is first eluted with a 0.1 N aqueous ammonia solution and then the quintomycin-B with an aqueous ammonia solution of the same normality. The quintomycin-C is eluted by a 0.15 N aqueous ammonia solution and then the quintomycin-D is eluted by a 0.3 N aqueous ammonia solution.



   It is also possible to break down the quintomycin into its individual components in this way that an aqueous solution of the quintomycin is obtained using a column of an anion exchange resin, advantageously a strongly basic anion exchange resin, such as. B. of the Dowex 1 x 2 OH type, Amberlite 400 developed and then eluted with water, first quintomycin-D, then the quintomycin-C, then the quintomycin-B and finally the quintomycin-A are eluted in succession.

 

   Each of the components separated in this way can be further purified by chromatographic treatment using an acid filled with activated carbon, alumina or cellulose and acidified by acetone or lower alcohol with a mineral acid such as hydrochloric acid, sulfuric acid or nitric acid, or a lower aliphatic acid such as acetic acid, as eluting liquid. These components can also be purified by converting them into their Picrat or Reinecke salts and converting them into an actual inorganic salt with the help of salt exchange agents.



   The new antibiotic substance produced according to the invention has new and useful antibiotic activities, as already mentioned at the beginning of the present description. These efficacies are described in more detail below.



   1. Characteristics
Quintomycin-A, Quintomycin-B and Quintomycin-D are basic substances. A free base, the hydrochloride and the sulfate of each of these substances is a white amorphous powder.



  2. Solubility
Quintomycin-A, quintomycin-B and quintomycin-D in the form of their free base, hydrochloride and sulfate salts are soluble in water, an aqueous sodium hydroxide solution, in a lower aliphatic acid and a mineral acid. They are soluble in methyl alcohol in their free basic form, but are very sparingly soluble or insoluble in C4 or higher aliphatic alcohols, ketones and ethers.



  3. Color reaction
Quintomycin-A, Quintomycin-B and Quintomycin-D are positive for the Molisch reaction, Elson-Morgan reaction, Abderhalden reaction (ninhydrin reaction) and Bile reaction, have a deep purple color in the Tollens' chemical reaction (phloroglucinol hydrochloride) and are negative for Fehling's reaction, Benedict's reaction, Biuret's reaction, Ehrlich's reaction and Sakaguchi reaction.



  Quintomycin-A and Quintomycin-B show a purple color in the Skatolian reaction, but Quintomycin-D does not show this color.



  4. Stability
Quintomycin-A, Quintomycin-B and Quintomycin-D in the form of their 1N aqueous hydrochloric acid solution are unstable when heated to 100 "C for 30 minutes, but are stable at room temperature. They are stable in their 1N aqueous sodium hydroxide solution, both when heated to 100 "C for 30 minutes than at room temperature.



  5. R-index F value
The R-index-f-value determined by an alumina thin-layer chromatography (Rr-values in the claims are determined with the aid of this method) using as the upper layer a mixed solvent of chloroform-methanol-17/0 ammonia water in a volume ratio of 2: 1 : 1 as developing liquid is 0.23 for quintomycin-A, 0.50 for quintomycin-B, and 0.75 for quintomycin-D. Some known antibiotic substances have the following Rr value determined by the above method: Kanamycin, product of Streptomyces kanamyceticus, 0.75; Paromomycin, product of Streptomyces rimosus f. paromomycinus, 0.70; Neomycin, product of Streptomyces fradiae or Streptomyces albogriseolus, 0.74; and gentamicin product from Micromonospora echinospora, 0.81.



   The Rr value determined by a paper chromatographic method using a methanol 3 / o NaCl aqueous solution in a volume ratio of 2: 1 as a developing liquid and Toyo filter paper No. 51A (product of Toyo Filter Paper Co., Ltd., Japan) is 0.25 for quintomycin-A and 0.27 for quintomycin-B. The Rr values determined by the same method of some known antibiotic substances are as follows: kanamycin, 0.36; Paromomycin, 0.31; Neomycin, 0.20; and gentamicin, 0.53.



   Other properties of the antibiotic substances produced in accordance with the invention together with the properties of other known antibiotic substances are listed in the following tables.



     Table I Antibiotic Substance Name Quintomycin-A Quintomycin-B Quintomycin-C Quintomycin-D Kanamycin-A Kanamycin-B Kanamycin-C Produced by S.lividus S.lividus S.lividus S.lividus S. Kanamyceticus S. Kanamyceticus S. Kanamyceticus Melting point (F) - C (decomp.) (Free) 197-203 (free) 197-203 (free) 178-184 (free) 263-268 (free) 170-190 (free) approx. 270 (-HCl) 195 (-HCl) 190 (-HCl) 203 [?] D + (-HCl) [?] 22 D = 59 (-HCl) [?] 22 D = 50 (same as the (-HCl) [?] D22-36 (free) [α] D24 = 146 (free) [α] D24 = 126 (free) [α] D20 = 126 Paromomuycin) (c = 1, H2O) (c = 1, H2O) ( c = 0.5, H2O) (c = 1.0, 1N H2SO4) (c-0.7, H2O) (c = 1, H2O) Molecular formula C29H55N5O19 C29H55N5O18 C23H45N5O14 C23H45N5O13 C18H36N4O11 C18H37N5O10 C18H36N4O117.77 Molecular weight (MG) 77.77 , 77 615.63 599.63 484.51 482.51 484.51 analysis% calc. Found Ber. Found Ber. Found.



  C: 44.77-C: 45.16 C: 45.72-C: 46.23 (same as the C: 46.07-C: 46.40 C: 44.62 C: 44.80 C: 44 , 62 H: 7.07-H: 7.05 H: 7.22-H: 7.70 Paromomycin) H: 7.56-H: 7.59 H: 7.49 H: 7.51 H: 7 , 49 N: 9.01-N: 8.73 N: 9.19-N: 9.14 N: 11.68-N: 11.33 C: 11.56 N: 14.52 N: 11.56 Components of the substances mannose + + (same as the - - - ribose + + paramomycin) + + - deoxystreptamine + + + + + + monoamino sugar + + + - + + diamino sugar + + + - + +
Table II Name of the anti-Quintomy-Quintomy-Quintomy-Quintomy-Kanamy-Kanamy-Kanamybiotic substance cin-A cin-B cin-C cin-D cin-A-cin-B cin-C Biolog.

  Activity M. l. C. mcg / ml Staph. the same as aureus 209p 1.56 0.8 Paromomycin 0.4 0.8 S. citreus 3.12 0.8 1.56 3.12 S. albus 0.8 0.4 0.1 0.8 Strept. faecalis 100 100 50 12.5 Sarcina lutea 100> 100 1.56 3.12 Bacillus subtilis 0.8 0.8 0.8 0.2 B. cereus 1.56 3.12 0.8 1.56 B. anthracis 1.56 1.56 0.4 Escherichia coli 6.25 6.25 3.12 1.56 Adhes. pneumoniae 6.25 3.12 6.25 3.12 Prot. vulgaris 12.5 12.5 12.5 12.5 Salmonella typhi 6.25 1.56 1.56 1.56 Shigella flexneri 6.25 12.5 12.5 6.25 pseudo. aeruginosa 100 3.12 3.12 25 Mycobacterium 607 5.12 1.56 1.56 1.56 M.

   phlei 3.12 1.56 1.56 1.56 Mycobacterium tuber 3.12 3.12 3.12
Table III Name of Anti-Quintomy-Quintomy-Quintomy-Quintomy-Kanamy-Kanamy-Kanamybiotic Substances cin-A cin-B cin-C cin-D cin-A cin-B cin-C Toxicity in Mice LD 50 mg / kg Intravenous 198 193 190 374.2 Intraperitoneal Subcutaneous 1878 1210 2282 Synonym Paromomycin Table IV Name of the antibiotic substance Neomycin-A Neomycin-B Neomycin-C Paramomycin-I Paramomycin-II Gentamicin complex Generated by S. fradiae S. fradiae S. fradiae S. rimosus f . S. rimosus f.

  Micromonospora echinospora S. albogriseolus S. albogriseolus S. albogriseolus paromomycinus paromomycinus Micromonospora purpurea FC (decomp.) (-H2SO4) 250-6 C1: 94-100, C2: 107-124 [?] D + (-H2SO4) [? ] 22 D = 123 (-H2SO4) [α] 22 D = 58 (-H2SO4) [α] D = 82 (-HCl) [α] D25 = 64 (-HCl) [α] D24 = 78 C: 1 [?] D25 = 158 (H2) (c = 0.7, H2O) (c = 0.5, H2O) (c = 0.5, H2O) (c = 1, H2O) C: [? ] D25 = 160 (H2O) Molecular formula C12H26N4O6 C23H46N6O13 C23H46N6O13 C23H45N5O14 C23H45N5O14 C17-18H34-36N4O7 M.

  G. 322.36 614.65 614.65 615.63 615.63 Analysis% Calculated Calculated Calculated Calculated C: 44.71 C: 44.94 C: 44.94 C: 44.87 C: 44.87 H : 8.13 N: 7.52 H: 7.52 H: 7.37 H: 7.37 N: 17.38 N: 13.67 N: 13.67 N: 11.38 N: 11.38 components of the substances mannose - - - -x - ribose - + + + + deoxystreptamine + + + + + + monoamino sugar - - - + + + diamino sugar + + + + + -
Table V Name of the anti-neomy-neomy-neomy-paromomy-paramomy-gentamicin biotic substance cin-A cin-B cin-C cin I cin-II complex biological activity M. l. C. mcg / ml Staph. aureus. 209p 0.8 0.8 0.8 S. citreus 0.4 1.56 3.12 S. albus 0.4 0.2 0.8 Strept. faecalis 12.5 25 25 6.25 Sarcina lutea 1.56 3.12 0.4 Bacillus subtilis 0.8 0.4 0.4 B. cereus 1.56 0.8 0.8 B.

   anthracis 1.6 0.1 Escherichia coli 1.56 3.12 3.12 Kleb-pneumoniae 6.25 3.12 0.8 Prot. vulgaris 6.25 6.25 1.56 Salmonella typhi 6.25 3.12 0.8 Shigella flexneri 6.25 6.25 3.12 Pseudo. aeruginosa 12.5> 100.0.8 Mycobacterium 607 0.8 1.56 M. phlei 3.12 0.4 Toxicity in mice LD50 mg / kg Intravenous 90 72 Intraperitoneal 433 Subcutaneous 220 423 484 Synonym Framycetin strepto-aminosidin,
Streptothricin thricin catenulin, BKK -B1 hydroxymycin,
Zygomycin
The above-described process according to the invention is explained in more detail below by means of a few examples.



   example 1
Streptomyces lividus strain (Streptomyces No.



     2230-Ní) was in a sterilized and to a pH of 7.0 sterilized medium consisting of 0.5% starch, 2.0% soybean meal, 0.1% dipotassium phosphate, 0.05 / O magnesium sulfate, 0.3 % Sodium chloride, fermented and was precultured at 27 "C for about 50 hours.



  Then 100-150 ml of this preculture broth were added to 10 liters of the same culture medium and this entire culture broth was cultivated at 27 ° C. with stirring at a speed of 250 rpm, with sterilized air being passed through at a speed of 10 liters / minute For forty hours, the pH of the culture broth was 7.2-7.4 and the production of quintomycin (Substance No. 2230) reached a maximum The culture broth was filtered using Celite trademark diatomaceous earth as a filter aid to give 1.2 liters of one The filtrate broth was then stirred with 300 ml of an Amberlite IRC-50 type resin for 10 minutes until the active components were completely adsorbed by the resin.

  The resin was then passed into an acid and washed thoroughly with water. The quintomycin was recovered by eluting with a 0.5N hydrochloric acid solution. The effective components were collected and adjusted to pH 7.6-7.8 with a 10N sodium hydroxide solution. Then they were adsorbed through a column filled with 10 g of activated carbon for the purpose of purification, the acid was washed with 0.01 / O ammonia water and then with water and treated with a 0.02 N hydrochloric acid / methanol solution in the ratio of 1: 1 eluted. The methanol was distilled under reduced pressure and subsequent drying by freezing gave quintomycin-A, quintomycin-B, quintomycin-C and quintomycin-D in the form of a hydrochloride complex in a yield of one gram.



   Example 2
A culture broth obtained by the method of Example 1 was passed through a column filled with a resin of the trademark Amberlite IRC-50 of the NH4 type, the active components being completely adsorbed by the resin.



  Thereafter, the resin was thoroughly washed with water and eluted with 1.0 N ammonia water to separate the effective components. These effective components were collected and treated with 2N hydrochloric acid to adjust the eluate to pH 6-7.8. The cleaning was carried out in the manner described in Example 1.



   Example 3
The strain Streptomyces lividus was in a sterilized and adjusted to a pH of 6.8, and 0.5% starch, 0.05 / o glucose, 2.0% soybean meal, 0.2 / O peptone, 0.05% Magnesium sulfate, 0.1 / o dipotassium phosphate and 0.3 / n sodium chloride containing medium fermented and then precultivated at 35 ° C for about 50 hours. Then 300 to 400 ml of this precultivated broth were added to 10 liters of the above medium and that Cultivation was carried out at 35 "C with stirring at 200
RPM and 14 liters per minute of sterilized air passed through. Within 96 hours the pH of the culture broth reached a value of 7.8 to 8.2 and the production of quintomycin reached a maximum.



  This culture broth was then mixed with 100 milliliters of a weak acidic ion-exchange resin of the trademark Amberlite IRC-84 of the NH4 type for 10 minutes, the active components being completely adsorbed by the resin. After the mycelium and the spent liquor broth were removed by decantation, the resin on which the active components are adsorbed was packed into a column; then this column was finally washed with water and then eluted with a 2.0 N ammonia water solution. The effective components were collected and concentrated. This solution was then adsorbed through a column of 30 x 2400 mm filled with a weak acidic cation exchange resin for use in chromatography. First a pigment was eluted with 0.08 N ammonia water and then with a 0.1 N ammonia water solution.

  This elution initially gives quintomycin-A and, after completion of this elution with 0.1 N ammonia water mentioned above, quintomycin-B. By eluting with 0.15N ammonia water after completion of the elution of quintomycin-B, quintomycin-C was obtained. After quintomycin-C was finished eluting with a 0.3N ammonia water solution, quintomycin-D started to elute.



   Pure quintomycin-A, quintomycin-B, quintomycin-C and quintomycin-D were obtained by concentration and freeze-drying in the form of free bases with yields of 650 mg, 4200 mg, 40 mg and 120 mg, respectively.



   Example 4
150 mg of the quintomycin complex obtained according to the procedure of Example 1 or 2 was dissolved in about 40 ml of distilled water and the solution was filled in a column of diameter filled with 30 ml of a Sephadex C-25 brand carboxymethyl resin of the NH4 type 1 cm adsorbed and then washed with water. Using 200 ml of a 0.05 N ammonia water solution and 200 ml of a 1.0 N ammonia water solution, it was carried out using a gradient method at a rate of 25 ml / hour. eluted. The eluate was successively collected in sections, each in an amount of 4 ml. The quintomycin-A was eluted in sections 45-50; the quintomycin-B in sections 53-58; and the quintomycin-C in sections 88-90. By continuing the elution with ammonia water having a concentration of 1.0 or more, quintomycin-D was obtained.

  Using freeze-drying techniques, pure quintomycin-A, quintomycin-B, quintomycin-C and quintomycin-D were found in free base yields of 31 mg, 49 mg, trace and 18 mg, respectively.



   Example 5
150 g of the quintomycin complex obtained according to the method of Example 1. were dissolved in 140 ml of distilled water and the solution was adsorbed through a column of 1 cm diameter; this column was filled with 30 ml of an ammonium formate buffered carboxymethyl resin of the trademark Sephadex C-25 of the NH4 type. After the adsorption was carried out, this packed column was washed with water. Using 200 ml of a 0.5 molar ammonium formate solution and 200 ml of a 3.0 molar ammonium formate solution, using a gradient method at a rate of 25 ml / hour. eluted.



  The eluate was then successively collected in sections each in an amount of 3 ml. The quintomycin-A was eluted in sections 29-37; Quintomycin-B in sections 40-45; and quintomycin-C in sections 78-80.



  By continuing the elution with 3.0 molar ammonium formate solution, quintomycin-D was obtained in the eluate.



  Freeze-drying gave pure quintomycin-A, quintomycin-B, quintomycin-C and quintomycin-D in free base form in yields of 35 mg, 56 mg, traces and 19 mg, respectively.



   Example 6
150 g of the quintomycin complex obtained according to the method of Example 1 or 2 were dissolved in 40 ml of distilled water. The solution was adjusted to pH 7.0 with a 0.1N ammonia water solution, and the quintomycin was completely adsorbed by a column 1 cm in diameter filled with 20 ml of Amberlite CG-50 NH4 type. Using 200 ml of a 0.02 N ammonia water solution and 200 ml of a 1.0 N ammonia water solution, using a gradient method at a rate of 25 ml / hour. eluted. The eluate was successively collected in sections each in an amount of 3 ml. The quintomycin-A was eluted in sections 29-35, the quintomycin-B in sections 40-50 and the quintomycin-C in sections 88-92.



  Continuing the elution with 1.0 N aqueous ammonia solution leads to the quintomycin D eluate. Freeze-drying gave pure quintomycin-A, quintomycin-B, quintomycin-C, quintomycin-D in the form of free bases in yields of 33 mg, 52 mg, traces or



  16 mg.



   Example 7
Each of the free bases of quintomycin-A, quintomycin-B, quintomycin-C and quintomycin-D obtained according to the method of Example 3 and the preceding examples were dissolved in methanol. An addition of a 2N sulfuric acid solution to the methanol solution thus obtained led to the precipitation of a sulfate of each of the quintomycins mentioned. The precipitate was thoroughly washed with methanol / acetone, and pure sulfate of each of the above quintomycin was obtained.

 

   Free bases of quintomycin-A, quintomycin-B, quintomycin-C and quintomycin-D were dissolved in methanol and acidified with a 2N hydrogen chloride solution. Addition of acetone to each methanol solution thus obtained resulted in precipitation of a hydrochloride of each of the quintomycins. The precipitate was washed thoroughly with acetone-ether and a pure pentahydrochloride of each of the quintomycins mentioned was obtained.

 

Claims (1)

PATENT CLAIM Process for the preparation of a mixture of the antibiotics quintomycin A, B, C and D, characterized in that Streptomyces lividus or its mutants are cultured under aerobic conditions in a medium containing a carbon source and a nitrogen source and the antibiotics are then obtained from the culture broth. SUBCLAIMS 1. The method according to claim, characterized in that said medium also contains a mineral substance. 2. The method according to claim and dependent claim 1, characterized in that the cultivation temperature is 20-39 "C, advantageously 25-37" C. 3. The method according to claim and sub-claims 1 and 2, characterized in that the pH of the culture medium is 6.6-7.5, advantageously 7.0 + 0.3. 4. The method according to claim, characterized in that the cultivation is carried out for two days to two weeks. 5. The method according to claim, characterized in that the carbon source used is starch, glucose, glycerol, maltose, inositol, fructose, dextrin, sucrose or galactose. 6. The method according to claim, characterized in that the nitrogen source used is yeast extract, polypeptone, dry yeast, meat extract, grain spring water, soybean flour, inorganic nitrate or ammonium salt. 7. The method according to dependent claim 1, characterized in that the mineral substance used is sodium chloride, dipotassium phosphate, magnesium sulfate, iron sulfate, calcium chloride, calcium carbonate, calcium hydroxide, iron chloride, zinc sulfate or cobalt chloride. 8. The method according to claim, characterized in that the product is obtained by adsorption and elution. 9. The method according to claim, characterized in that the product is obtained with the aid of a solvent extraction. 10. The method according to dependent claim 8, characterized in that the adsorption is carried out with the aid of a cation exchange resin and the elution is carried out with the aid of an aqueous solution of acidic or alkaline substances. 11. The method according to dependent claim 10, characterized in that weakly acidic cation exchange resins or carboxymethyl cellulose are used as the cation exchange resin. 12. The method according to dependent claim 8, characterized in that the adsorption with the aid of activated carbon and the elution with the aid of aqueous solutions of acids, acidified lower aliphatic alcohols or acidified acetone is carried out. 13. The method according to dependent claim 9, characterized in that the solvent extraction is carried out with the aid of a water-immiscible-aliphatic alcohol as the solvent. 14. The method according to dependent claim 13, characterized in that the aliphatic alcohol used is n-butanol, isobutanol or amyl alcohol. 15. The method according to claim, characterized in that the quintomycin obtained is adsorbed by a cation exchange resin and eluted with an aqueous solution of an alkaline substance, at least one of the quintomycins, d. H. Quintomycin-A ,. Quintomycin-B, Quintomycin-C or Quintomycin-D is obtained. 16. The method according to dependent claim 15, characterized in that the elution is carried out with the aid of a gradient method or a stepwise method. 17. The method according to claim, characterized in that the quintomycin obtained is adsorbed by an anion exchange resin, eluted with water, at least one of quintomycin-A, quintomycin-B, quintomycin-C and quintomycin-D is obtained.