DE1213568B - Use of Actinomyces varsoviensis for the production of oxytetracycline in the usual biological way - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. α .:

C12d

German class: 30h-6

Number: 1213568

File number: K 40466IV a / 30 h

Filing date: April 19, 1960

Opening day: March 31, 1966

The invention relates to the production of oxytetracycline by conventional biological means. • A strain of Actinomyces varsoviensis sp.nov. No. NCIB 9522 submers and aerobically in a conventional manner in a liquid sewing medium, which sources nitrogen, carbon, Contains mineral salts and trace elements, bred.

By using Actinomyces varsoviensis sp.nov. No. NCIB 9522 instead of the known Use of Streptomyces rimosus (German Patent 862 647) is compared to the known Working method achieved a much higher yield of Oxytetracyclm.

The new, so far not described species of radiation fungus, which is named Actinomyces varsoviensis sp.nov. has been given, produces the antibiotic mentioned above, oxytetracycline. . The new species of the microorganism Actinomyces varsoviensis sp.nov. became taxonomic investigated (cf. N. A. Krasilnikow, Opredielitiel bakterii i aktinomycetow, Izd. Ak. Nauk SSSr, Moscow, 1949, and Bergey a Manual of Determinative Bacteriology, Williams, Wilkins, Baltimore, 1948) and classified as a microorganism belonging to the genus Actinomyces (Krasilnikow).

The new species Actinomyces varsoviensis sp. nov. is deposited with the National Collection of Industrial Bacteria Torry Research Station, Aberdeen, Scotland, under NCIB 9522 dated 9/22/1964.

The antibiotic oxytetracycline is produced by numerous radiation fungi. The manufacture of Oxytetracycline has been described many times in the literature. For example, in the U.S. Patent 2516 080, in German patent specification 862 647 and in British patent specification 648 417 a process for the production of oxytetracycline by the biosynthetic route using the Radiation fungus strain Streptomyces rimosus described, which is in terms of breeding and morphological characteristics of the species Streptomyces albus (Rosse- T. Doria, Ann. Is. IG. Sper. Roma N. S., 1894, 1, p. 399) Waksman, Henrici, approaches.

British Patent 713 795 and Canadian Patent 520 836 describe a process for obtaining oxytetracycline from the fermentation liquid of the ray fungus called Streptomyces platensis. In the Swiss patent 331988 is a process for the production of oxytetracycline by means of a new use of Actinomyces varsoviensis for
Manufacture of oxytetracycline on usual
biological way

Applicant:

Dr. Wlodzimierz Kuryiowicz,

Dipl.-Ing. Franciszek Ulak, Warsaw

Representative:

Dipl.-Ing. F. Weickmann,

Dr.-Ing. A. Weickmann

and Dipl.-Ing. H. Weickmann, patent attorneys,

Munich 27, Möhlstr. 22nd

Named as inventor:

Dr. Wlodzimierz Kuryiowicz,

Dipl.-Ing. Franciszek Ulak, Warsaw

Claimed priority:

Poland of November 24, 1959 (92 482)

Radiation fungus, Streptomyces vendargensis sp.nov., Described.

There is also a new type of oxytetracycline in the French scientific literature producing ray fungus. This species was named Streptomyces armillatus (Mancy - Courtillet, Pinnert - Sindico, Ann. Inst. Pasteur, 1954, 87, p. 580). In the plant by S. A. Waksman and H. A. Lechevalier, "Actinomycetes and their Antibiotics," Williams, Wilkins, Baltimore, 1953, is mentioned by the authors when describing the species Streptomyces griseoflavus (Krainsky) Waksman, Henrici (cf. A. Krainsky, ZbI. F. Bakt. [II], 1914, 41, p. 639) whose strong antagonistic properties and state that this strain is a producer of oxytetracycline and des Rimocidins is. On isolating the strains of numerous oxytetracycline-producing radiation fungi also reports Silvestri (R. C. Ist. Sup. Sanita, Roma, 1955, 18, p. 1227). Both authors, in particular Silvestri, draw attention to the similarity that exists between the strain Streptomyces rimosus and the "ray fungus known since 1914 Streptomyces griseoflavus exists.

In the search for antagonistic ray fungi, a species of ray fungus has now been isolated, the differ in their morphological, breeding and physiological characteristics from those previously reported in the literature expressly differentiates the strains described.

609 540/375

This strain was subjected to very precise taxonomic investigations, with all modern tests for the identification of 'the'genus' and species being carried out. for diq the name Actinomyces varsoviensis sp.nov. was chosen. ■ '- ■ - "- ■ ^v

The spores of Actinomyces varsoviensis sp.nov. are usually globose or subglobose (0.6 / to 0.8-3 - 0.5), the 'number of spores' in '' den Sporophores usually more than 10.

Actinomyces varsovieosis sp.nov, has the following " Morphological.characteristics: Mohpodiale Ramifications, sporophores solitary or branched, usually scattered along the main fibrils, formation rudimentary in clusters. Spirals open, at first primitive, later with several (up to five) Loops; "often irregular." In the cultures of "des Actinomyces varsoviensis showed no cracks on the surface, the colonies. The differences

o decoration "was * with those in Table I below listed oxytetracycline-producing types of radiation fungus.

Table I.
Types of oxytetracycline producing ray mushrooms

No.

Art

literature

Streptomyces griseoflavus

(Krainsky) Wäksman, Hehrici

Streptomyces .rimosus
Streptomyces armillatus

Streptomyces platensis
Streptromyces vendargensis

Al. Krainsky, ZbI. f.Bakt. (II), 1914, 41, p. 639;

W a k s m a η, H e.n r i c i (Bergey's Manual of Determinative Bacteriology, Williams, Wilkins, Baltimore, 1948) '""

B.A. Sobin, A.C. Finlay, J.H. Kane, USA. * · Patent 2516080, 1950. ,

Mancy - D. Courtillet,
P.innert - S. Sindico, Ann. Inst.Pasteur, 1954, 87, p. 58O ^:

J. M, McGuire, British Patent 713,795, 1954

A. S the em an, A. P. S tr uyk-, Swiss patent 331988,: 1958 -

The following table II gives the breeding loan «nutrient medium; such as B-. Potatoes, potato

characteristics and some physiological features 35 agar, etc., but also well-defined »synthetic

of the strain Actinomyces varsoviensis sp.nov, so-Substrate «· ■ applied. Actinomyces varsoviensis

such as of the strain Streptomyces rimosus 2234. 'differs significantly from the species Strepto-

In the taxonomic investigations of Actinomyces rimosus; · · - ■

myces varsoviensis sp.nov. were not only »natural-. ;

Table Π growth Aerial mycelium White Green mycelium Soluble pigment fertile soil Actinomyces varsoviensis sp. nov. + + + White colorless is missing : Asparagine agar with glucose 120 + + + White colorless sulfur colored Sabouraud agar with malt sugar 120 (12L-4) + + + White colorless is missing Czapek Doxa agar with sucrose 120 + + / + + + White colorless is missing Agar with dextrin 48-72 + + + White linen color is missing potato 72 (12B-2) + + + White colorless is missing Potato agar with glucose 72 + + + White colorless is missing Agar with oatmeal 72 + + + White colorless is missing Solid triptone substrate 48 4.4.4. White colorless is missing Citrate substrate 72 4-4-4- White colorless is missing Linden leg substrate 72 4-4-4- colorless is missing Liquid triptone substrate 48. excretes hydrogen sulfide

.Tabel. II (continued).

fertile soil

growth

Aerial mycelium

Basic mycelium 'Soluble Pigment'

Actinomyces Varsoviensis sp. nov.

Liquid Emerson substrate
Synthetic agar with Peptqn.

Meatsoup -

Agar with starch

Ca malate agar

carrot

Milk with liquor

gelatin

Agar with blood
Agar with nitrates

48-72

72

24-48

+ ■ + + 120

72 72

72

+ 48-72-white
White

White
White

White
creamy white

colorless
colorless

cream colored
colorless

pale to brown olive

is missing

dark yellow (121/5) is missing
is missing *

pale brown is missing

colorless
slightly acidic, peptonized

does not evaporate,

after a while it partially liquefies

does not hemolyze

White

colorless

does not reduce nitrates

pyrite colored (12-1-3)

Asparagine agar with glucose

Sabouraud agar with malt sugar

Czapek Doxa agar with sucrose

Agar with dextrin

potato

Potato agar with glucose *

Agar with oatmeal

Solid triptone substrate

Citrate substrate.

Linden leg substrate

Liquid triptone substrate

Liquid Emerson substrate

Synthetic agar with peptone

Meatsoup
Agar with starch

Ca malate agar

carrot

Milk with liquor

gelatin

Agar with blood

Agar with nitrates

Streptomyces rimosus N.R.R.L. 2234 absent

yellow white

48-72

+ ■ ++ ■

120

120

72-96

72

+ + + 72

120

120

120

120

48

48-72

24-48

24-28

+ + 96

24-72 48 48

+. + 48 missing or white
Trace elements
is missing

is missing
White

beige

Fasanenfarbert

(13 L-12)
colorless

light brown

(13D-4)
yellow-brown

(13J-9)
Brown

brownish yellow

(12D-2)
yellow-green

(12F-2)
gray-yellow-green

(12E-5)
Hazelnut

(13J-9)

does not excrete hydrogen sulfide

Lemon pigment

(10J-2) Sienna

(13 L-10) is missing.,. _:

Peruvian

brown (13 L-10) yellow

(13 L-12) brown

(13 J-9) Isabella

(13K-7) yellow

is missing

antique gold (12L-2)

White
is missing

white

Trace elements
knows or

pale cinnamon pink
White

Brown
colorless

colorless
yellow-brown

cinnamon pale
yellow

yellow-brown

Isabella

(13 K-7) is missing

Tenis

(12L-6) pale brown

is missing

acidifies, peptonizes

liquefies
hemolyzes red blood cells

white
Trace elements

light brown
(13 D-3)

reduces nitrates to nitrites

Tenis
(12D-6)

In Table II:

+ 4- medium growth, + + + abundant growth.

The numbers indicate the breeding hours in which the specified growth was observed.

The colors are given according to the Dictionary of Color, March and Paul, McGraw-Hill, New York, 1950.

7 8

Below is the comparison table of the ■ microorganisms' Actinomyces varsoviensis, Streptomyces rimosus

and Streptomyces albus.

Tables!

Streptomyces rimosus Streptomyces albus Actinomyees varsoviensis Aerial mycelium weak (Rossi Doria emend. Krainsky)
Waksman, Henrici. Aerial mycelium weak Aerial mycelium quite white -.-. Aerial mycelium rich - Aerial mycelium white " Characteristic appearance of the Aerial mycelium white Colony and smooth appearance Colony and breeding ¹ ) Non-characteristic appearance of breeding Potato: basic mycelium yellow-brown the colony Potato: Base mycelium colored linen Milk: peptonization Potato: colonies white Milk: peptonization Milk: coagulation, peptonization Gelatin: liquefies tion Gelatin: no liquefaction or Gelatin: intensive liquefaction partial liquefaction according to length recently Nitrates: reduction to nitrites Nitrates: .does not reduce to nitrites Starch: slight hydrolysis Nitrates: reduction to nitrites Starch: lack of hydrolysis Starch: hydrolyzed _{separates H 2} S in the tryptone medium or does not hydrolyze _{Separates H 2} S in tryptone medium not off ² ) " the end hemolyzes red blood cells ³ ) Hemolizes red blood cells
not produces oxytetracycline Generates oxytetracycline generated-actinomycin, endomycin, Thiolutin

Remarks:
indicates that any information is missing.

*) A striking feature, the Streptomyces rimosus owes its name to the formation of numerous cracks on the surface of the colonies. This characteristic has been observed with Sabouraud's maltose agar, Emerson agar, iron peptone agar, Potato slice agar and base mineral medium + trehalose. In the corresponding cultures of the

Actinomyees varsoviensis no cracks were found.

² ) »jipd ³ ) indicate that the information relates to the strain Streptomyces rimqsus NRRL 2234. Other details regarding Strept. albus and strept. rimosus are taken from German Patent 862 647 and Bergey's Manual - of Determinative Bacteriology, Williams, Wilkins, Baltimore, 1948.

The information contained in Table IV relates to a strain of Streptomyces armillatus and are the work Mancy - D. Courtillet and Pinnert - S. Sindico, "Une nouvelle espece de Streptomyces: Streptomyces armillatus ", Ann. Inst. Pasteur, 1954, 87, p. 580.

Actinomyees varsoviensis ap. nov.

Nutrient medium Growth. Aerial mycelium Basic mycelium Soluble pigment Asparagine agar with glucose + + + White colorless. is missing Czapek Dox agar with sucrose + + + White colorless is missing Culture medium with glucose and peptone + + + White colorless dark yellow ^: (Pl, 12,1-5) Emerson culture medium + + / + + + - White colorless is missing or light brown potato + + + "; White linen color is missing (Pl, 12-B-2) Breeding ground with starch + + + White colorless is missing Culture medium with nitrates + + / + + + does not reduce nitrates to nitrites gelatin + ■ + + does not liquefy gelatine, after a long time it partially liquefies Meat at 26 ° C ; +++ ■ peptonized, acidified

In the cultivation of Actinomyees varsoviensis
sp. nov. come z. B. the following carbon sources in
Question :: Eructosis; Glucose ,. Galactose ,. Mannose; Trehalose, glycerine, mannitol, sodium succinate. Weakly exploited are: raffinose,, sucrose, lactose, inosite and sodium acetal ', .., .. -...._ ...

9 10

Actinomyces varsoviensis usually does not assimilate: rhamnose, maltose, dulcitol and starch

Table IV
Streptomyces armillatus

Nutrient medium growth Aerial mycelium Basic mycelium Soluble pigment Asparagine agar with glucose + + + weak, ent
wraps, white
is missing gray-yellow beige is missing Czapek Dox agar 4th developed colorless is missing Culture medium with glucose and peptone + + + weak, white gray-yellow, smooth is missing developed Emerson culture medium 4 4-4- weak, white yellow-gray, smooth pale pink and developed pale brown potato + + gray-yellow, smooth is missing Breeding ground with starch ± sciiwacn
does not hydrolyze starch Culture medium with nitrates + does not reduce nitrates to nitrites gelatin 4-4- partial liquefaction after 40 days Milk at 26 ° C 4-4- coagulates, peptonizes, acidifies

Remarks:
Growth: ± very weak, + weak,

+ + mediocre, + + + good to very lush (lush).

Streptomyces uses "synthetic" nutrient medium armillatus emits the following carbon sources: glucose, gelactose, mannose, glycerine, erytritol. Arabinose, raffinose, mannitol, sodium acetate, and sodium citrate are less used Strength.

Streptomyces armillatus does not use the following carbon sources: xylose, mannose, levulose,

Sucrose, maltose, lactose, sorbitol, dulcitol, inositol and sodium tartrate.

The physiological characteristics of the Actinomyces varsoviensis strain related to its Assimilation capacity of carbon sources from "synthetic" nutrient medium (mt. Priedham— Gottlieb) are given in Table V.

Table V

Use of carbon sources from "synthetic" agar by Act. varsoviensis, Strept. armillatus and Strept. rimosus

Carbon source Actinomyces
varsoviensis sp. nov. Steptomyces
armillatus Streptomyces
rimosus 2234 Glucose 4-4-4- + 4-4- 4-4-4- Galactose 4-4-4- 4-4-4- 4-4-4- Mannose 4 + + 4-4-4- + 4- 4- + 4- Fructose + + + 4-4- + + 4- lactose 4-4- Maltose . Sucrose + + 4- Raffinose + + Rhamnose 4-4-4 Trehalose strength + + + 4 ++ Glycerin Mannitol Dulcit χχχ XX Inositol Sodium succinate Sodium acetate

Remarks:

The signs +, +, ++, + + + indicate the degree of utilization of the carbon source, the specific degree of growth of the examined trunk.
- denotes a lack of growth,
■ indicates that no attempt has been made.

The information regarding Streptomyces armillatus, Streptomyces rimosus and Streptomyces griseoflavus come from the Mancy - D. Courtillet work

and Pinnert - S. Sindico (Ann. Inst. Pasteur, 1954, 87, p. 580). The information regarding Actinomyces varsoviensis sp. nov. suggest that

609 540/375

this strain approaches the strain Streptomyces armillatus with certain characteristics. Points to this also the lack of reduction of nitrates to nitrites, as well as the lack of hydrolytic properties for strength. However, it differs from this tribe by its behavior Gelatin and by growth character on certain nutrient media, such as those contained in Table VI Information.

The Actinomyces varsoviensis sp.nov strain differs on the basis of the information given. important from the strains Streptomyces rimosus and Streptomyces griseoflavus.

Table VI

Fertile soil Actinomyces Streptomyces Streptomyces Streptomyces ^ * III H \ JKJ UwU varsoviensis armillatus rimosus griseoflavus Synthetic nutrient weak wax weak wax no growth Basic mycelium orange floor tum, no pig tum, no pig or brown-pink, mentation mentation Pigment pale yellow potato Basic mycelium flax Basic mycelium gray Basic mycelium brown, Basic mycelium colors, light yellow, smooth, no Pigment bk yellow Creamy lichen type ruffled, no pigment color or brown pigment pink gelatin Liquefaction after slow liquefaction liquefaction weak liquefaction longer time gung, pigment gung, pigment brown-pink pale yellowish Milk at 26 ° C Peptonization, Coagulation, Peptonization rapid peptonisia slight acidity Peptonization, tion without coagu tion Acidification lation Emerson culture medium Basic mycelium colorless Basic mycelium gray Basic mycelium Basic mycelium or light brown, yellow, smooth, orange brown Lichen type, krem smooth, no pigment Pigment pale pink colors, cracked, pig colors or brown, or pale yellow ment yellow-brown Pigment yellowish Nitrates no reduction too no reduction reduction reduction Nitrites to nitrites to nitrites to nitrites strength no hydrolysis no hydrolysis weak hydro
lysis partial hydrolysis

The information on Streptomyces vendargensis and Streptomyces rimosus is taken from the Swiss Taken from patent 331,988. The descriptions of the tribes given there are, however insufficient as they do not have the characteristics of the basic physiological features. The differentiation between Strept. vendargensis and Strept. rimosus is in the cited patent mainly based on the fact that Strept. vendargensis another antibiotic besides oxytetracycline "Vengicid" produced while Strept. rimosus a second antibiotic besides oxytetracycline "Rimocidin" produced. The two antibiotics "Vengicid" and "Rimocidin" differ differ from each other through physiochemical and biological properties.

Based on the information given regarding the characteristics of the breeding of the three together The compared fungal strains can fundamentally differ between Actinomyces varsoviensis and the other two strains (see Tables VII and VIII). From the above By comparison, it can be seen that the strain Actinomyces varsoviensis sp.nov. in comparison with the other strains examined are distinguished by a lighter aerial mycelium and a colorless or light yellow base mycelium distinguishes; it also produces a yellow pigment that diffuses into the substrate. The differences between the compared strains, especially between Actinomyces varsoviensis and Streptomyces rimosus, but are significant and involve physiological traits. These differences are given in Table VIII.

Table VII

fertile soil wax
tum Actinomyces varsoviensis wax
tum Streptomyces platensis Asparagine agar with
Glucose
Czapek Dox agar with
Sucrose + + +
+ + + A knows
B colorless
P is absent
A knows
B colorless
P is absent + +
+ + A white-gray olive
B pale yellow
P pale brown
A white-gray
B olive yellow
P is absent

(Footnotes at the end of the table.)

Annotation:

A = aerial mycelium. B = basic mycelium.

13th

(Continuation of Table VII)

14th

fertile soil wax
tum Actinomyces varsoviensis wax
tum Streptomyces platensis potato _{+ + +} A knows
B linen color
P is absent _{+ + +} A white pale gray
B -
P brown Citrate medium + + + A knows
B colorless
P is absent + + A pale yellow
B pale salmon
P pale green yellow Starch agar + + + A knows
B colorless
P is absent + + A white-gray
B creamy yellow
P is absent Milk with liquor
gelatin peptonized, slightly acidic
partial liquefaction after
long time
does not hydrolyze weakly coagulates, peptonized
after 30 days
weak liquefaction strength hydrolyzed

P = soluble pigment. - = No information available.

fertile soil

Aerial mycelium

Basic mycelium

Soluble pigment

Actinomyces varsoviensis sp. nov.

Agar with oatmeal Emerson agar Sabouraud agar

Potato agar agar with sodium citrate Agar with glucose and nitrates

potato
Agar with starch

Agar with oatmeal

Emerson Agar Sabouraud Agar Potato Agar Agar with sodium citrate

Agar with glucose and nitrates

potato

Agar with starch

White colorless is missing White colorless or light brown is missing White colorless sulfur colored (12L-4) White colorless is missing White colorless is missing White colorless pyrite colored (12L-3) White linen color is missing (12B-2) know _t colorless is missing nyces venc
is missing largensis
light yellow is missing (11F-4) White light brown is missing White yellowish is missing is missing Brown is missing is missing White is missing (2 A-I) is missing light yellow is missing (10 B-I) White yellow or brown is missing (12D-5.13L-7) White colorless is missing

The description of the strain Streptomyces vendargensis sp. nov. comes from Swiss patent 331 998.

Table VIII

fertile soil Actinomyces varsoviensis White Streptomyces vendargensis is missing A. Streptomyces rimosus Agar with oatmeal A. colorless A. light yellow missing or white B. is missing B. is missing B. Trace elements P. P. P. green-brown-yellow White White A. dark yellow Emerson agar A. colorless or light brown A. light brown (13 D-5) B. White B. (13 D-5) B. is missing P. brown (15A-12) is missing P. yellow-brown P. White White A. Sabouraud agar A. colorless A. yellow (12E-3) B. yellow white B. sulfur colored B. is missing P. pheasant colors P. (L-412) P. yellow White is missing A. Potato agar • A colorless A. Brown B. White B. is missing B. is missing P. Brown P. White P. is missing A. Brown Agar with sodium citrate A. colorless A. white (2 A-I) B. is missing B. is missing B. is missing P. gray-yellow-green (13 J-5) P. White P. is missing A. is missing Agar with glucose A. colorless A. light yellow (10 B-I) B. is missing and nitrates B. pyrite colored (12L-3) B. is missing P. brown (13K-9) P. White P. White A. yellow potato A. linen color (12L-3) - A yellow or brown B. White B. B. (12 D-5,13L-7) P. yellow brown (12E-5) P. White P. White A. yellow Agar with starch A. colorless A. colorless White B. is missing B. is missing B. (Trace elements) P. P. P. yellow-brown yellow-brown

Remarks:
A = aerial mycelium, B = ground mycelium, P = soluble pigment.

When cultivating microorganisms, one never has to deal with a static state, but with to do with a weaker or more developed variability. As a result of this variability new shapes can arise, called variants, i.e. H. changeable forms,, which are not inherited, as well as mutants whose traits are inheritable. This requires a frequent Control the fertility of the strain and can even replace it with antibiotic. require more effective strains belonging to the same species. The new tribes must all possess the features characteristic of the species to which they belong. This behavior applies not only to Actinomyces varsoviensis, but also to other radiation fungi. Through many times carried out selections of variants or mutants of increased antibiotic yield it is possible to obtain stems with a very high productivity.

The mutants sometimes differ significantly from the original strain. the the most important differences concern the color of the basic mycelium, the pigment that is soluble in the substrate, the way of exploiting the carbon sources and nitrogen from the "synthetic" Culture media, and above all the differences concern antibiotic activity.

"Natural" and "artificial" mutants were selected for the first time with ray fungi applied a method to determine the trunk characteristic. It doesn't just allow an exact one Description of the population of the given mutants, but also samples to find the connection between the morphological characteristics of the population and its antibiotic activity. This method greatly facilitates the selection of antibiotic high extensive strains of Actinomyces yarsoviensis sp.nov.

A simple, inexpensive and quick method was also worked out that allows mutants of higher antibiotic capacity from such a population within 18 hours weed out. A detailed description of both selection methods is in the Biuletyn Informacyjny Instytutu Anytiotykow, Warszawa, 1959, 2nd volume 6, indicated.

On the basis of the comparative information mentioned regarding the strains of the ray fungi: Streptomyces albus griseoflavus, Streptomyces rimosus, Streptomyces armillatus, Streptomyces platensis, and Streptomyces

17 18

yendargensis it must be stated that actino- spread the spores over the entire spherical upper myces varsoviensis sp.nov. with none of the mentioned area of the Pfianzenkömer and can then in Genera is identical. To be suspended in water and used for inoculating the

Actinomyces varsoviensis sp. nov. is a new, liquid seed medium, So far not described, oxytetracycline producing 5 The mentioned working methods for storage Ray mushroom species. des, strain Actinomyces varsoviensis sp.nov.

■ For the purpose of producing oxytetracyelin, this strain is grown before changes in its physiology Strain Actinomyces varsoviensis sp.nov. in.an logical characteristics and antibiotic intrinsic properties Usually, submerged and aerobic in liquid shafts.

Culture medium, expediently on the shaker iq The sporulation medium for the strain Actino- and in closed vessels, which are protected from contaminants varsoviensis sp.nov. can except natural protect from other microorganisms; Nitrogen sources, such as potato extract, wheat at the same time is aerated, expediently with bran extract and other vegetable or animal extracts guidance of sterile air or sterile oxygen. Extracts, including amino acids, ammonium sulfate

In general, special conditions will include 15 or sodium nitrate. Appropriate carbon content, among which those for the biosynthesis of the sources are simple sugars or polysaccharides, Oxytetracycline cheap breeding of the strain higher alcohols, molasses or malt extract. Except Actinomyces varsoviensis sp. nov. takes place and to which the sporulation nutrient medium can be an additive the cycle of biosynthesis, the substrates, the cultivation of mineral salts and an addition of trace temperature, the actual fermentation and the 20 elements, e.g. B. cobalt salt in an amount of 0.1 Duration of the same relating to ventilation, etc. up to 1 mg%, contain 1.5 to 3% agar.

The Actinomyces varsoviensis sp. nov. Used to inoculate the seed medium

The 6- to 14-day cultivation of the culture medium or stored in lyophilized form. Actinomyces varsoviensis sp. nov. at an optimal 26 ° C

When storing, it is best to serve as follows, from which a spore suspension is made method: The strains of Actinomyces varsoviensis will.

sp. nov. sporulation medium can be applied to a sporulation medium without the composition of suitable sporulation media

Change about 6 weeks in the freezer for Actinomyces varsoviensis sp.nov .: (at -20 ° C) and in the cooler (at
+ 4 ° C) and about 3 to 6 months with room 30 nutrient medium 1

temperature (around + 18 ° C). be kept. Glucose 10 g

When stored at room temperature, CoCl ₂ becomes 0.0005 g

to prevent possible unfavorable agar 25 g

changes that may occur in the cultivation, aqueous potato extract (erhalin the sample glasses with inclined agar a few millimeters by extraction inside liter of liquid, sterile paraffin that is kept in flowing for 2 to 3 hours Must cover the cultivated area, poured. Steam) 1000 ml

Actinomyces varsoviensis sp. nov. can be in pH 6.6 to 6.8

a corresponding protein and carbohydrate

containing medium, in solutions of amino nutrient medium 2

acids or some organic acids, often with soft corn extract (50%) 4 g

would give a protective substance for the purpose of contraception potato starch 15 g

of loss of vitality when resuspending the (NH) SO 4 g

Lyophilize lyophilized material. It became solid- KH PO 2 g

put that the best medium for the spores of the 45 CaCO 3 g

Actinomyces varsoviensis a mixture of 10% agar? 15 g

Sucrose solution and 1% gelatin solution is. The distilled water 100.0 ml

The final moisture content of the lyophilized material must be pjf 6 _; 6 to 6.8

in this case be 0.2 to 1%. The ampoules or

Bottles in which the lyophilized spores of the 50 nutrient medium 3

Actinomyces varsoviensis sp. nov. Malt extract 10 ε is stored

den, must be reduced to 30 to 4 s of yeast extract

100 mm Hg pressure making tightly closed glucose 4 g

(melted) or filled with nitrogen. ^ 201

The spores of Actinomyces varsoviensis sp. nov. 55 Distilled water '.'. '.'. '.'. '.'. '.'. '.'. ' 1000 ml can also be used in a mixture of soil and ug 5 kj _s 6 8

Sand at room temperature (approx. + 18 ° C) or at '' '.'

Store at cold store ^{temperature (approx. +4 0} C). In the biosynthesis of oxytetracycline by the

Soil mixed with sand in which the spore pens strain Actinomyces varsoviensis sp.nov. has been introduced in large-scale, can be slowly dried on an industrial scale, one of three temperatures, or it can be used from the cultivation cycle consisting of the stages: drying in the freezing state in a high vacuum. . _Tr ,,. ^ · ■, *

Another storage ^{method of spores A} ^ ^orh % ^e ^ of ^the vegetative seed material on the strains Actinomyces varsoviensis sp.nov. is that of ^the knuttelapparat;

Storage of the same on previously sterilized and 65 B- preparation of a larger volume of vege- dried Plant products, e.g.-on millet-tative Saatinoculum in the fermentation tank and

groats. After several days of incubation at C. the actual fermentation, during which appropriate temperature (24 to 26 ° C) distribute the antibiotic oxytetracycline is formed.

■ ΪΦ13 & 68

19 2Ö

In all three stages of the large-scale industrial substrate must as a rule for each mutant

Standardized biosynthesis - the submers - will be determined separately; With simple sugars

Growing both on the shaker and it is 0.5 to 5%, for oils 0.5 to -3%>. · """'■''

in fermentation tanks of various volumes - '■' -

agile. The tanks must be equipped with devices - · 5 · Example 1 conditions that allow continuous ventilation

cultivation using sterile air, mixing of the ^: cultivation of a strain of Actinomyces medium, maintenance of a constant varsoviensis sp.nov. is carried out in three stages by allowing temperature and using devices.

Be prepared to allow the course of the fermentation to last the first stage of the fermentation cycle control and register mentation. This 24 to 48 hours, depending on the amount of for Devices allow the determination and application of the inoculation applied spores of the micro-preservation an optimal pH value of the nutrient organism, the composition of the nutrient medium, soil, the supplement of the fermentation temperature, the cultivation temperature, the intensity of the ventilation exhausted components of the nutrient medium, e.g. B. 15 tang, which in turn is of the shaker type, the carbon sources, the introduction of the number of shakes or revolutions as well as Creation of oxytetracycline stimulating compounds from the stroke, finally from the shape of the vessel fertilize, as well as means for preventing the sight and the ratio of the volume of the vessel mens of the nutrient solution and the like to the volume of the nutrient medium in it

The individual stages in the said work 20 depends.

for Actinomyces varsoviensis sp. nov. proven

different composition and by one the optimal conditions in the first stage

different pH of the nutrient solution, different the following:

Temperature and duration of the cultivation as well as by 2 to 5% suspension of the spores of a sloping spore different ventilation conditions. 25 agars a sub-die containing a corn soft extract Nutrient solution contains a source of nitrogen, e.g. B. strat, the peanut flour, ammonium sulfate, starch, an organic or inorganic nitrogen source sodium chloride and calcium carbonate in appropriate or a mixture of both, as well as a carbon containing the ratio, 80 ml of this nutrient medium quelle, a mineral salt source and a source for are placed in a 500 ml Erlenmayer flask, Trace elements; The nutrient solution can also become the nutrient medium after inoculation with spores Contain calcium carbonate. on a shaker with 180 revolutions per minute, with a stroke of 3 to 5 cm and as sources for mineral salts and trace elements In general, the "natural" components come at a temperature of 20 ° C, usually 24 degrees Nutrient medium, the plant or animal the long, hatched. It can be done at this stage Origin are to be considered. The main nitrogen source 35 performed two sieves from the poorer nutrient medium in the nutrient form z. B. soft corn extract, meat - containing a richer, carbon source Extract and the like. Nutrient medium can be made.

Source of the most widely used nitrogen sources

should be mentioned: meat extract, peptone, fish meal, nutrient medium 4

Yeast, yeast extract, yeast hydrolyzate, gasein, sour 40

Casein hydrolyzate with pancreatic enzyme, glucose 10 g

etched casein, corn soft extract, soy flour, cobalt chloride 0.0005 g

Extracts and hydrolysates from this flour, peanut calcium carbonate 4 g

flour, ground peanuts, extracts and hydro- Aqueous potato extract 1000 ml

lysates from peanuts, cotton seed flour, 45 soybean oil 5 ml

Flour made from sunflower seeds and other natural pH 6.6 to 6.8

Nitrogen sources of vegetable and animal origin as well as extracts produced from them, nutrient medium 5 swollen products and hydrolyzates; instead of

or in addition to the nitrogen sources mentioned, 50 soft corn extract 5 g

also uses ammonium sulfate or nitrates Ground arachid chips ... 10 g

will. The addition of some of the mentioned nitrogen potato flour 25 g

sources, e.g. B. soft corn extract, to the nutrient, in ammonium sulfate 4 g

where the main source of nitrogen is ground peanuts sodium chloride 5 g

have a stimulating effect on the 55 calcium carbonate 5 g

Generation of the oxytetracycline. Distilled water 1000 ml

The carbon sources can be soluble or pH 5.7 to 5.8

use insoluble carbohydrates, e.g. B. sucrose, maltose, glucose and other simple nutrient medium 6 Sugar or mixtures thereof, starch, potato flour, 60

Maize flour, malt extract or mixtures with the previously soft maize extract 7 g

mentioned simple sugars. You can also use higher arachid schnitzel 20 g

Use alcohols, e.g. B. glycerine, mannitol, and others. For potato flour 45 g

some "natural" and "artificial" mutants of ammonium sulfate 4 g

Actinomyces varsoviensis sp. nov. have plant 65 calcium carbonate 7 g

borrowed oils, e.g. B. arachis oil, soybean oil, sunflower oil cobalt sulfate 0.02 g

and others proven to be suitable sources of carbon. . Distilled water 1000 ml

The percentage of each component of the pH 6.0 to 6.2

21 22

Nutrient medium 7 The yield after the specified time of the

Fermentation in the flask on the shaker

Glucose 30 % is 282 meg per milliliter; pH 7.1.

Corn soft extract (50%) 20 g

Calcium carbonate 5 g ₅ nutrient medium 10

Soy flour 5g peanut flour 20g

Sodium chloride 5 g corn soft extract (50%>) 4 g

Ammonium sulfate 4 g sodium sulfate 2 g

Distilled water 1000 ml of malt extract, .. ■ 2 g

pH 6.6 ₁₀ sodium chloride 4 g

Calcium carbonate 10 g

The second stage of the breeding cycle takes soybean oil 10 g

also 24 to 48 hours, depending on the Fer- water 1000 ml

mentation conditions. Around 6001 ent pH, of course

speaking culture medium containing 1000-1-Fer- 15 ^ r- _{h A} - _ΛΛ

In the mentation tank, the cultivation time at 28 ° C is «anrmearam 11

about 30 hours. The nutrient medium contains a ground arachid schnitzel 22.5 g

Mixture of corresponding amounts of soft maize extract, soft maize extract 6.3 g

Ammonium sulfate, potato flour, sodium chloride, potato flour 40.0 g

Calcium carbonate and arachid oil (as anti-foaming ammonium sulfate 3.6 g

medium for the nutrient solution). The volume of cobalt sulfate .. 0.018 g

from the first stage of cultivation on shaking arachis oil 1.8 g

Apparatus-derived vegetative inoculum is calcium carbonate 6.3 g

more than 0.05%, e.g. 0.1 to 1% of the total water 1000ml

Volume of the nutrient solution of the 10004 tank. The 35 pH 6.3 to 6.5

Ventilation conditions for the cultivation are: 0.2 to 0.6

of the air volume to 1 volume of the nutrient medium In the 48th and 60th fermentation hour is each

per minute. The speed of the stirrer must add 120 to 2.5 g of soybean oil. Forms in the nutrient medium 10

360 per minute. the soybean oil, similar to the malt extract, a carbon

30 source of material.

Nutrient medium 8 ^ ^e yield after 120 fermentation hours in

Flask on the shaker is: 320E in

Peanut flour 20 g 1 ml; pH 6.8 for the nutrient medium 10 and 356 E in

Corn soft extract 4 g 1 ml; pH 6.9 for the nutrient medium 11.

Sodium chloride 4 g peanut flour 40 g

Calcium carbonate 10 g soy flour 20 g

Soybean oil 5 ml glucose 60 g

Distilled water 1000 ml 40 soft corn extract (50%) 5 g

pH 6.6 to 6.8 ammonium sulfate 5 g

Calcium carbonate 4 g

The third stage of the growth cycle of cobalt sulphate 0.05 g

Strain Actinomyces varsoviensis sp. nov., in the soybean oil 8 g

the actual fermentation takes place while 45 lard 3 g

which the oxytetracycline is generated takes, water 1000 ml

depending on the cultivation conditions, 100 to pH 6.6 to 6.8

150 hours. In the fermentation tanks with a volume of 5000 to 15000 1, containing 3500 to On the nutrient medium 12 in the fermentation tank of 10 0001 nutrient medium, composed of arachid 50 50001 volume, containing 35001 of the nutrient medium, Flour, soy flour, soft May extract, ammonium sulfate, after 100 hours of fermentation you have 2300 mcg / ml Potato flour, cobalt salt, calcium carbonate, glucose, obtained at pH 6.2.

Arachid oil, soybean oil or lard (as an anti-foaming agent) During the third stage of the biosynthesis of the

medium) the fermentation takes 100 to result in oxytetracycline, the first two stages are used

150 hours, at a temperature of 28 ° C and 55 for the preparation of the inoculum for inoculating the

in the case of cultivation aeration, the 0.6 to 1.0 of the nutrient medium of stage three, i.e. H. for inoculation

Air volume to a nutrient medium volume per the actual fermentation culture medium.

Minute. The inoculum is used in all stages of cultivation

controlled by cytochemical methods; in accordance

Nutrient medium 9 ^6o stained preparations using the gram method must

The seed inoculum should be 90% gram positive. An important

Soft corn extract (50%) 5 g also plays a role in the control and the 37.5 g on potato flour to maintain the corresponding pH value of the

Ammonium sulfate 6 g of medium in the individual stages, in particular

Sodium chloride 5 g 55 the pH of the Saatinoculum and the pH

Water 1000 ml of the nutrient solution during the actual fermentation

Soybean oil 2 tation. In the third stage, the pH must be the

Maintain pH 6.0 to 6.2 nutrient solution within the following limits

23 24

th are: before vaccination by means of the washed off, after which the adsorbed oxytetracyelin

Saatinoculum from an IQOO-l tank must be eluted using methanol saturated with HCl.

pH of the near medium within the limits of 6.2. The flow rate of the acidic methanol

up to 6.9, during fermentation it can be 30 ml / cm ² / hour. "■ ■. ■

to 6.8 to 8.8 increase, and at the end of the fer- -5 The yield of the elution process depends on

mentation must. it is within the limits of the initial values of the concentration of the adsorbed antibiotic

6.2 to .6.9. on the ion exchangers and is 50

Using paper chromatography in systems, up to 97%;

with water-saturated ethyl acetate with butyl ketone - The control of the adsorption and absorption

methyl isobutyl-n-butanol acetate in the ratio of io process is obtained by taking samples from the

5: 15: 2 were the individual fractions in the fermentation medium and by determining the in

Presence of only one antibacterial substance, antibiotic contained in them by means of general

the biological evolution of the chromatogram of known chemical and biological methods

by means of. des. Bacillus subtilis 6633 found. The carried out.

Position of this substance on the blotting paper strip 15 The one containing the adsorbed antibiotic

corresponded to the location of the methanol eluate acidic by the oxytetracycline can by a NaOH solution

evoked inhibition zone. be neutralized, but this process causes

After the end of the fermentation of the nourishment, an additional introduction of inorganic

The oxytetracycline can be added to the solution by salting it, thereby cleaning the medium

different known methods from the Fennen-ao antibiotic is made more difficult,

isolate the station liquid. It is beneficial to neutralize the new one

To separate the mycelium, the Fennen eluate is an anion exchanger, e.g. B. Amberlite IR

tation fluid. on.pH = 1.5 to 2.0, preferably or Dowex 1.2, of basic character to be used.

pH =. 1.8, - with oxalic acid with constant stirring. The neutralization on an anion expression

leavened. Within these pH limits, the decomposition exchange takes place together with the adsorption of

tion of the chlorine ions formed during fermentation from the solution and with the excretion

Oxytetracycline complex salts with calcium and magnesium of water. The neutralization is carried out

sium as well as .the ^ complete transition of the anti-led that the methanol solution of the antibiotic

biotikums in, the solution. Has a pH of 3.0 to 3.5 under these conditions.

• also partially suppresses protein 30 During the neutralization, the anti-

and other biotic losses contained in the mixture up to 7%. The neutralized eluate

impurities what. the filtering is made easier and the is in the vacuum evaporator at 35 to 40 ° C up to

Obtaining a transparent filtrate permitted. Thickened after a small volume and thereby the

the acidification is about 6% kieselguhr as filter hydrochloride of the oxytetracycline by means of concentrated

auxiliaries added; the whole thing is precipitated 30 meows 35 trated hydrochloric acid. The precipitated precipitate

vigorously stirred for a long time and then the mycelium is alternately closed after filtering and washing

Washed together with the precipitated calcium oxalate with methanol and acetone and then washed

filtered off, dried at a temperature of 40 ^{0 C.} the

From the clear filtrate you can see the final yield of the process is about 36%. the

Antibiotic by means of one of the well-known 40 activity of the obtained hydrochloride of the oxy-

Procedure for. Isolation and purification of the anti-tetracyclines is approximately 670 mcg / mg.

excrete biotics. In order to obtain a product with higher

The oxytetracycline has an amphoteric connective activity, the oxytetracycline obtained

formation of both acidic and basic groups with recrystallic

the dissociation constant pH 3.5, 7.6, 9.2 on, 45 size (see Example 5).

which the adsorptive capacity of the antibiotic -,. . ,

determine by ion exchanger. .Example I

For the chemical processing of the antibiotic 101 of a fermentation broth obtained according to Example 1, the following ion exchangers are used: A sulfonated 50 is acidified to a pH of 2.0 with constant stirring with oxalic acid to cation exchanger Dowex 50 W (then a polymerisation product of styrene and Butadiene), the 500 g of diatomaceous earth are added and the whole of the anion exchanger Amberlit IR 45 (a polymer stirred for 30 minutes, after which the nutrient medium is deposited on the sation product of melanin and formaldehyde), the centrifuge. If the filtrate is anion exchanger Dowex 1,2 (a polymerization cloudiness, the filtering must be repeated until a product of ethylenediamine and formaldehyde is obtained with a transparent filtrate,
which a column is filled to a height of 20 cm). The clear filtrate with a pH of 2.0. The ratio of the diameter of the column to its up to 2.5 is determined by a height with the cation exchanger is 1:10. The flow rate of the filled column with a rate of the nutrient medium filtrate through the ion exchanger - 200 ml / cm ² / hour. guided. After passing through the column is 200 ml / cm ² / hour. The yield of ad- 60 nutrient medium is the column with 500 ml of distilled sorbed antibiotic on the ion exchanger water and then with 250 ml of methanol depends to a large extent on the concentration of the wash. The on the cation exchanger adsor-Oxytetracyclins in the fermentation media and beer antibiotic is with 1500 ml N of half fluctuates within the limits of 200 to 1000 mg per an HCl solution in methanol with a flow-gram of the exchanger:. 65 speed of SOml / cm / hr. eluted.

After the end of the adsorption, the ion- The obtained oxytetracycline eluate contains 2.19 g

exchange column with the adsorbed antibiotic oxytetracycline, which is 49% of the original

alternating with. distilled water and. Methanol amount,.

25 26

The acidic eluate is filtered off by means of an anion, washed with alcohol and then with an ether exchanger up to a pH value of 3.0 to 3.5 and dried at 40 ° C,
neutralized and then in a vacuum evaporator at The potency of the hydrochloride obtained from 35 to 40 ° C up to a volume of 50 ml an oxytetracycline is 640 meg in 1 mg. The Thickened. 5 spoil of the process is 46%.

The oxytetracycline methanol concentrate is processed using a different procedure for excreting the

concentrated hydrochloric acid added and in the course of the oxytetracycline from the nutrient solution is based on the

In addition, the hydrochloride is salted out and the antibiotic is salted out by means of table salt

of oxytetracycline. The filtered precipitate is added to the alkaline medium. A small addition of butanol

is alternately mixed with methanol and ether. ίο The filtrate increases the purity

wash and then dried at 40 ° C. of depressed oxytetracycline.

It was 1.6 g of oxytetracycline with a potency. The filtered precipitate is after

of 670 mcg / mg gained. Washing and drying in a methanol solution of the

Another way of working for the isolation of oxy-calcium chloride, from which the precipitate is dissolved

tetracycline from the fermentation medium is based on 15 of the oxytetracycline hydrochloride with concentrated

on the extraction of the antibiotic from the hydrochloric acid is precipitated. The filtered precipitate

Water phase with an organic solvent, is alternately mixed with methanol and ether

Do not wash with water at the applied pH value and then dry at 40 ° C,
is miscible. Butyl and

Amyl alcohol and its esters and mixtures of 20 examples4

Alcohols and their esters with low organic ^p

see acids being applied. The clear Fermen- 101 of a fermentation medium obtained according to Example 1 will be with constant stirring with a tation broth with oxalic acid up to one Mixture of butanol and butyl acetate added, acidified pH 1.8. Then 500 g after which a 20% NaOH solution was added for the purpose of increasing diatomaceous earth and the whole thing lasted for 30 minutes setting a pH value of 9.5 is added. stirred for a long time, and then the medium is set up Under these conditions, the oxytetracycline is filtered off in a centrifuge. If the filtrate is cloudy from the water phase to the organic phase. the filtering process up to the achievement of a look-through The separation of the phases is repeated for the extracting filtrate.

hierte antibiotic-containing organic phase 30 The filtered nutrient medium are at constant

mixed with a sulfuric acid solution and thereby stirring 2.5 kg of common salt and 500 ml of butanol

the pH increases to 2.0. The antibiotic is given, followed by a NaOH solution up to

into the water phase, from which alkalization takes place after separation to a pH of 9.0 to 9.5,

of the organic phase, the hydrochloride of the oxy- The whole is stirred for 1 hour and for the purpose of

tetracycline with common salt at a pH of 35 phase separation. In between-

5.5 is salted out. The precipitated hydrochloride phase on the border of the water phase and the

Precipitation of the oxytetracycline is after the organic phase is the precipitation of the complex

Filtration with methanol and then suspended with ether salts of the oxytetracycline. After this

to wash. Separating the water phase becomes the organic

Example 3 ⁴ ° P ^nose mixed with kieselguhr together with the intermediate phase and, after stirring, the precipitate

101 a obtained according to Example 1 Fermen- the complex salts of oxytetracycline and des

tion broth are filtered off using oxalic acid to form a gravel.

pH 1.8 acidified with constant stirring. After washing and drying at 40 ° C Then 500 g of kieselguhr are added and the 45 g of complex salt precipitate became the anti-whole Stirred for V2 hours, after which the mycelium biotikums with a potency of 115 meg in 1mg and the filled calcium oxalate are filtered off. get what 72% of the original amount of the If a cloudy or opalescent filtrate is obtained, antibiotic corresponds. The Niederist won to repeat the filtering process. blow is made five times in 80 ml of a methanol solution

The clear filtrate with a pH of 50 of the calcium chloride is extracted, adding to the first 1.8 is with 1000 ml of a mixture of butanol extraction 30 ml and to the following second and and butyl acetate in a ratio of 93: 7 and the third 15 ml each, the fourth and fifth 10 ml each Continuous stirring will have a 20% turn.

NaOH solution added and the pH adjusted to 9.5 to that obtained in an amount of 58 ml

brought. After the pH has been adjusted, methanol filtrate becomes 6 ml of concentrated hydrochloric acid

the whole stirred for 1 hour and then added dropwise with constant stirring. Under these conditions

Separating the phases allowed to settle. conditions separate hydrochloride crystals of the

The yield from the nutrient medium down to the organic oxytetracycline, which after filtering and

see phase is 85%. Wash with 99% methanol at 40 ° C, dry

^{The organic 6} ° net separated from the water phase.

Phase is mixed with 0.1 nH ₂ SO ₄ with constant stirring. 1.78 g of the oxytetra hydrochloride were extracted and the pH was brought to 1.8. After cyclins with a strength of 860 meg in 1 mg adjusted the pH, the whole thing is won for 1 hour. In the known process, the win is stirred for a long time and then left to separate the phases between oxytetracycline and Streptomyces rimosus. The water phase is mixed with crystalline 65 (German patent specification 862 647), on the other hand, from NaCl, the pH is brought to 5.5 and 8 liters of fermentation liquid 1.69 g of product of a potency for the purpose of crystallizing out the hydrochloride of 625 meg in 1 mg received, which corresponds to oxytetracycline turned off. The precipitate becomes 211.2 mg of product from one liter of fermentation liquid.

- ■■■ - "- ■ - ■■ --- ■■ - 609540/375

After further purification, this product yields 61.25 mg, also with a potency of 860 meg in 1 mg.

In the process according to the invention, therefore, 178 mg of product are obtained from 11 fermentation broth compared to only 61.25 mg of product of the same potency in the known process.

Example 5

301 of the fermentation broth of 2300mcg / lml Activity are acidified with concentrated oxalic acid to pH 1.7 to 1.8, after which is. 0.5% kieselguhr added and the whole thing filtered off.

The filtrate obtained, the volume of which was 251, 1 tyoo potassium ferricyanide, 0.3% o zinc sulfate, 2% kafhexol and 3% kieselguhr were added. The precipitate formed was filtered off. to 1% cathexol, 1% magnesium sulfate, 1% ammonium arsenate, 0.3% sodium sulphate and 2% kieselguhr added. The pH of the mixture was diluted using a Ammonia solution adjusted to 8.5 to 8.7. The precipitate containing the oxytetracycline The complex is excreted, filtered off and washed with water. From the complex compound obtained the crude oxytetracycline base was recovered.

For this purpose, the moist complex precipitate was mixed with a mixture of 30 ml of n-butanol and 50 ml Water mixed and mixed well. The whole thing was then treated with a mixture of 30% sulfuric acid and concentrated oxalic acid acidified to pH 1.9 to 2.0 and after heating at 28 ° C. 1% ammonium arsenate and 0.5% sodium sulfite were added and then the pH adjusted to 4.5 with ammonia water. The resulting precipitate was filtered off, washed with n-butanol and water and dried.

In the further course of the procedure, the crude base was converted into the hydrochloride and using Crystallization purified. For this purpose, the oxytetracycline base obtained was mixed with a 4% strength solution of calcium chloride mixed in methanol and mixed well. The resulting precipitate was filtered off and washed with the methanol solution of calcium chloride. The basic feed and the Wash solutions are combined and vacuumed up to a concentration of the antibiotic of Evaporated 120000 mcg / ml.

The resulting solution is then concentrated with Hydrochloric acid acidified to pH 0.2 to 0.3 and turned off to crystallize. From-', separated crystals are filtered off, with metha-

ίο nol and then washed with acetone and im Vacuum dried at 50 ° C.

41 g of the hydrochloride of oxytetracycline were obtained of great purity as a yellow crystalline powder. This corresponds to a yield from the isolation process of over 70%.

Example 6

To recrystallize the Oxytetracyclirihydrochlorids obtained according to Examples 1 to 4 are 3.5 g of oxytetracycline hydrochloride with a potency of 670 U in 1 mg with 1.2 g of triethylamine and 30 ml the methanol solution of calcium chloride mixed and 4.5 ml of concentrated hydrochloric acid added. Of the Precipitation of the oxytetracycline hydrochloride is filtered off and then alternately with methanol and ether washed and dried at a temperature of 40 ° G.

2.6 g of oxytetracycline hydrochloride with a potency of 920 meg in 1 mg were obtained.

The control was carried out using well known chemical and biological methods.

Claims (1)

Claim: Use of Actinomyces varsoviensis sp.nov. No. NCIB 9522 for the production of Oxytetracycline by the usual biological route. Considered publications:
German Patent No. 862,647;
British Patent Nos. 648,417, 713,795; U.S. Patent No. 2516,080;
Canadian Patent No. 520 836;
Swiss Patent No. 331988. 609 S40 / 375 3.66 © Bundesdruckerei Berlin