DE1492137C - Process for the production of oxy tetracycline by biological means - Google Patents

Description

The invention. relates to a method of manufacture of oxytetracycline by biological means by cultivating a strain of Streptomyces in a conventional nutrient medium and working up the reaction mixture in a manner known per se, which is characterized in that the strain Streptomyces henetus nov. spec, at the Commonwealth Mycological Institute Ferry Lane, Kew, Surrey (England) under I.MI 109 532 and at the Institute of Microbiology of the Rutgers University (USA) deposited under I. M. 3872, used.

The antibiotic oxytetracycline, its production and its chemical, physical and · biological Properties are in the literature (W a k s man S. A., "The Actinomycetes", Vol. III, 1962, P. 329) has been described in detail.

It has been found that the method of the present invention can produce oxytetracycline in a very large amount high yield can be obtained.

The new oxytetracycline-producing microorganism, obtained from a near Ramera (Italy) • The soil sample taken has been isolated, has the following morphological, macroscopic, microscopic and biochemical properties.

Microscopic appearance

The vegetative mycelium that forms on potato agar and Czapek agar consists of 0.8 to 0.9 μ thick, more or 'less long and branched hyphae. From the same it becomes from 1.2 to 1.6 μ thick and not very long hyphae formed aerial mycelium. These hyphae branch out abundant and form straight conidiophores, which carry spiral hyphae, which in turn form spores pass over. ,

The conidia are predominantly oval with a smooth surface and have the following dimensions: 1.2 up to 1.6 μ χ 1.4 to 1.7 μ, first they are chain-wise and then freely arranged. Each chain has more than 10 spurs. The spore forming hyphae are on the Konidiophora arranged alternately or mutually.

The spiral-shaped Konidiophora are for the most part inserted like a whorl. The whisk can consist of two or several Konidiophora exist. The spirals are closed, whereby they can often form tangles.

Macroscopic appearance

In Table 1 the properties are corresponding

the culture soils indicated next to it are given, the microorganism growing at 28 ° C and made the observations on the 3rd, 8th, 16th and 21st days after inoculation.

Biochemical properties

The microorganism reduces nitrates to nitrites, hydrolyzes starch and gelatin, does not produce melanin and uses tyrosine, d-mannose, d-arabinose, d-glucose, meso-inositol and d-fructose. It produces acids from adonitol, maltose and ramnose, but not from d-xylose, sorbitol, sucrose. The microorganism is no sclerotia and can not grow at 5O ⁰ C.

Table 1

The characteristics of Streptomyces henetus nov. spec, at the Commonwealth Mycological Institute Ferry Lane, Kew, Surrey (England) under I.MI 109 532 and at the Institute of Microbiology

of the Rutgers University (USA) under I. M. 3872

medium growth Aerial mycelium Vegetative mycelium Soluble pigments Observations Bennet agar ¹ ) plenty, waiter
area shows
sublime
wrinkled folds Dirty white,
smooth, sparse from colorless to
Straw yellow up to
light brownish absent - Csapek agar ¹ ) sparse Dirty white,
with rectilinear
Sporophora,
which spiral
shaped hyphae
wear that of theirs
on the other hand in spurs
pass over colorless absent Asparagine
Glucose agar ¹ ) moderate whitish,
sparse colorless absent - ■ Glycerine
Glycine agar ') moderate Dirty white,
sparse colorless absent -

') Wa k s m a η, S.A., The Actynomycetes. Vol. II. The Williams and Wilkins Company, 1961, pp. 328-334.

continuation

medium growth Aerial mycelium Vegetative mycelium Soluble pigments Observations Emerson agar ¹ ) plenty, waiter
area shows
sublime
wrinkled folds moderate, of
Dirty white
to light gray
brownish bright absent - Strength- Priham-
Salts agar ² ) moderate mediocre from
dirtier whiter
Color, straight
lined conditional
phora which
spiral
Carry hyphae,
which in turn in
Spurs over
walk colorless absent Potato agar ⁴ ) plentiful of dirty
white to light
gray-brown of straw yellow
to light brown absent Oat agar ³ ) moderate sparse,
whitish of colorless
to yellowish absent - Asparagine
Glycerine agar ¹ ) moderate sparse,
whitish colorless yellowish absent - Peptone
Starch agar ¹ ) moderate sparse,
whitish slightly light brown absent positive
hydrolysis Tyrosine Agar ⁵ ) moderate sparse,
Dirty white colorless absent - Melanin agar ¹ ) sparse very sparse,
Dirty white colorless absent - Gelatine ¹ ) moderate absent Straw yellow absent - Peptone broth
with KNO ₃ ¹ ) moderate absent of honey colored
to yellowish absent - milk moderate absent Straw yellow absent -

') Wa ksman, S.A., The Actynomycetes, VoI. II, The Williams and Wilkins Company, 1961, pp. 328-334.

² ) P ridham TG, A η ders ο n, P., F ο 1 ey, C, L i η de η fe 1 ser, LA, H esse 11 i η e, CN and Benedict, R. b., Antibiotics Annual 1956 and 1957, pp. 947 to 953.

³ ) B a 1 dacci, E., G i ο 1 e 11 i, G., K ü ster, E., S c ο 11 i, T., 1961, Giornale di microbiologia, 9, p. 39.

⁴ ) To 200 g of potato stock poured through cheesecloth, 20 g of glucose and 20 g of agar are added. The volume of the mixture is then adjusted to 1000 cm ³ by adding water and sterilized for 20 minutes at 120 ^° C.

⁵ ) G ο rd ο n, RE, S mith, MM, J. Bact., 69, p. 147, 1955.

Table 2

Comparison between the Streptomyces used in the present case and other oxytetracycline producing streptomycetes

Streptomyces
according to the invention S. rimosus S. platensis S. armillatus S. vendargus-
vendargensis S. gilvus Sporophora ¹ ) Spiralata spiral spiral spiral just position Section S *) Section S *) Section S *) Section S *) Section S *) not known Spurs Oval with cylinder- oval not not not smooth upper shaped 0.7 to 0.9 described described described surface 0.6 to 0.7 x 0.8 to 1.2 to 1.6 x 0.8 to 1.2 µ x 1.4 to 1.4 µ 1.7 µ •

*) According to Pridham and coworkers, Applied Microbiology, Vol. 6; No. 1, 1958, p. 51.

') = According to the classification of Pridham and coworkers, Appl. Microbiol. 6, 1958, p. 52.

continuation

Streptomyces
according to the invention S. rimosus S. platensis S. armillatus S. vendargus-
vendargensis S. gilvus Vegetative
Mycelium of straw
yellow to
Ocher,
back
pale yellow of cream
colored over
Reddish
brown to
orange
coloured apricots
colored or
reddish ocher
colored, cinnamon
and apriko
mustard colored
back of colorless
to yellowish
Gray of pale yellow
until black
Brown Aerial mycelium Dirty
White,
sometimes
to gray
Brown of white
lich about
Mouse gray
to black of white
lich about
olive colored
or mouse
gray to
black whitish,
almost always
very sparse White almost always
absent Soluble
Pigments absent Yellowish,
sometimes
also brown of yellowish
over
Yellow green to
Brown sometimes
present
from pale
pink to
Brown absent not
described strength + ± ± - not
described not
described gelatin + ± ± - the same the same Nitrates + + + - the same the same Tryrosine + not
described not
described not
described the same the same Melanin - the same - - the same the same H ₂ S - - not
described not
described the same the same

+ = Positive reaction.

- = negative reaction.

± = Very sparse response.

Table 2a

Comparison between the Streptomyces and Streptomyces used in the present case varsoviensis according to French patent specification 1,279

S. varsoviensis French patent
font 1 279 456 Streptomyces
according to the invention Present description Aerial mycelium = white Table III Aerial mycelium: dirty white,
sometimes gray-brown Table 2,
Page 7 Does not utilize maltose and
Ramnose Table V utilizes maltose and
Ramnose Page 3,
Lines 1 to 2 Doesn't reduce nitrates too
Nitrites Table III reduces nitrates to nitrites Page 2,
Lines 3 from the bottom Gelatin: not hydrolyzed Table IV Gelatin: hydrolyzed Page 2,
Row 2 from the bottom Potato medium:
Aerial mycelium = white Table IV Potato medium:
Aerial mycelium = from dirty
white to light gray brown Table 1,
page 4 Emerson Medium:
Aerial mycelium = white Table IV Emerson Medium:
Aerial mycelium = moderate from
Dirty white to light gray
brownish Table 1,
page 4

Identification of the microorganism

According to the description, the microorganism examined belongs to the genus Streptomyces Waksman et Henrici (Berbey's Manual of Determinative Bacteriology, 7th edition, 1957, pp. 744 and 745). Examination of the species suggests the following.

Belongs in the classification system by P r i d h a m and coworkers, (Appl. Microbiol., 6, 1958, p. 52) the microorganism to the "Spira" section, White series; in the classification system of BaI-d a c c i (Giorn. Microbiol., 6, 1958, p. 10), the microorganism belongs to the series "Albidoflavus"; in Waksman's classification system (The Actinomyces, Vol. II, 1961, p. 123) belongs to the micro-organism the series »Flavus«.

A comparison between the properties of the microorganism and those of those specified species belonging to systematic groups (taxa) has shown that none of them have such properties which correspond to those of the microorganism examined.

The details of this comparison are given in Table 2, as far as they concern species, the substances generate which are similar to those examined by the applicant. Except for those given in Table II One can distinguish in particular a clearly different morphology between the Detect Streptomyces according to the invention and Streptomyces rimosus. Indeed, in the first case the hyphae transforming into spores alternately or mutually on long straight sporophores while in the latter case the spore-forming hyphae are bound to short Sporophora in more or less compact bundle system. The following are the differences with the species belonging to the specified orders which do not produce substances of the type mentioned above, cited.

The microorganism used according to the invention differs from the species • S. flavus, because this species generally does not produce spirals, differs in color from the vegetative one Mycelium and the air mycelium and does not reduce nitrates (S. A. W a k s m a n, The Actinomycetes, Vol. II, 1961, p. 210); it differs from the species S. flavovirens because it is this species in general no spirals created, soluble pigments formed, milk peptonized or curdled (W a k s ■ man, The Actinomycetes, Vol. II, 1961, p. 210); of the species S. flavogriseus, because this species has none Creates spirals and forms a mouse-gray aerial mycelium (W a k s m a n, The Actinomycetes, Vol. II, 1961, P. 209); of the species S. chrysomallus because this species does not form spirals and because it has soluble pigments forms (Waksman, The Actinomycetes, Vol. II, 1961, p. 193); of the species S. celluloflavus because of this Kind of soluble pigment produced and shows a different color of the air mycelium (Waksman, The Actinomycetes, Vol. II, 1961, p. 191); of the species S. viridans, because this species forms soluble pigments and because it has a different color of the air mycelium (Waksman, The Actinomycetes, Vol. II, 1961, P. 286); of the species S. albidoflavus, because it forms soluble pigments and has a different color from the Luftmycels shows (Waksman, The Actinomycetes, Vol. II, 1961, p. 168); of the species S. citreus, because this is a different color both of the veeetative Mycelium as well as air mycelium (Waksman, The Actinomycetes, Vol. II, p. 196); of the Species S. parvus because this forms soluble pigments and has a different color both from the vegetative Mycels as well as the Luftmycels (W a k s man, The Actinomycetes, Vol. II, 1961, p. 255); of the species S. griseoflavus, because these are a different color of both the vegetative mycelium and of the air mycelium and also forms soluble pigments (Waksman, The Actinomycetes, Vol. II, 1961, p. 222); of the species S. flavochromogenes, because these soluble pigments and are in the The color of the vegetative mycelium and the aerial mycelium are different (Waksman, The Actinomycetes, Vol. II, 1961, p. 209); of the species S. alboflavus because this species does not tend to form spirals and because it does differs in the color of the vegetative mycelium (Waksman, The Actinomycetes, Vol. II, 1961, p. 169); of the species S. hygroscopicus, because it forms soluble pigments and changes in color to the vegetative mycelium distinguishes (W a k s m a n, The Actinomycetes, Vol. II, 1961, p. 230); of the kind S. fimicarius because this forms soluble pigments and changes to the color of the vegetative mycelium and the aerial mycelium differs (W a k s m a n, The Actinomycetes, Vol. II, 1961, p. 208); of the species S. abikoeneum, because this is melanin-positive, does not form spirals and produces soluble pigments (Waksman, The Actinomycetes, Vol. II, 1961, p. 165); of the S. alboniger species because it does not form spirals and has a different color of the air mycelium (Waksman, The Actinomycetes, Vol. II, 1961, P. 170); of the species S. hachijoenais, because it does not form spirals and is the color of the air mycelium differs (Waksman, The Actinomycetes, Vol. II, 1961, p. 226); of the species S. eurocidicus because of this does not form spirals and produces soluble pigments (Waksman, The Actinomycetes, Vol. II, 1961, P. 205); of the S. mediocidicus species because it does not form spirals, is melanin-positive and nitrates are not reduced (Waksman, The Actinomycetes, Vol. II, 1961, p. 242); from the species S. albidus, v. invertens, because these belong to the species S. griseus (W a k s m a n, The Actinomycetes, Vol. II, 1961, p. 169); of the species S. albus, because it is a snow-white aerial mycelium produced, milk peptonized and shows various Sporophora (Waksman, The Actinomycetes, Vol. II, 1961, p. 172); of the S. cacaoi species, because it produces long and open spirals, a light gray to mouse gray Forms aerial mycelium and weakly reduces nitrates (Waksman, The Actinomycetes, Vol. II, 1961, P. 183); of the species S. erythreus, because it has a cream-colored to purple-red aerial mycelium and wine-red forms soluble pigments (Waksman, The Actinomycetes, Vol. II, 1961, p. 204); of the kind S. galtieri, because it forms a cream-colored aerial mycelium and a brown soluble pigment, and gelatin slightly liquefied (Waksman, The Actinomycetes, Vol. II, 1961, p. 215); of the species S. longisporoflavus, because these long, open spirals have a white up Forms lemon-yellow aerial mycelium, curdles and peptonizes milk, and weakly hydrolyzes starch (Waksman, The Actinomycetes, Vol. II, 1961, P. 37).

From this it can be concluded that the investigated Microorganism is different from the species known heretofore; therefore he is considered a new one Strain and is regarded as Streptomyces henetus

209 553/436

9 10

nov. spec, at the Commonwealth Mycological trium carbonate, urea, N ^ -Malonylurea,

Institute Ferry Lane, Kew, Surrey (England) under sulfone or sulfate derivatives. It can then

I. M. I. 109 532 and obtained at the Institute of Microbiology oxytetracycline by recrystallization.

of the Rutgers University (USA) under I.M. 3872 cleans or in its salts with an inorganic

deposited. . 5 or organic acid, such as hydrochloric acid, sulfuric acid,

The Streptomyces according to the invention can be converted to citric acid or tartaric acid,

both by repeated growth on a solid. Furthermore, the present invention encompasses

Culture medium (potato agar, Sabouraud agar) as well as a new method for extraction and purification

by freeze-drying using milk of the antibiotic described below

or milk serum as a suspension agent and is explained with examples,

be kept. The production of the antibiotic The pH 2 to 2.5 with an inorganic acid,

according to the method according to the invention, such as hydrochloric acid, sulfuric acid or phosphoric acid,

carried out the usual methods and made acidic culture broth is filtered, and the obtained

in that the Streptomyces filtrate according to the invention is first mixed with oxalic acid, again

filtered in a liquid culture medium under aerobic conditions to remove the calcium oxalate; on this

If the temperature is between 24 and 37 ° C, the pH of the filtrate is increased by the addition of alkali

preferably 28 ° C, adjusted over a period of time from 72 to 8.0 to 8.5, and this filtrate with a

144 hours is grown. The pH can de- mix of tricresol and carbon tetrachloride

extracted according to the fermentation media used,

vary from 6.1 to 6.5 to 8. 20 The tricresol used is the generally

The culture medium consists of a carbon and known and for industrial purposes applied

Nitrogen source and inorganic salts. As a carbon bond with a specific gravity of 1.030 to 1.038;

Sucrose, glucose, and dextrin can be a source of fuel; it consists of a mixture of three cresol isomers. fäj-y

. Starch, soy flour, peanut flour, cottonseed flour, It has been found that one prefers a '^

Corn steeple, soluble distillation residues, so- 25 mixture of tricresol and carbon tetrachloride

ya oil and pork fat are used. Should be used as a stick in a volume ratio of 2: 1 to 1: 2,

material source come except for some of the above-mentioned layers are obtained by centrifugal

nitrogenous substances also meat extract, fugate separately from one another, and to the organ-

Peptone, casein, casein hydrolysates and ammonium niches, the antibiotic-containing phase adds

salts, such as ammonium sulfate or diammonium, a mixture of acetone and aqueous salt

phat, in question. For the generation of the antibiotic acid, preferably 1N hydrochloric acid, in the same volume

tikum's useful mineral salts vary depending on the lumen ratio too. The obtained aqueous layer

the nutrient medium used. Calcium carbonate is made by adding an alkaline base such as alkali

almost always present, and sodium hydroxide or alkali carbonate, adjusted to pH 7, can also be used.

trium and potassium, magnesium, manganese, iron, 35 whereby the antibiotic oxytetracycline fails, wel-

Copper, cobalt, zinc chlorides and the sulfates, phosphes as such isolated and by recrystallization

phate of the same can be added. purified or in the salts with an inorganic

The fermentation can take place in Erlenmeyer flasks or organic acid according to the known techin Laboratory fermenters of various contents is transferred through niken.

be guided. The content of antibiotic in the 40 The following examples illustrate the invention:

Brewing is qualitative by means of paper chromatography ". . .

compared to a calibration sample of oxytetracycline e 1 s ρ 1 e

and quantitatively by means of spectrophotometric Ab- Two 300 ml Erlenmeyer flasks each contain 60 ml

reading or through the bio- of the following culture medium known in the literature: _#i ,

logical methods. 45 Destrin 3 ° / ^

The compound of the invention includes potassium carbonate 0 4 °° /

prey on oxytetracycline of more than 11,000 y / ml ". ". q-io /

to achieve. This means a considerable tecti- “ ^ais ° o'w °

niche progress, considering that one. ^as , V '"'. _{0 /} °

taking into account the literature, as far as this 50 Ammoniumsuiiat υ, i / o

Yield information includes, when using Strep tap water '. ".". ".". ".". ".".'. ". '.'.'ad 1000 ml °
tomyces rimosus a yield of only 1000 y / ml

achieved. The sterilization is done by heating in the autoclave

After the fermentation has ended, the culture is carried out at 120 ° C. for 20 minutes. To

broth adjusted to a pH of 2 to 2.5 and 55 sterilization the pH is 6.8 to 7.0.

remove the mycelium by filtering or centrifuging - each flask is filled with 1.5 ml of a spore pen-

separated. The sion contained in the clarified liquid is inoculated, which can be obtained by slurrying a

Oxytetracycline becomes 20 days old according to the known methods on the following solid nutrient medium

isolated and cleaned. Cultivated Streptomyces agar slants useful for this purpose

Methods are extraction with a suitable 60 according to the invention with 5 ml of sterile distilled

organic solvents such as butanol, pentanol, water.

Hexanol, amyl alcohol, methyl ethyl ketone, methyl 200 g of peeled potatoes are about 20 minutes

Isobutyl ketone or butyl acetate; Adsorb on solid boiled in 500 ml of water. One sets the volume

Substances such as activated charcoal and the subsequent egg of the potato mash to its original value

ate with water or an organic solution and filter it through cheesecloth. Then 2%

medium; Precipitation of the antibiotic in the form of a glucose and 2% agar added. You put that

its derivatives or complex salts by means of volume of the mixture to 1000 ml and sterile

Suitable substances such as amines, ammonia and nallized them in an autoclave at 120 ° C for 20 minutes.

The pH of the nutrient medium is 6.8 to 7.0. The flasks are left on for 24 hours at 28 ° C Shaker incubated at 220 rpm and an eccentricity of 60 mm; ImI one so won Culture is used to inoculate 300 ml Erlenmeyer flasks, each containing 60 ml of the following productive culture medium contain:

strength

Corn steep water.
Calcium carbonate

casein

Ammonium sulfate
K ₂ HPO ₄

strength

Corn spring water ..
Calcium carbonate.
Ammonium sulfate.
Manganese sulfate

Cobalt nitrate

Cottonseed meal
Pork fat

8th%

2.5%

1%

1%

0.015%

0.00075%

0.5%

3%

Production is in 300 ml flasks containing 35 ml of the following Nutrient medium, carried out:

3%

0.3%

0.4%

0.5%

0.1%

0.01%

Tap water to 1000 ml

Sterilization: 20 minutes at 12O ⁰ C. The pH is 6.7 to 7.0. Cultivation is carried out at 28 ° C. under the conditions already described for the vegetative nutrient medium.

After 96 hours of fermentation, a production of 4000 μg / ml oxytetracycline is achieved.

Example 2

The procedure is as in Example 1 with the difference that the culture to be inoculated is 10 days old and was grown on the following medium:

Peptone 0.8%

Glucose 4%

Difoo agar 2%

Tap water to 1000 ml

It is sterilized in the autoclave at 120 ^° C. for 1 minute. The pH is 6.8 to 7.0. In addition, the productive phase differs in the composition of the following nutrient medium:

Tap water to 1000 ml

It is sterilized in an autoclave at 120 ^° C. for 20 minutes. After the sterilization, the pH is 6.5 to 6.8. It is incubated at 28 ° C under the conditions specified in Example 1. After 144stündiger fermentation to reach a production of 8500 yj ml oxytetracycline.

Example 3

The procedure is as in Example 2 with the difference that the culture is 20 days old and that the vegetative nutrient medium has the following composition:

Corn steep water 3.5%

Calcium carbonate 1.8%

Sucrose 0.5%

Tap water to 1000 ml

Sterilization: 20 minutes at 120 ^° C

strength

Corn spring water ..
Calcium carbonate.
Ammonium sulfate.
Manganese sulfate

Cobalt chloride

Cottonseed meal

8th%

2.6%

1%

1%

0.014%

0.00078%

0.43%

Pork fat 1 ml per 35 ml culture medium

After sterilization (20 minutes at 12O ⁰ C) the pH is 6.5 to 6.7.

The amount of vegetative culture used for inoculating the productive nutrient medium is 4.3% of the first. The productive culture medium is incubated for 24 hours at 28 ° C and at 24 ° C up to a total of 120 hours carried out. The content of oxytetracycline is 7600 y / ml.

B ei s ρ i e 1 4

The procedure is as in Example 2, with the difference that production is consistently carried out at one temperature of 24 ° C.

After 96 hours of fermentation, a production of 6400 μg / ml oxytetracycline is achieved.

Example 5

With a 20-day-old culture of the Streptomyces to be used according to the invention, 60 ml of the inoculated nutrient medium described in Example 1, which is contained in 300 ml flasks. You incubate for 24 hours at 28 ° C under the conditions described in Example 1; 20 ml of the culture broth thus obtained serve to inoculate 3000 ml of the same liquid nutrient medium, which is made up in a 5-1 Fermeriter Neutral glass is located. This fermenter comes with a screw stirrer, an air inlet tube that goes under the said stirrer ends, a breakwater, an inoculation pipe, an air outlet pipe and a device for controlling the temperature. Ferment at 28 ° C with an air supply of 3 l per minute and a stirring speed of 400 rpm. After 24 hours you use 150 ml of the culture broth thus obtained for inoculating 1 of the following productive nutrient medium:

Defatted soy flour 3%

Soluble distillation residues 0.5%

Glucose 2%

Sodium chloride 0.25%

Tap water to 1000 ml

Sterilization: 20 minutes at 12O ⁰ C; after sterilization the pH value is 6.5. The fermentation is carried out in a similar 5-1 fermenter made of neutral glass as described above at a stirring speed of 500 rpm and with an air supply of 3 liters per minute, the foam formation being controlled by adding small amounts of silicone-containing antifoam agent. After 88 hours of fermentation, a maximum production of 5800 μg / ml oxytetracycline is obtained. ■

Example 6

After sterilization, the pH value is 6.2 65 The pH value of 33.3 1 culture broth with a

up to 6.3. Incubation is carried out for 26 hours at an antibiotic concentration of 4500 j- / ml, wel-

28 ⁰ C was obtained on a shaker with 220 rpm and ches according to one of the previous example,

an eccentricity of 60 mm. The pro is set to 2 with 25% sulfuric acid. Man

filtered with the help of 1 kg of kieselguhr and washes the filtration cake with so much aqueous dilute Sulfuric acid, the pH of which is not below 2, until the volume of the filtrate is 45 1, whereupon 2.2 kg of oxalic acid are added. The mixture is stirred for 30 minutes, filtered and the filter is washed with enough water until that the volume of the filtrate is 50 l. 50 l of this filtered and calcium-free broth, which 150 g Containing oxytetracycline are made with a mixture of 2.5 1 tricresol and 2.5 1 carbon tetrachloride shakes, the pH being adjusted to 8.3 with an aqueous sodium hydroxide solution. Through Centrifugation separates the two layers. The aqueous phase is mixed again with a mixture extracted from 11 tricresol and 11 carbon tetrachloride.

The lower organic layers are combined, poured into a mixture of 51% acetone and 51% aqueous 1N hydrochloric acid and shaken thoroughly, and the two layers are separated by centrifugation. The lower layer is extracted twice more with 3 liters of acetone and 2 liters of aqueous 1N hydrochloric acid. The pH of the combined aqueous layers is adjusted to 7 with 20% strength sodium hydroxide solution, a precipitate separating out, which is filtered off, washed with ether and ^{dried in vacuo at 40 °} C .; the product is then ground, washed again with ether and dried at 40 ° C.

150 g of crude oxytetracycline are obtained.

150 g of the product thus obtained are äufgesphlärnmt in 1.5 1 Methariöi '"and by the addition of 200 ml of a' dissolved 13% solution of dry hydrogen chloride in anhydrous methanol. To the solution is added 14 g of animal charcoal, the mixture is stirred for 30 minutes .filtered through kieselguhr and washed with methanol. · ■ .. ·. ·.

The filtrate is concentrated in vacuo to a volume of about 400 ml, whereupon 30 ml 13% solution of dry hydrogen chloride in anhydrous methanol can be added.

The crystallization of the product is initiated by friction on the walls of the container, which takes place overnight at room temperature. Then, the crystallized product is filtered, washed with methanol and then with ether and finally at 4O ⁰ C in vacuo. 95 g of oxytetracycline hydrochloride are obtained.

Example 7

The procedure is as in Example 3 with the difference that the vegetative phase and the practical phase of the process are carried out at 27 ° C and that the average amount contained in each container in the productive phase is 50 ml.

After 6 to 7 days of fermentation, a production of 11,000 y / ml oxytetracycline is obtained.

Claims (1)

Claim: Process for the biological production of oxytetracycline by growing a Streptomyces strain in a customary nutrient medium and processing of the reaction mixture in a manner known per se, thereby characterized in that the strain Streptomyces henetus nov. spec, at the Commonwealth Mycological Institute Ferry Lane, Kew, Surrey (England) under I. M. I., 109 532 and at the Institute of Microbiology of the Rutgers University (USA) under I.M. 3872 deposited.