DE1617814C - Process for the microbiological production of ergocryptine - Google Patents

Description

The invention relates to a method for the microbiological production of ergocryptine. Ergocryptine belongs to a group of ergot alkaloids; by hydrolysis it becomes lysergic acid, dimethylbrenzt acid, D-proline and L-leucine, the molecules of these substances are bound by amide bonds ; $ en connected. Ergocryptin is useful as a Hypertensive agent and used in the treatment of;) peripheral vascular disorders.

In the literature, various microbiological / experienced for the production of alkaloids of the Ergot -.- jruppe known (Dutch patents · ') 415 127 and 6 405 662; U.S. Patents i 117 917 and 3 110 651; Japanese patent specification IQ 250/64), according to which process it is only it is possible to obtain mixtures of such alkaloids.

It has now surprisingly been found that • nan in a process for microbiological production of ergocryptine by submerged aerobic cultivation of Claviceps purpurea into assimilable carbon and liquid nutrient media that release nitrogen sources and mineral salts in the case of these customary Only the ergocryptine alkaloid receives temperature and pH values in good yields and that afterwards In addition to the considerable advantages during the extraction and isolation processes, the alkaloid in of low purity and good yields is obtained -venn one as Claviceps purpurea Claviceps purpurea <\ TCC 20019 uses.

The new strain Claviceps purpurea ATCC 20019 A 'was isolated from Ergot sclerotia, which were obtained from a itogues had been collected; he shows the following norphological properties:

Microscopic appearance

On the usual solid culture media the vlycel shows hyphae, which when young are long and thin ind and little noticeable and well separated septa '. As you get older, the hyphae become larger; The septa become very noticeable, and the various parts which make up the hyphae swell. With boys Hyphae, the cell length is about 24 to 30 μ, and the diameter is 3 to 4 μ. Old cells are 10 to μ long and 4 to 5 μ in diameter. Sometimes the cells separate from the old ones Hyphae form, map and surround their end parts, creating arthrospores, their dimensions are not very different from those of the cells described above.

Form more or less quickly and more or less frequently, depending on the culture media used at the tip of numerous hyphae conidia, which in some culture media are rounded and in others are distinctly oval; the size of the former is about 5 to 7 µ (diameter) and that of the other up to 8 to 3 to 5 μ.

Macroscopic appearance

The macroscopic properties have been observed on the strip indicated in Table media, 8, 16 or 24Tage were incubated at 28 ⁰ C for.

TS medium:

Abundant growth; velvety appearance and white color that turns purple with age; that Aerial mycelium is plentiful. The back of the colony is colorless to purple. It won't be soluble Pigments formed. Abundant conidia.

Agar potato glucose:

Discrete growth, abundant aerial mycelium, white, often clustered like Coremia. The back of the Culture is unbranded. Soluble pigments are not present. Numerous conidia.

T36 medium:

Good growth with large wrinkles, moderate whitish aerial mycelium. The back is colorless; There are no soluble pigments. Numerous conidia.

T 31 medium:

Folded, light, whitish, moderate growth. No aerial mycelium present. No soluble pigments present. Sparse conidia.

T25S medium:

Sparse growth, irregular, in more or less dome-like colonies. Moderate aerial mycelium with a velvety appearance and pink color. Violet back. No soluble pigments present. Abundant conidia.

S. P. medium:

Good growth, evenly distributed, with a velvety appearance and whitish color. back colorless. No soluble pigments present. No conidia present.

T22 medium:

Good growth, uneven, whitish, with a colorless reverse. No soluble pigments and no conidia present.

TS table T 36 T 31 potato
glucose S. P. T 22 links 100 g
10 g T 25 p 200 g 300 g 20 g 300 g 25 g
100 g vlais source fluid 300 g Sucrose ilucose \ sparagine vlehlhydrolysatc

Table (continued)

links

TS T 25 p 0.1g 0.1g 15g 125 mg 0.5 g 0.5 g 0.3 g 0.5 g 7 mg 7 mg 6 mg 6 mg 20 g 20 g ad 11 ad U ad pH 5.2 ad pH 5.2

T 36 T 31 10 g 2g 60 mg 10 g 500 ml 80 mg 0.35 g 0.35 g 4 mg 3 mg 20 g 20 g ad 11 ad 11 ad pH 5.2

Potato glucose

S. P.

T 22

Cottonseed

Peptones

Yeast extract

citric acid

Potato infusion ( ¹ y ...
Chopped infusion ( ² ) ..

KCl

KH ₂ PO ₄

MgSO ₄ -7H ₂ O

FeSO ₄ -7H ₂ O

ZnSO ₄ -7.H ₂ O

Agar

Distilled water ..

tap water

aqueous ammonia

NaOH.

10 g
10 g

500 ml

20 g
ad 11

0.5 g
0.5 g
7 mg
6 mg
20 g
1000 ml

0.5 g 0.3 g

20 g 1000 ml

5.5

( ¹ ) 200 g of potatoes are boiled in 500 ml of tap water for 45 minutes. The whole is filtered through cheesecloth and the filtrate (infused) is brought to volume.

( ² ) 200 g of chopped grain (husks) are boiled for 45 minutes in 5 l of tap water, then filtered and brought to volume.

The invention used according to the microorganism is, during a period of 8 grown in particular in a liquid culture medium under aerobic conditions in submerged culture at a temperature of 20-30 ⁰ C, preferably 24 ° C to 16 days. The pH can vary depending on the fermentation media used. 4 to 6.5 fluctuate. Glucose, sucrose, mannitol, sorbitol, glycerine, citric acid and succinic acid can be used as raw materials for carbon. The raw material for nitrogen can be ammonia, asparagine, peptone, casein hydrolyzate and ammonium salts such as sulfate and chloride and other substances which are commonly used. As mineral salts, various salts are suitably used for the preparation of the alkaloid depending on the medium used, and chlorides, phosphates, sulfates and magnesium, iron, zinc, manganese and potassium can be used.

The strain Claviceps purpurea ATCC 20019 can be stored by freeze-drying, using as A liquid is used that consists of three parts of a 60% sucrose solution and suspending agent consists of two parts of milk. It can also be retransmitted on T36 medium. Fermentation can take place in Erlenmeyer flasks and in laboratory or industrial fermenters of various capacities.

The amount of alkaloid present in the nutrient broth can be determined qualitatively by paper chromatography can be determined in comparison with an ergocryptine standard and quantitatively by spectrophotometric Proceedings.

The following examples illustrate the invention:

example 1

In an 10-1 fermenter 61 TG medium are sterilized by heating at 30 minutes 12O ⁰ C. This medium has the following composition:

Glucose 100 g

Citric acid 10 g

Yeast extract 0.1g

KH ₂ PO ₄ 0.5 g

MgSO ₄ -7H _a O 0.3g

FeSO ₄ -7H ₂ O 7 mg

ZnSO ₄ • 7H ₂ O 6 mg

Distilled water to 1000 ml

pH 5.2, with

ammonia

Then 50 ml of a suspension of conidia and mycelium obtained by suspending five cultures of the strain Claviceps purpurea ATCC 20019 in a test tube in T36 medium, inoculated in water.

The culture is grown for 4 days at 24 ^° C., with aeration at a mixed flow of 41 per minute and shaking with a rotating shaker with six blades at a speed of 300 rpm. The cultures thus obtained are used for inoculation with a ratio of 10 ° / ₀ of 6 1 T 25 medium which had been produced and fermented in another 10 1 fermenter. This is then kept at 24 ^° C. with aeration corresponding to an air supply of 61 per minute and with a shaking stirrer, which is provided with six stirring blades, at a speed of 350 rpm. After 10 days of cultivation the culture contains

llOOy / ml ergocryptine. . .

In addition, the medium 668 was used instead of the T 25 medium, which has the following composition:

5 6

Sucrose 60 g. After 14 days of cultivation, the culture contains

Glycerin '. ... 60 ml 1500 y / ml ergocryptine. .

Glucose 80 g · _. . ,.

Yeast extract. ... 0.1g. . example

Citric acid 15 g 5 The procedure is as in Example 1, but with

KCl, 0.125 g from five cultures on S.F. medium in sample tubes

KH ₂ PO ₄ ... 0.5 g was assumed.

MgSO ₄ · 7H ₂ O 0.5 g 1300 μg / ml ergocryptine are obtained.

FeSO ₄ -7H ₂ O,. 7 mg

ZnSO ₄ -7H ₂ O 6 mg io Example 3

Distilled H ₂ O to 1000 ml

pH 5.2 with It is worked as described in Example 1,

Ammonia, however, from five cultures on T 22 medium in

Sterilization 20 minutes trial tubes has run out.

long at 10O ⁰ C 15 1250 μg / ml ergocryptin are obtained.

Claims (1)

Claim: Process for the microbiological production of ergocryptine by submerged aerobic cultivation from Claviceps purpurea into assimilable carbon and nitrogen sources and mineral salts containing liquid nutrient media at temperature and pH values customary for this purpose, d a d u r ch characterized in that one uses Claviceps purpurea ATCC 20019 as Claviceps purpurea.