CS239884B1 - A new industrial strain of the microorganism Claviceps purpurea (Fr.) TUL. CCM F-734 - Google Patents

Abstract

The purpose of the solution is to improve the raw material base for the production of preparations with a therapeutic effect, consisting of classical ergot alkaloids of the ergotoxine group and for the production of semi-synthetic therapeutic preparations. The raw material base is the sclerotia of this strain, containing ergot alkaloids. The stated effect is achieved by using sclerotia of a new industrial strain of the microorganism Claviceps purpurea (Fr.) TUL. CCM F-734, which contain ergot alkaloids in an amount of 0.32 to 0.93% (industrial content 0.51%) in the relevant productions. In sclerotia, at least 91.5% of the sum of alkaloids is formed by ergocornine, alpha-ergocryptine and beta-ergocryptine with an average mutual ratio of 3.3 : 2.1 : 1. The new industrial strain is further characterized by its ability to provide very high sclerotia yields per hectare in the range of 840 to 1270 kg when parasitically growing on rye using recent agricultural techniques. The solution concerns the fields of microbiology and the pharmaceutical industry.

Description

The present invention relates to a novel industrial strain Claviceps purpurea (Fr.) TUL. Characterized by field ergot sclerotia with high ergocornin, alpha-ergocryptin and beta-ergocryptin content and the ability to achieve very high ergot yields.

Ergocornin, alpha-ergocryptine and beta-ergocryptine are alkaloids of the ergotoxin group and are important therapeutic substances. They can also be used as starting materials for the preparation of semisynthetic ergot alkaloid derivatives with an extended spectrum of effects. The structure of ergot alkaloids alpha-ergocryptine and ergocornine was first described by Stoli and Hofmann (Helv. Chim. Acta 26, 1570, 1943), the structure of beta-ergocryptine was first described by Schlientz sp. (Experientia 23, 991 (1967)).

Presently, the pharmaceutical forms of ergot alkinoids, ergocornine, alpha-ergocryptine, beta-ergocryptine and ergocristin are used in the form of a mixture of ergot alkaloids mostly under the trade name DH-ergotoxin. These dosage forms contain ergocornin, alpha-ergocryptin, beta-ergocryptin and ergocristin in a well-defined ratio of 3: 2: 1: 3. Claviceps purpurea strains (Fr. ) TUL. CCM F-508 (Strnadova K., Kybal J., Author's Certificate No. 176 803, 1976), CCM F-672 (Malinka Z., Harazim'P., Spáčil J., Author's Certificate No. 222 150, 1982 ), CCM F-721 (Malinka Z., Harazim P., Spacil J., Authorial Certificate No. 225 272, 1983). Furthermore, a method of fermentation preparation of ergot alkaloids with controlled ratio of ergocornin, alpha-ergocryptine and beta-ergocryptine production is developed (Kybal J., Malinka Z., Harazim P., Stuchlik <J., Spacil J., Svoboda E., author's certificate no. 202, 613 (1980)) and bred Claviceps purpurea (Fr.) TUL. CCM F-725 producing a mixture of ergocornin, alpha-ergocryptin and beta-ergocryptin under saproMytic conditions

239 884

-3 (Strnadova K., Kybal J., Svoboda E., Spacil J., Author's Certificate No. 222 391, 1982).

The CCM F-508 strain is characterized by the production of sclerotia in anus cultivation which contain an average of 0.424% of the sum of alkaloids, of which ergocornins and ergocryptins make up 70 to 80%. The approximate ratio of ergocornin, alpha and beta-ergocryptin is 8: 5.5: 1.

In the manufacture of ergotoxin type ergot alkaloid dosage forms from sclerotia of this strain, the disadvantage is low content of desirable alkaloids, high amount of undesirable alkaloids (20-30% of the sum of alkaloids) and especially very low content of beta-ergocryptin. Another disadvantage is the low yield per hectare and the resulting high cost of ergot.

The CCM F-672 strain is characterized by the production of very pure alpha-ergocryptin during cultivation on rye. In the production of ergotoxin group ergot alkaloid dosage forms from this strain, it is necessary to add all the other ingredients and laboriously adjust the desired ratio. Because of the high purity of alpha-ergocryptine in the ecclerosis of this strain, it is economically more advantageous to use it for the manufacture of semisynthetic alpha-ergocryptine-based medicinal preparations.

The CCM F-721 strain is characterized by the formation of ergot with a high content of alpha-ergocryptine and beta-ergocryptine with an average ratio of 1.15 to 1 and a maximum content of unwanted accompanying alkaloids of 10%. To produce ergot alkaloid dosage forms of the ergotoxin group from this strain, ergocornin has to be added and it is difficult to adjust the exact ratio of ingredients required.

The fermentation process for the production of ergocornin, alpha and beta-ergocryptin according to the author's certificate δ · 202 613 of 1980 and using strain CCM F-725 is not yet economically accessible.

The above-mentioned shortcomings of the abovementioned strains of ergot for field production and fermentation process for the production of alkaloids of the ergotoxin group are largely eliminated by the invention, which is the new strain Claviceps purpurea (Fr.) TUL., Deposited in the Czechoslovak Collection of Microorganisms. Defenders of Peace 10, designated CCM F-734. This strain is an object of the present invention.

The main advantage of the new strain in the cultivation on rye is the high production of sum of alkaloids in sclerotia, which reaches an average of 0.51 # biomass dry matter, very low formation of undesirable accompanying alkaloids which make up 6% of sum of alkaloids on average and high production of desirable alkaloids ergocornine. , alpha-ergocryptin / and beta-ergocryptin (average 94% of the sum) in a very favorable ratio of average 3 »3 i 2,1: 1. Another advantage of the new strain is high / ability to infect the host plant, which allows the cultivation of ergot achieve an average hectare yield of ergot sclerotia of 982 kg. These characteristics of the strain allow sclerotia, which are a very advantageous raw material for pharmaceutical production, to be obtained in field cultivation. Due to the high yields per hectare, the purchase price of this ergot is lower than the price of the strains used to date. The use of this raw material will make it possible to streamline extraction and isolation processes in the manufacture of ergotoxin-type products, in particular by reducing raw material consumption, reducing the number of technological operations and labor. The properties of the strain also make it possible to use its sclerotia for the production of semi-synthetic preparations.

Description of new strain properties:

In saprophytic growth on agar-reinforced wort, the fungus forms a colony of typical Sphelium air mycelium. The mycelium is relatively richly branched and the lateral hybrids are terminated by conidiogenic cells. Conidia abundantly have an average size of 7 x 3 »5 micrometers. Young mycelium is almost white, older cultures have medium pink-violet pigmentation. In parasitic growth on rye, the fungus forms a medium dark, brown-violet pigmented sclerotia. On diploid fertile rye, the average weight of sclerotia is 0.106 g. Sclerotia of this strain, grown on rye, contain ergot alkaloids in an amount of 0.32 to 0.93% dry matter biomass with an average content of 0.51 At least 91.5% of the sum of alkaloids is consisting of ergocornin, alphaergocxyptin and beta-ergocryptin (94% on average). The ratio of ergocornin to ergocryptins is 0.95-1.6: 1 (mean

3.3 (3.1), the ratio of alpha-ergocryptine to beta-ergocryptine is

1.6 - 2.7 ί 1 (average 2.1: 1). The accompanying alkaloids are ergometrine, ergocornam and ergocryptam;

- iS 239,884

The new stem was bred from the originally grass stem of the ergot obtained from the forest cane. Cane sclerotia contained an average of 0.34% ergot alkaloids, consisting of 64% ergocornin, alpha and beta-ergocryptine, 19% ergosin, the remaining 17% ergocornam, ergocryptams and ergometrine. Sclerotia containing 0.38 95 alkaloids was obtained after inoculation of this grass ergot on rye, of which 73% were ergocornin, alpha-ergocryptin and beta-ergocryptin

5.3 to 3.6: 1; 12% were ergosin, 15% were ergocornam, ergocryptams and> ergometrine. Selected sclerotium was transferred to a saprophytic culture and the conidia formed were irradiated with UV radiation. The conidia suspension was irradiated with a UV source with a horsepower of 15 µm for 280 seconds while 0.15% of the conidia survived. After irradiation, the suspension was monoconidized. The whole procedure of UV-mutagenesis and isolation was repeated three times. Thereafter, cultures from individual monoconjugate isolates were seeded on rye and the resulting sclerotia was individually analyzed. Of these sclerotia, sclerotium with an alkaloid content of 0.41 95 was selected, of which 93% were ergocornin, alpha and beta-ergocryptine in a ratio of 4.2: 2.6: 1795 were ergocornam, ergocryptams and ergometrine. The suspension of conidia cultured from this sclerotia was irradiated with UV light and the irradiation procedure combined with monoconjugate isolation was repeated twice. Monoconidium isolate cultures were inoculated on rye. Based on the analysis of sclerotia derived from these isolates and the infectivity of the cultures, a strain having a very good spectrum of alkaloids, a minimum of accompanying undesirable alkaloids and a long-term stability of these properties was selected. This strain is characterized by very high hectare yields of sclerotia when growing on a host plant using recently used agrotechnics. The stability of these properties was confirmed by repeated cultivation on rye in 4 growing seasons. This strain is the subject of the invention.

The method of cultivating a new strain for obtaining a starting material for the isolation of ergocornin, alpha-ergocryptine and beta-ergocryptine and then producing the final products from these alkaloids, as well as obtaining the starting material for extracting the sum of alkaloids and subsequently producing semisynthetic preparations is exemplified.

239 884 without limiting in any way the properties of the new strain, the way it is cultivated and used.

Example of use:

An aqueous conidia suspension is prepared from the required volume of the saprophytic culture of the strain. Using the inoculation machine, ears of rye are inoculated in the methane phase over an area of 1 ha with an appropriately diluted conidia suspension. A minimum of 11 is used to inoculate this area. 10 konidií. After 35-60 days of growth on rye with chemically induced male sterility, the erect sclerotia are harvested. From one hectare of rye, 1,066 kg of dry sclerotia of this strain were obtained, containing 0.51% of the sum of alkaloids, of which 94% consisted of ergocornin, alpha and beta-ergocryptin in a ratio of 3 »3 ϊ 2.1: 1. the drug was extracted, the extracts partially isolated on individual alkaloids and used for the production of ergotoxin, partly used for the production of semi-synthetic preparations.

Claims (1)

SUBJECT OF THE INVENTION 239 884 A new industrial strain of Claviceps purpurea /Fr./ TUL. CCM F-734, generating at least 840 kg / ha of sclerotia in a field crop, with a sum of at least 0,32% of dry matter biomass, containing ergo-cornine, alpha-ergo-blood and beta-ergo-blood least 91.5% of the sum of alkaloids and having an ergocornin to ergocryptine ratio of at least 0.95: 1 and an alpha-ergocriptine to beta * ergocryptine ratio of at least 1.6: 1.