CS239883B1 - A new industrial strain of microorganisms of the species Claviceps purpurea (Fr) TUL. CCM F-745 - Google Patents

Abstract

The solution concerns the fields of microbiology and pharmaceutical industry. The purpose is to improve the raw material base for the production of argocornine in particular, as well as other alkaloids of the ergotoxine group and for the production of semi-synthetic alkaloids. The raw material base is ergot sclerotia containing ergot alkaloids. The stated purpose is achieved by using sclerotia of a new industrial strain of the microorganism Claviceps purpurea (Fr.) TUL. CCM F-745, which contains ergot alkaloids in an amount of 0.30 to 0.89% (average content 0.48 ;'.') in the relevant productions. In sclerotia, at least 70% of the alkaloid sum is formed by ergocornine and at least 12% of the alkaloid sum is formed by alpha-ergocryptine and beta-ergocryptine with an average mutual ratio of 1.8:1. The new industrial strain is further characterized by the ability to provide very high yields per hectare in the range of 692 to 1,118 kg when grown parasitically on rye using recent agrotechnology.

Description

(54) A new industrial strain of microorganisms of the species

Claviceps purpurea TUL. CCM F-745

The solution concerns microbiology and pharmaceutical industry. The purpose is to improve the raw material base for the production of mainly argocornin, further alkaloids of the ergotoxin group and for the production of semi-synthetic alkaloids. The sclerotia of ergot containing ergot alkaloids serves as a raw material base. This is accomplished by the use of sclerotia of a novel industrial strain of Claviceps purpurea (Fr.) TUL. CCM F-745, which contains ergot alkaloids in an amount of 0.30 to 0.89% (average content 0.48;) for the respective products. In sclerotia, at least 70% of the sum of alkaloids is ergocornine and at least 12% of the sum of alkaloids is alpha-ergocryptine and beta-ergocryptine with an average ratio of 1.8: 1. The new industrial strain is further characterized by the ability to Very high hectare yields in the range of 692 to 1,118 kg.

239 883

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The present invention relates to a novel industrial strain of the microorganism Claviceps purpurea (Fr.) TUL. Characterized by very high yields of ergotornin-containing ergot sclerotia and with the accompanying desired alkaloids alpha-ergocryptine and beta-ergocryptine in field rye cultivation. in a preferred ratio.

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 ergocornin and alpha-ergocryptin was first described by Stoli and Hofinann (Helv. Chim.

1570, 1943), the structure of beta-ergocryptine was first described by Schlientz et al. (Experiments 23 »991, 1967) ·

Ergocornin is currently used in therapeutic formulations in admixture with alpha and beta-ergocryptine and ergocristin mostly under the trade name DH-ergotoxin. Said alkaloids are contained in the dosage forms in a precisely defined ratio. Claviceps purpurea (Fr.) TUL has been used to produce ergocornin on an industrial scale. CCM F-508 (Strnadova K., Eybal J., Author's Certificate No. 176 803, 1976).

Further, a method of fermentation preparation of ergot alkaloids with controlled ratio of ergocornin, alpha-ergocryptin and beta-ergocryptin formation (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, and betaergocryptine under saprophytic conditions (Strnadova K., Kybal J., Svoboda E., Spacil J., 1982, 1982).

The CCM F-508 strain in soybean cultivation is characterized by sclerotia production with an average alkaloid sum of $ 0.424, of which

239 88!

Ergocornin, alpha and beta-ergocryptine make up 70-80% at an approximate ratio of 8: 5.5 ί 1 · Average hectare yields of sclerotia of CCM F-508 are around 120 kg. In the manufacture of ergot group ergotinic alkaloid dosage forms from sclerotia of this strain, the limiting factor is low alkaloid sum content, unfavorable alpha-beta-ergocryptine ratio, high content of unwanted accompanying alkaloids and high cost of ergot due to low hectare yields. The fermentation process for the production of ergocornin, alpha and beta-ergocryptin according to the author's certificate No. 202 613 (1980) and using the strain CCM F-725 is not yet economically accessible.

In addition to CCM F-508 strains, Claviceps purpurea (Fr.) TUL strains are currently used for the production of alpha and beta-ergocryptine. CCM F-672 (Malinka Z., Harazim P., Spacil J., Author Certificate No. 222 150, 1982), CCM F-721 (Malinka Z., Harazim P., Spacil J., Author Certificate No. 225 272) , 1983). The CCM F-672 strain is characterized by the production of sclerotia containing very pure alpha-ergocryptine in rye culture. All other ingredients need to be added in the manufacture of ergot group ergotinic alkaloid dosage forms from this strain. The use of this strain for the manufacture of semisynthetic medicinal preparations is more economical. The CCM F-721 strain is characterized by high hectare yields of ergot sclerotia with a high content of alpha and beta-ergocryptine and low content of accompanying alkaloids. Ergocornin must be added in the manufacture of ergot-alkali ergot groups from this strain.

These deficiencies of the CCM F-508 strain and the fermentation process for the production of ergocornin are largely overcome by the invention, which is a new strain of Claviceps purpurea (Fr.) TUL., Deposited in the Czechoslovak Collection of Microorganisms, University of J. E. Purkyně, Brno. Defenders of Peace 10, designated CCM F-745. This strain is the subject of the 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 0.48% biomass dry matter, with the sum of alkaloids being the predominant constituent of ergocornins contained in proportion to the desired accompanying alkaloids alpha-ergocryptin The ratio of alpha-ergocryptine to betahergocryptine is preferably 1.8: 1.

The advantage is the low formation of undesirable accompanying alkaloids (ergocornam, ergocryptams, ergometrine), which make up an average of 12% of the sum of the alkaloids. Another advantage is the very high hectare yields of sclerotia, which, with the use of recent agrotechnics, grow ergot with an average of 847 kg. These characteristics of the strain make it possible to obtain sclerotia in field cultivation at a lower purchase price than the cultivation of the previously used GCM F-508 strain. These sclerotia are a very preferred raw material for the pharmaceutical industry due to the content and spectrum of alkaloids. Especially when used concomitantly with CCM F-721 strain in the manufacture of ergotoxin-type preparations, the strain provides an effective source of ergocornin and the ratio of accompanying desirable alpha and beta-ergocryptine alkaloids greatly simplifies work operations by accurately adjusting the desired ratio of individual components. The benefits of using this strain translate primarily into streamlining extraction and isolation procedures for the production of ergotoxin-type products. The properties of the strain also make it possible to use its sclerotia as a raw material for the production of semisynthetic preparations.

Description of new strain properties:

Upon saprophytic growth on wort agar-reinforced wort broth, the fungus forms colonies of typical Sphelia-like air mycelium. Mycelium. it is relatively richly branched and the lateral hyphae are terminated by conidiogenic cells. Conidia have an average size of 6.6 x 3.5 microns. 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 0.113 g. The sclerotia of this strain, grown on rye, contain ergot alkaloids in an amount of 0.30 to 0.89% dry matter biomass, with an average content of 0.48 S. At least 70% of the sum of alkaloids is ergocornine, at least 12% of the sum of alkaloids is alpha-ergocryptine and beta-ergocryptine in the ratio 0.6 - 2.3: 1 (average 1.8: 1). The ratio of ergocornin to alpha © and beta-ergocryptin is 2.3 7 7.8 ί 1 (average 4.2: 1). Other accompanying alkaloids are ergocornam, ergocryptams and ergometrine; their content is not more than 18% of the sum of the alkaloids (average 12%).

The new production strain was bred from the original grass strain

- 4 239 883 ergot obtained from 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% were ergocornam, ergocryptams and ergometrine. Sclerotia containing an average of 0.38% alkaloids was obtained after seeding this grass strain on rye. Sclerotium containing 0.38% of the sum of alkaloids was selected for further breeding, of which 73 So were ergocornin, alpha-ergocryptin and betaergocryptin in a ratio of 5.3: 3.6: 1; 12% were ergosin, 15 95 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

-2 -1 UV radiation source with output 15 J.o. s for 280 seconds when 0.15% conidia survived. After irradiation, the conidia suspension was monoconidally isolated. The entire UV mutagenesis procedure 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 0.43 was selected for further breeding, of which 79% were ergocornin, alpha and beta-ergocryptin in a ratio of 4.9:

2.3% and 21% 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 four times. Monoconidium isolate cultures were inoculated on rye. Based on an analysis of sclerotia derived from individual isolates and the rye culture infectivity, a strain characterized by high ergocornin production, the formation of desirable water-containing alpha and beta-ergocryptine alkaloids in a preferred ratio, low content of undesirable accompanying alkaloids and long-term stability of these properties was selected. This strain is characterized by very high yields of sclerotia when grown on a host plant using the recently used ergot cultivation technique. The stability of these properties was confirmed by repeated cultivation on rye in three growing seasons. This strain is the subject of the invention.

A process for cultivating a new strain to obtain a starting material for the isolation and extraction of ergocornin, alpha-ergocryptin and beta-ergocryptin and for the subsequent production of the final products from these alkaloids

No. 5,239,883 for obtaining a starting material for the sum of alkaloids and subsequent production of semi-synthetic preparations is exemplified in the use example, without limiting in any way the properties of the new strain, the method for its cultivation and use.

Example of use:

An aqueous conidia suspension is prepared from the required volume of the saprophytic culture of the strain. An appropriately undiluted conidia suspension is used to inoculate the ears of rye in the sweeping phase on an area of one hectare using a seeding machine. A minimum of 5 · 10 ^ conidia was used to inoculate this area. After 35-60 days of growth on rye with chemically induced male sterility, harvested sclerotia of ergot is performed. From one hectare of rye, 927 kg of dry sclerotia of this strain were obtained, containing 0.48% of the sum of the alkaloids, of which ergocornine, alpha-ergocryptine and beta-ergocryptine were 7.6: 1.8: 1 relative to each other. The drug obtained was extracted, the extracts used in part for the production of ergotoxin, in part used for the production of semi-synthetic preparations.

Claims (1)

SUBJECT OF THE INVENTION 239 883 Novel industrial strain of Claviceps purpurea (Fr.) TUL. CCM F-745, wji-vetrtfj / ct 'in field sclerotia sieve at a minimum of 692 kg / ha, with an alkaloid content of at least 0,30% based on biomass dry matter, an ergocornin content of at least 70% of the alkaloids, s an ergocornin to alpha and beta-ergocryptine ratio of at least 2.3: 1 and an alpha-ergocryptine to beta-ergocryptine ratio of at least 0.6 ί 1.