CS242138B1 - A new industrial strain of Claviceps purpurea (Pr.) TUL. CCM P-755 - Google Patents

Abstract

The invention relates to the fields of microbiology, mycology and pharmaceutical industry. The purpose of the invention is to optimize the raw material base for the production of ergotamine in particular and for the production of semi-synthetic alkaloids. The raw material base is ergot sclerotia containing ergot alkaloids. The stated purpose is achieved by growing and using sclerotia of a new industrial strain of the microorganism (pyrenomycetes) Claviceps purpurea (Pr.) TUL. CCM P-755, which contain ergot alkaloids in an amount of 0.51 to 1.73% of the dry biomass (average content 0.74 #), in the relevant production. In sclerotia, at least 90.5 1° of the gum alkaloids is formed by ergotamine and at most 1.6% of the sum of alkaloids is formed by ergostin. The new industrial strain is also characterized by its ability to provide very high yields per hectare, ranging from 516 to 954 kg, when growing parasitically on rye using recent agricultural techniques.

Description

(54) New industrial strain of Claviceps purpurea (Pr.) TUL. CCM P-755

The invention relates to the fields of microbiology, mycology and the pharmaceutical industry.

The purpose of the invention is to optimize the raw material base for the production of ergotamine in particular and for the production of semi-synthetic alkaloids. Sclerotia ergot containing ergot alkaloids serves as a raw material base.

This purpose is achieved by cultivating and applying sclerotia of a new industrial strain of Claviceps purpurea (Pr.) TUL microorganism (pyrenomycetes).

CCM P-755, which contain ergot alkaloids in an amount of 0.51 to 1.73% biomass dry matter (average content 0.74 #), for the respective productions. In sclerotia, at least 90.5 1 ° of the alkaloid gum is ergotamine and at most 1.6% of the sum of the alkaloids is ergostin. The new industrial strain is further characterized by the ability to provide very high hectare yields ranging from 516 to 954 kg during parasitic growth on lived using recent agrotechnics.

242 138 Int. ⁴

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242 138

The present invention relates to a novel industrial strain of the microorganism Claviceps purpurea (Pr.) TUL, characterized by a very high ergotamine content, a minimum representation of accompanying and undesirable alkaloids in ergot sclerotia, and very high yields of ergot in field cultivation on rye.

Brgotamine is a ergot alkaloid with significant physiological and medicinal effects. It can also be used as a starting material for the preparation of semi-synthetic alkaloid derivatives with an extended spectrum of effects. The structure of ergotamine was first described in 1918 by A. Stoli (Swiss Patent No. 79879) ·

Brgotamine is currently used in therapeutic practice mainly in the hydrogenated state (DK-ergotamine) alone or in dosage forms mixed with other active substances. Ergotamine can be obtained from sclerotia of a fungus cultivated parasitically in a field culture on rye (Békésy von N., in Genetics of Industrial Microorganisms, 375-392, Academia, Prague, 1973) Breuel K. et al., Planta Med. 1982} Grzybowska T. et al., Herba Pol. 26, 95 (1980)} Kosir B. et al., Parm.

32, 21 (1981)} Kybal J., Stary P., Planta Med. 6, 406, 1958. Paadotra V.R. et al., Cultiv. Util. Copper. Aromat. Plants 97, 1977} Ramstad E., Gjerstad G., J. amer. Ass. 44, 741, 1955} Salgina Al et al., Microbiologia 39, 67, 1970) or by fermentation procedures (Amici AM et al., Experientia 22, 415, 1966} Appl. Microbiol. 15, 597, 1967} Appl. Microbiol. 18, 464, 1969} Arcamone P. et al., Can J. Microbiol., 16, 923, 1970} Stoli A. et al., Helv Chim Acta 37, 1815, 1954} Šalagina AI et al., Microbiology 34 »901, 1965} Tonolo A, Nature 209, 1134» 19665 (US Patent 104 6U5 Swedish Patents 310 477 and 354 673} French Patents 1 108 120 and 1 404 533 and 1 576 678; US Patent 2 809 920). For the production of ergotamine in Czechoslovakia was earlier

I ² 242 138 used sclerotia of the strain PLA 4 (Hroneš J., Kybal J., Z. Pflanzenzttcht. 48, 73, 1922j Strnadova K., Planta Med. 12, 521, 1964) and more recently sclerotia of the strain FBA 4 (Strnadova Κ. , Kybal J., Folia Microbiol. 19, 272 (1974).

The FBA 4 strain is characterized by the production of sclerotia in rye cultivation which contain an average of 0.531% of the sum of alkaloids, based on the bioraase dry matter, of which the desired ergotamine is an average of 87.1%. The accompanying alkaloids ergometrine (6.9%), ergotoxin group alkaloids (1.7%), piano alkaloids (1.9%) and especially unwanted ergostin (2.4%) are also represented. growing on rye 249 kg.

The main disadvantage of FBA 4 strain is the relatively low ergotaraine content in sclerotia compared to CCM F-755 strain. Another disadvantage of the FBA 4 strain is the higher content of accompanying alkaloids and in particular the relatively high proportion of undesired ergostin alkaloid. In extraction and isolation procedures to obtain pure ergotamine, ergostin is very difficult to separate. For therapeutic practice, ergotaraine dosage forms with an ergostin content of no more than 0.5% of the active ingredient are now required (British Pharmacopoeia 1980, Vol. I, p. 157, Her Majestys Stationery Office London,

1980). In order to achieve this quality of the substance, repeated and demanding isolation and purification operations are required in the manufacture of ergot FBA 4. Another disadvantage of the FBA 4 strain is the lower yields per hectare. This property, together with the lower ergotamine content, required the need for considerable arable land in field cultivation of FBA 4.

All other strains for field production described in the above references are characterized by a significantly higher proportion of accompanying and undesirable alkaloids in total, lower ergotamine content, lower total alkaloid content and lower yields per hectare compared to the new CCM F-755 strain.

For the ergotamine fermentation production described in the above references, a special process equipment is necessary. The strains and procedures described in these references produce relatively low amounts of ergotamine, often with a very high proportion of accompanying and undesirable alkaloids.

These shortcomings of ergot strains for field and fermentation production of ergotamine are eliminated to a great extent by the invention, which is a new strain of microorganism (pyrenomycetes) Claviceps purpurea (Fr.) TUL., Deposited in the Czechoslovak Collection of Microorganisms, University of JE Purkyně, Brno. Defenders of Peace 10, under the designation GGM P-755. ^{242 138}

The main advantage of the new strain in the cultivation on rye is very high production of alkaloid sum in sclerotia, which reaches an average of 0.74% biomass dry matter, high ergotamine in the sum of alkaloids (92.1%) and low content of accompanying alkaloids (7.9% sum) ), of which especially unwanted ergostin (1.3% of the sum). Another advantage of the new strain is the high ability to infect host plants, which allows for the recently used optimal agrotechnics of ergot cultivation to achieve a hectare yield of 516 - 954 (average 713) kg ergot sclerotia. This characteristic of the strain makes it possible to obtain sclerotia in field cultivation at a lower cost than in the PBA 4 strain used so far. The use of sclerotia CCM P-755 in the production of ergotamine-type products makes the extraction and isolation procedures more efficient. operations and laboriousness. The properties of the strain also make it possible to use its sclerotia as a raw material for the manufacture of semi-synthetic ergot alkaloids.

Description of properties of the new strain Claviceps purpurea (Pr.) TUL.

CCM P-755

Upon saprophytic growth on wort agar-reinforced wort broth, the fungus forms colonies from a typical sphcelium air mycelium. The mycelium is richly branched _with . side hyphae terminated by conidiogenic cells. Conidia have an average size of 7.3 x 3 »9 micrometers · Young mycelium is almost white, older cultures are strongly violet pigmented. During parasitic growth on rye, the fungus forms dark, brown-violet pigmented sclerotia. On dipsloid fertile rye, the average weight of sclerotia is 0.296 g. The sclerotia of this strain, grown on rye, contain ergot alkaloids in an amount of 0.51 to 1.73% dry matter biomass, with an average content of 0.74% · Minimum 90.5% sum alkaloids consist of ergotamine (an average of 92.1% of the sum). The remainder consists of the accompanying alkaloids ergometrine (on average 6.3% of the sum), ergostine (on average 1.3% of the sum), clavin alkaloids (on average 0.3% of the sum) and traces of ergotoxin alkaloids.

The new production strain was bred from the originally grass stem of ergot, obtained from the grass Melica uniflora Retz .. in September 1978 on the Plešivecká plain. Grass sclerotia contained on average 0.41% ergot alkaloids consisting of 62% ergotamine,

242 138% ergosin, 9% ergometrin, 4% ergostin, 1.5% ergotoxin alkaloids and 0.5% clavin alkaloids · Selected sclerotium was transferred to saprophytic culture and conidia were inoculated on rye. Sclerotia was obtained containing 0.39% alkaloids consisting of 74% ergotamine, 12% ergosin, 7.5% ergometrine, 3.5% ergostin, 2% ergotoxin alkaloids and 1% clavin alkaloids. 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 of výkonu2-1

J.m.s. for 300 seconds when 0.32% conidia survived. After irradiation, the conidia suspension was isolated in a raonoconidial manner. The UV mutagenesis and isolation procedure was repeated three times. Thereafter, cultures from individual monoconjugate isolates were seeded on rye and the resulting sclerotia was individually analyzed. From sclerotia obtained, sclerotium with an alkaloid content of 0.52% was selected for further breeding, of which 76% were ergotamine, 16% ergometrine, 5% ergosin, 1.3% ergostin, the rest were ergo-toxin and clavin alkaloids. The conidia suspension cultured from this sclerotia was irradiated with UV-irradiation and the irradiation procedure combined with monoconidium isolation was repeated five times. alkaloids 0.81%, of which 88% were ergotamine, 6% ergosin,% ergometrine, 1% ergostin, the rest were ergotoxin and clavin alkaloids. The suspension of conidia cultured from this sclerotia was irradiated with UV-radiation and the irradiation procedure combined with monoconjugate isolation was repeated twice. Monoconidium isolate cultures were inoculated on rye. On the basis of the analysis of sclerotia obtained from these isolates and infectivity of cultures, a strain designated CCM P-755, which has the above-mentioned properties, was selected. The stability of the strain properties was confirmed by repeated cultivation on lived in 3 growing seasons. This strain is the subject of the invention.

The method of cultivation of a new strain for obtaining a starting material for the isolation of ergotamine and subsequent production of final products as well as for obtaining a starting material for extracting the sum of alkaloids and subsequent production of semi-synthetic preparations is exemplified in this application. cultivation and use in production.

Application example: 242 138

An aqueous conidia suspension is prepared from the required volume of the saprophytic culture of CCM F-755 strain. The ears of rye are inoculated with an appropriately undiluted conidia suspension in a sweeping phase on an area of one hectare. At least 5 · 10 ¹¹ conidia should be used to inoculate this area. After 35-60 days of growth on male sterile veins, the sclerotia of the ergot is grown. From one hectare of rye, 825 kg of dry ergot sclerotia were obtained, containing 0.83% of the sum of alkaloids, of which 91.6% were ergotamine, 6.5% ergometrine, 1.1% ergostine, 0.7% clavin alkaloids and 0%. , 1% ergotoxin alkaloids. The obtained drug was extracted, the extracts were used in part for the production of ergotamine-type substances, in part for the production of semi-synthetic ergot alkaloids.

Claims (1)

BACKGROUND OF THE INVENTION A new industrial strain of Claviceps purpurea (Fr.) TUL.CCM F-755, characterized in that it produces sclerotia in a minimum of 516 kg / ha, with a sum of alkaloids of at least 0.51% of biomass dry matter, in field rye culture with an ergotamine content of not less than 90,5% of the sum of alkaloids and with an ergostin content of not more than 1,6% of the sum of alkaloids.