CS276359B6 - The new industrial microorganism strain Claviceps purpurea (Fr.) TUL.CCM 8057 - Google Patents

Abstract

The purpose of the solution is to provide a suitable raw wine base for the production of ergot alkaloids of the ergotoxin group, in particular the gerocornine, or for the production of semi-synthetic lysergic acid derivatives. The sclerocia of ergot obtained by parasitic cultivation is the raw material for these production. This purpose is achieved by growing sclerotia of the new industrial strain Claviceps purpurea (Fr.) Tul. CCM 8057 on a suture of at least 960 kg / ha and using such sclerotia to produce substances of native or semi-synthetic ergot alkaloids. Sclerotia contains ergot alkaloids in the amount of 0.52% to 1.25% biomass dry matter, at least 93.5% of the alkaloid sum consists of a mixture of ergocornine, alpha-ergocryptineU and beta-ergocryptine in a ratio of 4.2: 1.6: 1.

Description

The invention relates to a new industrial strain of the microorganism Claviceps purpurea (Fr.) Tul. CCM 8057, characterized by very high hectare yields of ergot sclerotia with a high content of ergocornine, alpha-ergocryptine and beta-ergocryptine and a minimal proportion of undesired concomitant alkaloids during field cultivation on rye. ’

Ergokornin, alpha-ergocryptine and beta-ergocryptine are ergotoxin alkaloids with significant therapeutic effects. These alkaloids can be used in therapeutic practice alone or in mixtures. Ergokornin is used in current therapeutic practice in a mixture with alpha-ergocryptine, beta-ergocryptine and ergocristine in a precisely defined ratio under the trade name Secatoxin. Advantageously, the therapeutic properties of ergocornine alone or in a mixture with ergocristine can be exploited. A mixture of alkaloids can be used as a starting material for the production of semi-synthetic derivatives with a wide range of effects.

A number of strains of Claviceps purpurea (Fr.) Tul., Described in the technical and patent literature, are currently used for the production of ergocornine in a mixture with alpha-ergocryptine and beta-ergocryptine. The main disadvantages of these strains are the low content of desirable ergot alkaloids, the unfavorable ratio of ergocornine to alpha and beta-ergocryptine and the high content of undesired concomitant alkaloids.

These shortcomings were to some extent eliminated by the invention (Czechoslovak author's certificate No. 239 884), which is a new industrial strain CCM F-734, producing a ergot drug with a more favorable ratio of individual components in a mixture of alkaloids and a high yield per hectare of at least 840 kg / ha. The high yields are due to the high ability of this strain to infect the host plant. However, the disadvantage of this strain is the relatively low content of ergot alkaloids (at least 0.32%), causing higher costs for obtaining alkaloids from the ergot drug.

These shortcomings of ergot strains for the field production of ergotoxin alkaloids are largely eliminated by the invention, which is a new industrial strain of the microorganism Claviceps purpurea (Fr.) Tul., Deposited in Cs. collection of microorganisms J. E. Purkyně University, Brno, cl. Defenders of Peace 10, under the designation CCM 8057.

The main advantage of this strain is the high production of the sum of alkaloids in sclerotia when cultivated on rye, which reaches an average of 0.91% (minimum 0.52%) of biomass dry matter. Another advantage is the very low formation of undesired concomitant alkaloids, which make up at most 6.5% of the total alkaloids. Ergotoxin alkaloids make up at least 93.5% of the total sum of alkaloids in the relative average proportion of ergocornine: alpha-ergocryptine: beta ergocryptine = 4.2:: 1.6: 1. also for the isolation of ergocornine and ergocryptine separately. The properties of the new strain make it possible to streamline the extraction and isolation processes in the production of ergot alkaloids, in particular by reducing the consumption of raw materials, energy and labor. Sclerotia of this strain can also be used as a starting material for the production of semi-synthetic derivatives.

Description of the properties of the new strain Claviceps purpurea (Fr.) Tul. CCM 8057:

During saprophytic growth on agar-hardened wort soil, the fungus forms colonies of typical spherical air mycelium. The mycelium is richly branched, white and lateral hyphae are terminated by conidiogenic cells. Conidia have average dimensions of 7.4 x 3.8 micrometers. Young cultures are white, older ones are slightly pinkish-purple pigmented.

In parasitic cultivation on rye, the fungus forms dark, violet-pigmented sclerotia weighing between 0.016 and 0.105 g (average 0.046 g). Sclerotia of this strain contain ergot alkaloids in an amount of from 0.52% to 1.25% of the biomass dry matter, on average 0.91%. At least 93.5% of the sum of alkaloids consists of ergocornine, alpha-ergocryptine and beta-ergocryptine. The ratio of ergocornine to ergocryptines is 1.62: 1, the ratio of alpha-ergocryptine to beta-ergocryptin. tinu is 1.6: 1. The accompanying alkaloids are mainly ergometrine, ergocornam and clavine alkaloids.

CS 276359 B6. 2

The new production strain was bred from the original production strain CCM F - 734. 2-1 cultures and formed conidia were irradiated with UV radiation at 15 J ms for 185 s, when 0.50% of the conidia survived. After irradiation, the spore suspension was isolated monosporically. The procedure of UV-mutagenesis and monoconidium isolation was repeated three times under the same conditions. Cultures from individual monoconidium isolates were inoculated on rye. The resulting sclerotia was isolated individually. From the analyzed sclerotia, sclerotia with a total alkaloid content of 1.05%, ergocornine, alpha-ergocryptine and beta-ergocryptine in a ratio of 3.5: 1.1: 1 was selected for further breeding. culture. The prepared conidia suspension was subjected to UV mutagenesis and subsequent monoconidium isolation three times. Cultures of monoconidium isolates thus obtained were inoculated on rye. Based on the evaluation of individual mutated monoconidium isolates, ie determination of their infectivity and analysis of ergot alkaloids in a set of sclerotia, a strain designated CCM 8057 was selected, which is characterized by the preferred spectrum of alkaloids, minimum concomitant undesirable alkaloids and very high infectivity. The stability of these properties was confirmed by repeated cultivation on rye in two growing seasons in six different localities.

The method of culturing a new strain to obtain a starting material for the isolation of ergocornine, alpha-ergocryptine and beta-ergocriptine and the subsequent production of final products from these alkaloids is documented in the example of use, without limiting the properties of the new strain, its method of cultivation and use. production.

Example of use:

An aqueous suspension of conidia is prepared from the required volume of saprophytic culture of the CCM strain. Rice ears are inoculated with the conidia suspension using a seeding machine in the throwing phase, so that 5.10 live conidia are used on an area of 1 ha. After 35 to 60 days of growth on the zite, the grown sclerotia of the ergot is collected. 1 020 kg of dry sclerotia of ergot was obtained from one hectare of rye, containing 0.91% of the sum of alkaloids, of which 97.5% was ergocornine, alpha and beta-ergocryptine in a ratio of 4.2: 1.6: 1, the remainder it consisted of ergometrine, ergocornam and clavin alkaloids.

The drug obtained was extracted, the extracts were partly isolated into individual alkaloids and used for the production of an ergotoxin preparation, partly used for the production of a mixture of alkaloids with a high content of ergocornine and partly used for the production of semisynthetic derivatives.

Claims (1)

PA TENTAL CLAIMS ’ A new industrial strain of the microorganism Claviceps purpurea (Fr.) Tul. CCM 8057 producing ergocornine, alpha-ergocryptine and beta-ergocryptine.