CZ2023105A3 - An industrial production strain of microorganism Claviceps purpurea (Fr.) Tul., CCM9309 - Google Patents

Abstract

The solution concerns the industrial production strain of the microorganism Claviceps purpurea (Fr.) Tul., CCM9309, which forms sclerotia containing ergot alkaloids, especially α-ergocryptine, during parasitic growth on cereals, especially on rye or triticale and on their sterile analogues. The purpose of the invention was to obtain a producer of the starting raw material for the production of ergot alkaloids α-ergocriptine and their semi-synthetic derivatives with a significantly higher yield compared to the previously known strains.

Description

Industrial production strain of the microorganism Claviceps purpurea (Fr.) Tul., CCM9309

Field of technology

The invention generally relates to the field of biotechnology, microbiology, mycology and the pharmaceutical industry, specifically to the industrial production strain of the fungus Claviceps purpurea (Fr.) Tul., CCM9309, which forms sclerotia containing alkaloids during parasitic growth on rye and other cereals, especially a- ergocriptine.

Current state of the art

The structure of α-ergocryptine was first described by Stoll and Hofmann (Helv. Chim. Acta 26, 1570, 1943):

It is an important medicinal substance. In human medicine, α-ergocryptine is used in pharmaceutical forms either alone or in mixtures with other active substances. They can also be used as a starting material for the preparation of semi-synthetic derivatives of ergot alkaloids with a specific spectrum of effects, such as nicergoline, pergolide, cabergoline, bromocriptine.

α-ergocryptine can be obtained from the sclerotia of the fungus Claviceps purpurea (Fr.) Tul., cultivated parasitically in field culture on rye or other cereals. For the production of α-ergocryptin, the strain described in e.g. patent CZ 279533 and stored in the Czech Microorganism Collection (Masaryk University, Faculty of Science, Kamenice 5, 625 00 Brno) under number CCM8178 has been used in the Czech Republic. When cultivated on rye, the mentioned strain produced sclerotia with an average content of 1.3% of the sum of alkaloids, based on the biomass dry matter. An average of 90% of the total amount of alkaloids is made up of α-ergocryptine, the rest is made up of accompanying alkaloids ergometrine and valinamide. The overall low content of alkaloids and the relatively high content of accompanying alkaloids is the main disadvantage of this strain and other known strains.

Furthermore, from the sclerotia of the fungus Claviceps purpurea (Fr.) Tul., cultivated parasitically in field culture on rye or other cereals, ergolene and ergoline derivatives such as lysergic acid, nicergoline, pergolide, cabergoline and others can be prepared by standard procedures.

- 1 CZ 2023 - 105 A3

lysergic acid

The essence of the invention

The essence of the present invention is an industrial strain of the microorganism Claviceps purpurea (Fr.) Tul., deposited in the Czech Collection of Microorganisms (Masaryk University, Faculty of Science, Kamenice 5, 625 00 Brno) under accession number CCM9309 (hereinafter referred to as strain CCM9309), forming growth on cereals, especially on rye (Secale cereale) or triticale (hybrids of rye (Secale cere ale) and wheat (Triticum aestivum)) and their sterile analogues of sclerotia containing alkaloids, especially α-ergocryptine. The subject strain makes it possible to make the extraction and isolation procedures in the production of α-ergocryptine and their semi-synthetic derivatives more efficient by substantially increasing the yield of these procedures. The increased yield is due to the high content of α-ergocryptine in the sclerotia of the subject strain compared to the previously used strain CCM8178. The content of alkaloids in individual sclerotia of strain CCM9309 reaches values of 1.2% to 2.1% by weight with an average content of 1.6% of the sum of alkaloids in the biomass dry matter in the average spectrum of alkaloids: 98% α-ergocryptine, 0.6% galline and 1, 4% of other alkaloids. At the same time, there was an increase in drug yields per hectare for the strain in question, compared to other high-production strains of Claviceps purpurea (Fr.) Tul. (CCM8057, CCM8405 and CCM8178). These yields are on average 1100 kg/ha for the CCM9309 strain in question with the recently used agrotechnics of growing ergot.

Examples of implementation of the invention

Example 1

Preparation of strain CCM9309

The industrial strain of the microorganism Claviceps purpurea (Fr.) Tul., CCM9309 was bred from strain CCM8178. In 2018, the sclerotium of the aforementioned strain was transferred to a saprophytic culture and this was exposed to UV radiation with a wavelength of 250 to 270 nm at a dose of 40 J/m ² . The irradiated isolates were injected onto the swept ears of rye and the grown sclerotia were analyzed for the qualitative and quantitative content of ergot alkaloids. The most mature was selected from them

-2CZ 2023 - 105 A3 sclerotia with the maximum amount of alkaloids, which was transferred to a saprophytic culture, which was tested on the scale of a small plot experiment in 2019. From this plot, the largest and most mature sclerotia were harvested, which were again analyzed for qualitative and quantitative content ergot alkaloids. In the following year, 2020, the highest content sclerotium was passaged on rye on the scale of a semi-operational trial (approx. 1 ha). The highest-content sclerocium with the maximum content of α-ergocryptin harvested from this area was transferred to a saprophytic culture and used for the production of ergot inoculant applied to operational areas in the 2021 growing season. The highest-content sclerotium was again transferred to a saprophytic culture and this culture was stored in the Czech collection of microorganisms (Masaryk University, Faculty of Science, Kamenice 5, 625 00 Brno) under accession no.

CCM9309. As a result of the aforementioned mutation intervention, strain CCM9309 increased the average content of ergot alkaloids in sclerotia by 20% compared to the original strain CCM8178. At the same time, the spectrum of individual alkaloids was improved by increasing the content of α-ergocryptine from 90% to 98% and reducing the content of impurities. There was also a decrease in the content of the problematic impurity ergogalin, which causes problems in the production of ergot alkaloids. The content of ergogalin in this strain is represented by an average of 0.6% of the sum of defined alkaloids. Furthermore, there was an increase in the yield of ergot sclerotia from 770 kg/ha to 1,100 kg/ha, i.e. by 30%.

Example 2

Description of CCM9309 strain properties

During saprophytic cultivation on wort medium solidified with agar, strain CCM9309 forms colonies from typically sphacelial aerial mycelia. The mycelium is initially white and smooth, after 28 days of cultivation velvety wrinkled, white-grey. The mycelium consists of branched hyphae and oval-shaped conidia approximately 4 to 10 micrometers long and approximately 3 to 6 micrometers wide.

During parasitic growth on rye, the fungus forms dark brown pigmented sclerotia with an average weight of 30 mg. Individual sclerotia contain ergot alkaloids in the amount of 1.2% to 2.1% by weight. biomass dry matter at an average content of 1.6 wt.% sums of alkaloids with a predominance of αergocryptine.

Example 3

Examples of use of strain CCM9309

a) An aqueous suspension of conidia was prepared from the required amount of durable dry silt of strain CCM9309, produced according to the procedure according to patent CZ 276530. With this suspension of conidia, rye ears were inoculated with the help of an inoculating machine in the sweeping phase, so that 5x1011 germinating conidia were used on an area of 1 ha divided into two doses with an interval of at least two days during sweeping. At the time of ripening of the rye (drying of the ears), when the sclerotia begin to be released from the bed, i.e. in about 55 days, the grown sclerotia were harvested with a combine harvester. An average of 1,100 kg of brown-pigmented sclerotia containing an average of 1.6% by weight was obtained from 1 ha. sums of alkaloids and this sum is made up of 98% αergocryptine. The obtained drug was extracted and the extracts were used for the production of ergotoxin-type substances and for the production of semi-synthetic ergot alkaloids.

b) An aqueous suspension of conidia was prepared from the necessary amount of durable dry silt of strain CCM9309, produced according to the procedure according to patent CZ 276530. This conidia suspension was used to inoculate rye ears in the flowering phase with the help of a spraying machine, so that 5x1011 germinating conidia divided into at least three doses during flowering were used on an area of 1 ha. At the time of ripening of the rye (drying of the ears), when the sclerotia begin to be released from the bed, i.e. in about 55 days, the grown sclerotia were harvested with a combine harvester. From 1 ha, 1100 kg of brown pigmented sclerotia were obtained, containing an average of 1.6% by weight. sums of alkaloids and this sum was made up of 98% α-ergocryptine. The obtained drug was extracted, the extracts were

- 3 CZ 2023 - 105 A3 used for the production of ergotoxin-type substances and for the production of semi-synthetic ergot alkaloids.

c) Using triticale and sterile analogues of both rye and triticale in experiments arranged identically to those described above, identical yields of alkaloids, specifically α-ergocryptine, were achieved.

d) From the sclerotia of strain CCM9309, natural ergot alkaloids such as α-ergocryptine, or others, which were further used for the preparation of ergotoxin-type medicinal products, or for the preparation of lysergic acid and semi-synthetic derivatives of ergot alkaloids, were isolated from the sclerotia of strain CCM9309 using standard procedures known to the expert.

Industrial applicability

The mushroom strain Claviceps purpurea (Fr.) Tul., CCM9309 can be used in the pharmaceutical industry as a new source of ergot alkaloids with a high content of α-ergocryptine for the production of both ergotoxin substances and pharmaceutical forms, as well as for the production of semi-synthetic derivatives.

Claims (9)

1. An isolated strain of the fungus Clavicepspurpurea (Fr.) Tul., deposited in the Czech collection of microorganisms of the Masaryk University in Brno under accession number CCM9309. 2. An isolated strain of the fungus according to claim 1, forming during parasitic growth on cereals, especially rye, Secale cereale, or triticale, hybrids of rye and wheat, Triticum aestivum, and their sterile analogs, sclerotia, which contain a sum of alkaloids from 1.2% by weight up to 2.1% by weight, on average 1.6% by weight, based on the dry matter of the sclerotia biomass. 3. An isolated mushroom strain according to claim 1 or 2, where on average 98% of the sum of alkaloids is formed by α-ergocryptine. 4. An isolated mushroom strain according to any one of claims 1 to 3, wherein an average of 0.6% of the sum of alkaloids consists of ergogalin. 5. Culture from the fungus strain according to any one of claims 1 to 4 on rye, its cultivars and genetic lines, or on triticale, their other hybrids, cultivars and genetic lines. 6. The sclerotia of a fungal strain according to any one of claims 1 to 4. 7. Use of the mushroom strain according to any one of claims 1 to 4 or the culture according to claim 5 or the sclerotia according to claim 6 for the production of natural ergot alkaloids and/or their semi-synthetic derivatives. 8. Use according to claim 7, where ergot alkaloids are selected from the group consisting of αergocryptine, lysergic acid and semi-synthetic derivatives prepared from them. 9. Use according to claim 8, where the ergot alkaloid is α-ergocryptine.