DE3006989C2 - - Google Patents

Description

The invention relates to a new for ergot alkaloids production, especially ergocristine production able Claviceps purpurea virulent strain on rye and its use (see the claims).

To produce ergot alkaloids are used for Time 2 different ways known, on the one hand the classic methods in which those on rye plants grown and collected drugs as raw materials used and the alkaloids from this through Extraction can be obtained, and on the other hand, that on the sterile fermentation or fermentation with suitable Claviceps strains based alkaloids production. There are several processes that can be carried out on a large scale known of the latter type (compare for example Hungarian patents 1 50 631, 1 51 724, 1 52 238 and 1 64 816), the breeding of ergot drugs has theirs Meaning not lost; a substantial part The world production of peptide alkaloids is still today still on the use of bred, at different Alkaloid-type fortified drugs. This is especially in the production of ergocristine and ergotamine the case. The side chain of the peptide alkaloids consists of Proline, a group of characteristic amino acids and one for the alkaloid in question (within the mentioned group) specifically characteristic amino acid. The for the two alkaloids mentioned (Ergocristin and Ergotamine) characteristic amino acid is phenylalanine. But it is extremely difficult to get education respectively the overproduction of phenylalanine during the fermenting that takes place within a few days or fermentative biosynthesis, because during the same time the biosynthetic structure of the lysergic acid part of the alkaloid molecule takes place and also from the synthesis of aromatic amino acids  significant amounts of tryptophan are required. in the Course of maintaining the tribe and successive Vaccination can therefore be the original one Proportion of the strains producing ergotamine and ergocristine through the balancing control of the metabolic pathways in the direction of producing ergocornin and then moved by water-soluble alkaloids. Also this may have contributed to the fact that certain Not later fermentation or fermentation processes could be reproduced more successfully (cf. for example the Czechoslovak patent 1 04 613, German Patent 12 15 637 and British Patent Specification 11 92 912). As for the reprocessing drug-based procedures with such difficulties Such procedures will not continue to be expected used on a large scale and it continues to exist a need for process engineering respectively technological development of such processes. The is a fundamental condition for such further development but the production of strains of Claviceps purpurea with a correspondingly high ability to generate alkaloids.

There have been various manufacturing processes of Claviceps purpurea strains with good production ability with regard to ergoline alkaloids in several patent addresses described; there were different ways to Extraction of strains with higher production capacity proposed. So it was after in the Soviet patent 2 32 468 a method described by certain colonial morphological properties and spore dimensions Strain selected for the generation of ergotoxin.  In Soviet patent 2 32 467 there is a on artificial medium almost with the morphological Characteristics of the above strain identical morphological properties showing 80% ergocryptine producing strain described. In the Hungarian patent specification 1 54 035 is a quick and effective one Process according to which the production of Cultures capable of ergotoxin due to the color of the Colonies to the ergotamine producing individuals can be enriched.

For the production of strains with high alkaloid production the following more important levels are required:

• A) breeding a grafted strain of Claviceps purpurea,
• B) the manufacture of for infecting Suitable spores by rye plants fermenting or fermentative Propagation of the tribe and
• C) the conservation of these spores up to their use.

The invention has for its object to one Ergocristine production in a simple and superior way, especially with the generation of Ergocristin in higher Yield capable, virulent on rye, compared to the Strains of the prior art have more advantageous properties Strain Claviceps purpurea with good ability  for the formation of spores with advantageous properties and create its use.

The above was surprisingly accomplished by the invention reached.

The invention relates to a for the production of ergot alkaloids, especially capable of producing ergocristine, deposited on rye virulent strain Claviceps purpurea on May 2, 1977 under the designation OKI 00163 at the national collection of the Hungarian State Institute for health care (Országos Közeg´szs´gügyi Int´zet). This strain has the following characteristics:

• a) Malt agar: After 28 days, white, 0.5 to 0.6 cm large colonies with short aerial mycelia; from 12th day of conidia formation; Size distribution the conidia: 1.3 µm to 3.8 µm, most common size: 2.5 µm.
• b) Mannitol / citrate agar: after 28 days flat, cream-colored, 1.0 to 1.5 cm large, wrinkled Colonies; Conidia formation from the 15th day.
• c) Average size of the conidia (out of 100 unselected grains): 2.5 µm; smooth surface.

The strain of the invention shows the following advantageous Characteristics:

• α ) Good spore formation on artificial nutrient media, both on agar surfaces and in submerged aerated cultures.
• β ) The spores are sufficiently resistant and therefore durable.
• γ ) The spores are viable and virulent, so that when they return to a nutrient medium they germinate 98 to 100% and effectively infect the plant when applied to plants.
• δ ) The amount of mother grain drug produced per unit area of the spores is high.
• ε ) The ergot drug produced by the spores of the strain shows a high content of main alkaloids and contains only insignificant amounts of secondary alkaloids in addition to these main alkaloids.

The strain according to the invention can be combined by the Application of sexual and selective finishing can be produced as follows:

A strain of Claviceps purpurea, deposited in the collection of Gyógynöv´ny Kutató Intez´t (Medicinal Plant Research Institute) under the name 661/1974, which is described in Hungarian patent 1 54 035  with the filing date of September 19, 1965, with the following characteristic properties:

• a) Malt agar: after 28 days gray-white, 0.8 to 12.0 cm large colonies with full grown or spread aerial mycelia; Conidia formation from day 15; average Conidia size: 3.4 µm.
• b) Mannitol / citrate agar: after 28 days, white, 1.5 to 2.0 cm large, felt-like, extensive or spreading colonies; Conidia formation from day 20; average Conidia size: 4 µm.
• c) Average size of the sclerotia (out of 100 unselected grains): 18 mm; cracked surface.

From this parent strain were considered the morphological point of view well developed, externally flawless sclerotia with smooth surface selected. These sclerotia were placed on damp sand and Maintained at 0 to 5 ° C for 6 weeks, then rinsed and placed on a damp sterile sponge. The development of currents began after 12 days; the published currents developed fully in 6 days. The currents of those were used to obtain spores Sclerotia selected, which only some (3 to 4) mushroom heads have driven; such currents were taken from them whose diameter was larger than 4 mm and on their surface  the largest bulges by microscopic examination were sighted by peritetia. These currents were then placed on a liquid malt culture medium sterile conditions due to heat to form spores or sporulation brought. From the microscopic controlled containing ascospores Liquid was then placed on solid malt / agar medium Growing or spreading made. From the colonies formed from 1 spore each became a priority after the greater spore formation ability and subordinate selected according to the lower development speed. The Spores of the selected colonies were spawned after their multiplication isolated and placed under parasitic conditions, that is, the ears of those in the pre-condition Rye plants with the help of vaccination needles Spores have been infected. From the rye plants were then selected sclerotia for which the Sclerotic approaches within the shortest time after Appearance of honeydew have appeared. The on this Grains were collected by thin layer chromatography analyzed. The further selection was made due to the ergocrist content and the amount of those present contaminating side alkaloids.

That way through sexual reproduction Sclerotic cultures obtained were then another Subjected selection series, in which from the sclerotic particles Colonies grown on solid culture medium and with these liquid culture media have been vaccinated; from the so produced Submerged cultures were then field tested  the best spores, the most viable and the cultures showing highest virulence due to the quantity and the average alkaloid content of those produced Ergot selected.

Following this procedure, a number of selections were made in the successive stages of 2 - also parasitic Growth in closed - propagation cycles performed, whereby the strain of Claviceps according to the invention purpurea could be generated.

The invention also relates to the use of this Strain for breeding for the production of ergot drugs, especially those with a high ergocrist content Infecting rye, usable spore cultures in usual way.

The strain according to the invention is particularly advantageous suitable for use in the production of vaccine spore preparations, since he spores the fermentation respectively Fermentation increased. But it also shows when it is used beneficial in the field production of ergot alkaloid drugs Properties, with it the amount of produced Drug increased and its quality are significantly improved.

In Table 1 below are the results of the the strain Claviceps purpurea according to the invention on May 2, 1977 under the designation OKI 00163 at the national collection of the Hungarian State Institute for health care (Országos Közeg´szs´gügyi Int´zet) (hereinafter referred to as Claviceps purpurea OKI 00163) and  with that known from the Hungarian patent specification 1 54 035 Parent strain Claviceps purpurea for its production, deposited in the collection of Gyógynöv´ny Kutató Int´zet (Medicinal Plant Research Institute) under the Designation 661/1974 (hereinafter briefly Claviceps purpurea 661/1974) as a comparison material Fermentation, fermentation and field trials compiled.  

Table 1

By using the strain according to the invention So the inoculation spore generation of the fermentation respectively Fermentation increased to 2 times that Ergot alkaloid drug production increased by 50% and the Alkaloid content of the ergot obtained almost by 100% increased. Due to the much higher alkaloid content the alkaloid yield is also influenced favorably. Another advantage is that the Increasing the alkaloid content does not result from an increased Accumulation of fatty substances is accompanied so that the relative amount of fatty substances in the ergot is lower.

The invention is illustrated by the following examples explained in more detail.

example 1

It became a 24 day old malt / agar culture of the Strain Claviceps purpurea OKI 00163 in a 5 liter fermenter glass on a culture medium with the following Components vaccinated:

Malt extract2.0% by weight Potato powder 1.0% by weight Glucose 0.5% by weight pH of the culture medium: 6.0

The culture was stirred at 24 ° C with 440 revolutions / minute with a ventilation of 0.5 l Air / l fermentation or fermentation liquid / minute Incubated for 3 days.

With the vaccine culture obtained in this way (inoculum culture) then became 100 l culture medium with the following components vaccinated:

Mannitol 8.0 wt% citric acid 0.7 wt% corn steep liquor
(based on dry matter) 0.1% by weight monopotassium phosphate 0.025% by weight magnesium sulfate 0.025% by weight

The fermentation or fermentation was carried out in one equipped with an agitator, acid-proof 160 l fermenter carried out.

The incubation was carried out at 24 ° C. with stirring 230 revolutions / minute with a ventilation of 1.0 l Air / l fermentation or fermentation liquid / minute and continued at pH 4.5 to 5.0 for 6 days. The pH value of the fermentation or fermentation liquid was by adding ammonium hydroxide in specified range.  

At the end of fermentation or fermentation showed the fermentation or fermentation liquid a spore count of 4.2 x 10⁸ / cm³.

The biomass obtained was separated off with 30 l diluted in a 10% cane sugar solution, in bottles bottled and stored under deep freezing.

The vaccine spore preparation thus obtained showed one Germination rate of 99%. This vaccine spore preparation was then diluted to a concentration of 30,000 spores / mm³ and in this condition for infecting rye plants used before ear formation. From the crop 250 kg of ergot / ha were obtained in this way; their fat oil content was 20% by weight.

Ergocrist content: 6.2 mg / g Ergotamine content: 0.1 mg / g Ergometrine content: 0.2 mg / g  

Example 2

The strain Claviceps purpurea OKI 00163 incubated on malt / agar at 24 ° C for 21 days; the received Spores were treated with sterile physiological saline washed off the surface of the culture. With this Spore-containing saline was then placed in a phytotron grown rye plants infected; the spores were with With the help of an injection syringe through 5 stitches per ear introduced the plant body. The same procedure with the parent strain Claviceps purpurea 661/1974 carried out as reference material. With the 100 strains of both strains were infected.

The fully developed sclerotia were in maturity Condition collected. The results obtained are in the following table 2 compiled.  

Table 2

Example 3

She became 28 days old on malt / agar Culture of the strain Claviceps purpurea OKI 00163 with washed off sterile physiological saline and the number of spores of the washing liquid to 30,000 spores / mm³ set. With this liquid were in 30 m² large trial plots of rye plants in 4 parallel experiments infected. There were also identical ones Conditions parallel experiments with one on the same Wise prepared culture of the parent strain Claviceps purpurea 661/1974 performed.

The infection was cleared by hand with the help of Infection plates carried out. The mature sclerotia were also collected by hand without loss. Those with the strain according to the invention and the starting strain income obtained as comparative material summarized in Table 3 below.  

Table 3

Claims (2)

1. For ergot alkaloids production, in particular Ergocristine production, more capable, on rye virulent strain Claviceps purpurea, deposited on May 2, 1977 under the designation OKI 00163 at the Hungarian national collection State Institute for Health Care (Országos Közeg´szs´gügyi Int´zet).   2. Use of the strain according to claim 1 for Breeding for the production of ergot drugs, especially those with a high ergocrist content by infecting rye Spore cultures in the usual way.