CS222689B2 - Process for preparing spores of Claviceps purpurea strain - Google Patents

Abstract

The invention relates to a method for preparing spores of the Claviceps purpurea strain, the essence of which consists in selecting developed sclerotia from the starting strain 661/1974, allowing them to germinate, separating the largest bundles of sclerotia and cultivating them under sterile conditions in malt nutrient medium under microscopic control. The resulting ascospores are transferred to malt agar, after which rye plants are infected with the developed vital, virulent and well-sporulating colonies and the whole procedure is repeated if necessary.

Description

The invention relates to a method for producing a spore culture of a new strain of Claviceps purpurea virulent on rye, producing ergocristine.

Two different routes are known today for the production of ergot alkaloids: the classical method, which uses drugs cultivated and collected on rye as the starting material, from which the alkaloids are obtained by extraction, and the production of alkaloids based on sterile fermentation of suitable Claviceps strains. Several methods are already known for the implementation of the latter method, which can be carried out on a large scale (cf. e.g. MaSar patent specifications No. 150,631,

151 724, 152 238 and 164 816), but the cultivation of ergot drugs has not lost its importance; a substantial part of the world production of peptide alkaloids is still based on the use of cultivated drugs enriched with various types of alkaloids. This is especially the case for the production of ergocristine and ergotamine. The side chains of peptide alkaloids consist of proline, a group of characteristic amino acids, and an amino acid specific to the alkaloid in question (within the range of the mentioned group). The amino acid characteristic of both mentioned alkaloids (ergocristine and ergotamine) is phenylalanine. However, it is extremely difficult to ensure the formation or overproduction of phenylalanine during fermentative biosynthesis, which takes place over several days, because during the same time the biosynthetic construction of the lysergic acid part of the alkaloid molecule also takes place, and for this purpose considerable amounts of tryptophan from the synthesis of aromatic acids are necessary.

During strain maintenance and subsequent re-inoculations, the initially present proportion of strains producing ergotamine and ergocristine can therefore be shifted towards the production of ergocornine and then water-soluble alkaloids by a balancing regulation of the metabolic pathway.

This circumstance could also contribute to the fact that certain fermentation methods could no longer be successfully reproduced later (cf. e.g. Czechoslovak patent specification 104 613, German Federal Republic patent specification 1 215 637, British patent specification 1 192 912). Since methods based on drug processing do not have to face similar difficulties, these methods continue to be used on a large scale and there is still a need for such methods to be further developed technologically. However, the basic condition for such further development is the production of Claviceps purpurea strains with a reasonably high ability to produce alkaloids.

Various methods of producing strains of Claviceps purpurea with good production capacity of ergoline alkaloids have already been described in a number of patent documents; various ways of obtaining strains with higher production capacity have been proposed. According to the method described in Soviet patent document 232 488, a strain characterized by certain morphological characteristics of colonies and spore dimensions is selected for the production of ergotoxine. Soviet patent document 232 467 describes a strain showing almost identical morphological characteristics to the above-mentioned ones on artificial nutrient media, producing 80% of ergocryptine. Hungarian patent document 154 035 describes a rapidly feasible and effective method according to which cultures capable of producing ergotoxine can be enriched against individuals producing ergotamine based on the color of the colonies.

The following important steps are required to produce strains with high alkaloid production:

1. Cultivation of an improved strain of Claviceps purpurea;

2. Production of spores suitable for infecting rye by fermentative propagation of the strain;

3. Preserving spores until they are used.

The aim of the present invention was to produce a strain producing ergocristine in increased yields, which exhibits the following advantageous properties in particular:

- the strain shows good spore formation on artificial nutrient media, both on the agar surface and in submerged, aerated cultures;

- the spores are sufficiently resistant and can therefore be stored well; ,

- the spores are viable and virulent, so when they come back into the nutrient soil environment, they germinate 98 to 100% and, when transferred to plants, effectively infect them;

- the quantity of drug produced per unit area is high;

- the drug has a high content of main alkaloids and, in addition to these main alkaloids, contains only small amounts of minor alkaloids;

The new strain was produced according to the invention by the combined use of sexual and selective breeding in the following manner:

From the initial strain, which was registered in the own collection of the Hungarian Research Institute for Plant Medicine under No. 661/1974, well-developed, externally flawless sclerotia with a smooth surface were selected, taking into account morphological aspects. These sclerotia were placed on moist sand and kept for 6 weeks at a temperature between 0 and 5 °C, then rinsed and applied to a sterile sponge, which was kept moist. After 12 days, the development of filament bundles began; the filament bundles that appeared were fully developed in 6 days. For the collection of spores, those filament bundles that had only produced a few pistils (3 to 4) were selected; from these, those filament bundles were selected whose diameter was greater than 4 mm and on whose surface the largest fruiting bodies with perithecia were found based on microscopic examination. These fiber bundles were then brought to sporulation on liquid malt broth under sterile conditions by the action of heat.

From the microscopically controlled liquid containing ascospores, streaks were then made on solid malt-agar medium. Colonies formed from each spore were selected based on their rate of development and their ability to form spores. After their inactivation, the spores of the selected colonies were isolated and parasitic conditions were created for them, i.e., they were infected with these spores using inoculation needles into rye ears before anthesis. Those sclerotia were then selected from the rye in which the sclerotia germs appeared within the shortest time after the appearance of the honeydew. The grains collected in this way were analyzed by thin-layer chromatography. Further selection followed based on the ergocristine content and the amount of contaminating secondary alkaloids present.

The sclerotia cultures obtained in this way by sexual reproduction were then subjected to another series of selection, in which colonies were cultivated from sclerotia particles on solid nutrient medium and liquid nutrient mediums were inoculated with these; from the submerged cultures produced in this way, the best sporulating, vital and most virulent cultures were then selected in field trials on the basis of the amount and average content of alkaloids of the drug produced.

According to this method, a series of selections were carried out in successive steps of two reproduction cycles - including parasitic growth - which allowed the production of a new strain of Claviceps purpurea, which exhibited more advantageous properties than previously known strains.

The new strain, deposited in the Hungarian Provincial Institute of Public Hygiene (OKI) under the number 00163, is particularly well suited for the industrial production of spore vaccination preparations, as it increases spore production during fermentation; however, this strain also shows advantageous properties in field production of the drug, increasing the amount of drug produced and significantly improving the quality of the drug.

The table below summarizes the results of fermentation and field experiments conducted with the new strain OKI 00163 (for comparison) with the starting strain No. 661/1974 of the Hungarian Research Institute for Plant Medicine.

222689 4

Table

OKI 00163 661/1974 spore production capacity on the surface, number of spores/tube 1 to 2.10 ^seconds 0.5 to 1.10’ number of spores in fermentation broth, spores/ml ' 4 to 5.10® 1 to 2.10® germination rate of the preparation for spore inoculation, % 95 to 99 90 to 96 virulence (concept in phytotron, in % of injections) 70 to 80 50 to 60 mass of 1,000 grains, g 175 to 200 140 to 170 drug production kg/ha (in trials) 200 to 350 150 to 220 ergocreatine content, mg/g 5.0 to 6.2 2.6 to 3.6 minor alkaloids: ergometrine mg/g 0.2 to 0.3 0.1 to 0.2 ergotamine mg/g footprints 0.05 to 0.1 fatty oil content, % 15 to 20 16 to 18 ergocreatine production, g/ha (average kg/ha.average mg/g) 1,540 590

Morphological characteristics of the new strain obtained by selection according to the invention and the starting strain:

OKI strain 00163:

a) Malt agar: after 28 days white, 0.5 to 0.6 cm colonies with short aerial mycelia; starting from the 12th day, conidia formation according to size: 1.3 µm to 3.8 µm, most common size: 2.5 µm.

b) Mannitol-citrate-ager: after 28 days flat, cream-coloured, 1.0 to 1.5 cm large, ciliated colonies; conidia formation starting from the 15th day.

c) Average size of conidia (100 unselected grains) 2.6 µm; surface smooth.

Initial strain 661/1974:

a) Malt agar: after 28 days, gray-white, 0.8 to 12.0 cm large colonies, with expanded aerial mycelia; conidia formation starting from the 15th day; average size of conidia: 3.4 /un.

b) Mannitol-citrate agar: after 28 days white, 1.5 to 2.0 cm large, tomentose, expanded colonies;. formation of conidia starting from the 20th day; average size of conidia: 4/im.

o) Average size of sclerotia (100 unselected grains) 18 mm; surface cracked.

The production of inoculum spores from fermentation is increased by using the new strain by twofold; the production of the drug is increased by 50% and the alkaloid content of the obtained drug is almost 100% higher. The substantially higher alkaloid content also has a positive effect on the alkaloid yield; as a further advantage, the increase in the alkaloid content is not accompanied by an increased accumulation of fatty substances, so that the relative amount of fatty substances in the drug is lower. The method according to the invention is explained in more detail by means of the following examples.

Example 1

A 24-day-old malt-agar culture of strain OKI 00163 is inoculated into a 5-liter glass fermenter on a nutrient medium with the following composition:

malt extract 2.0% potato powder 1.0% glucose 0.5% pH value of the nutrient medium 6.0. The culture is incubated at 24°C, with stirring at 440 rpm, aeration of 0.5 liters of air per liter of fermentation liquid per minute, for 3 days.

The inoculum culture obtained in the above-mentioned manner is then inoculated into 100 liters of nutrient medium of the following composition:

mannitol 8.0% citric acid 0.7% swelling corn water (calculated on dry matter) 0.1% potassium dihydrogen phosphate 0.025% magnesium sulfate 0.0025%

Fermentation is carried out in a 160-liter fermenter, equipped with a stirrer and resistant to acids.

Incubation is continued at 24°C, with agitation at 230 rpm and aeration at 1.0 liter of air per liter of fermentation broth per minute, at a pH of 4.5 to 5.0 for six days. The pH of the fermentation broth is maintained within the specified limits by the addition of ammonium hydroxide.

Q

At the end of fermentation, the fermentation liquid shows a spore count of 4.2:10/ml.

The obtained biomass is separated, diluted with 30 liters of 10% sodium hydroxide solution, filled into bottles and stored in deep cooling.

The inoculum spore preparation obtained in the above-mentioned manner shows 99% germination. This inoculum spore preparation is diluted to a concentration of 30,000 spores/mm ³ and in this state is used to infect rye before ear formation. From the harvest yield in this manner, 250 kg of drug per hectare is obtained; the fatty oil content in the drug is 20%.

Ergocristine content: 6.2 mg/g

Ergotamine content: 0.1 mg/g

Ergometrine content: 0.2 mg/g.

Example 2

The strain OKI 00163 is incubated on malt agar at 24 °C for 2. days; the spores obtained are washed off the surface of the culture with sterile physiological saline solution. This saline solution containing spores is then used in a phytotron to infect cultivated rye plants; the spores are injected into the plant body with a syringe by five punctures into each ear. For comparison, the same procedure was performed with the starting strain 661/1974, in the same way. 100 ears were infected with both strains.

Fully developed sclerotia were collected at maturity. The results obtained are summarized in the following table:

Tribe Sclerocia (pieces) Total weight of sclerotia Ergocristine content mg/g OKI 00613 470 79 grams 5.9 661/1974 420 57 grams 3.0

Example 3

A 28-day-old culture of strain OKI 00163, cultivated on malt agar, was washed with sterile saline solution and the spore count was adjusted to 30,000 spores/mm 2 with liquid. Rye plants were infected with this liquid in four parallel experiments on a 30 m2 experimental plot. For comparison, parallel experiments were carried out under identical conditions with a culture of the starting strain 661/1974 prepared in the same way in advance.

Infection was performed manually using infection plates; mature sclerotia were also collected manually without loss. The results obtained with the new strain and for comparison with the initial strain used are summarized in the table below.

Tribe Weight 1,000 grains g Content of ergocristine mg/g Harvest yield kg/ha Ergocristine yield g/ha OKI 00163 190 6.10 390 2,380 661/1974 160 3.26 210 685

SUBJECT OF THE INVENTION

Claims (1)

Process for the preparation of spores of Claviceps purpurea strain, virulent on rye and producing ergocristin, characterized by selecting from the strain Claviceps purpurea 661/1974 developed sclerotia which are allowed to germinate, the largest bunches of sclerotia are separated and cultivated under sterile conditions in a sweetening medium , the ascospores formed are transformed into malt agar, whereupon the vital, virulent and well sporulating colonies are infected into the rye plants and the whole cycle is repeated if necessary.