CS222689B2 - Method of preparation of the spore of the claviceps purpurea strain - Google Patents

Abstract

The invention relates to a process for the preparation of spores strain of Claviceps purpurea, whose essence it consists in getting out of the initial strain 661/1974 selects developed sklerooia, this they germinate, the largest clusters of sclerotia are separated and under sterile conditions are cultivated in the sweetening medium under microscopic control. The ascospores emerged are transferred to a sweetening agar followed by with developed vital, virulent and well sporulating colonies infect the rye plants and the whole process if necessary repeats.

Description

The invention relates to a method for producing a spore culture of a novel strain of Claviceps purpurea virulent on rye producing ergocristin.

Two different routes are known for the production of ergot alkaloids, the classical methods in which the cultivated and harvested drugs are used as the starting material, from which the alkaloids are obtained by extraction and the production of alkaloids based on the sterile fermentation of suitable strains of Claviceps. For the implementation of the latter method, several methods are known which can be carried out on a large scale (cf., for example, Hungarian patents 150,631,

151 724, 152 238 and 164 816), but the development of ergot drugs has not lost its importance; a substantial part of the world's production of peptide alkaloids is still based today on the use of cultured drugs enriched in various types of alkaloids. This is particularly the case for the production of ergocristin and ergotamine. The side chains of peptide alkaloids consist of proline, a group of characteristic amino acids, and an amino acid specific for the alkaloid in question (within the range of said group). The amino acid characteristic of the two alkaloids (ergocristin and ergotamine) is phenylalanine. However, it is most difficult to ensure the formation or overproduction of phenylalanine during fermentative biosynthesis, which takes place over a period of several days.

Therefore, during maintenance of the strain and successive revaccination, the initially present proportion of ergotamine and ergocristin producing strains may be shifted towards the production direction of ergocornin and then water-soluble alkaloids by compensating regulation of the metabolic pathway.

Even this circumstance could contribute to the fact that certain fermentation processes can no longer be reproduced successfully (cf., for example, U.S. Patent 104,613, Germany Patent 1,215,637, British Patent 1,192,912). Given that similar difficulties do not have to be envisaged for drug-based methods, these methods continue to be used on a large scale and there is a continuing need to further develop such methods. However, the basic conditions for such further development are the production of Claviceps purpurea strains with reasonably high alkaloid production capacity.

Various processes for the production of Claviceps purpurea strains with good ergolin alkaloid production capability have already been described in numerous patents; various ways of obtaining strains with higher production capacity have been proposed. According to the method described in Soviet Patent 232 488, a strain characterized by certain morphological properties of colonies and spore dimensions is selected for the production of ergotoxin. Soviet Patent 232 467 discloses a strain exhibiting identical morphological properties, producing 80% ergocryptine, on artificial culture media almost identical to the above. Hungarian Patent 154,035 discloses a rapidly feasible and efficient method according to which cultures capable of producing ergotoxin can be enriched against individuals producing ergotamine by color colony.

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

1. Cultivation of an upgraded strain of Claviceps purpurea;

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

3. Preserving disputes until their use.

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

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

- disputes are sufficiently resistant and can therefore be kept well; ,

- the spores are viable and virulent, so that when they come back into the culture medium they germinate 98 to 100% and effectively infect these plants on the plants;

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

- the drug has a high content of major alkaloids and contains only minor amounts of minor alkaloids besides these major alkaloids;

According to the invention, the new strain was produced by the combined use of sexual and selective refining in the following manner:

From the starting strain, which was registered in its own collection of the Hungarian Research Institute for Plant Treatment under No. 661/1974, well-developed, superbly smooth sclerotia with a smooth surface was selected taking into account morphological aspects. This sclerotia was laid on moist sand and kept at a temperature between 0-5 ° C for 6 weeks, then rinsed and deposited on a sterile sponge that was kept moist. After 12 days, fiber bundle development began; the fiber bundles that appeared completely developed in 6 days. To obtain the spores, those fiber bundles were selected that drove only a few sticks (3-4); fiber bundles with a diameter of more than 4 mm were selected from these and on the surface of which the largest fruiting beds with peritecia were found by microscopic examination. These fiber bundles were then fed on liquid malt broth under sterile conditions by heat to sporulation.

Microscopically controlled liquids containing ascospores were then made into solid malt-agar broth. The colonies formed from each dispute were selected on the basis of their pace of development and their ability to create disputes. The spores of the selected colonies were isolated after they were prevented and parasitic conditions were created for them, ie they were infected with the ears of rye ear before the anesthetic using these spores. The sclerotia in which the sclerotia embryos appeared within the shortest time after the appearance of the honey tendon was then selected from the rye. The collected grains were analyzed by thin layer chromatography. Further selection was made based on the ergocristin content and the amount of polluting minor alkaloids present.

Sclerotia cultures obtained by sexual reproduction in this manner were then subjected to a further series of selections in which the sclerotia particles were cultured on a solid colony broth and inoculated with the liquid broth; Submerged cultures produced in this manner were then selected in the field trials for the best-sparing, vital and greatest virulence-exhibiting cultures based on the amount and average alkaloid content of the drug produced.

According to this method, a number of selections were carried out in successive steps of two - even parasitic growth involving inherent reproductive cycles - thereby producing a new, more advantageous property exhibiting a strain of Claviceps purpurea than previously known strains.

The new strain, deposited at the Hungarian Provincial Institute for the Hygiene of the People (OKI) under number 00163, is particularly well suited for the operational production of spore vaccination products as it increases spore production during fermentation; however, this strain shows advantageous properties even in field drug production, increases the amount of drug produced and greatly improves the quality of the drug.

The following table summarizes the results of the fermentation and field trials conducted with the new strain OKI 00163 (for comparison) with the starting strain No. 661/1974 of the Malar Research Institute for Plant Treatment.

222689 4

Table

OKI 00163 661/1974 production ability of spores on the surface, number of spores / tube 1 to 2.10 ^s 0.5 to 1.10 ’ number of spores in fermentation slurry, spores / ml ' 4 to 5.10® 1 to 2.10® germination of spore vaccination product,% 95 to 99 90 to 96 virulence (concept in phytotron, in% of punctures) 70 to 80 50 to 60 weight 1 000 grains, g 175 to 200 140 to 170 drug production kg / ha (in experiments) 200 to 350 150 to 220 ergokrietin 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 tracks 0.05 to 0.1 Fatty oil content,% 15 to 20 16 to 18 ergokrietin production, g / ha (average kg / ha.Ag mg / g) 1 540 590

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

Tribe OKI 00163:

(a) Malt-agar: after 28 days white, 0,5 to 0,6 cm large colonies with short aerial mycelia; starting on day 12, conidia formation by size: 1.3 µm to 3.8 µm, most common size: 2.5 µm.

b) Mannit-citrate-ager: after 28 days, flat, cream-colored, 1.0 to 1.5 cm large, cilia-like colonies; conidia formation starting on day 15.

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

Default 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 on day 15; average conidia size: 3.4 / un.

(b) Mannitol-citrate-agar: after 28 days white, 1.5 to 2.0 cm large, tomentose, extended colonies; conidia formation starting on day 20; average conidia size: 4 µm.

o) Sclerotia average size (100 unselected grains) 18 mm; cracked surface.

The production of inoculation spores from fermentation is doubled by using a new strain; drug production increases by 50% and the alkaloid content of the drug obtained is almost 100% higher. A significantly higher alkaloid content will also favor the alkaloid yield; as a further advantage, the increase in 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 process according to the invention is explained in more detail by means of the following examples.

Example 1

In a 5-liter glass fermenter, a 24-day-old malt-agar culture of OKI 00163 is inoculated on a culture medium of the following composition:

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

The inoculum culture obtained as above was then inoculated with 100 liters of nutrient medium as follows:

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

The fermentation is carried out in a 160-liter fermenter equipped with an acid-resistant stirrer.

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

Q

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

The biomass obtained is separated, diluted with 30 liters of a 10% strength solution of harnose, filled into bottles and stored under deep cooling.

The vaccine spore preparation as described above has a germination rate of 99%. This vaccine 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. This yields 250 kg of drug per hectare from the harvest yield; the content of fatty oils in the drug is 20%.

Ergocristin content: 6.2 mg / g

Ergotamine content: 0.1 mg / g

Ergometrine content: 0.2 mg / g.

Example 2

The OKI 00163 strain is incubated on malt-agar at 24 ° C for 2 days; the spores obtained are washed off the surface of the culture with sterile saline. Cultured rye plants are then infected with the spore-containing salt solution in phytotron; spores are injected, injected with a syringe, five punctures into each ear into the body of the plant. For comparison, the same procedure was performed with the starting strain 661/1974, in the same manner. Both strains were always infected with 100 ears.

Fully developed sclerotia was collected in the mature state. The results are summarized in the following table:

Strain Sclerocia (incomplete) Total weight of sclerotia Ergocristin content mg / g OKI 00613 470 79 g 5.9 661/1974 420 57 g 3.0

Example 3

The culture of the OKI 00163 strain, 28 days old, cultured on malt-agar, was washed with sterile sodium chloride solution and the spores count was set to 30,000 spores / mm with liquid. Rye plants were infected with this kappa line in four concurrent experiments on a 30 m large plot. For comparison, parallel experiments were carried out under identical conditions with a previously prepared culture of the starting strain 661/1974.

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

Strain Mass 1,000 grains G Content ergocristine mg / g Yield harvest kg / ha Yield ergokristinu 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.