HU193311B - Process for the parasitic preparation of ergot alkaloids, particularly ergotamine - Google Patents

Abstract

The prods. are prepd. in parasitic conditions on rye by using as microorganism the Claviceps Purpurea strain variant deposited under number 00320 at the National Institute for Public Health. The new variant of the Claviceps Purpurea strain is prepd. by mutation enhancing radiation and selective breeding.

Description

The present invention relates to a process for the production of ergot alkaloids, in particular ergotamine by the parasitic route, with a novel variant of the Claviceps purpurea strain.

There are two different ways of producing nutmeg alkaloids; processes based on the extraction of the cultivated drug; and sterile fermentation alkaloid production with Claviceps strains. However, there are known methods for sterile fermentation alkaloid production (Hungarian Patent Nos. 150631, 151724, 152239, 164816), however, drug production has not lost its importance and a significant proportion of the world's peptidal alkaloid production is increasingly using drugs enriched with various alkaloid species. This is especially true for the production of ergotamine and ergocristine.

The production of ergoline-based alkaloid-producing strains of Claviceps purpurea has been the subject of several prior patents which sought to obtain higher yielding strains by varying methods. Thus, U.S. Pat. U.S. Patent No. 4,193,125 to Chrysepsia describes a process for producing a strain of Claviceps purpurea with spore size and colon morphology for the production of ergot toxin. No. 155148. According to the method of the Hungarian patent, strains capable of producing ergotamine can be produced on a special medium based on the color of the colonies.

Hungarian Patent No. 186823, issued May 9, 1984, describes a process for the production of parasitic alkaloids, especially ergotamine, by parasitic activity.

. SUMMARY OF THE INVENTION It is an object of the present invention to provide a method of producing ergotamine on a parasite rack in which a novel variant of the parasitic strain Claviceps purpurea spores well on artificial media, agar surfaces and immersed culture;

- the spores are well preserved;

- its spores are viable and virulent, 90 to 95% germinated when re-introduced into the medium and infested with rye; - high drug yield; - the drug contains 99,5% ergotamine as the main alkaloid;

It is an object of the present invention to provide a strain variant having a higher ergotamine content.

The new strain was prepared using the selection breeding method and the radiation treatment to promote the mutation.

The starting material is disclosed in U.S. Patent No. 186,823. Drug substance deposited with OKI under number 00208, filed October 8, 1984, mainly on ergotamine ter5 working strain.

From the parent strain, large-scale, smooth-surface sclerotia was selected, taking into account morphological aspects. The highlighted plurality of sclerotia salts were analyzed by thin layer chromatography. Based on the results of the study, individuals containing the most ergotamine and the smallest contaminating secondary alkaloids were highlighted. Sterile fragments of sclerotia were inoculated onto 10% malt agar medium. Adult cultures were exposed to 1200-1500 Gray doses of radioactive gamma radiation, which were delivered to the cultures over 12-15 hours. The cultures were then plated on 10% malt-agar medium. Colonies developed from the irradiated surviving conidia were selected for sporulation and morphological aspects. Selected batteries microscope! Submersible cultures were assayed from assayed spores using the medium described in Example 1, fel3Q. The viability and virulence of the cultures that sporulated best in liquid culture were tested in greenhouse experiments. In isolated conditions, the ears of rye plants in pre-anesthesia condition were stained with fer35 and the propagated spores of the spore cultures giving the best infection result were tested in the open air. Parcels of the same size were infected with the same spore number. For further selection, the yield of the parcel and the ergotamine content of the drug were taken into account. Most ergotamine-containing drugs were selected for sclerotia designated for further vaccination. The selection work was continued for a further 3 years, taking into account the above selection points.

The new strain bearing the institute designation Bk4 / S / 1/1964. On December 19, 1984, we deposited it at 0050 with the Or50 National Institute of Public Health. The new strain gave the following results in spore fermentation and drug cultivation compared to the parent culture:

New tribe

Old tribe

Institution Indication OKI Deposit Number

OKI deposit time Fermentation juice spore number / spore / mm ¹ /

Bk4 / S / 1/1984

00320

1984.dec.19.

6-7 *! 0 ⁵

Bk4 / S / 64/1981

00208

1981.okt.8.

6-7x10 ⁵

New tribe Old tribe Germination of preserved vaccine spores Z 95-99 95-99 Virulencia Z 85-90 80-90 Ezerszklerócium- wt / g / 180-200 211 crop result / Kg / ha / 300-350 280-350 Ergotamine yield / g / ha / / average kg / haxgmg / g / 373? 2504 Ergotamine content / mg / g / 11-12 7.8-8.1 Secondary alkaloids, impurities / a. alkaloid content in Z / 0.3 0.5 al; Green oil content 7, 18-22 18-20

The following examples illustrate the process of the invention in more detail:

Example 1

The OK1 strain deposited under number 00320 was inoculated with 21 days of malt agar in a 500 ml Erlenmeyer flask with the following medium:

malt extract 2 g / g% sucrose 5 g / g% succinic acid 1 g / g% calcium nitrate 0.1 g / g%

Potassium dihydrogen phosphate 0.025 g / g% magnesium sulphate 0.025 g / g% potassium chloride 0.0125 g / g% ferric sulphate 0.0008 g / g% zinc sulphate 0.0003 g / g% ₃ QA medium with 25% ammonium hydroxide 5.4- . Cultivation was carried out at 24-26 ° C on a shaker (230 rpm) for 144 hours. Spore number of fermentation broth: 6.5x10 ⁵ spores / mm ³ . The biomass was separated and then homogenized in 6% glucose solution at -40 ° C. The deep-frozen seed spore is prepared by disintegration into an infectious fluid whose spore number is adjusted to a concentration of 30,000 spores / mm ³ . The infectious liquid prepared in this manner was used to inoculate a tetraploid rye of Sopronhorpác on 30 m plots. The infection was carried out with manual inoculum.

The results for the new and the ₄₅ old strains tested for comparison are shown in the table below:

Tribe Ezerszkleró- ciumtömeg / g / Yield / kg / ha / ergotamine / Mg / g / Ergotamine yield / g / ha / New tribe OKI 00320 200 350 12 4200 Old strain, 0ΚΪ 00208 211 320 7.8 2496

Example 2

OKI No. 00320 strain prepared nagymenynyiségű vaccine tenyészt- ^{6 (5} then, purified Biostat fermenter broth and as described in Example 1. Centrifuge separated biomass is preserved by deep freezing at -40 ° C in the fermentation broth. The fermentation is obtained from the composition frozen oltóspóra 65 pots of liquid, 4 replicates in block system “On French rye varieties 2500 m ² plots were infested with the same technical parameters as the industrial infection.

The results for the new strain and the old strain used for comparison are shown in the table below:

Tribe Ezers'zkleró- ciumtömeg / g / Yield / kg / ha / ergotamine / Mg / g / Ergotamine yield / g / ha / New tribe OKI 00320 195 320 11.5 3680 Old strain OKI 00208 205 300 8.1 2430

Patent Claim Point

Claims (1)

A process for the production of ergot alkaloids, mainly ergotamine, by spore culture of Claviceps purpurea under parasite conditions, characterized in that the strain Cla-I5 viceps purpurea, deposited with the National Institute of Public Health, is 00320.