DD156190A5 - METHOD FOR THE REGULATION OF THE QUANTITY AND QUANTITY OF LIQUID OF ALKALOIDS ARISING FROM FERMENTATION - Google Patents

Abstract

The invention relates to a method with which the amount and the quantity ratio of the ergot alkaloids produced by variant strains Claviceps purpurea under saprophyte conditions can be regulated. In particular, the method is applied to strains that produce mainly ergokornin, alpha and beta ergocryptine. According to the invention, in the fermentation known per se, a compound which acts as a precursor in the biosynthesis of isoleucine or a compound which stimulates the formation of isoleucine as a result of biochemical regulation is used as regulator. This precursor, preferably methyl oxaloacetic acid, alpha-ketobutyric acid, threonine and / or homoserine or homocysteine and / or methionine, is used in an amount of from 0.01 to 10 kg / m 3 of fermentation broth. In the fermentation according to the invention, a higher total alkaloid concentration in the ferment broth is achieved, and the quantitative ratio between alpha and beta-ergokryptin is more favorable.

Description

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Regulation of the ratio of alkaloids formed during fermentation

Field of application of the invention:

The invention relates to a method for regulating the amount and the ratio of alkaloids formed during fermentation.

Characteristic of the known technical solutions;

It is known that the dihydroergotoxine methane or ethane sulfonate is used in medicine to regulate the central metabolism and the central and peripheral circulation. The preparation enhances the protein synthesis of the central nervous system and inhibits the adenyl cyclase stimulated by the catecholamines weakly sedative, inhibits reflex tachycardia, improves cerebral blood flow and lowers blood pressure. In medicine, the agent is used primarily for the treatment of diseases that are based on peripheral or cerebral circulation disorders.

The dihydroergotoxin is produced by hydrogenating the ergotoxin

The resulting product is a mixture of dihydroergokristin, dihydroergokornin and dihydroergokrypsin (HU-PS 129 061) _β The dihydroergokryptin is present in two different modifications (<a and / 3 form) (Eixpe- • imentia 2J | , 991 / ^ 9 & /) t wad according to the latest studies, the pharmacological effect of the two forms are not completely identical, the <Λ shape is active in a contraceptive test in rats, while the β-form has to deeerebrierten cats a stronger Vasopresscr effect (Experientia 21t 1552/1977 /) "For this reason, the regulations for dihydroergotoxin-containing preparations set the ratio of <λ- and β- dihydroergokryptin. A ratio of d ^ "shape is considered ideal considered to β form of 2i1 and the dihydroergotoxine optimal composition contains the components Dihydroergokristin, Dihydroergokornin ρ o <-Dihydroergokryptin and β -Dihydroergokryp- tin relative 3ί3 £ 2ϊ1 <> The permissible tolerances wherein the ratio of o (- and β ~ type are in different countries different · ^A in the United States and several European countries, the allowed limit values of the content of fi> "Dihydroergokryptin at 26 _S from 7 to 44% of the total content of Dihydroergokryptin _s while in other European Ιι'έω .- * countries and in Japan the tolerance is 28.6 to 40% _Φ

The initial step in the preparation of dihydroergotoxin methane or ethane sulfonate is the preparation of. unhydrogenated ergot alkaloids (Ergo _Kristin? Ergokornin, <^ -and /? -Ergokryptin) *

These alkaloids are produced by biological means _e One possibility is the parasitic Biosyntheeej by infected rye and the alkaloid produced is recovered by extraction drugs. " This method is mainly used at

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used in the production of ergocristin (uses strains: the strain deposited under the number ATCC-20103 in the American Type Culture Collection, Societa Farmaceutici Italia strain described in GB-PS 1 192 912 and the judge Gedeon Vegyeszeti Gyar ET under the number MNG 00163 deposited in the Hungarian National Tribe Association) *

Furthermore, the biosynthesis can also take place in a saprophytic way, with the alkaloids being isolated from the fermentation broth. The best-known strains for this procedure are the ergocryptine and ergotamine-producing strain ATCC 20102 (GB-PS 1 158 380), the ergokornin and ergosin-producing strain. , ATCC 20106 (GB-PS 1 184 039), the ergocryptin-producing strain ATCC 20019. (US Pat. No. 3,485,772), the β- ergocryptine and ergosin-producing strain of Soc. Farmaceutici Italia (in their own collection under No ₀ FI 7374 deposited, described in the BE-PS 824 987) ο

It can thus be seen that these selected strains produce important alkaloids not only for the production of the ergotoxin, but also other ergot alkaloids, so that their fermentation is not selectively oriented to ergotoxin.

Hungarian by researchers for the first Man strains of Claviceps purpurea were selected mainly o \ - and β -Ergorkryptin produce (HU-PS 152,238), deposit number: MNG 0022, HU-PS No. 164 816 _S deposit no .: MNG 0088). "Even with these strains, however, the adjustment of the ratio of alkaloids to one another during fermentation is not adjustable. The ratio of the three

alkaloids produced to each other can be nor subsequently adjusted * by decomposing the resulting Alkaloidgemisch with the available cumbersome methods into its components and these mixed again in the appropriate ratio »

Also known is a method in which two strains are fermented simultaneously (CB-PS 577 556), of which one Ergokornin _s ergocryptine and their isomers _s produces the other Srgokristin From the common culture fluid of the two strains, the alkaloids listed in one The problem is that fermentation can be much more difficult to control when two strains are used at the same time than a procedure in which only the breeding optimum of a single strain is taken into account must ©

Η ", Kobe! set (held in his 1977 in Basel at the meeting of the Federation of European Microbiological Societies Lecture) found that the biosynthesis of the peptide alkaloids may be affected by certain amino acids _o The biosynthesis of the. Peptide part containing valine ergokornins can be promoted by the addition of valine ,, the biosynthesis of leucine-containing c * -Ergokryptins by the addition of leucine. On the other hand, the biosynthesis of isoleucine-containing ergocryptine can not be affected by the addition of isoleucine. As a probable cause of this peculiar phenomenon, Kobel indicated that the growth of the strains capable of producing β- ergocryptine was inhibited by isoleucine.

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Since in the culture broths of the known ergokornptin or ergokornin and Ergokryptin producing strains just the proportion of β -ergokryptins is smaller than necessary, the appropriate ratio could not continue to be set »

Aim of the invention; ·

The aim of the invention is therefore to influence the fermentation of variants of the strain Claviceps purpurea in a favorable direction,

Explanation of the essence of the invention:

Surprisingly, it has now been found that the proportion of β- bromo-cryptin in the fermentation broth increases if the alkaloid production of the variant strains of Claviceps purpurea, instead of isoleucine, is influenced by a compound which acts as a precursor in the biosynthesis of isoleucine. The precursors of isoleucine therefore do not inhibit the growth of the strains.

It has also been found that alkaloid production can also be controlled with compounds that stimulate the formation of isoleucine as a result of biochemical regulation. The precursors and regulatory compounds are keto acids, hydroxy acids and amino acids of 4 to 6 carbon atoms, whose biosynthesis starts from aspartic acid or its phosphate «

To influence the fermentation in a favorable direction and new variant strains were selected. In the course of these experiments, a new variant strain Claviceps purpurea was selected, which was listed on May 9, 1979 under the number MNG 00186.

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was panned. The strain has a yeast-like morphology and produces mainly ergokornin and P- JSrgokryptin and is therefore suitable for increasing the ß- ergocryptin content.

Includes ⁰ * ¹ has been found that the ratio of the alkaloids produced hardly changes _t the total amount of alkaloids found in the fermentation broth, however, increases if one uses the new strain to Aikaloidppoduktion and Fermentbriüie a functioning as a precursor in the biosynthesis of isoleucine -y _{erbindi ; in} g _{0 (ier a} result of biochemical regulation of the formation of isoleucine zusetzte stimulating compound,

The invention accordingly provides a process for regulating the amount and the ratio of the alkaloids _β J ¹ produced by the variant strains of Claviceps purpurea produced by the under saprophytic conditions, especially ergokornin, ^- and β- ergokryptine. The method according to the invention is characterized in that the per se known per se as a regulator acting in the biosynthesis of isoleucine precursor compound or a biochemical regulation the formation of isoleucine stimulating compound used as a regulator »

According to the invention, in the method mainly Ergokornin ₉ O ^ - and β -Brgokryptin producing variant strains of Glaviceps purpurea used preferably are those available under the numbers MG 0022, 0088 and MNG MNG 00186 deposited strains _o

The fermentation is carried out in submerged culture under aerobic conditions on a carbon dioxide and nitrogen source.

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ralsalze and optionally other additives containing liquid nutrient medium at 20 to 26 ⁰ O and a pH ve 5.2 to 6.8 4 to 8 days made long

The fermentation is carried out in the presence of a ratio of the alkaloids regulating additive (precursor)

When controlling agent is used in a Biosynthe se des'Isoleueins as a precursor acting connection (Lehningerj Biochemistry, Worth Publ _o Inc. NY, p 704 or the biosynthesis of isoleucine by biochemical regulation stimulating compound »

The precursors of isoleucine in its biosynthesis are ~ in the order of their formation - the following: via homoserine and threonine or 3-methylaspartic acid ^ -Amino ^ -methylsuccinic acid) and methyl oxalacetic acid (2-methyl-3-oxosuccinic acid) -Λ-keto-butyric acid subsequent ^ -Acetyl- ^ -hydroxyb utter acid, o ^, / 3-dihydroxy- β- methyl-valeric acid and <A -Keto- β- methyl-valeric acid.

The biochemical regulation of isoleucine stimulation by biochemical regulation belong biochemically to the so-called aspartates, i. Compounds whose biosynthesis starts from aspartic acid or its phosphate. First and foremost are ketonic acid, hydroxy acids and amino acids with 4 to 6 carbon atoms. Homocysteine and / or methionine are preferred.

The alkaloid production regulating compound is added to the fermentation broth in an amount of 0.01 to 10 kg / m, preferred

from 0.05 to 5 kg / m, added "The optionally weakly acidic aqueous solution of the regulatory compound is added after sterilization, at one time or divided into several portions or even in one of the periods of fermentation continuously"

In previous patent applications, the content of the fermentation broth of alkaloids is often indicated by spectrophotometrically measured values. The total alkaloid content was also measured spectrophotometrically in the present case, but the measurement results were not specific.

The individual ergotoxin components were analyzed by elution chromatography. or quantitative liquid chromatography determined separately *

Embodiments;

The invention will be explained in more detail with reference to the following examples, but is not limited to the examples.

Example 1

200 ml culture medium GK in a 500 ml Erlenmeyer flask are inoculated with the culture grown on agar medium AIC strain Claviceps purpurea MNG 0022. The Erlenmeyer flask is placed at 24 ⁰ on a shaking table (frequency ~ 1 N

300 min) for 3 days. The inoculum obtained in this way is inoculated onto 5 liters of nutrient medium ST in a fermenter of 10 liters volume. The culture is at 24 ⁰ O and. a stirring

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speed of 24-0 min under aeration with 0.5 liter of air per liter of fermentation broth and 7 days of culture bred "In the 40," 64, 88 and 112 "hours, the fermentation broth precursor is a 10% aqueous solution of Methyloxalessigsäure

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(0.5 S precursor / liter of fermentation broth) was added. The ratio of ergokornin and ergokryptine alkaloids reaches the desired level on the 7th day of fermentation. The total content of alkaloids measurable by color reaction is 1150 ^ T / ml. The thin - layer chromatographic determination (J. of Chrom. 82, 433/1973 /) shows 300 Γ / ml ergokornin, 180 / / ml ergokryptin and 110 f / ml of their rightful epimers ο The ratio between c - - and / -E - ergokryptine is 66:34 as determined by high pressure liquid chromatography (J · Phärm, vol. 62, 98/1978 /).

The alkaloids are isolated in a conventional manner from the fermentation broth.

The nutrient media used have the following composition: agar medium AIC

Mannitol 40.0 g

Citric acid 7 »0 g

Corn steep liquor 2.0 g

Potassium dihydrogen phosphate 1.0g

Magnesium sulfate 0.3 g

Agar powder (Difco) 25.0 g ammonia ad pH 5 »2-5» 3 water ad 1000 ml

The desired pH of the nutrient medium is set during the boil-up. Then the nutrient medium is filled in portions of 6 ml in test tubes and allowed to solidify by tilting the glasses as a slant agar.

Nutrient medium GK

Trypkasin - 7 * 0 g

Citric acid. 4.1 g

Potassium dihydrogen phosphate 0.3 g

Magnesium sulfate _ 0.3 g

Ammonia ad pH j? »7 to 5» 8 water ad 840 ml

The nutrient medium is filled into flasks of 84 or 168 ml each. In each flask add four 16 or 32 ml of 50% glucose solution ».

Sucrose 100.0 g

Succinic acid 10.0 g

Potassium dihydrogen phosphate. 0.25 g

Magnesium sulfate 0.25 g

ammonium nitrate 1.0 g

Calcium chloride 1.0 g. Ammonia ad pH 5 $ 2-5 $ 3 drawers ad 1000 ml

The nutrient medium is sterilized in portions of 0.1 or 5 or liters

Example 2

200 ml of nutrient medium GK in an Erlenmeyer flask of 750 ml volume are the grown on agar medium AIO culture of the variant strain Glaviceps purpurea IMG 0088 geimpfto The culture (frequency 300 min ") is incubated for 3 days at 24 ⁰ O on a shaker. The culture obtained is inoculated to 5 liters of nutrient medium TG 54 in a laboratory fermentor of .10 1 volume ₀ The culture is incubated at 24 ⁰ G and a

With a speed of 240 minutes under aeration with 0.5 liters of air per liter of fermentation broth and minute for 3 days.

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cubed. With the obtained inoculum 100 liters of nutrient medium St are inoculated in an acid-fast, equipped with a stirrer equipped fermentor, and the culture is at 24- ⁰ O and

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fermented with a stirring rate of 120 minutes under aeration with 0.3 liters of air per liter of IPerment broth and for 6 days. In the first 5 days of fermentation, a 5% aqueous cn -Ketobuttersäurelösung at a metering rate of 20 ml / h is added to the culture as a precursor.

After the termination of the fermentation, the Gesamtalkaloidgehalt 920 § * / ml · With the liquid chromatographic method indicated in Example 1 260 7 ^ / ml Ergokornin, 155 / ^ Ml <k -Ergokryptin and 95 vVml β -Ergokryptin demonstrated «, Moreover is still a content of 80 T ^ / ml Ergokornininkryptinin measurable.

The nutrient medium TC 54 · has the following composition:

Sucrose 100.0 g

Citric acid 10.0 g

Sodium chloride 10.0 g

Potassium dihydrogen phosphate 0.5 g

, Magnesium sulfate 0.5 S ammonia ad pH 5.7-5.8 water ad 1000 ml

The nutrient medium is sterilized in 5 liter portions in a laboratory fermentor.

Example 3

100 ml of nutrient medium GK in an Erlenmeyer flask of 500 ml volume are cultured with the agar medium St

Culture of the variant strain Claviceps purpurea MG 0088 vaccinated The culture is incubated at 24 ⁰ O on a shaking table (frequency 300 min °°) for 3 days »10 ml of the resulting culture are inoculated on 100 ml of nutrient medium T 25 This culture is at 20 ⁰ O also incubated on the shaker table for 5 days _e In the 20 _? Hour, the fermentation broth is treated with O ₅ 5 g of threonine as precursor "Am. 5 »day the total alkaloid concentration is 1200 / v / ml. With the method given in example 1 260 / ^ / ml ergokornin _? 140 P / ml o \ »Ergokryptin and 80 If ^ / ml β- ergokryptin detected * The total content of ergokorninin and ergokryptinin is 130

The Agarnährmedium St differs from the specified in Example 1 liquid nutrient medium St in that he per liter of 25 g of agar powder (Difco) are added, "After the addition of the agar, the nutrient medium is boiled, then filled in portions of 6 ml in test tubes , sterilized and allowed to solidify by tilting the jars into a slanting agar *

The nutrient medium T 25 has the following composition: sucrose 300.0 g

Citric acid 15 _f 0'g

Yeast Extract 1,0g

, Potassium dihydrogen phosphate 0.5 g

Magnesium sulfate 0.5 g

Ammonia ad pH 5.2-5 _s 3 water ad 1000 ml

The nutrient medium is filled and sterilized in 100 ml portions in flasks.

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Example 4

The procedure is similar to that described in Example 3, but using 0.5 g of homoserine instead of threonine as precursor. »

At the 3c day of fermentation, the total content of alkaloids is 8Q0f / mL * The fermentation broth contains 220 f / ml ergokornih, 150 Ζ ¹⁷ / ml cc / -ergokryptin 'and 80 ^ / ml /? -Ergocryptin · Ergokorninin and Ergokryptinin make up ml

Example 5 ₍

The procedure is similar to that described in Example 3, but uses 0.05 g of homocysteine as a precursor. On 5 days, the total alkaloid concentration is 700 μg of VmI. The fermentation broth contains 190A ¹ ZmI ergokornin, 85 ^ / ml <? C ergokryptine and 90 / VmI / 2 ^ ergokryptin · Ergokorninin and ergokryptinin together make up 150 y * Vml »

Example 6

100 ml of nutrient medium GK in an Erlenmeyer flask of 500 ml volume are inoculated with the culture grown on agar medium St of the strain Claviceps purpurea MG 00186. The culture is at 24 ⁰ C on a shaking table (frequency

-1 300 min) for 4 days. 10 ml of the culture obtained are given in 8 flasks containing 100 ml of nutrient medium containing St · The cultures are also fermented at 24 ⁰ C on the shaker 7 days Long. In the other four flasks, 0.02 g of methionine, dissolved in 2 ml of water, are added per hour to the fermentation at 24 hours and 48 hours, and the fermentation is stopped on 7 days. · · ·.

The fermentation broths cultured without precursor addition are combined and their alkaloid content is determined: Ergokorninj 80 y / ml, <A -Ergokryptin: 15 / VmI »/ ^ - Ergokryptins 30

The fermented broths cultured with precursors are also combined and examined »250 / ^ / ml average ergokorning content 50 / ^ / ml <^ - ergocryptin and 100 / ^ / ml 5" ergokryptin are detected "

Claims (4)

- 22 74 6 9 4 Inquiry: Verfahren _Φ Method for the regulation of the quantity and the ratio of quantities of fermentation. Alkaloids, and that of the under saprophytic conditions especially Ergokornin, <* · - and β- Ergokryptin produ-. variant alkaloids of Glaviceps purpurea produced alkaloids, geke η η ze i. This is achieved by using, as a regulator, a compound acting as a precursor in the biosynthesis of the isoleucine or a compound stimulating the formation of the isoleucine as a result of biochemical regulation, 2 "Procedure according to item 1, characterized in that the iso-leucine precursor used is methyloxaloacetic acid, <λ- ketpbutyric acid, threonine and / or homoserine © Method according to item 1 or 2, characterized in that the biochemically stimulating compound homocysteine and / or methionine is used as the formation of the isoleucine. Method according to one of the items' 1 to 3, characterized in that the regulator is used in an amount of 0.01 to 10 kg / m fermentation broth, preferably 0.05 to 5.0 kg / mr fermentation broth.