CS236660B2 - A method for controlling the amount and relative alkaloid content - Google Patents

Abstract

Method for regulating the amount and mutual ratio of the content of alkaloids produced under saprophytic conditions by variants of the Claviceps purpurea strain, producing during fermentation carried out on a liquid nutrient medium containing a source of carbon, nitrogen, mineral salts, or other nutrients, in a certain temperature range and at a certain pH value, especially ergocornine and α-ergocryptine. The essence of the solution lies in the fact that for fermentation carried out in a submerged culture under "aerobic conditions" on a liquid nutrient medium containing a source of carbon, nitrogen, and mineral salts at 20 to 26 °C and a pH value of 5.2 to 6.8 for a period of four to eight days, Claviceps purpurea strains deposited under the numbers MNG 0022, MNG 0083, MNG 00186 at the Hungarian State Institute of Hygiene are used, while the regulator is used in an amount of from 0.01 to 10 kg/m3 of fermentation broth, preferably 0.05 to 5.0 kg per m3 of fermentation broth, compounds active in biosynthesis as intermediates, namely α-ketobutyric acid, threonine and/or homoserine or compounds of homocysteine and/or methionine stimulating the biochemical formation of isoleucine.

Description

The invention relates to a method of regulating the amount and mutual ratio of the content of alkaloids produced under saprophytic conditions by variants of the Claviceps purpurea strain producing, during fermentation carried out on a liquid nutrient medium containing a source of carbon, nitrogen, mineral salts, or other nutrients, in a certain temperature range and at a certain pH value, in particular ergocornine and α- and β-ergocryptine.

It is known that dihydroergotoxine methane or ethanesulfonate is used in medicine as a regulator of central metabolism and also in the treatment of central and peripheral circulatory system. The preparation increases the synthesis of central nervous system protein and inhibits the formation of catecholamine-stimulated adenyl cyclase. The preparation also acts as a weak sedative, inhibits reflex tachycardia, improves cerebral blood flow and reduces blood pressure. In medicine, the preparation is used mainly in diseases to calm peripheral or cerebral circulatory disorders.

Dihydroergotoxine is prepared by hydrogenation of ergotoxine. The resulting product is a mixture of dihydroergocristine, dihydroergocornine and dihydroergocryptine. Dihydroergocryptine occurs in two modifications (α- and β-forms) and according to the latest research, the pharmacological effect of the two forms is not completely identical.

The α-form is more active in the contraceptive test in rats, while the β-form has a stronger effect in decerebrate cats. For these reasons, the regulations for preparations containing dihydroergotoxine specify the ratio of α- and β-dihydroergocryptine. The ideal ratio of α- and β-forms is 2:1, and the overall optimal composition of dihydroergotoxine corresponds to the components dihydroergocristine, dihydroergocornine, α-dihydroergocryptine and β-dihydroergocryptine in the ratio 3:3:2:1.

The respective tolerances for the ratio between α- and β-forms vary from country to country. In the USA and most European countries, the respective values for β-dihydroergocryptine content are between 26.7 and 44 percent of the total dihydroergocryptine content, while in other European countries and Japan, the tolerance is between 28.6 and 40 percent.

The first step in the production of dihydroergotoxine methane or ethanesulfonate is the preparation of non-hydrogenated ergot alkaloids (ergocristine, ergocornine, α- and β-ergocryptine).

These alkaloids are prepared biologically. One option is parasitic biosynthesis, in which rye is infected and the resulting alkaloid is obtained by drug extraction. This process is mainly used in the production of ergocristine.

Saprophytic synthesis can also be used, in which the alkaloids are isolated from the fermentation broth. The best-known strains for this method of preparation are ATCC 20102, which produces ergocryptine and ergotamine, ATCC 20106, which produces ergocornine and ergosine, ATCC 20019, which produces ergocryptine, and Soc. Farmaceutici Italia, which produces «-ergocryptine and ergosine. «

From the above list, it is clear that these selective strains do not only produce alkaloids suitable for the preparation of ergotoxine, but also other ergot alkaloids, meaning that their fermentation is not exclusively oriented towards ergotoxine.

Hungarian researchers were the first to cultivate strains of Claviceps purpurea that produce mainly «- and β-ergocryptine. In these strains, too, the ratio of alkaloids to each other cannot be regulated during fermentation. The ratio of the three alkaloids produced can only be adjusted subsequently, so the obtained mixture of alkaloids must be separated into its individual components using lengthy, still known procedures and then mixed together again in the desired ratio.

Furthermore, a procedure is known in which two strains are fermented simultaneously, one of which produces ergocornine, ergocryptine and its isomer, the other strain produces ergocristine. The above-mentioned alkaloids cannot be obtained in a more precisely defined ratio from a joint solution of cultures of both strains. Also, in this method of preparation, it is not possible to regulate the mutual ratios of alkaloids. It is also problematic and more difficult to manage fermentation when two strains are used simultaneously than in a procedure in which optimal conditions are required for the growth of only one strain.

It has been found that the biosynthesis of peptide alkaloids can be influenced by certain amino acids. The biosynthesis of ergocornine, which contains valine in its peptide part, can be promoted by the addition of valine, the biosynthesis of «-ergocryptine, which contains leucine, can be promoted by the addition of leucine. Although β-ergocryptine contains isoleucine, the biosynthesis in this case cannot be influenced by the addition of isoleucine. To explain the probable cause of this peculiar phenomenon, it is stated that the growth of a strain capable of producing β-ergocryptine is inhibited by isoleucine.

Although in the broth of known strains producing ergocryptine or ergocornine and ergocryptine, the proportion of β-ergocryptine is smaller than desired, we have not been able to influence the mutual ratio so far. The purpose of the invention was therefore to influence the fermentation of the Claviceps purpurea strain in the desired direction.

However, it was surprising to find that the proportion of β-ergocryptine in the fermentation broth increases when the alkaloid production of the Claviceps purpurea strain variant is influenced by a compound that acts as an intermediate in biosynthesis instead of isoleucine. Therefore, the intermediates of isoleucine do not inhibit the growth of the strain.

It was further found that the formation of alkaloids can also be regulated by compounds that stimulate the formation of isoleucine through biochemical regulation. Intermediates and regulatory compounds

36600 of them are ketoacids, hydroxysulfur compounds and amino acids with 2 to 4 carbon atoms, whose biosynthesis is based on aspartic acid or its phosphate.

To influence the fermentation in the desired direction, new strain variants were also isolated by selection. During these experiments, a new variant of the Claviceps purpurea strain was isolated, which was deposited under No. MNG 00186. The strain has a morphology close to yeast and mainly produces ergocornin and β-ergocryptin, making it suitable for increasing the proportion of ,6-ergocryptin.

The current method of fermentation of Claviceps purpurea strain variants cannot influence either the content or the mutual ratio of the resulting ergot alkaloids. The proportion of β-ergocryptine in the fermentation broth is low.

The above-mentioned shortcomings are eliminated by the method of regulation according to the invention, the essence of which consists in that in the fermentation carried out in a submerged culture under aerobic conditions on a liquid nutrient medium containing a source of carbon, nitrogen and mineral salts at 20 to 26 °C and a pH value of 5.2 to 6.8 for a period of four to eight days, strains of Claviceps purpurea deposited on April 22, 1963 under number MNG 0022, on January 25, 1972 under number 0083, on May 9, 1979 under number MNG 00186 with the Hungarian State Institute of Hygiene are used, while as a regulator, a compound active in biosynthesis as an intermediate product, namely ω-ketobutyric acid, is used in an amount of cd 0.01 to 10 kg/m ³ of fermentation broth, preferably 0.05 to 5.9 kg/m ³ of fermentation broth. threonine and/or homoserine or homocysteine compounds stimulating the chemical formation of isoleucine.

Fermentation is carried out in the presence of additives (precursors) that regulate the ratio of alkaloids.

A compound that acts as an intermediate or compound in the biosynthesis of isoleucine can be used as a regulatory additive.

The intermediates in such isoleucine biosynthesis are — in the order in which they are formed — the following: via homoserine and threonine or 3-methylaspartic acid (2-amino-3-methylsuccinic acid) and methyloxaloacetic acid (2-methyl-3-oxo-succinic acid, α-ketobutyric acid and finally to α-acetyl-α-hydroxybutyric acid, α,β-dihydroxy-j3-methylvaleric acid and α-keto-(5-methylvaleric acid.

Compounds that stimulate the formation of isoleucine by biochemical regulation belong biochemically to the so-called aspartates, i.e. compounds whose biosynthesis is based on aspartic acid or its phosphate. These are primarily ketoacids, hydroxyacids and amino acids with four to six carbon atoms. They are primarily homocysteine and/or methionine.

Compounds that regulate the formation of alkaloids are added to the fermentation broth in an amount of 0.01 to 10 kg/m ³ , preferably

0.05 to 5.0 kg/m ³ . In this case, the weakly acidic aqueous solution of the regulating compound is added after sterilization, either all at once or divided into several doses, or continuously during the entire fermentation.

Previously, the alkaloid content of fermentation broth was often reported in values measured spectrophotometrically. The total alkaloid content was measured spectrophotometrically, although the measurement results are not sufficiently specific;

The individual components of ergotoxin were also determined by elution chromatography or quantitatively by liquid chromatography.

The invention is further illustrated by the following examples, but is not limited thereto. Example 1

200 ml of GK nutrient medium in a 500 ml Erlenmeyer flask is inoculated with a culture of Claviceps ¹ purpurea MNG 0022, pre-grown on AIC agar nutrient medium. The Erlenmeyer flask is tempered at 24 °C on a vibrating table 8 with a frequency of 300 min ^-1 for three days. The resulting embryo is sub-inoculated into 5 liters of St nutrient medium in a 10 liter fermenter.

The culture is grown at 24 °C and 240 rpm with shaking at 0.5 liters of air per liter of fermentation broth per minute for seven days. At 40, 64, 88 and 112 hours, a 10% aqueous solution of methyl oxaloacetic acid is added to the fermentation broth as a precursor (0.5 g of precursor ^per liter of fermentation broth). The content of ergocornine and ergocryptine alkaloids increases to the desired value after seven days of fermentation.

The total content of alkaloids according to the color reaction is 1150 y/ml. Thin layer chromatography (J. of Chrom. 87, 433—1973) revealed; 300 y/ml ergocornine, 180 y/ml ergocryptine and 110 y/ml of their dextrorotatory epimers. The ratio between α- and S-ergocryptine, determined by high-pressure liquid chromatography, was determined to be 66 : 34.

Alkaloids were isolated from the fermentation broth by a known method.

Composition of the nutrient media used:

AIC agar medium

Mannitol 40.0 g citric acid 7.0 g water swollen corn 2.0 g potassium dihydrogen phosphate 1.0 g magnesium sulfate 0.3 g powdered agar (Difco) 25.0 g ammonia to pH 5.2 to 5.3 water to 1000 ml

The desired pH value is adjusted by heating to boiling. Then 6 ml portions of the nutrient medium are added to the test tubes and left to solidify in the inclined test tubes.

Nutrient medium GK trypcasein 7.0 g citric acid 4.1 g potassium dihydrogen phosphate 0.3 g magnesium sulfate 0.3 g ammonia to pH 5.7 to 5.8 water to 840 ml

Fill flasks of 84 ml or 168 ml with nutrient medium. Add 16 or 32 ml of 50% glucose solution to each flask.

Nutrient medium St

sucrose 100.00 succinic acid 10.00 dihydrogen phosphate potassium 0.25 magnesium sulfate 0.25 ammonium nitrate 1.00 calcium chloride 1.00 ammonia to pH 5.2 to 5, water up to 1000 n

The nutrient medium is sterilized in batches of 0.1 or 5 or 100 liters.

Example 2

200 ml of GK nutrient medium in a 750 ml Erlenmeyer flask is inoculated with the Claviceps purpurea MNG 0088 culture variant, grown on AIC nutrient medium. The culture is incubated for three days at 24 °C on a vibrating table at a frequency of 300 min ^-1 .

The obtained culture is transferred to five liters of TC 54 nutrient medium, which is in a 10-liter laboratory fermenter. The culture is then blown through at 24 °C and a stirrer speed of 240 min ^-1 · 0.5 liters of air per liter of fermentation broth per minute, for three days. The obtained seed culture is inoculated into 100 liters of ST nutrient medium in an acid-resistant fermenter equipped with a stirrer.

The culture is maintained at 24 °C with stirring at a rate of 120 min ^-1 and aeration at 0.3 liters of air per liter of fermentation broth per minute for six days. During the first five days of fermentation, a 5% aqueous solution of α-ketobutyric acid is added to the culture as a precursor at a rate of 20 ml h ^-1 .

After the fermentation was complete, the alkaloid content was 920 χ/ml. The chromatographic method described in Example 1 revealed 260 γ per milliliter of ergocornine, 155 χ/ml of α-ergocryptine and 95 χ/ml of ,<3-ergocryptine. In addition, 80 χ/ml of ergocornine creatine was found.

The TC 54 nutrient medium has the following composition:

sucrose citric acid sodium chloride dihydrogen phosphate potassium magnesium sulfate ammonia water

100.0 g

10.0g

10.0g

0.5g

0.5 g to 5.7 to 5.8 to 1000 ml

The nutrient medium is sterilized in batches of five liters in a laboratory fermenter.

Example 3

100 ml of GK nutrient medium in a 500 ml Erlenmeyer flask is inoculated with a culture of the Claviceps purpurea variant MNG 0088, propagated on agar nutrient medium St. The culture is then tempered at 24 °C on a vibrating table at a frequency of 300 min ^-1 for three days. 10 ml of the obtained culture was sub-inoculated into 100 ml of nutrient medium T 25.

This culture was also incubated for five days at 20 °C on a shaking table. At 20 hours, 0.5 g of threonine was added to the fermentation broth as a precursor. On day 5, the total alkaloid concentration was 1200 χ per milliliter. By the method given in Example 1, 260 χ/ml of ergocornine, 140 χ per milliliter of α-ergocryptine and 80 χ/ml of á-ergocryptine were found. The total content of ergocornine and ergocryptine was 130 χ/ml.

The nutrient medium St differs from the liquid nutrient medium St, given in Example 1, in that 25 g of powdered agar (Difco) was added to the solution. After adding the agar, the nutrient medium is heated to boiling, then divided into 6 ml portions into test tubes, sterilized and left to solidify in an inclined position.

The T 25 nutrient medium has the following composition:

sucrose 300.0 citric acid 15.0 yeast extract 1.0 dihydrogen phosphate potassium 0.5 magnesium sulfate 0.5 ammonia to pH 5.2 to 5 water to 1000 π

The nutrient medium is filled into flasks in 100 ml portions, in which it is sterilized.

Example 4

The procedure is the same as in Example 3, but instead of threonine, 0.5 g of homoserine is used as the precursor. After the 5th day of fermentation, the total alkaloid content was 880 χ/ml.

The fermentation broth contained 220 χ/ml ergocornine, 150 χ/ml α-ergocryptine and 80 χ per milliliter (3-ergo.cryptine. Ergocornine and ergocryptine together were found to be 180 χ per milliliter.

Example 5

The procedure is the same as in Example 3, but 0.05 g of homocysteine was used as a precursor. After the 5th day of fermentation, the total concentration of alkaloids was 700 χ/ml. The fermentation broth contained 190 χ/ml of ergocornine, 85 χ/ml of α-ergocryptine and 90 γ per milliliter (3-ergocryptine. The total amount of ergocornine and ergocryptine was 150 χ/ml.

Example 6

100 ml of GK nutrient medium in a 500 ml Erlenmeyer flask is inoculated with a culture of the Claviceps purpurea strain MNG 00186, grown on agar nutrient medium St. The culture is tempered at 24 °C on a vibrating table with a frequency of 300 min ^- l for four days. The culture thus obtained is transferred in 10 ml doses to eight flasks, each containing 100 ml of nutrient medium St. The cultures are also fermented at 24 °C on a vibrating table for seven days. No precursor was added to four cultures. 0.02 g of methionine, dissolved in 2 ml of water, was added to the remaining four cultures at the 24th and 48th hours of fermentation. After seven days, the fermentation was interrupted.

Fermentation broths, cultured without precursor, were combined together and the alkaloid content was determined: 80 χ/ml ergocornine, 15 χ/ml α-ergocryptine, 30 χ/ml (3-ergocryptine.

The fermentation broths cultured with the precursor were mixed together and the alkaloid content was determined: the average content of ergocornine was 250 χ/ml, 50 χ/ml α-ergocryptine and 100 χ/ml (3-ergocryptine.

Claims (1)

Subject Process for controlling the amount and ratio of alkaloid content produced under saprophytic conditions by variants of the strain Claviceps purpurea producing on fermentation carried out on a liquid nutrient medium containing a source of carbon, nitrogen, mineral salt or other nutrients within a certain temperature range and pH, especially ergocornins; α- and β-ergocryptine, characterized in that in fermentation carried out in submerged culture under aerobic conditions on a liquid nutrient medium containing a source of carbon, nitrogen and mineral salts at 20 to 26 ° C and a pH of 5.2 to 6 , For a period of four to eight days, use the strains Claviceps purpurea MNG 0022, MNG 0083 and MNG 00186, using as fermenter a quantity of from 0.01 to 10 kg / m ^{3 of} fermentation broth, preferably 0.05 to 5.0 kg / m ³ fermentation broths, compounds active in biosynthesis as an intermediate, namely α-ketobutyric acids, threonium and / or homoserine or a homocysteine and / or methionine compound stimulating the biochemical production of isoleucine.