CS252603B1 - Industrial strain of fungi Claviceps purpurea (Fr.) Tul. 59 CC 5/86 - Google Patents

Abstract

The solution concerns a new industrial strain of the fungus Claviceps purpurea (Fr.) Tul 59 for the preparation of the alkaloids chanoclavine and chanoclavine-1-aldehyde by submerged fermentation, which is stored in the collection of microorganisms of the Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague under the designation CC 5/86. The strain produces high amounts of clavine alkaloids under conditions of deep cultivation with a favorable representation of chanoclavine and chanoclavine-1-aldehyde at a low level of accompanying clavine alkaloids and negligible formation of exopolysaccharides.

Description

The invention relates to a new industrial strain of the fungus Claviceps purpurea (Fr.) Tul. 59, stored in the collection of microorganisms of the Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague under the designation CC 5/86, characterized by high production of chanoclavine alkaloids of structural formula I during submerged cultivation.

and chanoclavin-1-aldehyde of structural formula II

Both alkaloids are present only in trace amounts in wild and industrial strains of the genus Claviceps. For these reasons, their pharmacological effects have not yet been determined. Chanoclavine is thought to have a hallucinogenic effect in mixtures with other clavine alkaloids produced by plants of the Convolvulaceae family.

Chanoclavine-l-aldehyde can be considered a suitable substrate for the preparation of new semi-synthetic derivatives of ergot alkaloids. The invention will enable a thorough investigation of the biological effects of both alkaloids and the preparation of their semi-synthetic derivatives.

The advantages of the new strain according to the invention are: high submerged production of clavine alkaloids (3 to 6 g/L), rich presence of chanoclavine (40 to 60%) and chanoclavine-l-aldehyde (20 to 30%), low content of accompanying alkaloids (10 to 15% elymoclavine, 5 to 10% agroclavine), intensive sporulation, low occurrence of exopolysaccharides.

Characteristics of the new strain C. Purpurea 59

The patented strain 59 forms on agar nutrient medium T2 (C. Spalla, in Genetics of Industrial Microorganisms, eds. Z. Vaněk, Z. Hošfálek, J. Cudlín, Elsevier, Amsterdam 1973, p. 393) a continuous, locally lumpy, wrinkled flat layer of gray-beige myoelia with drops of light brown exudate on the surface of the culture.

It secretes a brown pigment into the agar medium. The aerial mycelium of the culture sporulates profusely. It mainly forms microconidia. Submerged cultures are characterized by shorter hyphae of branched multicellular mycelium initially consisting of sphacelial cells.

The discharge of the culture under submerged incubation conditions is accompanied by the formation of macro- and microconidia. The mycelium of older submerged cultures (over 10 to 12 days) thickens and breaks up into arthrospores and chlamydospores. The formation of clavine alkaloids is accompanied by a wavy red exocellular pigment, which turns black in older cultures.

Origin of the new tribe

The new production strain 59 was bred from the industrial strain C. purpurea 35 (AO 246 704), derived from the industrial strain CP7 (AO 204 301), by UV light mutation in three steps. All cultivations during strain breeding were carried out in the dark at 24+1 °C. Spores of the mother culture of the strain

35, which had been cultured for 3 to 4 weeks in Endo tubes on T2 agar slants, were suspended in saline.

The resulting suspension contained ^3.9.107 spores/ml. From isolates after UV irradiation at a dose of 2

000 erg/mm, strain 41 was selected, which formed flat colonies, cooperated well, and whose spore germination was 67%. Under submerged fermentation conditions, it produced 3 to 4 g/l of alkaloids.

In the spectrum of these alkaloids, agroclavine predominated, as in the case of the parent strain 35.

By mutating spores of strain 41 with a UV light dose of 2,000 erg/mm using the above procedure, isolate 37 was obtained with an alkaloid spectrum changed in favor of chanoclavine and chanoclavine-l-aldehyde „ 2 with a yield of 2 to 3 g/1. By mutating spores of strain 37 with a UV light dose of 2,000 erg/mm, strain 59 was obtained with a higher production of total alkaloids (3 to 6 g/1). A comparison of the new strain 59 with the starting strains is shown in Tab. 1.

The method of cultivating the new strain C. purpurea 59 to obtain the starting material for the isolation of chanoclavine and chanoclavine-l-aldehyde and for the subsequent production of final products and medicinal preparations is illustrated in the example of use, without this example in any way limiting the properties of the new strain, the method of its cultivation and use in industrial production.

Example 1

In the short term, the new strain 59 is stored in the dark at 4 °C with protection against drying out of the medium in the form of 21-day cultures grown on slants of T2 agar medium in Endo tubes at 24-1 °C. The stored cultures are regularly re-inoculated at three-month intervals. In the long term, strain 59 is stored by a specific lyophilization procedure.

The initial inoculum for submerged fermentation of clavine alkaloids is prepared by washing the spores of a maximum four-week-old culture of strain 59 grown on T2 agar medium. From this suspension 7, approximately 5.10 spores in a volume of 2 ml are transferred to 60 ml of liquid nutrient medium T1 (Spalla C., in Genetics of Industrial Microorganisms, eds. Z. Vaněk, Z. Hošřálek, J. cudlín, Elsevier, Amsterdam 1973, p. 393) in Erlenmeyer flasks (300 ml).

The inoculated medium is incubated for 10 days in the dark at 26 - 1 °C on a rotary shaker (240 rpm, eccentric 5.5 cm). 4.5 ml of the inoculum thus obtained is transferred to Erlenmeyer flasks (300 ml) each containing 60 ml of CS2 fermentation medium (AO 199 986). The inoculated fermentation medium is incubated for 21 days in the dark at 26 - 1 °C on a rotary shaker (240 rpm, eccentric 5.5 cm).

Alkaloids secreted into the fermentation medium are determined colorimetrically (G. J. Banks et al., J. Gen. Microbio. 82, 345, 1974) and the composition of the alkaloid mixture densitometrically, i.e. by liquid chromatography in a developing mixture of chloroform-methanol-NH^ (8:2:0.02) on Silufol plates without UV detection. After spraying the chromatogram with Ehrlich's solution and drying with hot air, the spots were analyzed on a Shimadzu densitometer at a wavelength of 585 nm.

Table 1: Comparison of the new strain C. purpurea (Fr. ) 59 sec by the original tribes 35, 41 and 37 Culture Sign 59 37 41 35 surface gray-white gray-white gray-white gray-white mycelium flat flat flat wrinkled x0 > 0 h (0 pigment brown reddish brown reddish brown reddish brown the conidiation good good good good germination conidia (%) 10 25 67 10

continuation of table 1

Culture Sign 59 37 41 35 Total alkaloids (14 d/mcg/ml) 3,760 2,500 4,850 2,800 I dominant chanoclavine chanoclavine agroclavin agroclavin (0 alkaloids chanoclavin-l- chanoclavin- elymoclavin elymoclavin and -aldehyde -1-aldehyde ω ' exocellular glucans footprints footprints 10 12 g/1 pigment wine to black reddish brown reddish brown reddish brown

Chanoclavine-l-aldehyde has an azure (greenish) blue color after spraying with Ehrlich's reagent, which distinguishes it from the blue-violet stains of other clavine alkaloids.

Claims (1)

Industrial strain of Claviceps purpurea (Fr.) Tul. 59 CC 5/86 for the preparation of chanoclavin and chanoclavin-1-aldehyde alkaloids by submersible fermentation.