CS209783B1 - Method of cultivating coprinus sp. strains - Google Patents

Description

BACKGROUND OF THE INVENTION The present invention is directed to a method of breeding strains of basidiomycetes of Coprinus sp. Capable of degrading cellulose present in a straw substrate while increasing the content of nitrogen and free glucose in the cultured substrate.

The original strain of Coprinus sp. isolated from Jáchymov mines grew very slowly on the straw substrate, and after 12 to 14 days of superficial activation, the total nitrogen content, expressed as total nitrogen x 6.25, increased from 2 to 3 Z, to 12 to 14 with the final substrate containing only traces of monosaccharides. Feeding experiments with the substrate thus obtained stimulated the growth of fed animals when used as a supplement to the crude protein in traditional feed. By passive selection of spontaneously forming variants of Coprinus sp. it is not possible to achieve an economically advantageous t. j. 18-20% of crude protein containing straw substrate, prepared industrially.

Active breeding was used, especially for strains of the genus Penicillium / Μ. P. Backus, J.F. Staufer, Mycologia, 477, 446, 1955; J. Fuška, E. Welwardová: Biology 2 4, 69 1, 1 969) and of the genus Cefalosporium / R. P. Elander: Microbiologia p. 517, 1976 / UV wavelength 2530 A.

Spores of the strain Coprinus sp. like other basidiomycetes, it is relatively highly resistant to UV and X-rays / M. Semerjieva, M. Blumaver: Studia Biophys, 36/37, 183 (1973). A better mutant formation effect has been achieved with the mutagens applied by us, which react with cellular desoxyribonuclein as alkylating agents, prevent the replication of desoxyribonucleinic acid, which at the same time cannot serve. as a matrix for ribonucleic acid synthesis. Thus, tris (3-chloroethylamine) hydrochloride and the mutant TS 160, which, in conjunction with the addition of substances which react with thiol groups blocking SH-enzymes such as iodoacetamide, increased the mutagenic effect induced by radiation and alkylating agents / E. J. Grajevsky, A. G. Tarasenko; Radilogija 6, 983 (1972). This is shown in Table 1.

T a b u I k a 1

Surviving Disputes Mutants with prod. N-substances over / Z / 120% control / 2 /

inspection 100 - Iodoacetamide / 0.1 rAM / 85 - TS 160 / 0.004M / 70 1.96 Iodoacetamide / 0.1 mM / TS 160 /0.004M/ 55 13.76

These deficiencies are eliminated by the method of breeding of the basidiomycetes Coprinus sp. , the essence of which is that the spores of the crust are affected. mutagenic agents reactive with its desoxyribonucleic acid in terms of alkylation reactions such as 0.005 to 0.04 M bis / beta-chloroetamine / hydrochloride or 0.005 to 0.04 M N-mustard and SH-blocking agents such as 0.005 to 0 100 µl of iodoacetates or N-ethylraaleimide, for 10 to 30 minutes, an aliquot of the spore suspension is diluted with a 2% glycerin solution and sown on an agar plate and grown on a straw substrate after growth.

A strain of the basodimycetes Coprinus sp. Czq »CCMF-674, with increased production of crude protein while enriching the cultured straw substrate with monosaccharides.

In addition, the strain of the basidiomycetes Coprinus sp, C2Q-CCCF-674 can be cultivated in a straw substrate, treated with the addition of nitrogen, potassium and phosphorus-containing substances such as urea and potassium salts of phosphoric acid, and cultivated at 28-36 ° C. to obtain a strain with a higher proportion of crude protein and monosaccharides in the straw substrate.

The advantage of this procedure is that both the variation margin of the obtained mutants (from 140-170% of the N-substances compared to the control) is increased and the number of mutants capable of increasing the total N-content above 120% relative to the control in the culture substrate. trunk. The new strains obtained by this process, in addition to increasing the total N-content, leave a portion of biotrans-formed cellulose in the form of monosaccharides, predominantly glucose, in the culture process, as shown in Table 2.

Table 2

tribe

The content of N-substances in the substrate? Z increases relative to the initial circulation

Concentration of reducing sugars in the substrate (Z)

Coprinus sp. authentic Coprinus sp. 12-14 - 2-3 CCMF-674 -21- 64 4-6 Coprinus sp. θ20 lá according to experiment 3 1 8-19 28 to 35 3-5

Monosaccharides are readily available for the sphincter metabolic processes, thereby increasing the energy effect of the cultured substrate. After cultivation of the substrate, the strains of Coprinus sp. there was no production of substances with potential mutagenic or carcinogenic effects as evidenced by the Amesa / B tests. N. Ames, J. Mc. Cann, E. Yamasaki Mutation Res ,,

31, 347 (1975). In in vivo experiments, no negative symptoms were seen in the test animals after administration of the enriched substrate.

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In this way, the whole process of cellulose biotransformation is more economical and attractive to use. The strains of Copri3 nus sp. C ^ g "CCCF-674 _° to the verification of the operational scale created on average 18 to 21 from the N-compounds in the substrate, a 28-64% increase over the control strain.

The preparation of the strains by this procedure and the verification of their properties are given in the examples.

Example 1

Disputes of Coprinus sp. were suspended in water and filtered through a cotton wool pad to remove spores and vegetative mycelium fragments, the number of spores being 3.10.

The tris mutagen solution (beta-chloroethylamine) was added in a sterile manner to the spore suspension.

HCl in an amount such that its final concentration in the solution was 0.008 M. The prepared spore suspension was mixed in a 1: 1 sterile manner with an aqueous solution of iodoacetamide at a concentration of 0.2 mH. After 20 min. For mutagenic treatment with constant agitation, an aliquot of the spore suspension was removed and transferred to a 1% glycine solution. The spore suspension was further diluted and the spores were sent to the wort agar surface by 0.1 ml spores per 10 cm diameter agar surface. After 48 to 72 hours By cultivation at 34 ° C, the grown monocoloniums were transferred to sloping wort agar and gradually evaluated by both submerged and surface cultivation on a straw substrate. Coprinus strain, when evaluated on a commercial scale, after 12 days of culture yielded a product containing up to 21.7 N-substances per gram dry matter.

Example 2

Disputes of Coprinus sp. suspended in water were discarded by filtering the mycelium fragments and mixed with the tris (beta-chloroethylamine) mutagen solution. HCl in such a ratio that the final mutagen concentration was 0.008 M. The prepared spore suspension was mixed in a 1: 1 ratio with a 0.01 M aqueous solution of N-ethylmaleimide. After 20 min. For mutagen treatment, an aliquot of the spore suspension was collected and diluted with 1% glycine solution. After a thinner dilution chosen to produce monocolonies on the surface of the agar plates, 0.1 ml of the spore suspension was spread over the surface of the wort agar (as in Example 1). After 48 to 72 hours by cultivation at 34 ° C, the grown monocolonies were transferred to sloping wort agar and gradual after growth was evaluated by surface cultivation on a straw substrate. Strain Coprinus sp. The conditions of this experiment achieved a production of 19.8% N-substances per gram dry matter under operating conditions.

Example 3

To 1 kg of hot-95 ° C steamed straw substrate with water for 60 min. 3.5 liters of an aqueous solution containing 32 g of urea and 1.6 g of K 2 PO 4 are added. The substrate is inoculated with a vegetative inoculum of 7.5 to 10 7 volumes per volume of substrate with one of the strains of Coprinus sp. The inoculated substrate is spread in a 5 to 8 cm thick layer on the culture dishes and allowed to cultivate at 28 to 36 ° C and high humidity for 10 to 12 days. The product obtained contains 18 to 21% N-substances per gram of dry matter and 3.5 to 4.5% water-soluble reducing sugars.

Claims (2)

209783 nus sp. The CCCF-674, which generated an average of 18 to 21% of the substrate in the assay at 28 to 64% increase over the control strain. The preparation of strains by this procedure and verification of their properties are given in the examples. Example 1 Coprinus sp. spore agglomeration and vegetative mycelium fragments were added to the spore suspension with a sterile solution of the tris / beta-chloroethylamine mutagen. HCl in such a quantity that the final concentration in the solution was 0.008 M. The prepared spore suspension was sterically mixed 1: 1 with an aqueous solution of iodoacetamide at a concentration of 0.2 mH. After 20 min. Mutation of the mutagen under stirring was an aliquot of the spore suspension removed and transferred to a 1% glycine solution. The spore suspension was further diluted and the spores sown on the surface of the wort agar, 0.1 ml of spore on a 10 cm diameter agar surface. After 48 to 72 hours at 34 ° C, the grown monoclonal plants were transferred to the slant wort and sequentially subjected to submerged and surface culture on a straw substrate. The Coprinus strain, on an operational scale, after 12 days of culture, yielded a product that contained up to 21% of N-substances per gram of dry matter. Example 2 Coprinus sp. suspended in water were freed from the mycelia fragments and mixed with the tris / beta-chloroethylamine mutagen solution. HCl in such a ratio that the final concentration of the mutagen was 0.008 M. The prepared spore suspension was mixed 1: 1 with 0.01 M aqueous N-ethylmaleimide. After 20 min. The mutagen was treated with an aliquot of the spore suspension and diluted with 1% glycine solution. After a dilute dilution chosen to produce monocolonies on the surface of the agar plates, 0.1 ml of the spore suspension was ground to the surface of the wort agar (as in Example 1). After 48 to 72 hours at 34 ° C, the monocolonies grown were transferred to sloping wort agar, and were grown by surface-cultivation on a straw substrate after growth. Strain Coprinus sp. The results obtained under the conditions of this experiment yielded 19.8% N-substances per gram of dry matter under operating conditions. Example 3 To 1 kg of straw substrate boiled with hot - 95 ° C - water for 60 min. 3.5 liters of an aqueous solution containing 32 g of urea and 1.6 g of Kl 2 PO 3 are added. The substrate is an inoculum-inoculum inoculum of 7.5-10% by volume per substrate volume of one of Coprinus sp. The inoculated substrate is blotted in a 5-8 cm-thick layer onto culture dishes and allowed to cultivate at 28-36 ° C and high air humidity for 10-12 days. The product obtained contains 18-21% N-substances per gram-dry and 3.5-4.5% water-soluble reducing sugars. SUBJECT OF THE INVENTION 1. A method of breeding a strain of the basidiomycetus Coprinus sp. characterized in that the sporycene is caused by the mutagenic reactants with its cellular desoxyronuclein acid in the sense of alkylation reactions such as 0.005-0.04 M tris / beta-chloroethylaraine hydrochloride or 0.005-0.04 M N- yperite and SH-group blocking agents such as 0.005-0.01M iodoacetamide or N-ethylmaleimide for 10-30 minutes, dilute an aliquot of the spore suspension with 1 µL of glycine solution and aspirate to the agar surface valid after the growth is cultivated on a straw substrate or cultivated at 28-36 ° C under high humidity on a treated straw substrate by adding a solution of nitrogen-containing, potassium-containing substances to the straw substrate 209783 4 with 95 ° C water. and phosphorus, preferably in the urea and potassium salts of phosphoacetic acid » 2. Basidiomycetes strain Coprinus sp. CCMF-674, degrading cellulose in a straw substrate obtained according to the method of Severopafia. n. p .. eivod 7. Most