RU2717974C1 - Nutrient medium for cultivation of chaga fungus - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to the biotechnology. Disclosed is a nutrient medium for cultivation of a fungus Inonotus obliquus based on potato decoction, containing (g/l) glucose 20.0; glycine 0.1–0.5; melanin complex with iron 0.1–0.5; polyphepan 0.1.

EFFECT: invention provides higher output of the chaga fungus biomass.

1 cl, 1 tbl, 3 ex

Description

The invention relates to biotechnology, namely, nutrient media for the cultivation of the basidiomycetes Inonotus obliquus (chaga).

Inonotus obliquus birch fungus is a medicinal raw material, since in addition to the main active component of melanin, it also includes biologically active compounds such as polysaccharides, phenolic compounds, terpenoids, steroids, carbohydrates, etc. (Sysoeva M.A. : monograph / M.A. Sysoeva. - Kazan: Publishing House of KNITU, 2013 - 226 p.). On its basis, various drugs are produced that are used for the prevention and treatment of gastrointestinal diseases, postoperative treatment of malignant neoplasms. The long time of renewal of this fungus in natural conditions makes the development of biotechnologies for the cultivation of fungus chaga, which will replace natural raw materials, relevant. During artificial cultivation of the fungus, it is necessary to select the conditions for its cultivation, including the choice of a nutrient medium, as well as growth stimulants, which will provide a large amount of biomass, as well as target products.

A known nutrient medium for growing the fungus Inonotus obliquus, which includes glucose, potassium dihydroorthophosphate, potassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, peptone, copper sulfate, tyrosine, thiamine, corn extract (patent BY10670, C12N 1/14, С12Р 19/19 00, published on June 30, 2008).

A known nutrient medium for growing the fungus Inonotus obliquus, which includes: glucose, malt extract, peptone, yeast extract, magnesium sulfate, potassium hydrogen phosphate, potassium dihydroorthophosphate, thiamine-HCl, calcium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, sulfate copper, cobalt chloride, sodium hydrogen phosphate, potassium tetraborate (Kim, Y. O. Immunostimulating effect of the endo-polysaccharide produced by submerged culture of Inonotus obliquus / OY Kim, et al. // Life Sciences. - 2005. - V. 77. - P. 2438-2456).

The disadvantages of known nutrient media include the multicomponent composition and the use of a large number of inorganic substances. In addition, the authors do not give the amount of seed, which does not allow us to evaluate the efficiency of the use of media, for example, by the yield of biomass and / or melanin.

As a prototype, a nutrient medium was selected for growing chaga mushroom based on potato broth containing glucose and yeast extract (Cho, N.-S. Optimization of in Vitro cultivation of Inonotus obliquus / N.-S. Cho, Y.-S. Shin // Mokcae Konghak. - 2005. - V. 33. - P. 92-98.).

The disadvantage of this nutrient medium is to obtain the biomass of the fungus in low yield.

The technical problem of the alleged invention is the development of a nutrient medium that will allow you to get the biomass of chaga mushroom in high yield.

The technical problem is solved by a nutrient medium for growing chaga mushroom based on potato broth containing glucose, glycine, a complex of melanin with iron and polyphepan at a mass concentration of components in g / l: glucose - 20.0; a complex of melanin with iron - 0.10-0.50; glycine - 0.10-0.50; polyphepan - 0.1.

Using the proposed nutrient medium allows you to get the biomass of fungus chaga with a yield of 2 times greater compared to the prototype.

In the proposed nutrient medium, the following stimulants are added to the potato broth containing glucose: glycine, a complex of melanin with iron and polyphepan.

Glycine is an aliphatic amino acid found in proteins that act as enzymes for the fungus Inonotus obliquus. The enrichment of the nutrient medium with the amino acid glycine will stimulate the growth of the fungus and, therefore, increase the amount of biomass accumulated by the fungus chaga.

The use of a complex of melanin with iron will enrich the nutrient medium with iron ions, which serve as cofactors of the enzymes peronoxidase and catalase of the fungus Inonotus obliquus, which are necessary for the biosynthesis of melanins and other biologically active substances, as well as for its active growth. For better assimilation of iron ions, a complex of melanin with iron was used, since iron ions are better absorbed by living organisms if they enter into complexes with organic substances. The melanin complex with iron is obtained as a result of the interaction of freshly precipitated melanin of the fungus Inonotus obliquus with a saturated solution of FeSO ₄ . The content of iron ions Fe ²⁺ and Fe ³⁺ in the resulting complex according to x-ray fluorescence analysis is 3.7% (Urazlina, L.N. Modification of chaga melanin / L.N. Urazlin, PP Garaev, V.R. Khabibrakhmanova, M. A. Sysoyeva, A.V. Pyatayev // IV International Baltic Maritime Forum: abstracts - Kaliningrad, 2016.S. 93-94).

Polyphepan is a hydrolytic lignin, which is a source of phenolic compounds and lignin necessary for the growth of the fungus Inonotus obliquus.

To obtain the biomass of the fungus Inonotus obliquus used strain SUB2092728. The proposed strain of the fungus was isolated by tissue from fruit bodies growing in the Zvenigovsky district of the Mari El Republic and deposited in the GenBank database. The strain is stored at 4-6 ° C in glucose-potato medium. The fungus Inonotus obliquus SUB2092728 is an aerobic, the optimum growth temperature is 24-27 ° C and a pH of 6.0-7.0.

The nutrient medium was prepared as follows. 5: 1 water was added to the chopped potato, boiled and filtered. The resulting broth was brought up to 1 liter with water, poured into 500 ml vials and the components of the nutrient medium were added to it: glucose, a complex of melanin with iron, glycine and polyphepan. Nutrient medium was autoclaved at 1 atm. within 30 minutes.

The invention is illustrated by the following examples.

Example 1. The culture of the strain of the fungus Inonotus obliquus was grown on agarized glucose-potato medium for 5-7 days. The resulting inoculum in an amount of 0.001% was seeded with a bacteriological loop into a 500 ml vial with a nutrient medium of the following composition in g / l: glucose - 20.0; a complex of melanin with iron - 0.1; glycine - 0.1; polyphepan - 0.1. Cultivation was carried out for 12 days under stationary conditions, at a temperature of 27 ± 2 ° C. The grown biomass of the fungus was separated by filtration from the culture fluid, washed repeatedly and dried at room temperature.

Examples 2-3 are similar to example 1. The compositions of the nutrient media are shown in the table.

The determination of biomass yield was carried out gravimetrically. The results of repeated experiments were processed using the "Statistics 10.0" program (at p = 0.95, n = 5) and are shown in the table.

Analysis of tabular data shows that the use of the proposed nutrient medium for growing chaga mushroom leads to an almost 2-fold increase in biomass yield compared to the prototype. A decrease and / or increase in the concentration of substances introduced into the nutrient medium is inefficient, since it does not lead to a change in the biomass yield.

Obtained by the proposed method, the biomass of the fungus Inonotus obliquus is a promising object for the development on its basis of medicinal, cosmetic and biologically active additives.

Claims (5)

Nutrient for cultivation of chaga fungus based on potato broth containing glucose and stimulating additives, characterized in that glycine, a complex of melanin with iron and polyphepan are used as stimulating additives at a mass concentration of components in g / l: glucose - 20.0 glycine - 0.1-0.5 complex of melanin with iron - 0.1-0.5 polyphepan - 0.1.