RU2808749C2 - Nutrient medium for cultivating basidiomycete fungus inonotus obliquus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient medium for cultivating the basidiomycete fungus Inonotus obliquus. A nutrient medium for cultivating Chaga mushroom based on potato broth contains glucose, glycine, melanin-iron complex, polyphepane and enzistal at a mass concentration of components in g/l: glucose - 20.0; glycine - 0.3; melanin complex with iron - 0.1; polyphepane - 1.9; enzistal - 0.6.

EFFECT: use of the proposed nutrient medium in comparison with the prototype leads to an increase in the yield of fungal biomass by 2.3-2.7 times.

1 cl, 1 tbl, 5 ex

Description

The invention relates to biotechnology, namely to nutrient media for cultivating the basidiomycete fungus Inonotus obliquus (chaga).

The mushroom Inonotus obliquus (chaga) is a valuable natural raw material because it contains various biologically active substances, including the main active component - melanin (Szychowski, KA Inonotus obliquus - from folk medicine to clinical use / KA Szychowski et al.// Journal of Traditional and Complementary Medicine. - 2021. - V.11. - P. 293-302 doi:10.1016/j.jtcme.2020.08.003). Today, the drug Befungin and dietary supplements based on crushed raw materials, liquid and dry extracts are produced from natural chaga raw materials in Russia and other countries. The spectrum of therapeutic activity of chaga preparations is very wide. They are known to have antioxidant, protective, antitumor, and immunomodulatory properties. It has been shown that chaga extracts and melanin exhibit antiviral activity against influenza viruses, herpes simplex type 2, immunodeficiency (HIV-1) and vaccinia (Patent RU 2375073, IPC A61K 38/06, A61P 31/18, publ. 10.12. 2009, Patent RU 2480227, IPC A61K 36/06, B01D 11/02, A61P 31/14, published 04/27/2013). The effectiveness of aqueous extracts and chaga melanin against a new coronavirus infection deserves special attention; it has been proven that they inhibit the replication of the SARS-CoV-2 coronavirus (Patent RU 2741714, IPC A61K 36/06, B01D 11/02, A61P 31/14, publ. 01/28/2021; Patent RU 2747018, IPC A61K 36/06, B01D 11/02, A61P 31/14, published 04/23/2021).

Large volumes of harvesting of the mushroom Inonotus obliquus , taking into account the long renewal time (at least 4-5 years), have led to the depletion of its reserves in nature. In this regard, it is relevant to develop biotechnology of the fungus Inonotus obliquus , which will allow replacing scarce natural raw materials with biomass obtained under artificial conditions. When cultivating a mushroom, the selection of conditions is important. The main requirement for a nutrient medium for cultivating a fungus is to provide the biomass with all the necessary components for its intensive growth and accumulation of target metabolites.

A liquid nutrient medium for surface cultivation of chaga mushroom based on potato broth containing glucose, glycine, melanin-iron complex and polyphepane is proposed at a mass concentration of components in g/l: glucose - 20.0; melanin complex with iron - 0.10-0.50; glycine - 0.10-0.50; polyphepane - 0.1. When surface cultivation of the fungus on this nutrient medium for 12 days, the biomass yield does not exceed 5 g/l. The low productivity of the fungus may be due to a long period of adaptation of the culture to the nutrient medium, as well as ineffective consumption of the substrate due to its growth only on the surface of the nutrient medium.

The deep method, when the culture is grown in a layer of nutrient medium, has higher productivity compared to the surface cultivation method.

A known liquid nutrient medium for deep cultivation of the basidiomycete fungus strain Inonotus obliquus TV-18, which contains, g/l: glucose - 30; tryptone - 10.0; yeast extract - 5.0; KN ₂ RO ₄ - 1.1; K ₂ HPO ₄ - 4.4; MgSO ₄ ×7H2O - 0.25; water - up to 1 l. The yield of biomass in dry matter on the 10th day of cultivation averages 18.2 g/l (Patent RU 2716590, IPC C12N 1/14, A61P 31/00, A61P 35/00, published 03/12/2020).

A nutrient medium for growing the fungus strain Inonotus obliquus BIM F-350 D has been proposed, which contains, g/l: glucose-30; peptone-3.0; corn extract - 2 ml; KN ₂ RO ₄ - 1.0; K ₂ HPO ₄ - 1.0; (NH ₄ ) ₂ SO ₄ - 1.4; MgSO ₄ - 0.25; CuSO ₄ ×5H ₂ O - 0.08; tyrosine - 0.2; thiamine - 300 mcg; water - up to 1 l. The biomass yield during deep cultivation for 10 days was 12.5 g/l (Patent BY 10670, IPC C12N 1/14, C12P 19/00, publ. 06/30/2008).

Cultivation of the fungal strain Inonotus obliquus F-1244 in flasks on a rocking chair was carried out on a nutrient medium with the composition, g/l: glucose - 30; tryptone - 2.5; yeast extract - 1.25; KH ₂ PO ₄ - 1.1; K ₂ HPO ₄ - 4.4; MgSO ₄ - 0.25; water - up to 1 l. The accumulation of biomass within 12-15 days is 17 g/l (Ananko G.G. Melanins from deep culture of Inonotus obliquus and their antiviral activity against herpes simplex virus type 2 / G.G. Ananko, T.V. Teplyakova, A .V. Bardasheva, T.N. Ilyicheva // Advances in medical mycology. - 2015. - pp. 384-388.).

A review of the literature shows many more studies on the deep cultivation of various strains of the fungus Inonotus obliquus , while the nutrient media always have a complex composition. Thus, the main disadvantages of nutrient media proposed for the cultivation of various strains of the fungus Inonotus obliquus include multicomponents, the use of expensive stimulating additives (vitamins, amino acids, protein hydrolysates) and a large amount of inorganic substances. In addition, the authors do not always provide the amount of seed material, which does not allow assessing the efficiency of using media, for example, by the yield of biomass and/or melanin. It is also worth noting that the existing research lacks focus on reducing the adaptation period of the culture of the fungus Inonotus obliquus when sown on the nutrient media used.

As a prototype, a nutrient medium was chosen for the surface method of growing chaga mushroom based on potato broth containing glucose, glycine, melanin-iron complex and polyphepane (Patent RU 2717974, IPC C12N 1/14, C12R 1/645, published 03/27/2020) .

The disadvantage of this nutrient medium is the production of fungal biomass with low yield.

The technical problem of the proposed invention is the design of a liquid nutrient medium that will allow obtaining biomass of the fungus Inonotus obliquus with high yield using the submerged cultivation method.

The technical problem is solved by constructing a liquid nutrient medium for deep cultivation of the fungus Inonotus obliquus based on potato broth containing glucose, glycine, melanin-iron complex, polyphepane and enzistal at a mass concentration of components in g/l: glucose - 20.0; glycine - 0.30; melanin complex with iron - 0.10; polyphepane - 1.9; enzistal - 0.60.

The use of the proposed nutrient medium makes it possible to increase the amount of biomass obtained from the fungus Inonotus obliquus by 2.3-2.7 times compared to the prototype (Table Examples 2 and 3).

In the proposed nutrient medium, enzistal is added to the potato broth containing glucose and growth-stimulating components: glycine, melanin-iron complex and polyphepane. This is a medicinal enzyme preparation (registration number PN015075/01) containing 192 mg/g pancreatin, which has amylolytic, lipolytic and proteolytic effects. When using it, enzymatic hydrolysis of various components of the nutrient medium is carried out to simple compounds. In this regard, the use of an enzyme preparation will make it possible to enrich the nutrient medium with easily digestible and accessible components for the fungus, mainly di- and monosaccharides, which are sources of energy, amino acids necessary for the synthesis of proteins, and fatty acids for the formation of lipids. When cultivating a fungus, a lag phase is observed at the beginning, in which the culture adapts to new environmental conditions, and the main adaptation process is the synthesis of the necessary enzymes for the absorption of nutrients. The addition of enzistal will reduce the lag phase during deep cultivation of the fungus Inonotus obliquus , because the bulk of the nutrients in the medium will be hydrolyzed by pancreatin. The absence of the need for the fungal culture to synthesize enzymes to break down the substrate will allow it to switch to active growth and synthesis of target products at the metabolic level.

It is worth noting that, by analogy with the prototype, the same stimulating additives are used in the constructed liquid nutrient medium for deep cultivation of the fungus Inonotus obliquus , but their dosages are changed. The exception is glycine; its content in the constructed nutrient medium is 0.30 g/l, the same as in the nutrient medium according to the prototype. Despite the fact that glycine is an important amino acid in the metabolism of the fungus, and under conditions of deep cultivation the need of the fungal culture for it may increase, its content in the proposed nutrient medium was left unchanged. This is due to the fact that due to the enzymatic hydrolysis of potato broth proteins under the influence of enzyme, the constructed nutrient medium has a richer amino acid composition compared to the prototype.

The dosage of the melanin complex with iron was reduced from 0.3 to 0.1 g/l compared to the prototype based on the results of experimental studies. The complex of melanin with iron is the initiator of its active growth, as well as an important source of available iron ions, which the fungus uses for the biosynthesis of the enzymes peroxidase and catalase necessary for the synthesis of melanins and other secondary metabolites. Typically, the need for metal ions and biosynthesis initiators in producers is low and does not exceed 0.1 g/l (Zheng W. Effects of culture media and three metal ions on the accumulation of lanosterol and ergosterol in cultured mycelia of Inonotus obliquus / W. Zheng et al.// Mycosystema. - 2008. - 27. - R. 126-139).

It is proposed, in comparison with the prototype, to increase the dosage of polyphepane in the constructed nutrient medium from 0.1 g/l to 1.9 g/l. Polyphepane is a hydrolytic lignin. Its introduction into the nutrient medium makes it possible to provide the fungal culture with phenolic compounds, which it usually utilizes from wood during development in natural conditions. In addition, the fungus can immediately use the components of hydrolyzed lignin as structural units in the biosynthesis of melanin.

To obtain biomass of the fungus Inonotus obliquus, strain SUB2092728 was used. The proposed strain of the fungus was isolated by the tissue method from fruiting 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 on glucose-potato medium. The mushroom Inonotus obliquus SUB2092728 is an aerobe, the optimal growth temperature is 24-27°C and pH 6.0-7.0.

The nutrient medium was prepared as follows. Water was added to the crushed potatoes in a ratio of 5:1, boiled for 30 minutes and filtered. The resulting broth was brought to 1 liter with water, poured into 500 ml flasks, and the components of the nutrient medium were added to it: glucose, melanin-iron complex, glycine and polyphepane. Enzistal in powder form was added to prepared flasks with a nutrient medium, mixed thoroughly and incubated at a temperature of 37±1°C for 1 hour. After this, the nutrient medium in the flasks was sterilized at a temperature of 115±3°C for 40 minutes.

The invention is illustrated by the following examples.

Example 1. A culture of the fungus Inonotus obliquus strain SUB2092728 was grown on glucose-potato agar medium for 5-7 days. The mycelium was selected from the growth zone of the grown culture colony with a scalpel and added in an amount of 0.03±0.001 g/l into flasks with a nutrient medium in a volume of 200 ml of the following composition, in g/l: glucose - 20.0; melanin complex with iron - 0.1; glycine - 0.3; polyphepane - 1.9. Cultivation was carried out in an Infors AG CH-4103 Bottmingen (USA) shaker-incubator for 12 days under submerged conditions with a stirring speed of 200 rpm, at a temperature of 26±2°C. The grown fungal biomass was separated by filtration from the culture liquid, washed several times and dried at a temperature of 40±2°C.

Example 2. A culture of the fungus Inonotus obliquus strain SUB2092728 was grown on glucose-potato agar medium for 5-7 days. The mycelium was selected from the growth zone of the grown culture colony with a scalpel and added in an amount of 0.03±0.001 g/l into flasks with a nutrient medium in a volume of 200 ml of the following composition, in g/l: glucose - 20.0; melanin complex with iron - 0.1; glycine - 0.3; polyphepane - 1.9; enzistal - 0.6. Cultivation was carried out in an Infors AG CH-4103 Bottmingen (USA) shaker-incubator for 12 days under submerged conditions with a stirring speed of 200 rpm, at a temperature of 26±2°C. The grown fungal biomass was separated by filtration from the culture liquid, washed several times and dried at a temperature of 40±2°C.

Example 3 is similar to example 2. The duration of cultivation is 15 days.

Example 4. A culture of the fungus Inonotus obliquus strain SUB2092728 was grown on glucose-potato agar medium for 5-7 days. The mycelium was selected from the growth zone of the grown culture colony with a scalpel and added in an amount of 0.03±0.001 g/l into flasks with a nutrient medium in a volume of 200 ml of the following composition, in g/l: glucose - 20.0; melanin complex with iron - 0.1; glycine - 0.3; polyphepane - 1.9; enzistal - 2.7. Cultivation was carried out in an Infors AG CH-4103 Bottmingen (USA) shaker-incubator for 12 days under submerged conditions with a stirring speed of 200 rpm, at a temperature of 26±2°C. The grown fungal biomass was separated by filtration from the culture liquid, washed several times and dried at a temperature of 40±2°C.

Example 5 is similar to example 4. The duration of cultivation is 15 days.

Biomass yield was determined gravimetrically. The results of multiple experiments were processed using the “Statistics 10.0” program (at p=0.95, n=5) and are shown in the table.

Table
Biomass yield with deep cultivation method
mushroom Inonotus obliquus strain SUB2092728 Example Composition of the nutrient medium Duration
cultivation,
days Exit
biomass,
g/l Components Mass concentration, g/l Prototype Glucose
Melanin Iron Complex
Glycine
Polyphepan 20.0
0.30
0.30
0.10 12 4.90±0.23 Example 1 Glucose
Melanin Iron Complex
Glycine
Polyphepan 20.0
0.10
0.30
1.90 12 5.31±0.36 Example 2 Glucose
Melanin Iron Complex
Glycine
Polyphepan
Enzistal 20.0
0.10
0.30
1.90
0.60 12 11.28±0.32 Example 3 Glucose
Melanin Iron Complex
Glycine
Polyphepan
Enzistal 20.0
0.10
0.30
1.90
0.60 15 13.43±0.24 Example 4 Glucose
Melanin Iron Complex
Glycine
Polyphepan
Enzistal 20.0
0.10
0.30
1.90
2.70 12 3.92±0.08 Example 5 Glucose
Melanin Iron Complex
Glycine
Polyphepan
Enzistal 20.0
0.10
0.30
1.90
2.70 15 5.52±0.35

Analysis of the tabular data shows that despite the high productivity of deep cultivation, mainly due to more efficient mass transfer, its use for growing the fungus Inonotus obliquus in example 1 increases the biomass yield compared to the surface method of cultivating the fungus in the prototype slightly - no more than 0 .4 g/l. This indicates that the fungal culture, when reseeded from an agar nutrient medium into a liquid nutrient medium, experiences significant stress, to overcome which it needs a long period of time. To reduce the duration of the lag phase during deep cultivation of the fungus Inonotus obliquus , it is proposed to balance the nutrient medium in terms of the composition of easily digestible and accessible nutritional components. This is achieved by introducing enzistal into the nutrient medium at the stage of its preparation; under the influence of this enzyme preparation of complex action, enzymatic hydrolysis of the components of the nutrient medium takes place with the accumulation of simple compounds that can be immediately used by the culture of the fungus Inonotus obliquus for nutrition. As can be seen from the table, during deep cultivation of the fungus Inonotus obliquus on the proposed nutrient medium with the addition of enzyme in examples 2 and 3, the biomass yield increases by almost 2.5 times compared to the prototype and example 1. Active growth of biomass on the proposed nutrient medium is observed at the dosage enzistal 0.6 g/l. The addition of enzistal to the nutrient medium at a high dosage of 2.7 g/l led to a noticeable inhibition of the growth of the culture of the fungus Inonotus obliquus and, accordingly, a low biomass yield. Apparently, when enzistal is used in large quantities in the nutrient medium, a significant amount of simple compounds accumulates during enzymatic hydrolysis. This creates unfavorable conditions for the growth of the culture. For example, it is known that a high content of simple carbohydrates in the medium can cause osmotic shock in a cultivated biological object.

The biomass of the fungus Inonotus obliquus obtained by cultivation on the proposed nutrient medium is a promising object for the development of medicinal and cosmetic products and dietary supplements based on it.

Claims (1)

Nutrient medium based on potato broth for deep cultivation of the fungus Inonotus obliquus , containing glucose - 20 g/l and stimulating additives: glycine - 0.3 g/l, melanin complex with iron - 0.1 g/l, polyphepane - 1.9 g/l, enzistal - 0.6 g/l.