RU2699991C1 - Substrate for culturing xylotrophic basidiomycetes and a method for production thereof using methods of chemical modification of lignocellulose material - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology and can be used to produce fruit bodies of basidiomycetes. Disclosed are a method of preparing selective substrates and a selective substrate for producing fruit bodies of basidiomycetes. Method involves acid modification of lignocellulose raw material - agricultural wastes and wood processing industry – with aqueous solutions of mineral or organic acids with concentration of 0.5–5 wt%. at temperature of 80–135 °C and ratio of raw material to acid solution from 1:2 to 1:8 for 30–60 minutes, washing with water and adding to modified lignocellulose material nitrogen source in concentration of 10–20 wt% and chalk or gypsum in concentration of 2 wt% The selective substrate is obtained using said method.

EFFECT: inventions provide higher yield of fruit bodies of basidiomycetes.

8 cl, 3 tbl, 4 ex

Description

The invention relates to biotechnology. The invention can be used to obtain fruiting bodies of edible, medicinal-edible and medicinal mushrooms, as well as to produce a substrate for producing fruiting bodies of edible, medicinal-edible and medicinal mushrooms.

Known substrate (RF patent No. 2436283) for growing fruit bodies of mushrooms consisting of birch sawdust 96%, gypsum 1% and chalk 3%. The disadvantage of this substrate is the use of only birch sawdust as a basis, because This greatly limits the raw material base of production. In addition, in the description of the invention there is no source of nitrogen and the yield of fruiting bodies will greatly depend on the time of harvesting and freshness of the sawdust.

A known substrate for producing fruiting bodies of Ganoderma lucidum consisting of hydrolyzed diluted hydrochloric acid rye straw - 55%; oak sawdust - 25%; rye bran - 10%; cornmeal - 8%; chemically precipitated chalk - 1%; natural gypsum - 1% described in the patent of the Russian Federation No. 2453105. The specified substrate is prepared by preliminary hydrolysis of rye straw with a 0.5-1% hydrochloric acid solution, and then mixing hydrolyzed rye straw, oak sawdust with nutrients - rye bran, corn flour, chemically precipitated chalk, natural gypsum. The disadvantage of this method is the duration of the acid hydrolysis process (2 hours), as well as the use of valuable raw materials - oak sawdust, which are used to produce composite building materials, smoked gourmet foods, etc. In addition, the acid hydrolysis described in the RF patent No. 2453105 leads to an increase in the proportion of free mono- and oligosugars, which leads to a high risk of contamination by mold, yeast and bacteria.

Closest to the claimed invention is a method of obtaining a substrate (RF patent 2140145 C1) for growing edible mushrooms, including grinding vegetable raw materials, moistening them and sterilizing, characterized in that the raw materials are moistened with a solution of acetic acid to simultaneously hydrolyze the raw materials, where as vegetable raw materials use a mixture of cereal straw and coniferous waste in an amount respectively 35-60 and 40-65 wt. %, and the concentration of acetic acid is 1.5-2%.

The disadvantage of this method is the high content of free Sugars that are formed during the partial hydrolysis of plant materials, which leads to a significant increase in the risk of contamination.

The inventive method for producing substrates for the cultivation of xylotrophic basidiomycetes using methods of acid modification of lignocellulosic raw materials and substrates obtained by the proposed method includes the step of modifying lignocellulosic agricultural wastes and timber processing with acids. For this, lignocellulosic raw materials (mono- or multicomponent) are loaded into acid-resistant containers lined with fluoroplastic and acid solution is added in a concentration of 0.5-5%. Pretreatment is carried out at a temperature of 80-135 ° C, the ratio of raw materials to acid solution from 1: 2 to 1: 8 and the duration of the process is 0.5-1 hours. After completion of the process, the resulting pulp is washed with 1-4 volumes of water to remove sugars and tar as well as residues of unreacted acid. Next, an additional source of nitrogen is introduced, as well as chalk and lime. Acid pretreatment reduces the amount of sugars in the substrate, increases the lignin content, making such a substrate selective for “white rot” xylotrophic fungi, including edible, edible, and medicinal.

For the pretreatment of lignocellulosic raw materials, both organic and mineral acids can be used.

The proposed substrate may contain lignocellulosic raw materials from the list including waste from the wood processing industry and agriculture, such as sawdust of deciduous trees, sawdust of conifers, bark, leaves, needles, cereal straw, corn cob, flax bonfire, rice husk, etc. the list does not limit the types of lignocellulosic raw materials that can be used in the claimed method.

The proposed substrate may contain a nitrogen source selected from wheat, rye or oat bran, corn, soy or fish flour, different in source for the production of meal and bagasse. This list does not limit the types of nitrogen sources that can be used in the claimed method.

The inventive method is intended for the cultivation of medicinal and edible xylotrophic fungi from a list that includes the following species: Agrocybe aegerita, Flammulina velutipes, Ganoderma lucidum, Grifola frondosa, Hericium erinaceus, Hypsizygus marmoreus, H. ulmarius, Lentinususo citrin, eringii, P. djamor, P. ostreatus, P. pulmonarius, P. sajor-caju, Trametes versicolor. This list does not limit the types of xylotrophic fungi that can be used in the claimed method.

Example 1

Pretreatment was carried out in a sterilizing autoclave. To do this, lignocellulosic raw materials were loaded into chemically resistant containers, moistened with a counseling solution of mineral or organic acid according to Table 1, and heated to the appropriate temperature for 1 hour. The content of cellulose, hemicelluloses and lignin was determined by the methods described in NREL LAB-002, LAB-003.

Carrying out the acid pretreatment helped to remove the easily hydrolyzable polysaccharides (hemicelluloses and the amorphous part of cellulose), partially destroyed the intermolecular bonds between the lignin and cellulose molecules.

Example 2

The rate of development of modified substrates was determined by the following method: a mixture of lignocellulosic substrate, wheat bran and chalk taken in the ratio of 7.8: 2: 0.2 was placed in Petri dishes, moistened with 80% water and sterilized. Inoculation with a fungal culture was carried out using agar blocks with a fungal culture of 2 * 2 * 2 mm in size. The size of the fungal colony was measured on day 8.

Acid modification of lignocellulosic raw materials led to an increase in the growth rate of xylotrophic basidiomycetes by 20-50%, which significantly reduced the time for the development of blocks by the fungus during cultivation. An increase in the growth rate not only reduces the time of technological cycles, but also reduces the likelihood of contamination by other microorganisms - competitors for the substrate.

Example 3

Obtaining substrates for solid-phase cultivation.

Substrate 1. Coniferous and deciduous sawdust in a ratio of 2: 1 was placed in chemically resistant containers and mixed with a solution of hydrochloric acid with a concentration of 1.5% in a ratio of 1: 2 and placed in an autoclave. Processing time 0.5 hours, temperature 135 ° C. The feed was cooled to room temperature, transferred to a filter and washed once with water and allowed to drain excess moisture. In the resulting base was made 15% of the mass. wheat bran, 2% chalk and thoroughly mixed. The resulting substrate was packaged in polypropylene bags with glued filters and pasteurized.

Substrate 2. Tree bark and wheat straw in a 1: 4 ratio were placed in chemically resistant containers and mixed with a 2% sulfuric acid solution in a 1: 3 ratio and placed in an autoclave. Processing time 1 hour, temperature 121 ° C. The feed was cooled to room temperature, transferred to a filter and washed once with water and allowed to drain excess moisture. 10% of the mass, rye bran, 2% of chalk were added to the resulting base and mixed thoroughly. The resulting substrate was packaged in polypropylene bags with glued filters and pasteurized.

Substrate 3. Needles and flax bonfire in a ratio of 1: 8 were placed in chemically resistant containers and mixed with a solution of phosphoric acid with a concentration of 2.5% in a ratio of 1: 4 and placed in an autoclave. Processing time 1 hour, temperature 135 ° C. The feed was cooled to room temperature, transferred to a filter and washed once with water and allowed to drain excess moisture. In the resulting base was made 20% of the mass. cornmeal, 2% chalk and thoroughly mixed. The resulting substrate was packaged in polypropylene bags with glued filters and pasteurized.

Substrate 4. Corn cob and pine sawdust in a ratio of 1: 5 were placed in chemically resistant containers and mixed with a solution of acetic acid with a concentration of 5% in a ratio of 1: 6 and placed in an autoclave. Processing time 0.5 hours, temperature 110 ° C. The feed was cooled to room temperature, transferred to a filter and washed once with water and allowed to drain excess moisture. 10% of the mass of fish meal, 2% of chalk were mixed into the resulting base and mixed thoroughly. The resulting substrate was packaged in bags of polypropylene with glued filters and sterilized.

Substrate 5. Flax campfire and hardwood sawdust in a ratio of 1: 6 were placed in chemically resistant containers and mixed with a solution of hydrochloric acid with a concentration of 2% in a ratio of 1: 8 and placed in an autoclave. Processing time 0.5 hours, temperature 121 ° C. The feed was cooled to room temperature, transferred to a filter and washed once with water and allowed to drain excess moisture. In the resulting base was made 20% of the mass. sunflower meal, 2% chalk and thoroughly mixed. The resulting substrate was packaged in bags of polypropylene with glued filters and sterilized.

Substrate 6. Wheat straw and tree bark in a ratio of 4: 1 were placed in chemically resistant containers and mixed with a solution of hydrochloric acid with a concentration of 1.5% in a ratio of 1: 8 and placed in an autoclave. Processing time 1 hour, temperature 80 ° C. The feed was cooled to room temperature, transferred to a filter and washed once with water and allowed to drain excess moisture. 20% of the mass, beet pulp, 2% of chalk were added to the obtained base, and thoroughly mixed. The resulting substrate was packaged in bags of polypropylene with glued filters and sterilized.

Example 4

The cultivation of P. ostreatus, G. lucidum, H. erinaceus on the substrates obtained according to example 3. The following substrate served as control: hardwood sawdust 80%, 20% of the mass. wheat bran, 2% chalk and thoroughly mixed and wetted with water to 80% humidity. The resulting substrate was packaged in polypropylene bags with glued filters and pasteurized.

From the results given in the examples, it follows that the technical task has been solved - selective conditions have been created for the development of edible and medicinal xylotrophic mushrooms “white rot” by increasing the lignin content in the substrate, which increased the rate of substrate development, reducing the time for fruit bodies to reach marketable maturity and yield increase.

Claims (12)

1. A method of preparing selective substrates for obtaining fruiting bodies of medicinal and edible xylotrophic basidiomycetes, including: - acid modification of lignocellulosic raw materials with aqueous solutions of mineral or organic acids with a concentration of 0.5-5% wt. at a temperature of 80-135 ° C and a ratio of raw materials to an acid solution from 1: 2 to 1: 8 for 30-60 minutes; - washing the modified lignocellulosic raw materials with water; - adding to the modified lignocellulosic raw materials a nitrogen source in a concentration of 10-20% wt. and chalk or gypsum at a concentration of 2% wt .; - packaging, sterilization or pasteurization of substrates. 2. The method according to p. 1, where the medicinal and edible xylotrophic basidiomycetes are selected from the list including the species Agrocybe aegerita, Flammulina velutipes, Ganoderma lucidum, Grifola frondosa, Hericium erinaceus, Hypsizygus marmoreus, H. ulmarius, Lentinotus citrinotus, Pleinota citrinotus, Pletotus phantomus, ulentius , P. eringii, P. djamor, P. ostreatus, P. pulmonarius, P. sajor-caju, Trametes versicolor. 3. The method of claim 1, wherein the mineral or organic acids are selected from hydrochloric, sulfuric, phosphoric, and acetic acids. 4. The method according to p. 1, where the lignocellulosic raw materials mono - or multicomponent. 5. The method according to p. 1, where the lignocellulosic raw materials relates to agricultural waste and timber processing industry. 6. The method according to p. 5, where the waste from agriculture and forestry industry is selected from the list including sawdust of deciduous trees, sawdust of coniferous trees, bark, leaves, needles, cereal straw, corn cob, flax bonfire, rice husk. 7. The method according to p. 1, where the source of nitrogen is selected from the list including wheat, rye or oat bran, corn, soy or fish flour, different in the source of the meal and bagasse. 8. Selective substrate for obtaining fruiting bodies of medicinal and edible xylotrophic basidiomycetes, prepared by the method according to claims. 1-7.