RU2734079C1 - Growth activator of yeast, fungi, microorganisms and crops - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Disclosed is use of a concentrate of biologically active substances of soya, obtained by preliminary barothermic treatment of raw material with subsequent aqueous extraction and evaporation, as growth activator of microorganisms, agricultural crops. Concentrate is applied in an effective amount in a nutrient or fermentative medium, a substrate, soil or foliar treatment.

EFFECT: invention increases productivity and biomass of cultivated crops, provides faster growth of fungi, bacteria, yeast and other microorganisms, faster processes of composting and decomposing cellulose.

1 cl, 4 dwg, 7 tbl, 4 ex

Description

Application area

The invention relates to the field of biotechnology, and can be widely used in various industries: processing, food, feed, pharmaceutical and microbiological, biotechnological, agriculture (agrochemistry), namely, the use of a biologically active drug based on plant raw materials to activate the growth of microorganisms and crops and can be used in the production of mushrooms: champignons, oyster mushrooms and others, as components of fermentation media for the cultivation of various microorganisms, in aerobic composting, as an organic fertilizer in the cultivation of crops.

Prior art

Soybeans are widely used for the production of protein products (flour, concentrates, isolates). At the same time, to meet the demand for protein ingredients with advanced functional and technological properties and improved biomedical characteristics, new technological solutions are needed. In the last 10-15 years, the preventive and therapeutic properties of soy peptides, flavonoids, oligosaccharides and other biologically active compounds contained in soy seeds and their processing products have been investigated and confirmed. In this regard, at present, there is a growing interest in the content of the corresponding biologically active factors in consumer products, in increasing their specificity, and in creating industrial technologies for their extraction, purification and concentration.

For the preparation of nutrient media in the microbiological industry, raw materials of mineral, animal and plant origin are used, as well as chemically synthesized. The most accessible for microorganisms are carbohydrates, therefore, in laboratories, as well as in many industrial biotechnological processes (in the production of enzymes, antibiotics, amino acids, etc.), glucose, sucrose, lactose and other carbohydrates are used. However, the listed carbohydrates are valuable food raw materials and are quite expensive. In this regard, in most large-scale microbiological industries, individual carbohydrates are replaced with more affordable products: waste of starch and syrup production (molasses, hydrol), hydrolysates of peat and plant waste, by-products of the dairy industry, etc. As a substrate for the growth of microorganisms, molasses can be used for the production of microbial protein, lactic and citric acids, for the cultivation of bifidobacteria in order to obtain probiotic preparations, bioethanol and biobutanol.

Thus, soybean molasses is, on the one hand, a valuable product due to the content of a large amount of carbohydrates (which are a source of carbon when used as a component of a nutrient medium), on the other hand, an inexpensive source of isoflavonoids that can be isolated from molasses.

Soybean molasses is a brown viscous syrup with a bittersweet odor. Typically soybean molasses contains 50% solids. It is a collection of various soy nutraceuticals, as well as oligosaccharides, consists of carbohydrates (60%), proteins and other nitrogenous substances (10%), mineral components (10%), fats and lipid compounds (20%). The main components of soy molasses are sugars, which include oligosaccharides (stachyose, raffinose), disaccharides (sucrose), small amounts of monosaccharides (fructose, glucose). Soy molasses are often used as components of culture media for the cultivation of microorganisms that produce various solvents (acetone, ethanol, butanol), organic acids, including lactic acid, polyesters, glycolipids, etc.

Traditionally, soy products with a high protein content are obtained using extraction processes with solutions of ethyl alcohol or weak alkalis, isofocusing in an acidic pH zone, washing with water; more modern technologies include the use of membranes.

As an alternative to these methods, the main disadvantages of which are the complexity of instrumental circuits, the use of reagents (organic solvents, acids, alkalis), and large protein losses, a 3D structuring technology was developed using the thermobaromechanical method. The 3D structuring technology consists in the transfer of soy proteins into a single three-dimensional structure - a porous gel, followed by the extraction of low-molecular substances with water. The result is a protein product with a crude protein content of 75%. The developed method allows to reduce protein losses, to increase the efficiency of extraction, to exclude the use of chemical reagents [Khabibulina NV, Bikbov TM, Ponomarev VV. 3D structuring is an innovative approach to the intensification of biotechnological processes. / Project culture and quality of life. 2015. No. 1, S. 473-491].

When obtaining soy protein concentrates by the extraction method, a concentrate of low molecular weight biologically active substances of soy or so-called soybean is formed as a secondary product. aqueous extract of 3D granules, with a high content of low molecular weight carbohydrates, amino acids, minerals, vitamins, growth factors.

According to the method disclosed in the RF patent for invention No. 2636047, publ. 11/17/2017, the feedstock is soy flour with a bulk density of 500-600 g / l, mixed with water until the concentration of raw materials reaches 65-75%. The resulting moist mass is subjected to stepwise heating from 20 to 140-220 ° C for 60-90 seconds at a pressure of 50 to 120 atm. Then the pressure is abruptly released to 1 atm and dried to a residual moisture content of 10%, obtaining a granular porous product with a bulk density of 50 to 350 g / l (in the form crushed to flour particle size, the resulting product has a bulk density of 200 to 700 g / l) ... Further, flour is extracted with water at neutral pH at a total flour: water ratio of 1: 5-1: 10 to obtain an aqueous extract. The extract is evaporated to a dry matter content of 50%, obtaining a concentrate of biologically active substances of soybean (CBVS) (total sugars - 60-70% of dry matter, crude protein - 10-15% of dry matter, isoflavonoids - 0.7-1.0 % of dry matter).

When using 3D structuring technology, a concentrate of low molecular weight soybean substances (KNVS) is obtained as a by-product, containing 10-15% of crude protein, 60-70% of carbohydrates, 0.7-1.0% of total fat, as well as mineral and biologically active substances. Based on the successful experience of using traditional soy molasses in the cultivation of microbial strains, the concentrate of biologically active substances of soy can also have prospects as a component of fermentation media, as a source of carbohydrates and growth factors that stimulate the synthesis of target products.

The known method RU 2603088, publ. 20.11.2016, stimulation of plant growth and development, providing for the treatment of seeds with a biological product, which is used as a culture of lactobacilli Enterococcus durans VKPM B-10093, mixed with beet molasses. The invention makes it possible to increase the productivity of agricultural crops and reduce their morbidity.

From patent RU 2227806, publ. 04/27/2004, the use of soy molasses is known as stimulants for the growth of bifidobacteria. Waste from the production of soy protein isolate - sour soy molasses - is subjected to electrochemical activation in the cathode zone until the specified parameters are reached, the liquid phase is separated and subjected to freeze-drying. The resulting preparation is mixed with the nutritional base "Epidermat-O", sodium chloride and distilled water to obtain a nutrient base for the cultivation of bifidobacteria. The invention makes it possible to utilize the waste from the production of soy protein isolate - sour soy molasses.

From RU 2698213, publ. 08/23/2019, a known medium for obtaining a probiotic additive for quail feed, consisting of a mixture of beet and corn molasses, taken in equal parts, K ₂ HPO ₄ , microorganisms Lactobacillus salivarius. The invention improves the productivity of quails by growing them using the developed probiotic additive.

Enzymes widely used in various industries and agriculture are in demand products of microbial synthesis. One of the dominant groups on the industrial enzyme market is carbohydrases, which accounted for 24% of the total enzyme market in 2018. Commercial carbohydrases include mainly amylases, cellulases and hemicellulases.

The ability of cellulases and hemicellulases to hydrolyze non-starch polysaccharides (NSP) determines their widespread use in the bioconversion of plant materials in the food, textile, pulp and paper industries, in the feed industry, in the production of biofuels and detergents [Alimova F.K. Industrial use of fungi of the genus Trichoderma. / F.K. Alimova. - Kazan: Kazan State University. IN AND. Ulyanov-Lenin, 2006.209 p., Mikhailova R.V. Macerating enzymes of filamentous fungi in biotechnology / R.V. Mikhailova. - Minsk: Belarusian. Science, 2007. 407 p.].

The most intensively growing demand for enzyme preparations (FP) of cellulases and hemicellulases in feed production, where hydrolysis of NCP in grain raw materials leads to a decrease in the viscosity of feed mixtures and digestible mass, increases the availability of nutrients for the action of digestive enzymes, contributes to the normalization of intestinal microflora, increases energy value and the digestibility of feed, allows you to replace expensive components of the ration with cheaper cereals.

Filamentous fungi are preferably used as industrial producers of carbohydrases, which are distinguished by high productivity and rather simple cultivation schemes and preparation of finished form preparations. It is known that soy and its products are often used for the cultivation of filamentous fungi. Thus, the T. reesei WX-112 strain, a producer of cellulases, was grown superficially on a medium containing wheat bran, Avicel, and soybean meal as the main components. To induce the synthesis of T. reesei carbohydrases during submerged cultivation, soy husks are added to fermentation media.

At VNIIPBT, together with the NRC "Kurchatov Institute" by the method of gamma mutagenesis on a cobalt source, a strain of the mycelial fungus Trichoderma reesei Co-44 (VKM F-4789D) with increased activity of xylanase and endoglucanase for the production of enzyme preparations for feed purposes was obtained [Kostyleva E.V. , Tsurikova N.V., Sereda A.S., Velikoretskaya I.A., Veselkina T.N., Lobanov N.S., Shashkov I.A., Sinitsyn A.P. Increase in the activity of endodepolymerase carbohydrases in the Trichoderma reesei strain by mutagenesis. Microbiology. 2018.Vol. 87.No.5. S. 530-540].

Disclosure of invention

The technical result of using a preparation based on a concentrate of biologically active substances of soy as an activator of the growth of yeast, fungi and other microorganisms, by introducing it into a substrate, nutrient medium, soil or by foliar treatment, is, first of all, in the environmental friendliness of the preparation used, obtained without using chemical reagents, as well as in accelerating the growth of microorganisms, increasing the accumulation of metabolic products, increasing the yield of biomass, accelerating biotechnological processes.

The claimed technical result consists in the use of a concentrate of biologically active substances of soy obtained by preliminary barothermal treatment of raw materials, followed by water extraction and evaporation, as an activator of the growth of yeast, bacteria, fungi, strains of microorganisms, agricultural crops by introducing into a nutrient or fermentation medium, substrate, soil or foliar treatment in an amount of 0.5-10 wt. %.

Preferably, the concentrate of biologically active substances of soy is used in the form of a concentrate of 50 wt. % or in dry form with a dry matter content of 90-95 wt. %.

As a component of the fermentation medium during the cultivation of fungal enzyme producers, the concentrate of biologically active substances of soy is used in an amount of 0.5-10 wt. %.

As a component of the nutrient medium for the cultivation of baking and alcoholic yeast, the concentrate of biologically active substances of soy is used in an amount of 1.0-3.0 wt. %.

As a component of the nutrient medium for the cultivation of yeast, lactic acid bacteria, the concentrate of biologically active substances of soy is used in an amount of 1.5-5.0 wt. %.

As a component of the substrate for growing mushrooms, oyster mushrooms or champignons, the concentrate of biologically active substances of soy is preferably used in an amount of 1.5-3.0 wt. % of the substrate.

As a component in aerobic composting and decomposition, the concentrate of biologically active substances of soy is used in an amount of 2.0-4.0 wt. %.

As an organic fertilizer of prolonged action when growing crops by adding to the soil at the rate of 50 mg / kg of soil in the form of a solution with a concentration of 0.5-1.2 wt. % or foliar treatment with a concentrate of soybean biologically active substances of 0.5 wt. %.

Examples of implementation of the invention

Example 1.

Investigation of the effect of soybean biologically active substances concentrate (CBVS) contained in the fermentation medium on the production of target enzymes by a highly active industrial strain T. reesei.

The following materials were used: a concentrate of biologically active substances of soybeans with a protein content of 14.5 ± 0.3% of dry matter, total sugars - 53.1 ± 1.5% of dry matter, mass fraction of moisture - 4 ,8%. To determine the activity of the target enzymes, we used xylan from birch wood, Na-salt of carboxymethylcellulose (Sigma, USA).

T. reesei cultivation was carried out in flasks on a thermostatted shaker (250 rpm) at 30 ° C for 72-120 h in 750 ml Erlenmeyer flasks with 50 ml of fermentation medium of the following composition, wt. %: lactose - 2.0; yeast extract - 1.0; malt sprouts - 1.0; KH ₂ PO ₄ - 0.2; (NH ₄ ) ₂ SO ₄ 0.6; CaCl ₂ - 0.06; tap water; pH of the medium 5.0 ± 0.2. Soybean biologically active substances concentrate (CBVS) was introduced into the medium at a concentration of 1 to 9 wt. % by experience options. Sterile culture media were inoculated with a microbiological loop, using as an inoculum a culture grown on a modified medium SM of the following composition (wt%): KH ₂ PO ₄ - 0.2; glucose - 1.0; yeast extract - 0.4; peptone - 0.6; malt wort - 2.0; agar-agar - 2.0. The biomass of the culture grown by the submerged method was separated by centrifugation at 10750 g for 5 min. The culture fluid (CL) was used to determine the activity of xylanase, endoglucanase, and the concentration of soluble protein.

Periodic T. reesei cultivation in fed-batch mode was performed in laboratory fermenters CF 108/3 with a working volume of 1.5 liters, equipped with automatic control systems pH, PO ₂ and temperature. Inoculum for inoculation of fermenters was obtained by culturing the fungus in shaking flasks (250 rpm) at 30 ° C for 48 hours in a medium of the following composition (wt%): glucose - 2.5; corn extract (40% DM) - 2.0; KN ₂ PO ₄ - 0.5, tap water. Inoculation of the fermenter was carried out by introducing a liquid inoculum in an amount of 8 vol. % of its working volume.

Fermentation was carried out under the following conditions: temperature - 30 ± 2 ° C, air flow (aeration) - 0.5 l / min, the oxygen partial pressure (pO ₂₎ - 30 ± 5% - the regulation is effected by changing the agitator speed, pH of the medium was maintained at the level of 5.5 ± 0.1 by means of titration with solutions of 5% H ₂ SO ₄ and 10% NH ₄ OH. Used a fermentation medium of the following composition (wt.%): Lactose - 2.0; microcrystalline cellulose - 1.0; malt sprouts - 1.0; yeast extract - 1.0; KH ₂ PO ₄ - 0.2; (NH ₄ ) ₂ SO ₄ 0.6; CaCl ₂ - 0.06; antifoam propinol - 0.1; tap water. The concentrate of biologically active substances of soy was introduced into the initial fermentation medium at a concentration of 5 wt. %. Feeding was carried out after 48 h of cultivation in an automatic mode at a rate of 5 ml / h with 41% aqueous solution of lactose, glucose and xylose in a ratio of 6: 3: 1. The duration of cultivation was 120 h. min. In the CL, the activities of the target enzymes were determined, as well as the total content of soluble protein.

The activity of xylanase and endoglucanase was determined by the rate of formation of reducing sugars by the Shomody-Nelson method during the hydrolysis of xylan from birch wood and Na-salt of carboxymethylcellulose, respectively. A unit of activity was defined as such an amount of enzyme that leads to the formation of 1 μmol of reducing sugars in 1 min at pH 5.0; 50 ° C at a substrate concentration in the reaction mixture of 0.5 wt. %.

Investigation of the effect of the introduction of a concentrate of biologically active substances of soy on the biosynthesis of xylanases and endoglucanases during cultivation of T. reesei in flasks.

T. reesei synthesizes a wide range of cellulolytic and hemicellulolytic enzymes and is characterized by increased activity of endodepolymerase carbohydrases - xylanases and endoglucanases, which are key in the composition of feed enzyme preparations, contributing to a rapid decrease in the viscosity of grain mixtures, an increase in the availability of nutritional components of grain raw materials, an increase in energy value and assimilation feed.

The introduction of a preparation based on a concentrate of biologically active substances of soybeans into the fermentation medium during the cultivation of T. reesei in flasks led to a significant increase in the activity of the target enzymes and to the acceleration of the dynamics of their biosynthesis. Thus, the endoglucanase activity of the strain increased by an average of 50 U / ml with an increase in the concentration of the concentrate of biologically active soybean substances in the medium by 2 wt. %, and the maximum accumulation of the enzyme shifted from 120 h to 72 h of cultivation. At a concentration of 3 wt. % and higher, a decrease in endoglucanase activity was observed by 120 h of growth. This was probably due to high CL pH values (7.0-7.3), at which endoglucanase is unstable and loses some of its activity, while the expression of xylanases II and III of T. reesei is favored by pH values in the neutral zone. ... The maximum endoglucanase activity - 489 ± 25 U / ml (220% of the maximum endoglucanase activity in the control) was obtained at 72 h of cultivation with 9 wt. % concentrate of biologically active substances of soy.

Similarly, the addition of a concentrate of biologically active substances of soy influenced the biosynthesis of xylanase. The greatest increase in xylanase activity, almost 2 times in relation to the maximum xylanase activity on the initial medium, was observed at 72-96 h of growth in the variant with 9 wt. %.

In Fig. 1 shows the effect of the concentration of the concentrate of biologically active substances of soy on the biosynthesis of endoglucanase (A) and xylanase (B) T. reesei during cultivation in flasks. When making 5 and 7 wt. % of the concentrate of biologically active substances of soybeans, the activity of endoglucanase increased by 1.8 times, xylanase - by 40-50%.

Thus, during cultivation in flasks, the concentrate of biologically active substances of soy promoted an increase in the xylanase and endoglucanase activity of T. reesei, in addition, an acceleration of the dynamics of biosynthesis of target enzymes was observed, which makes it possible to reduce the cultivation time in flasks to 72-96 h instead of 120 h.

Cultivation of T. reesei in laboratory fermenters with the addition of a concentrate of biologically active substances of soy.

To check the results of submerged cultivation of T. reesei with the addition of a concentrate of soybean biologically active substances, carried out in flasks, then the strain was cultivated in laboratory fed-batch fermenters. The soy preparation was added to the test medium at a concentration of 5 wt. %, since 7 and 9 wt. % of the drug contributed to an increase in the viscosity of the fermentation medium.

In Fig. Figure 2 shows the dynamics of accumulation of endoglucanase (A) and xylanase (B) during cultivation of T. reesei in laboratory fermenters in the fed-batch mode. The data obtained showed that during cultivation in the fed-batch mode, the introduction of 5 wt. % of the concentrate of biologically active substances promoted an increase in the activity of endoglucanase and xylanase by 11 and 28%, respectively. In the variant with the addition of a concentrate of biologically active substances, a more intensive accumulation of endoglucanase was observed in the first 2-3 days of growth. On the contrary, xylanase biosynthesis in the variant with the addition of a concentrate of biologically active soybean substances intensified after 96 h of growth, while in the control variant the accumulation of xylanase slowed down after 120 h of growth.

It should be noted that, in addition to the productivity of the strains, the fermentation efficiency is determined by such an important criterion as the total removal of the target enzymes from the fermenter. The calculation of this parameter includes the activity of the target enzymes and the amount of the obtained QOL supernatant. During cultivation with the addition of a concentrate of biologically active substances of soybeans, the viscosity of the plum decreased by more than three times (Table 1), which in turn contributed to an increase in the yield of the supernatant by 55%, therefore, the removal from the fermenter by the activity of endoglucanase increased by 73%, and by the activity xylanase - twice. The results obtained confirm the significant positive effect that the introduction of 5 wt. % of concentrate of biologically active substances of soy and the feasibility of its use as a component of nutrient media.

Example 2. The use of a concentrate of biologically active substances of soybeans when growing mushrooms in a growing experiment in a protected soil.

Experiments were laid with oyster mushrooms and mushrooms with different concentrations of the concentrate of biologically active soybean substances in the substrate. The rate of growth of the substrate with mycelium was monitored. Coconut fiber was used as a substrate for oyster mushrooms, and a mixture of leaf compost and peat was used for mushrooms. The following soy preparation concentrations were tested: 2.0; 3.0; 4.0 g per liter of coconut substrate (1.7-3.3 wt%). In the experiment with champignons, the concentration of the soy preparation (CBVS) was: 3.5; 7.0; 10.5 g per liter of substrate (1.2-3.5 wt%).

On coconut fiber, the fastest overgrowing of the substrate was observed in variants with a concentration of 2.0 grams of concentrate of biologically active substances per liter of coconut substrate (1.7 wt.%); Higher concentrations inhibited mycelium growth. Laboratory studies of coconut fiber showed a low content of the main nutrients, for further experiments it was decided to use a substrate richer in chemical composition: a mixture of sawdust and crushed corn shafts. On the substrate for champignons, the variant with 7.0 g of concentrate of biologically active soybean substances per liter (2.3 wt%) was the best in terms of mycelium growth rate.

Experience in growing Oyster mushrooms (Pleurotus ostreatus).

To grow oyster mushrooms (Pleurotus ostreatus), a substrate of the following composition was used: pine sawdust, washed from resinous substances, and chopped corn rods. The ratio of the components in the substrate is 3: 1 by volume. 15 g of lime was added per kg of dry substrate. The chemical composition of the resulting substrate (wt.%): Total ash - 2.3, cellulose - 36; hemicellulose - 24; lignin - 28.4; extractives - 9.3. Content in wt. %: total nitrogen - 1.5, total phosphorus - 0.45, total potassium - 2.0. After mixing all the components of the substrate (sawdust, corn rods, lime), the resulting substrate was sterilized - autoclaving at 1 atm for 20 minutes. Part of the sterile substrate was mixed with a dry concentrate of biologically active substances of soy at the rate of 2 grams per 1 liter of dry substrate. Then the substrate was moistened with sterile water at the rate of 2.5 parts of water per 1 part of the substrate, which corresponded to 70% humidity. The content of the concentrate of biologically active substances of soybeans in the finished substrate was 2 wt. %.

The wet substrate was inoculated with oyster mushroom grain mycelium (Pleurotus ostreatus,) at the rate of 25 grams of grain mycelium per 1 liter of wet substrate. The substrate was placed in 1.2 liter ziplock polyethylene bags. The bags with substrates were kept in a lighted room at a temperature of + 23 ° C. Intensive overgrowth of the substrate with cherry mycelium was observed after 12 days. After 24 days from the beginning of the experiment, when the mycelium completely occupied the entire surface of the substrate, the bags were placed in a climatic chamber with an air temperature of + 12 ° C for "cold stress" for 7 days. After the "cold stress" bags with oyster mushroom mycelium were placed in a climatic chamber with an upward current of warm humid air (temperature + 21 ° C). A sterile blade made incisions in the bags every 3.5 cm. A week later, the formation of primordia began on the variant with a concentrate of biologically active substances of soy. The first fruiting bodies were obtained 57 days after inoculation. By this time, fruiting bodies had not yet formed in the control. Packages of the control variant (without concentrate of biologically active substances of soybeans) were repeatedly subjected to "cold stress" for 7 days. Fruit bodies on the variant without soy preparation were obtained almost a month later. Oyster mushrooms, grown on a substrate with a concentrate of biologically active substances of soybeans, were cut and weighed after 62 days from inoculation, and from the control variant after 86 days. The average raw weight of mushrooms in the variant with the concentrate of biologically active soybean substances (4 replicates) is 43.7 g / bag, in the control variant (4 replicates) - 6.4 g / bag. The cut fruiting bodies of oyster mushrooms were dried in a thermostat at a temperature of + 40 ° C to constant weight, and ground. In dry matter, the content of basic nutrients and protein was determined. In terms of chemical composition, that is, in terms of the content of nitrogen, phosphorus and potassium in dry matter, as well as in terms of protein content, fruit bodies in the control and in the variant with a concentrate of biologically active substances did not differ significantly. Taking into account that the weight of fruit bodies in the control was about 7 times lower than in the variant with a concentrate of biologically active substances, then the protein yield when using the concentrate as a component of the substrate was proportionally higher.

Thus, the addition of a concentrate of biologically active substances of soybeans to the substrate made it possible to obtain the production of oyster mushrooms almost a month earlier than in the variant without the addition of the concentrate, the weight of fruit bodies and the yield of protein were 7 times higher.

Experiment with mushrooms (Agaricus bisporus). The substrate for growing champignons was a 4-year-old compost sheet. Content in wt. %: organic matter in the compost was 45, total nitrogen - 1.7, phosphorus - 0.8, potassium - 3.8. Before starting the experiment, the substrate was mixed with beet molasses or a concentrate of biologically active substances of soybeans and autoclaved in 0.5 liter flasks with cotton-gauze stoppers to maintain sterility. The substrate layer in the flasks is 5 cm. After autoclaving the substrate for 20 min at 1 atmosphere using tyndalisation methods (within 3 days), it was inoculated with fungal mycelium. The experiment consisted of three options: Option 1 - Control, substrate without adding additional components, Option 2 - substrate + 7 g of dry concentrate of biologically active soybean substances per 1 liter of dry substrate or 3% of the finished substrate mass, Option 3 - substrate + 1 g beet molasses per 1 liter of dry substrate. After overgrowing the substrate after 12 days with mushroom mycelium, the mycelium was backfilled with a cover substrate (1 cm of peat).

On variants with additives, the formation of mushroom fruiting bodies was noted after 60 days. The size of mushroom caps did not exceed 1.5-2.0 mm. On the control, mushroom fruiting bodies were absent. Visually, the number of fruiting bodies was about 10-12 times higher in the variant with the concentrate of biologically active substances of soybeans than in the variant with beet molasses.

Thus, a concentrate of biologically active substances of soy in an amount of 1.2-3.5 wt% is a more effective stimulator of the formation of mushroom fruiting bodies on the selected substrate than beet molasses, and its content in an amount of 2.0-3.0 wt%, close to the optimal concentration range of its use for the cultivation of these types of mushrooms ..

Example 3. Research of aerobic decomposition (composting) of beech sawdust using a concentrate of biologically active substances of soybeans, a biological product and fertilizers.

6 variants of the experiment were carried out in 3 replicates (Table 2), the composting time was 48 days. Sawdust - recycled beech trunk, peeled from bark, measuring ≈1.5 × 1.5 cm

The experiment was carried out in vessels of 500 ml, sawdust weighing 50 g dried to an air-dry state was introduced into them, then moistened to 70%, pouring in water, 35 ml, or a nutrient solution of the same volume. The nutrient solution was prepared by analogy with the nutrient medium for the cultivation of aerobic cellulitis. The composition of the Hutchinson and Clayton medium for the cultivation of aerobic cellulitis (g / l), where no cellulose was introduced: NaNO ₃ - 2.5; K ₂ HPO ₄ 1.0; MgSO ₄ × 7H ₂ O - 0.3; NaCl - 0.1; CaCl ₂ × 4 H ₂ O - 0.1; FeCl ₃ × 6 H ₂ O - 0.01.

In embodiments experiment 3 and 4 were added dry concentrate biologically active substances soybean mass 1 In experiment variants 5 and 6 were added culture of microorganisms isolated from the gastrointestinal tract of an elephant, the volume of - 3 ml, the number of microorganisms - October ⁵ cells / ml. Next, the sawdust was composted with daily stirring.

After the completion of composting, the sawdust was washed on a sieve with a mesh of 5 mm, air dried, and weighed. At the same time, it was noted that the sawdust of options 3 and 4 was washed more difficult than in other options, the wash water was darker in color, the wood easily crumbled, which is the criterion for the greatest decomposition of organic matter. Weighing showed that the greatest loss of cellulose was noted in option 4, where a concentrate of biologically active substances and a nutrient solution were added simultaneously.

As follows from table 3, in the presence of a nutrient solution (fertilizer), a concentrate of biologically active substances of soybeans with its content in an amount of 2-4 wt. % provides the best result: the decomposition of cellulose is accelerated (in comparison with the control) by 1.34 times, and in comparison with a biological product mixed with fertilizer - by 1.16 times.

Example 4. Study of the effect of a concentrate of biologically active substances of soybeans as an organic fertilizer in the cultivation of crops.

Soil (soil type - illuvial-ferruginous sandy loam podzol) was sampled in the area of Malakhovka, Moscow region, pH - 5.6, mobile phosphorus - 330 mg / kg air-dry soil, exchangeable potassium - 110.0 mg / kg air - dry soil (potassium and phosphorus were determined by the Kirsanov method).

sp.), сорт «Михайловский» и редис (Raphanus sativus), сорт «Дуро». Опыт состоял из пяти вариантов в 3-х повторностях, вариант 1 (К) - Контроль, в почву ничего не вносили, вариант 2 (N) - вносили в почву аммиачную селитру (NH₄NO₃) в концентрации 100 мг/кг действующего вещества (азота) или 300 кг/га, вариант 3 (М) - вносили в почву концентрат биологически активных веществ сои, масса препарата - 2,73 г/сосуд, содержание азота - 100 мг/кг, вариант 4 (О) - одновременное внесение минерального азота (NH₄NO₃) и азота концентрата биологически активных веществ сои в дозе 50 мг/кг почвы каждого, вариант 5 (Ф) - фолиарное (на лист) внесение концентарта биологически активных веществ сои, всего было проведено 3 обработки его водным раствором до полного смачивания растений с концентрацией 1 г/300 мл воды, т.е. с разведением 1:300. Почву, перед тем, как набить сосуды, перемешивали в лотке с точной навеской минерального или органического удобрения.The vegetation experiment was carried out in a phytotron at a temperature of 22 ° C and a lighting intensity of 10,000 lux. The vessels with a volume of 1 liter were filled with soil (soil type - illuvial-ferruginous sandy loam podzol) and agricultural crops were sown in them: peas (Pisum sp.), Variety "Ambrosia", barley (

sp.), variety "Mikhailovsky" and radish (Raphanus sativus), variety "Duro". The experiment consisted of five options in 3 replicates, option 1 (K) - Control, nothing was added to the soil, option 2 (N) - ammonium nitrate (NH ₄ NO ₃ ) was added to the soil at a concentration of 100 mg / kg of active substance (nitrogen) or 300 kg / ha, option 3 (M) - a concentrate of biologically active substances of soy was introduced into the soil, the mass of the preparation was 2.73 g / vessel, the nitrogen content was 100 mg / kg, option 4 (O) was a simultaneous application mineral nitrogen (NH ₄ NO ₃ ) and nitrogen concentrate of biologically active substances of soy at a dose of 50 mg / kg of soil each, option 5 (F) - foliar (per leaf) application of a concentrate of biologically active substances of soy, a total of 3 treatments were carried out with an aqueous solution until the plants are completely wetted with a concentration of 1 g / 300 ml of water, i.e. with a dilution of 1: 300. The soil, before filling the vessels, was mixed in a tray with an accurate weight of mineral or organic fertilizer.

The illuvial-ferruginous sandy loam podzol, before setting up the experiment, had the following characteristics: pH - 7.1, exchangeable (available to plants) potassium - 109.3 mg / kg air-dry soil (low supply), mobile (available to plants) phosphorus - 330 mg / kg air-dry soil (very high degree of supply), zinc (Zn) - 6.5 mg / kg air-dry soil, magnesium (Mg) - 63.0 mg / kg air-dry soil, copper (Cu) - 0.75 mg / kg air-dry soil, sodium (Na) - 12.0 mg / kg air-dry soil.

Analysis of the dry concentrate of biologically active substances showed that its moisture content was ≈5%, water-soluble potassium - 14285.7 mg / kg of the drug (photometrically with staining according to Denizhe), water-soluble phosphorus - 1297.5 mg / kg of the drug (on a plasma photometer). C / N ratio - 11.27 (carbon - 41.24%, nitrogen - 3.66%).

The content of elements was determined by inductively coupled plasma atomic emission spectrometry on an Agilent 7700x ICP-MS instrument. The content of elements in a dry concentrate of biologically active substances, mg / kg: P (according to P ₂ O ₅ ) - 18902; S - 2118; K (for K ₂ O) - 54056; Ca - 3264; Cr 137.9; Co - 0.5; Mn 31.8; Fe - 936; Ni 89.2; Cu - 14; Zn 55.3; Mo <0.1; As 0.32; Na - 206; Ca - 3114; Mg - 4500; C - 41.24%; N - 3.66%.

The results of the growing experience. After 42 days, the experiment was completed, the green mass of crops was weighed. The results are presented in Table 4 and Fig. 3. In Fig. 3 shows the green mass of one hundred plants according to the variants of the experiment. The addition of green mass in the variant (M) with the addition of a concentrate of biologically active substances of soy was: radish - 98.4%, peas - 33.5%, barley - 18.6%; on option (O) with the introduction of a concentrate of biologically active substances of soybeans and mineral fertilizers: radish - 138.1%, peas - 39.3%, barley - 0.5%, on option (F) with foliar treatment of plants with an aqueous solution of the concentrate biologically active substances: radish - 138.1%, peas - 10.1%, barley - 18.6%.

However, more objective data are obtained when comparing the mass of dried plants. After drying the plants in the air and taking into account the mass, it was shown that the increase in green dry mass in the option (M) with the addition of a concentrate of biologically active soybean substances was: radish - 92.4%, peas - 49.6%, barley - 19.9% ; on option (O) with the introduction of a concentrate of biologically active substances of soybeans and mineral fertilizers: radish - 153.6%, peas - 11.8%, barley - 32.1%, on option (F) with foliar treatment of plants with an aqueous solution of the concentrate biologically active substances (F): radish - 46.0%, peas - 4.4%, barley - 55.6% (table 5, fig. 4). In Fig. 4 shows the dry weight of 100 plants according to the variants of the experiment in grams.

Determination of pH and macroelements in the soil, after harvesting the experiment, showed that: total nitrogen (determined by the Kjeldahl method after wet ashing with sulfuric acid) on the Control variants of the experiment - 0.34%, on the option (N) - 0.28%, on option (M) - 0.31%, on option (O) - 0.20%, on option (F) - 0.36%, with a significant removal of nitrogen from options N, M, O and F in comparison with Control, remains about the same. Significant removal of available forms of phosphorus and potassium by plants was noted (Table 6).

Determination of macro- and microelements (zinc and copper) in plants has shown that when a concentrate of biologically active soybean substances is introduced into the soil, the N: P: K ratio in plants becomes more optimal compared to other experimental options (Tables 6 and 7). Nitrogen, phosphorus, and potassium in plants were determined after wet ashing in sulfuric acid: nitrogen - by the Kjeldahl method, phosphorus - photometrically with Denige staining, potassium - using a flame photometer. Zinc and copper, after dry ashing and dissolution of a sample with 1 m nitric acid on an atomic absorption spectrometer.

The vegetation experience showed that the concentrate of biologically active substances of soybeans can be introduced into the soil as an organomineral fertilizer of prolonged action, increasing the biomass of cultivated crops in the amount of 0.5-1.2 wt. %.

With the introduction of a concentrate of biologically active substances of soybeans together with a mineral fertilizer, the increase in the dry weight of a radish plant in comparison with the use of only a mineral fertilizer was 20%. Hence, we can draw a preliminary conclusion about high responsiveness to the concentrate of biologically active substances of root crops. For cereals (barley), the additional responsiveness was 10%. When a concentrate of biologically active substances of soy is introduced into the soil, the N: P: K ratio in plants is optimized. The best results for raising the level and optimizing N: P: K were achieved with radishes (root vegetables).

Despite the high content of microelements with controlled levels of MPC in the initial concentrate of soybean biologically active substances, the concentration of zinc and copper in all plant samples did not exceed the established standards, which indicates the safety of the concentrate of biologically active substances as a fertilizer.

Claims (1)

The use of a concentrate of biologically active substances of soy obtained by preliminary barothermal treatment of raw materials, followed by water extraction and evaporation, as an activator of the growth of yeast, fungi, microorganisms and agricultural crops by introducing into a nutrient or fermentation medium, substrate, soil or foliar treatment in an amount of 0.5 -10 wt. %.

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