RU2696074C1 - Trichoderma reesei mycelial fungus strain - producer of endoglucanase, xylanase and pectinase complex for production of protein additives based on cereal and legume raw material for use in fodder production - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Disclosed are a Trichoderma reesei VKM F-4789D strain, a method for producing a fodder complex enzyme preparation and a method for increasing the feed value of cereal and legume mixtures. Micellial fungus strain Trichoderma reesei VKM F-4789D is a producer of endoglucanase, xylanase and pectinase, which decompose non-starch polysaccharides.

EFFECT: invention is used to produce an enzyme preparation for destroying non-starch polysaccharides of cereal and legume raw material in fodder production for increasing feed value of rations and in production of protein fodder additives for farm animals, poultry and aquaculture.

3 cl, 5 tbl, 5 ex

Description

The invention relates to the field of biotechnology and can be used to destroy non-starch polysaccharides (NCP) of grain and leguminous raw materials in feed production to increase the feed value of diets and to obtain protein feed additives for farm animals, poultry and aquaculture.

One way to solve the problem of feed protein deficiency is the rational use of vegetable protein-containing substrates. The assimilation of protein from plant sources is hindered by the high content of NKP in plant cell walls, which have an anti-nutritional effect on the body of monogastric animals, birds, and aquaculture. NKP increase the viscosity of the digested mass, impede the hydrolysis of nutrients and the absorption of hydrolysis products by the intestinal walls, form complexes with the protein, reducing its digestibility, promote the development of pathogenic microflora in the intestine, alter the physiology and morphology of the digestive tract [1]. In Russia, cereals form the basis of the diets of farm animals. LCPs of cereals are mainly represented by arabinoxylans, cellulose and β-glucans [2]. Most diets include legumes, such as soy, lupine, and peas, as additional sources of protein. Legumes are characterized by a high pectin content, mainly cellulose is contained in the shells, and arabinoxylan in a number of crops [3-5].

In order to increase the feed value of plant-based rations, fodder production widely uses enzyme preparations (FP) of carbohydras capable of hydrolyzing NCP. The use of AF cellulases, hemicellulases and pectinases eliminates the anti-nutritional properties of water-soluble NCPs, destroys the matrix of plant cell walls, which reduces the viscosity of the digested mass, increases the availability of nutrients for the action of digestive enzymes, releases sugars necessary for the growth of lactic acid bacteria, and helps to normalize intestinal microflora , increases the energy value and digestibility of feed [1, 6]. In addition, protein is released, which in plant raw materials is bound to hemicellulose and pectins, and the degree of utilization of nutrient components of the feed increases [1]. In recent years, the aquaculture feed market has been developing rapidly. The most pressing issue in the development of technologies for obtaining feed additives for aquaculture based on plant-based protein-containing raw materials is to increase the protein content by reducing the amount of NKP, which is solved, first of all, by enzymatic hydrolysis of NKP with subsequent separation of water-soluble hydrolysis products [7].

Due to the fact that NKP of plant protein-containing raw materials are represented by cellulose, hemicellulose and pectin substances, complex FPs containing cellulases, xylanases and pectinase enzymes are actively used in feed production. As a rule, endo-depolymerase action hydrolases are the key enzymes in the feed use of AF [8]. Endoglucanase (endo-β-1,4-glucanase, CMCase, EC 3.2.1.4) hydrolyzes internal β-1,4-glycosidic bonds removed from the ends of the polymer chain of cellulose and β-1,4 (1,3) -glucans, which leads to a decrease in the degree of polymerization of these polysaccharides and a decrease in the viscosity of their aqueous solutions. Xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) performs the main function in the hydrolysis of arabinoxylans of grain and leguminous crops. Xylanase leads not only to a decrease in the degree of polymerization of xylans and a decrease in their viscosity, but also to the formation of xylo-oligosaccharides with prebiotic properties [1, 9, 10]. The main depolymerases involved in the degradation of pectin substances include: polygalacturonases (PG) and polymethylgalacturonases (PMG), catalyzing the hydrolytic cleavage of α-1,4-glycosidic bonds in pectin and pectic acid, respectively, as well as pectin / pectatliases that cleave α- 1,4-glycosidic bonds during the trans-elimination reaction to form galacturonides. PG and SGP are of two types: endodepolymerase action, catalyzing the disordered hydrolysis of α-1,4-glycosidic bonds in the corresponding substrates with the formation of oligogalacturonates or methylated / acetylated oligogalacturonates, and exodepolymerase action, catalyzing the sequential hydrolysis of the corresponding substrates to form monogonal PMG methylated / acetylated. In the processing of pectin-containing raw materials, polygalacturonases and pectin / pectatliases, preferably of endodepolymerase action, are most widely used [11, 12].

The following FPs are presented on the feed enzyme market, which contain enzymes of cellulolytic, hemicellulolytic and pectinase action:

- VGPro feedbest - a drug manufactured by a domestic enterprise - PO Sibbiofarm LLC, contains xylanase, β-glucanase, pectinase and protease as the main components (http://www.sibbio.ru/catalog/ptitsevodstvo/fermentnyy-preparat-fidbest-vgrro /);

- Feedbest VGPro G (Sibbiopharm software LLC) - a new multi-enzyme AF containing xylanase, β-glucanase, pectin lyase, pectinase and amylase from fungal and bacterial producers (http://www.sibbio.ru/catalog/ptitsevodstvo/fidbest-vgpro- g- /);

- Yudozyme AF 831 manufactured by VTR BioTech (China) is standardized for xylanase, α-amylase, cellulase and pectinase activity, and also additionally contains galactosidase, cellobiase, protease [13];

- Pectinex 5XL (Novozymes A / S, Denmark) contains pectinase, hemicellulase, and cellulase as the main enzymes [14];

- Ladozim Respect Ultra (Concern Microbioprom, Russia) contains an active complex of cellulases and hemicellulases with a high level of pectinase activity [15].

However, all such preparations are obtained by mixing enzymes produced by different producers. In addition, the presence of proteolytic enzymes in enzyme complexes in some technologies for the preparation of protein feed additives leads to protein loss at the stages of removal of water-soluble products of NKP hydrolysis [15].

The main industrial producers of cellulases and hemicellulases at the moment are mycelial fungi of the genus Trichoderma, and for the production of AF pectinases, in most cases, fungi of the genus Aspergillus are used [16, 17]. It is economically feasible to obtain complex preparations with the required ratio of the main components based on a single producer strain. The enzyme complex of cellulase and hemicellulase producers, including fungi of the genus Trichoderma, often contains trace amounts of pectinases insufficient for the effective hydrolysis of pectin substances in leguminous raw materials. Pectinase producers, on the contrary, are characterized by a low content of xylanases and endoglucanases, which break down the main NKP of grain raw materials.

So known strains A. foetidus 379-K (VKPM F-962) and A.foetidus 379-K-5-1 (RCAM 01136) [18, 19] with activity in the culture fluid (QL) during deep cultivation in flasks: β β-glucanases 6.5 and 13.3 units / cm ³ , cellulases (according to CMC) - 2.6 and 6.1 units / cm ³ , endopolygalacturonases - 2.01 and 3.7 units / cm ³ , respectively. The preparations obtained on the basis of these strains are intended for the processing of pectin-containing raw materials and microbial biomass, while the activity of cellulases and hemicellulases is insufficient to produce AFs capable of effectively hydrolyzing grain raw materials.

Known strain T. longibrachiatum VKM F-3865D [20] with activity in QOL during deep cultivation in flasks: cellulase (according to CMC) - 8-14 u / cm ³ , β-glucanase - 80-105 u / cm ³ , xylanase - 95-250 units / cm ³ and pectinases - 9-11 units / cm ³ . AF based on this strain are mainly intended for the processing of cellulose-containing plant materials, in particular, cereals; pectinase activity in the strain is low.

A known strain of A. aculeatus VKM F-3766D is a producer of a complex of carbohydrases containing xylanases, β-glucanases, pectinases and xyloglucanases [21]. However, the strain has low activity with respect to cellulose, which is one of the main components of plant cell walls, which reduces the effectiveness of the preparations obtained on its basis in the hydrolysis of grain raw materials.

The strain A.foetidus BKM F 3890D [22] produces acid protease and polygalacturonase as main enzymes, and in lesser amounts xylanase, β-glucanase, arabinase, xyloglucanase, and other concomitant carbohydrase complex enzymes. This ratio of components makes the strain promising for use mainly in the food industry. To increase the feed value of grain raw materials in the enzyme complex of the strain A. foetidus BKM F 3890D, insufficient cellulases and hemicellulases are present.

Known mutant strain of micellar fungus: T. longibrachiatum TW-14-220, deposited in VKM under the number F-4642D, is a producer of the complex of enzymes endoglucanases, beta-glucanases and xylanases that destroy NKP, characterized by increased activity in CMC, β-glucan and xylan , and reduced microcrystalline cellulose activity [23]. However, this producer does not have the ability to biosynthesize enzymes with pectolytic action.

Thus, it remains an urgent task to obtain an industrial strain - a producer of a balanced complex of carbohydrases for the effective hydrolysis of basic NKP in grain and leguminous raw materials in order to increase the nutritional value of feed.

The technical result consists in obtaining a new strain of mycelial fungus - a producer of a balanced enzyme complex of carbohydras of endodepolymerase action, which has high activity of endoglucanase, xylanase and pectinases for use in feed production.

The invention is a new strain of the mycelial fungus Trichoderma reesei K-4, a producer of endoglucanase, xylanase and pectinases. Strain T. reesei K-4 deposited in VKM at the Institute of Biochemistry and Physiology of Microorganisms named after G.K. Scriabin RAS under the number VKM F-4789 D.

This strain can be used to ensure high processing efficiency of cellulose-, hemicellulose- and pectin-containing substrates, including the hydrolysis of NKP grain and leguminous feed materials for farm animals and birds, as well as aquaculture.

The invention also relates to a method for producing complex AF with endoglucanase, xylanase and pectinase activity.

The method for obtaining AF involves deep cultivation of the producer strain T reesei K-4 on an appropriate medium, followed by concentration of QOL and drying.

Another aspect of the invention is a method of increasing the feed value of cereal and leguminous feed mixtures, comprising treating grain and leguminous feed mixtures obtained from the T. reesei K-4 AF strain.

Thus, the invention provides a complex AF with high activity of endoglucanase, xylanase and pectinases, the use of which will increase the feed value of diets based on grain and leguminous raw materials for farm animals and poultry, as well as to obtain protein supplements with a high content of crude protein from leguminous raw materials for animals, birds and aquaculture without the use of solvents, acids and alkalis.

The invention is implemented as follows.

The strain T. reesei K-4 is obtained using multistage mutagenesis and selection from the strain T. reesei BCM 18.2 / KK (VGNKI28), an industrial producer of cellulolytic and hemicellulolytic enzymes [24]. For mutagenesis using γ-radiation, which has a high penetrating ability, causes ionization of atoms and is a powerful mutagenic factor.

The technique of mutagenesis. The strain is grown on an agarized modified medium SM,%: KH ₂ PO ₄ - 0.2, glucose - 1.0, yeast extract - 0.4, peptone - 0.6, malt wort - 2.0, agar-agar - 2 , 0, for 7 days at 30 ° C and 7 days at room temperature in the light. The spores of the fungus grown on the surface of the agar medium are suspended in distilled water with the addition of Twin-80 (0.01%), filtered through a glass filter and mutagenized on a γ-chamber type GUT-200 M installation at the Kurchatov Institute ( Russia). The irradiation dose is from 1500 to 2500 Gy, the processing time is 4-8 hours, the irradiation temperature of the spores is 20-23 ° C, the irradiation medium is air. Irradiated spores are plated on Petri dishes with selective medium.

Strain storage. On an agarized modified SM medium at 4 ° C with mandatory reseeding at least once for 3-6 months; in a lyophilized state, spores using protective media.

Cultural and morphological characteristics of the strain. At 30 ° C on a modified SM medium, before the sporulation, the colonies are white, transparent, flat with a smooth surface. Subsequently, the formation of conidia (phialospores) occurs after induction by visible light, and on day 3 the colonies acquire a greenish color. The formation of air (bulk) mycelium is not observed. The mycelium is septic and highly branched. After 5-6 days of growth, the diameter of the colonies is about 40 mm, the colonies are dense, compact, concentric, with uneven edges, yellow pigment is observed in the medium. The conidial zone is formed by numerous compact pads with a diameter of up to 2-2.5 mm, initially white in color, green with age. The reverse side is light greenish yellow.

Conidiophores are unpainted, smooth-walled, formed mainly on the substrate mycelium, the main branches are long and straight, sometimes crimped, up to 4-5 mm wide at the base, gradually tapering to the apex to 2 microns wide; lateral branches form at unequal intervals, usually short, are formed at an angle to the apex of the conidiophores. The phialides are single, club-shaped or broadly bottle-shaped, often curved, size - 5-10 × 2-3 microns. Conidia are unicellular, greenish, smooth-walled, ellipsoidal, 4-7 × 2.5-4 microns, collected in small heads at the tops of the phialids.

When cultivated in deep conditions using soluble substrates (glucose, fructose, lactose), a loose branched mycelium with weak pelletization is formed.

Physiological and biochemical characteristics of the strain. Mesophilen. The optimum mycelium growth temperature is 32 ° C (29-34 ° C), the optimum for cellulase formation is 28 ° C (26-29 ° C), and the optimum for xylanase formation is 32-34 ° C. The optimal pH of growth and secretion of cellulases is 3.5-5.0. The optimal pH for the secretion of xylanases and polygalacturonases 5.5-7.0. The growth of mycelium is also observed at pH 2.5, but there is a very weak formation of cellulases and other carbohydrases.

Nystatin resistance is weak. During surface cultivation, it is resistant to a concentration of up to 0.5 μg / cm ³ ; at a concentration of 2.5 μg / cm ^3, growth is completely inhibited.

It is a prototroph. It is able to assimilate glucose, lactose, glycerin, galactose, xylose, D-mannitol, mannose, trehalose, L- and D-arabinose, sorbose, sorbitol, ribose. Uses ammonium and organic nitrogen, assimilates the nitrate form of nitrogen very poorly.

It forms enzyme systems that allow it to grow on the corresponding complex substrates: cellulose, starch, xylan, β-glucan, pectin, laminarin, lichenin, galactomannan and chitin. The catabolite repression of the biosynthesis of carbohydrases is practically absent.

Difference from the original strain. The mutant T. reesei K-4 obtained by its morphological characteristics when growing on a modified SM medium differs from the original T. reesei BCM 18.2 / KK strain (VGNKI28) by increased sporulation rate, compact, dense mycelium, and faster growth on solid media. In addition, it is distinguished by its enhanced ability, during deep cultivation on liquid media, to biosynthesis of CMC-ase, xylanase, and pectinase.

Pathogenicity. This type of mycelial fungus belongs to the IV group of pathogenicity in accordance with the sanitary and epidemiological rules “Safety of work with microorganisms of the III-IV pathogenicity (danger) groups and pathogens of parasitic diseases” (SP 1.3.2322-08), approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation No 4 on January 28, 2008

The cultivation of the strain T. reesei K-4 is carried out under aerobic conditions in a nutrient liquid aqueous medium containing one or more substrates - carbon sources, which are the inducers of enzyme biosynthesis. The strain is capable, under appropriate conditions of the cultivation process using soluble substrates, for example, lactose, glucose, xylose, to secrete a complex of enzymes - carbohydrases (cellulases, xylanases, pectinases and related enzymes) in the QOL.

The following substrates are used to determine the activity of the target enzymes: for endoglucanase (CMC-ase) activity - water-soluble Na-salt of carboxymethyl cellulose (CMC), for xylanase activity (Xyl) - birch xylan, for polygalacturonase activity (PG) - polygalacturonic acid K-salt, for pectin lyase (PL) activity, highly methoxylated pectin. The hydrolysis of CMC and xylan is carried out in 0.1 M Na-acetate buffer, pH 5.0, at 50 ° C for 5 and 10 minutes, respectively, at a concentration of polysaccharide substrates in the reaction mixture of 5 g / L. The hydrolysis of the K-salt of polygalacturonic acid is carried out at 50 ° C, pH 5.0, highly methoxylated pectin at 40 ° C, pH 5.0, for 15 minutes As products of hydrolysis of the Na salt of CMC, xylan and the K salt of polygalacturonic acid, reducing sugars (BC) are determined by the Somogy-Nelson method. A unit of endoglucanase, xylanase, and polygalacturonase activity is taken to be such an amount of enzyme that within 1 minute under the above hydrolysis conditions it releases 1 μmol of BC equivalent to 1 μmol of glucose [25, 26]. The unit of pectin lyase activity is taken as the amount of the enzyme, which, when applied to pectin for 1 min under the above conditions, forms 1 μmol of 4,5-unsaturated reaction products, increasing the optical density of the reaction mixture at 232 nm by 0.1 [26].

AF obtained using the proposed strain can be used in the form of QOL, in the form of concentrated liquid preparations obtained by ultrafiltration or evaporation of QOL, or in the form of dry preparations obtained by drying or granulation.

The possibility of using the invention is illustrated by examples, which do not limit the scope and essence of the claims associated with them.

Example 1

The cultivation of the strain T. reesei K-4 VKM F-4789 D is carried out under aerobic conditions in 750 ml Erlenmeyer rocking flasks containing 50 ml of a liquid aqueous medium of the following composition, in g / l: amorphous cellulose - 10, lactose - 20, malt sprouts - 10, soy husk - 10, yeast extract - 10, (NH ₄ ) ₂ SO ₄ - 6, KH ₂ PO ₄ - 2, CaCl ₂ - 0.6, pH 6.0. To obtain inoculum (inoculum), T. reesei K-4 fungus culture was grown on modified SM-arape at 30 ° С for 7 days and then at room temperature in the light for 7 days.

Inoculation of flasks is carried out with 1 ml of a suspension of spores washed with agar with water containing 0.1% Tween-80. The flasks are incubated on a shaker at 30 ° C and 250 rpm for 120 hours.

The activity of the new T. reesei K-4 strain for CMC, xylan, K-salt of polygalacturonic acid, and highly methoxylated pectin in QOL are 390, 500, 150, and 100 u / ml, respectively. When cultivating the initial strain of T. reesei BCM 18.2 / QA in rocking flasks under the above conditions, the activity of CMCase, xylanase, PG and PL is 60, 70, 10, and 7 units / ml, respectively (Table 1).

Example 2. The process of cultivation of the strain T. reesei K-4 in laboratory KF 104/3 fermenters with a geometric volume of 3 l and a working volume of 1.35 l, equipped with automatic control systems for pH, pO ₂ and temperature, as well as air bubblers and 2-tier stirrer. The inoculum for inoculation of fermenters is obtained by culturing the fungus in rocking flasks (250 rpm) at 30 ° C for 48 hours in the following composition,%: glucose - 2.5, corn extract (40% CB) - 2, KH ₂ PO ₄ - 0.5, tap water. The dose of inoculum inoculation of fermenters is 8%. Fermentation is carried out for 120 h on a medium of the following composition,%: lactose 2.0, soybean husk 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. pH is maintained at a level of 6.0-6.5. Fermentation is carried out at 30 ° C, with aeration, 1 volume of air per 1 volume of medium per min, maintaining pO ₂ above 30% due to the mixing speed. The substrates are fed continuously after 24 hours growth for 72 hours with a 30% aqueous solution of lactose and xylose in a ratio of 5: 1. The volume of recharge is 0.5 liters. Fermentation ends after 96 hours.

The activities of CMC-basics, Xyl, PG and PL in QOL for 96 hours of fermentation are 1170, 1500,450 and 220 u / ml, respectively.

When cultivating the initial strain of T. reesei BCM 18.2 / KK in a laboratory fermenter, the activity of CMC-basics, Xyl, PG and PL in QOL for 96 hours of cultivation under similar conditions is 560, 1000, 29 and 15 units / ml, respectively.

Example 3. The process of culturing strains of T. reesei K-4 and T. reesei BCM 18.2 / KK in laboratory fermenters KF 104/3 is carried out (as in example 2). At the end of the fermentation, the fungal biomass is removed by centrifugation (4000 rpm for 20 min on an Avanti JXN-26 centrifuge, Beckman coulter, USA), free cell free QOLs are concentrated by ultrafiltration (with a cutoff limit of 10 kDa), the ultra-concentrate is frozen at a temperature of -70 ° C and dried on a Benchtop 2K ES freeze dryer (VirTis, USA) or dried on a Buchi Mini Soray Dryer B-290 spray dryer (Buchi? Switzerland) to obtain a dry AF (which is yellow brown powder, easily soluble in water). AFs are a tan powder readily soluble in an aqueous medium. The characteristics of the resulting AF are presented in table 3.

From the data in Table 3, it is obvious that in AFs based on a new mutant strain they have increased activity of cellulolytic complex enzymes - endoglucanase and xylanase (220% and 160%, respectively), as well as high polygalacturonase and pectinliase activity (1300% and 1200%, respectively, relative to the original strain).

Thus, the proposed strain has the ability to simultaneously produce high endoglucanase, xylanase and pectinase activity, which creates the possibility of obtaining AF with a balanced complex of enzymes for the efficient processing of NKP cereals. For the cultivation of a new strain can be used nutrient media, traditionally used in industrial technologies for obtaining this kind of technical AF.

The advantage of the new complex AF is the synergistic effect of specific enzymes of the complex on grain and leguminous mixtures, which eliminates the anti-nutritional properties of water-soluble NCPs, effectively destroys the matrix of cell walls and increases the concentration of soluble substances, which improves the rheological properties of feeds, increases their energy value and digestibility .

Example 4. The use of AF based on a new strain for the hydrolysis of grain mixtures when receiving feed.

A mixture of wheat (50%) and oat (50%) flour was used as substrates. For enzymatic treatment, experimental samples of AF based on T. reesei K-4 were used, and a sample of AF 18.2 / KK obtained on the basis of the initial strain, the characteristics of which are shown in Example 3. The hydrolysis of the substrates was carried out in 750 ml rocking flasks at 40 ° C at 220 rpm The concentration of the substrate was 250 g / l, AF was introduced at a dosage of 0.025 mg of protein AF / g of the substrate (raw material). After 3 hours of incubation, the viscosity was measured in the obtained hydrolysates using a SV-10 vibrating viscometer, A&D (Japan).

In the table. 4 presents data on changes in viscosity of grain kneading under the influence of AF T. reesei.

It can be seen that treatment of a mixture of wheat and oat flour with a new complex AF K-4, when added in an amount of 0.0025% by weight of the substrate, contributes to a maximum decrease in the viscosity of the mixture, which is a very important aspect of the positive “feed” action of enzymes.

Example 5. The use of AF based on a new strain for the hydrolysis of protein-containing mixtures when receiving feed.

(Германия) при температуре 120-130°С.The tested soybean meal extruded using a twin screw extruder was used as a substrate.

(Germany) at a temperature of 120-130 ° C.

As AF used:

- integrated FP K-4 (characteristics are shown in example 3);

- Ladozyme Respect Ultra (Microbioprom, RF) with CMCase activity - 350 units / ml, Xyl - 240 units / ml, PG - 340 units / ml, protein content - 30 mg / ml.

Complex AFs were dosed according to the activity of CMC-basics at the rate of 5 units. per 1 g of raw materials. Hydrolysis was carried out for 5 h at a temperature of 40 ° C at a natural pH of an aqueous suspension of extruded soybean meal in the range of 6.0-6.2, with a water ratio of 1: 6. The resulting hydrolyzate was centrifuged on a TsLS-3 with a rotor RU 8-90 (separation factor 6000) for 20 minutes, the precipitate formed was dried to constant humidity and the crude protein content was determined (according to GOST 13496.4-93). The control variant was incubated under similar conditions (5 h, 40 ° C, 220 rpm) without AF.

The data obtained show that the processing of a protein-containing substrate - soybean meal, FP K-4 provides the maximum yield of crude protein in the resulting products, which indicates the effectiveness of its use for the hydrolysis of NKP soy.

Thus, the proposed method creates the possibility of obtaining complex feed enzyme preparations with a balanced composition, simultaneously containing high activity of endoglucanase, xylanase and pectinase, the use of which leads to an increase in the nutritional value of rations.

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Claims (3)

1. The strain of mycelial fungus Trichoderma reesei K-4, deposited in the All-Russian collection of microorganisms at the Institute of Biochemistry and Physiology of Microorganisms named after G.K. Scriabin RAS No. VKM F-4789D is a producer of endoglucanase, xylanase and pectinases that destroy non-starch polysaccharides. 2. A method of obtaining a feed complex enzyme preparation with activity of endoglucanase, xylanase and pectinases, comprising cultivating a strain of Trichoderma reesei K-4, deposited in the All-Russian collection of microorganisms at the Institute of Biochemistry and Physiology of Microorganisms named after G.K. Scriabin RAS under the number VKM F-4789D, according to claim 1, followed by concentration of the culture fluid and drying. 3. A method of increasing the feed value of grain and leguminous feed mixtures, which consists in processing grain and leguminous feed mixtures with a complex enzyme preparation obtained by the method according to p. 2.