EA033616B1 - Method of producing high-protein biomass of yeast - Google Patents

Abstract

The invention relates to biotechnologies, agriculture, namely to the production of yeast from hydrolysates produced while processing vegetable raw materials. The essence of the invention is as follows: in order to obtain high-protein yeast biomass with high content of crude protein, essential amino acids in parallel with simultaneous deep complex processing of starch-containing grain raw materials and simplify the process flow, starch-containing grain raw materials are crushed, subjected to high temperature single-stage enzymic hydrolysis without separating grain shells followed by the consequent separation of grain shells and the fermentolysat (hydrolysat) is used as the base to produce the medium to cultivate and subsequently incubate two yeast strains: Pichia guilliermondii VKPM Y-248 and Pichia guilliermondii VKPM Y-1858. The invention also ensures obtaining the fodder, pharmaceutical or cosmetics product that contains the produced high-protein yeast biomass or its autolyzates, as well as a fodder and/or veterinarian product - grain-shell based fodder fibers.

Description

Technical field

The invention relates to biotechnology, agriculture, in particular to technologies for the production of fodder yeast from hydrolysates obtained from the processing of plant materials. Obtained by this method, a high-protein biomass of yeast based on starch-containing grain raw materials can be used as a pharmaceutical, cosmetic product, as well as in the feed industry as a feed product and protein and vitamin supplements in compounding for various types of farm animals and aquacultures (pigs, birds , cattle, young farm animals, valuable fish species) as a protein component as a substitute for fish uki.

State of the art

The issue of feed supply for livestock, poultry and fisheries is the main and determining one in the formation of the structure of agricultural livestock and pricing policies for meat and fish products.

Currently, the world deficit of fishmeal is 6 million tons / year and is constantly increasing. As a substitute for fish meal in fodder production, genetically modified products are used - soy flour, soybean meal and soy isolate. The main way to reduce this deficit is the production of high-protein biomass using microbiological synthesis, including the use of grain such as wheat, triticale, rye, barley, and waste from flour milling as raw materials.

At the same time, all countries have and constantly accumulate large reserves of underutilized or generally unused wastes from agriculture, crop production, animal husbandry, grain processing and other industries, which due to their physical and mechanical properties, as well as due to the fact that they are characterized low feed value due to the presence of difficultly hydrolyzable polysaccharides and a low content of digestible protein cannot be used for feed for farm animals, however, after appropriate vuyuschey processing fodder can acquire properties, 1.5-3.0 superior cornmeal good quality, and also have a number of essential and necessary properties.

Therefore, the problem of finding new and alternative ways of obtaining feed products, improving quality while reducing the cost of their production is relevant and is one of the main for the agricultural sector of the economy.

The disadvantage of grain raw materials for feeding farm animals is the low availability of the nutrients contained in it, high fiber, poor digestibility, and the lack of many essential amino acids and vitamins, which makes it impossible to use it for fattening farm animals and fish.

Fodder yeast protein is added to the diet of animals as a protein-vitamin supplement. According to the total nutritional value, 1 kg of fodder yeast contains 1.03-1.16 of fodder units (the unit of measurement of the nutritional value of agricultural feed, adopted in Russia; 1 fodder unit corresponds to the nutritional value of 1 kg of medium dry oats sown Avena sativa) and especially a lot of digestible (true) protein : 380-480 g. In terms of amino acid composition and crude protein content in the product, fodder yeast is as close as possible to fishmeal (animal protein), soybean and sunflower meal (vegetable protein). However, animal protein is much better absorbed by animals than vegetable protein.

Therefore, the need for fodder yeast is constantly increasing and methods for producing fodder yeast are one of the areas of large-scale industrial production of fodder protein and vitamins.

One of the directions for producing fodder yeast is the technology for producing yeast biomass obtained at hydrolysis and cellulose enterprises and using carbohydrates for this purpose contained in hydrolysates and sulfite liquors.

The prior art methods for producing fodder yeast from grain raw materials, including the hydrolysis of raw materials of plant origin with mineral acids, followed by the cultivation of yeast cultures - protein producers (RU 2113490, EP 0169068, RU 2560987). A common disadvantage of these methods is the lack of fractionation of the crushed grain mass, because of which the finished product has a high concentration of cellulose and a low concentration of crude protein.

Known methods for producing protein biomass by cultivating the yeast Saccharomyces cerevisiae (RU 2562146, WO 2009129320, RU 2220590,), joint incubation of lactic acid and propionic acid bacteria (RU 2391859, RU 2243678), as well as using yeast of the genus Candida, 2160 as producer strains RU 2041946, RU 2042331). A common drawback of these methods is the use of cultures of microorganisms that do not allow to obtain a high (more than 60%) level of accumulation of intracellular nitrogen compounds (crude protein).

In terms of the essential features, the closest prototype analogue is the Method for producing a protein-vitamin supplement from starch-containing grain raw materials (RU 2613493), in which the grain raw material is fractionated, followed by two-stage enzymatic hydrolysis of starch to glucose and the subsequent cultivation of three different yeast strains - producers of a protein of the genus Hansenula: Hansenula polimorpha Y-314, Hansenula jadinii VKPM Y797, Hansenula petersonii VKPM Y-1012. The finished product obtained in this way - a protein-vitamin supplement contains up to 65% of crude protein, B vitamins, amino acids (lysine - 3.6, methionine - 1.1, valine - 1.0), fiber content up to 1%.

The disadvantage of this method is that the culture of Hansenula polimorpha Y-314 uses only glucose as a carbon source. From literature data it is known that glucose enters the cell in the form of glucose-6-fostat (G6F), as it is phosphorylated by enzymes attached to the outer part of the cell wall. Next, 1 mol of G6F in the cell is divided in half into two synthesis pathways: pentose phosphate (synthesis of nucleic acids) and the Krebs cycle (synthesis of amino acids, that is, proteins). Based on this, when using glucose as the sole source of carbohydrates, it is impossible to obtain more than 50% of intracellular true protein (Skurida G.I. Calculation of the minimum cost of substrates for energy metabolism in microorganisms. / Skurida G.I., Mironov V.A., Drozdov- Tikhomirov L.N. // Microbiology. T.53. - B.2. - 1984. - p. 187-193).

If, however, di-, tri- and tetrasugars are used as a carbon source, which enter the yeast cell by diffusion and active transport, then they are hydrolyzed to glucose inside the cell by enzymes attached to the inner membrane. Phosphorylation of the resulting glucose also occurs inside the cell. Moreover, one mole of G6F is also halved into two synthesis pathways: pentose-phosphate and Krebs cycle, and the second mole completely enters the Krebs cycle. Theoretically, the maximum possible percentage of obtaining intracellular protein in this case increases to 75%.

Disclosure of Invention

The problem solved by the invention is aimed at obtaining high-protein yeast biomass with a high content of crude protein and essential amino acids with simultaneous deep complex processing of starch-containing grain raw materials, simplification of the technological scheme.

The problem is solved by developing a method for producing high-protein yeast biomass on a cultivation medium obtained from starch-containing grain raw materials, comprising the following stages:

a) crushing starch-containing grain raw materials;

b) a high-temperature one-stage enzymatic hydrolysis of starch-containing grain raw materials obtained in stage a), without separation of the grain shells at a temperature of at least 90 ° C to obtain an enzymeolysate;

c) separating the grain shells from the fermentolizate;

d) processing the grain shells obtained in stage c) with a solution of hydrogen peroxide at a temperature of not more than 75 ° C, extraction to obtain a drain;

e) preparation of a culture medium based on the fermentolizate obtained in step c) with the addition of mineral salts, including cobalt (II), as well as thiamine;

f) the sequential cultivation of two strains of yeast, Pichia guilliermondii VKPM Y-248 and Pichia guilliermondii VKPM Y-1858, namely:

1) growing a strain of Pichia guilliermondii VKPM Y-248 on the culture medium obtained in stage e);

2) separation of the biomass of the strain Pichia guilliermondii VKPM Y-248 from the culture medium to obtain a filtrate of the culture fluid;

3) the cultivation of the strain Pichia guilliermondii VKPM Y-1858 on a culture medium containing a filtrate of the culture fluid obtained in stage f.2), and the stock obtained in stage d);

4) separation of the biomass of the strain Pichia guilliermondii VKPM Y-1858 from the medium for cultivation;

g) combining the biomass of the strains obtained in steps f.2) and f.4) to produce the finished product.

In some embodiments of the invention, the crushing in stage a) is carried out in a wet manner with a 1: 3 hydraulic module.

Also in some particular, but not limiting embodiments of the invention, hydrolysis in step b) is carried out with an enzyme preparation containing alpha-amylase.

In some particular, but not limiting embodiments of the invention, hydrolysis in step b) is carried out at a pH of 5.5-6.5.

In some private, but not limiting embodiments of the invention, the culture medium obtained in stage e) contains the following components:

(NH4) 2SO4 - 0.5%;

MgSO4 - 0.05%;

NaCl - 0.01%;

CaCl2 - 0.01%;

Thiamine - 50 mcg / L;

CoSO4 · 7H2O - 25 mg / l;

fermentolizate to the content of total reducing substances (ODS) - 1-6%;

- 2 033616 water - the rest.

In some embodiments of the invention, in step f.1), the cultivation is carried out by the weaning-refilling method and the topping is carried out by the culture medium obtained in step e).

In some embodiments of the invention, in step f.3), the cultivation is carried out by a weaning-refilling method and the topping is carried out with a culture medium containing the filtrate of the culture fluid obtained in step f.2) and the stock obtained in step d).

Also in some embodiments of the invention, in step f.1), the cultivation is carried out with a carbohydrate feed.

In some private, but not limiting embodiments of the invention, the feed is carried out by a fermentolizate with a content of total reducing substances 25-35%.

In some particular, but not limiting embodiments of the invention, in step f), the pH of the medium is maintained at a predetermined level with a 12% solution of ammonia water or ammonia.

To obtain the finished product in some embodiments of the invention, the biomass obtained in stage f.2) and f.4) is subjected to concentration, after which the biomass is combined, dried and optionally granulated.

In some other embodiments, the biomass obtained in steps f.2) and f.4) is combined and concentrated, dried, and optionally granulated to obtain a finished product.

To obtain the finished product, autolysis of the combined biomass of the strains obtained in stages f.2) and f.4) is additionally carried out, combined with fermentolysis with a proteolytic enzyme at a temperature of 50-55 ° C and a pH of 6.5 for 6 hours, while increasing the concentration of free amino acids to a content of at least 45%, and the autolysate is dried.

In some embodiments of the invention, the grain shells obtained in stage d) are dried in a vacuum dryer at a temperature of 40 ° C and crushed to a size of not more than 80 microns or to a size of not more than 200 microns to obtain a finished feed and / or veterinary product.

The invention also relates to a feed, pharmaceutical or cosmetic product containing high protein yeast biomass.

As a result of the invention, the following technical results are achieved: the crude protein content in the yeast biomass is increased to 70% or more and the true protein is up to 55-65%, and the composition of the essential amino acids in the yeast biomass is improved;

the stages of enzymatic hydrolysis and the consumption of enzyme preparations during the pre-treatment of starch-containing raw materials are reduced;

an additional feed and veterinary product was obtained from grain shells, as well as an autolysate of yeast biomass with a free amino acid content of at least 45%;

ensured the use of substandard starch-containing grain raw materials, as well as the disposal of industrial effluents.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to obtaining high protein yeast biomass with a high content of crude protein, essential amino acids: lysine, methionine and valine, with simultaneous deep complex processing of raw materials and simplification of the technological scheme.

To obtain high protein biomass of yeast with a crude protein content of at least 70%, with a high content of essential amino acids lysine, methionine and valine (at least 6, 2.1, 3%, respectively) with simultaneous deep complex processing of starch-containing grain raw materials and simplification of the technological scheme the process of production of yeast biomass, pre-treatment of starch-containing grain raw materials, including substandard raw materials, preparation of a culture medium, a sequential process of growing two tamma of microorganisms of protein producers: Pichia quilliermondii VKPM Y-248, consuming di-, tri- and tetrasaccharides, and Pichia quilliermondii VKPM Y-1858, utilizing organic acids, separation of the obtained yeast biomass.

The pretreatment of starch-containing grain raw materials consists in crushing the starch-containing grain raw materials by high-temperature single-stage enzymatic hydrolysis at a temperature of not less than 90 ° C of the grain raw material, without separating the grain shells to obtain an enzyme lysate (starch-containing hydrolyzate from the grain feed and the following).

Grain shells obtained after separation from the fermentolizate are treated with a solution of hydrogen peroxide at a temperature of not more than 75 ° C, followed by the extraction of grain shells. In the process of squeezing grain shells, a drain is formed - liquid technological waste, which is used in the subsequent stages of growing yeast strains as a component of the medium for cultivating one of the yeast strains. The use of runoff in the subsequent stages of cultivation allows us to solve the problem of disposal of technological runoff.

As raw materials, bran and other starch and cellulose-containing wastes, post-alcohol distillery stillage are used, including substandard grain raw materials that can be used: basic grains

- 3 033616 crops, beaten, corroded by pests; grains sprouted with a sprout that has emerged or lost a sprout; feeble, underdeveloped (grains are small, with an underdeveloped endosperm); frost;

green grains of the main crop (immature); crushed grains; puny grain; grains with a kernel completely eaten away by pests.

The fermentolizate (hydrolyzate) obtained from the pretreatment of grain raw materials is used to prepare a medium for culturing yeast.

According to this invention, the culture medium contains a carbohydrate and salt portion and is used to cultivate a first yeast strain, a protein producer. The carbohydrate portion of the culture medium is a starch-containing grain feed fermentolizate (hydrolyzate) obtained in the pre-treatment of the grain feed, and / or its solutions. The salt portion of the culture medium is mineral salts. The culture medium also contains additional components (additives) in the form of cobalt (II) and thiamine (vitamin B1). The addition of additives to the main nutrient medium contributes to an increase in biomass accumulation (thiamine is used as a growth factor for microorganisms) and an increase in the content of essential amino acids: lysine, methionine and valine, since the enzymes involved in the synthesis of amino acids are cobalamin-dependent, their coenzyme is vitamin B12, which includes cobalt.

The cultivation (cultivation) of two yeast strains, the first of which uses di- and triri tetrasaccharides, and the second utilizes organic acids from the previous fermentation (cultivation) process, is carried out sequentially. In the framework of this invention, the strain of Pichia guilliermondii VKPM Y-248 is preferably used as the first strain, and the protein producer consuming organic acids is used as the second strain of the yeast strain. Most preferably, the use of the yeast strain Pichia guilliermondii VKPM Y-248 consuming di-, tri- and tetrasaccharides (carbohydrates: disaccharides and oligosaccharides, namely tri- and tetrasaccharides), and the strain of the yeast Pichia guilliermondii VKPM Y-1858, utilizing organic acids forming at the first stage of the growing process, and the nutrient medium for the cultivation of the second yeast culture is the combined runoff after processing the grain shells and the filtrate of the culture fluid from the previous fermentation process. For this, after the stage of growing the strain of Pichia guilliermondii VKPM Y-248 yeast on the main culture medium, the biomass of the strain Pichia guilliermondii VKPM Y-248 is separated from the culture medium to obtain a filtrate of the culture fluid, which is combined with the effluent obtained from the processing stage of the grain shells. The combined effluent of the culture fluid filtrate and the effluent from the processing stage of the grain shells are used as a medium for cultivating the yeast strain Pichia guilliermondii VKPM Y-1858. After the stage of growing the strain of Pichia guilliermondii yeast VKPM Y-1858, the biomass of the strain is separated from the culture medium.

The yeast strains used are not pathogenic, prototrophic and heat resistant.

The obtained biomass of yeast strains, both individually and jointly, are used to obtain the finished product - high-protein yeast biomass.

In some of the most preferred embodiments of the present invention, starch-containing, including substandard, grain raw materials are subjected to wet crushing during a pre-treatment with a 1: 3 hydraulic module. In the case of using a hydromodule of more than 1: 3, an additional concentration step is carried out (for example, by evaporation) of the enzyme obtained in the subsequent stages of the fermentolizate (hydrolyzate) to obtain the specified concentrations of total reducing substances (ODS).

High-temperature enzymatic hydrolysis of starch-containing grain raw materials is carried out by enzyme preparations hydrolyzing the grain raw materials to di- and oligosaccharides, preferably di-, tri- and tetrasaccharides. When using tetrasaccharide dihydria (for example, maltose, maltotriose, and maltotetraose) as a carbon source, part of the sugars supplied to protein synthesis increases to 70-75%, resulting in more than 50-55% of intracellular true protein, since When using di-, tri- and tetrasugars as carbon sources, they enter the yeast cell by diffusion and active transport; their hydrolysis to glucose occurs inside the cell under the action of enzymes attached to the inner membrane. Phosphorylation of the resulting glucose also occurs inside the cell. In this case, one mole of glucose-6-fostat is also divided in half into two synthesis pathways: pentose-phosphate and Krebs cycle, and the second mole completely enters the Krebs cycle. Thus, theoretically, the maximum possible percentage of intracellular protein production in this case increases to 75%.

Amylolytic enzyme preparations, for example, amylase enzyme preparations, can be used as enzyme preparations. In some particular, but not limiting embodiments of the invention, alpha-amylase enzyme preparations are used. The pH range is selected depending on the enzyme preparation used. In some private, but not limiting embodiments of the invention, the hydrolysis of starch-containing grain raw materials is carried out at a pH of 5.5-6.5.

An example of a medium for culturing a strain of the yeast Pichia guilliermondii VKPM Y-248 according to the invention can be a medium of the following composition:

(NH ₄ ) ₂ SO ₄ - 0.5%;

- 4 033616

MgSO4 - 0.05%;

NaCl - 0.01%;

CaCl ₂ - 0.01%;

thiamine - 50 mcg / l;

CoSO ₄ -7H2O - 25 mg / l;

fermentolizate to the content of total reducing substances (ODS) - 1-6%; water is the rest.

The carbohydrate portion of the culture medium (culture medium) is a fermentolizate (hydrolyzate) obtained by enzymatic hydrolysis of starch-containing grain raw materials.

In some private, but non-limiting embodiments of the invention, the cultivation of the yeast strain Pichia guilliermondii VKPM Y-248 is carried out by the weaning method and topping up is carried out with the main culture medium consisting of carbohydrate and salt parts.

In some particular, but non-limiting embodiments of the invention, the cultivation of the Pichia guilliermondii yeast strain VKPM Y-1858 is carried out by the weaning method and the topping is carried out with a culture medium containing the filtrate of the culture fluid obtained in the step of growing the Pichia guilliermondii yeast strain VKPM Y-248, and runoff obtained at the stage of processing grain shells.

The cultivation of the yeast strain Pichia guilliermondii VKPM Y-248 in some embodiments of the invention is carried out with the supply of carbohydrate feed. As a feed, it is preferable to use a fermentolizate with a content of total reducing substances (ODS) of 25-35%.

During the cultivation process, the pH of the medium is maintained at a predetermined level, most preferably the pH of the medium is maintained with a 12% solution of ammonia water or ammonia.

To obtain the finished product in some embodiments of the invention, the biomass of the yeast Pichia guilliermondii VKPM Y-248 and Pichia guilliermondii VKPM Y-1858, obtained during cultivation, is separated from the culture medium and separately subjected to concentration, then combined, dried and optionally granulated. Within the framework of the present invention, it is also envisaged to separate, combine the obtained biomass of the two yeast strains and then concentrate, dry and optionally granulate.

To obtain the finished product, the combined biomass of two yeast strains is additionally carried out combined with fermentolysis with a proteolytic enzyme at a temperature of 5055 ° C and a pH of 6.5 for 6 hours, while increasing the concentration of free amino acids to at least 45% and drying the autolysate .

The obtained grain shells are subjected to drying in a vacuum dryer at a temperature of 40 ° C, grinding to a size of not more than 80 microns or to a size of not more than 200 microns to obtain a finished feed and / or veterinary product. The resulting feed and / or veterinary product - dietary fiber has an enterosorbing, detoxifying, antidiarrheal, antioxidant and lipid-lowering effect in gastrointestinal diseases of farm animals.

To obtain the finished product, both high-protein yeast biomass and yeast autolysates can be used. Based on high-protein biomass, a feed product, a pharmaceutical product of both veterinary and medical use, as well as a cosmetic product for use in cosmetology are obtained.

The method is as follows.

In one of the embodiments of the invention, the phased cultivation of two strains of microorganisms is carried out - protein producers under aeration conditions, by a detachable-refill method, on a nutrient medium consisting of carbohydrate and salt parts.

At the first stage of growing yeast cultures, the first, giving 85% of the total biomass, used the strain of yeast Pichia guilliermondii VKPM Y-248, consuming di-, tri- and tetrasaccharides. The process of growing this strain of yeast culture in the first stage consists of the accumulation of biomass for 12-18 hours with the supply of carbohydrate feed at pH above 6.5 in the amount of 6.8-7% of the working volume of the fermenter, preferably with a total amount of reducing substances ( ODS) 25-35%, most preferably with an ODS content of 30%. At the end of the feed, the cultivation process is carried out by the weaning-filling method. The total number of draining-topping operations is 5. Weaning of the culture fluid and adding the main culture medium (culture medium), preferably with an ODS content of 1-6%, most preferably ODS of 5-6% of additional components (vitamins and trace elements: thiamine and cobalt, 50 μg / L and 25 mg / L, respectively) are carried out in an amount of 65-70% of the working volume of the fermenter. The time of each operation is 12-14 hours. The process of growing yeast culture is with the supply of additional carbohydrate nutrient medium with an ODS content of 30%. Carbohydrate replenishment is given with an increase in pH above 6.5 every 6-8 hours of operation. During the cultivation of yeast cultures of microorganisms, a pH of 5.5-6.5 is maintained with a 12% solution of ammonia water or ammonia.

At the second stage of production of high-protein yeast biomass, cultivation is carried out in

- 5 033616 in a volume-filling mode during 56-60 hours of the strain of Pichia guilliermondii yeast VKPM Y-1858, consuming metabolites - organic acids formed during the cultivation of the previous yeast culture. The nutrient medium at this stage is the combined runoff obtained after processing the grain shells and the filtrate of the culture fluid from the previous fermentation process of the yeast strain Pichia guilliermondii VKPM Y-248. The pH of the process is maintained with a 12% solution of ammonia water or ammonia. The yeast biomass is concentrated, combined, and the finished product is dried in a granulating dryer.

To prepare a nutrient medium, starch-containing grain raw materials and / or bran are subjected to wet crushing with a 1: 3 hydraulic module, after which enzymatic hydrolysis of thermostable alpha-amylase is carried out without preliminary separation of the grain shells, preferably at a temperature of at least 90 ° C, most preferably at a temperature of 90 ° C and pH not more than 5.5-6.5.

Carrying out the described enzymatic hydrolysis allows you to get the fermentolizate, consisting of 80% or more of di-, tri- and tetrasugars, such as maltose, maltotriose and maltotetraose.

After enzymatic hydrolysis, the grain shells are separated and subjected to treatment with a concentrated solution of hydrogen peroxide, creating a concentration of hydrogen peroxide in the solution of 1.5%, at a temperature of preferably not more than 75 ° C, most preferably 70 ° C for 2 hours with vigorous stirring, after which the grain the shells are squeezed in a press, dried in a vacuum dryer at a temperature of 40 ° C, crushed to a particle size of preferably not more than 80 μm and / or not more than 200 μm in diameter, most preferably 80 μm and 200 μm.

Get the finished target product of feed and / or veterinary use - dietary fiber.

The proposed method for producing high-protein yeast biomass from starch-containing grain raw materials is implemented as follows.

Starch-containing grain raw materials are subjected to crushing with a hydraulic module 1: 3 by any wet method (using a cavitation apparatus, ultrasonic, rotary pulsating equipment), an aqueous suspension of crushed raw materials is prepared without preliminary separation of grain shells with a dry matter content of 20-25% and enzymatic hydrolysis by amylolytic with thermostable alpha-amylase at a temperature of 90 ° C, pH 5.5-6.5 for 2 hours. As a result of enzymatic hydrolysis, a fermentolizate is obtained, the content of total edutsiruyuschih substances (ODS) in which 15-16% consisting of 80% or more of di-, tri- and tetrasaharov, wherein 45% of maltose.

Conducting high-temperature enzymatic hydrolysis of crushed grain raw materials without preliminary separation of the grain shells allows the use of substandard grain raw materials and receive dried and ground grain shells of feed and / or veterinary use as a finished product (dietary fiber).

After enzymatic hydrolysis, the grain shells are separated on rotor screens.

To use the obtained fermentolizate as an additional carbohydrate feed, the ODS content in it is increased to 25-35% by evaporation in a vacuum evaporation plant of 50% of the volume of the obtained fermentolizate.

The remaining volume of the fermentolizate is used without evaporation to prepare the main nutrient medium as its carbohydrate part (the ODS content is 1-6%), which allows to halve the evaporated volume and, accordingly, reduce the costs of this process.

The isolated grain shells are treated with a concentrated solution of hydrogen peroxide, creating a concentration of hydrogen peroxide in a solution of 1.5% at a temperature of 70 ° C for 2 hours with vigorous stirring, then squeezed in a screw press, dried in a vacuum dryer at a temperature of 40 ° C, crushed to particle sizes with a diameter of 80 or 200 microns and get the finished target product of feed and / or veterinary use - dietary fiber.

The drain after pressing the processed grain shells is directed to the preparation of a nutrient medium for the second stage of cultivation — the yeast strain Pichia guilliermondii VKPM Y-1858.

The main nutrient medium consists of carbohydrate and salt parts. The ODS content in the main nutrient medium is 5-6%. To increase the content of essential amino acids (lysine, methionine, valine), additional components (vitamins and trace elements: thiamine and cobalt (II) in the form of cobalt salts) in the amount of 50 μg / l and 25 mg / l, respectively, are added to the nutrient medium. After that, the cultivation of the strains of the yeast Pichia guilliermondii VKPM Y-248 and Pichia guilliermondii VKPM Y-1858 is carried out sequentially.

The method for producing high protein yeast biomass consists of two stages: cultivating the yeast strain Pichia guilliermondii VKPM Y-248 and cultivating the yeast strain Pichia guilliermondii VKPM Y-1858.

The cultivation process of the yeast strain Pichia guilliermondii VKPM Y-248 is aerobic, consists of the accumulation of biomass for 12-18 hours with the supply of carbohydrate feed containing 30% ODS, with an increase in pH above 6.5, in the amount of 6.8-7% of the working fermenter volume. At the end of the feed (after 6-12 hours), the cultivation of the yeast strain is carried out by the weaning-filling method,

- 6,033,616; the total number of weaning and refilling operations is 5. Weaning of culture fluid and adding the main nutrient medium with an ODS content of 5-6%, additional components (vitamins and trace elements: thiamine and cobalt) 50 μg / l and 25 mg / l, respectively, in the amount of 65-70% of the working volume of the fermenter. The time of the first operation is 14 hours, the time of the next four operations is 12 hours each. The process of growing yeast culture is with the supply of carbohydrate feed (fermentolizate) with an ODS content of 30%. Carbohydrate replenishment is given with an increase in pH above 6.5 every 6-8 hours of operation. During the cultivation of yeast cultures of microorganisms, the pH is maintained with a 12% solution of ammonia water or ammonia. The concentration of yeast biomass at the end of each operation is 35-45 g / l. Concentration of biomass is carried out in any possible way (separation, centrifugation, ultrafiltration). The filtrate of the culture fluid is sent to combine with the effluent obtained after squeezing the grain shells, the combined effluent serves as a nutrient medium for the second culture of the yeast Pichia guilliermondii VKPM Y-1858, utilizing the organic acids remaining after the first stage of cultivation.

The cultivation of the yeast strain Pichia guilliermondii VKPM Y-1858 in the second stage is also carried out in the detachable-topping mode, with the preliminary accumulation of biomass up to 6-7 g / l for 16 hours under aerobic conditions at a temperature of 40 ° C with the number of drain-topping operations, equal to 5. The concentration of biomass at the end of each operation is 8-10 g / l, the operation of the drain-topping operation is 8 hours each.

During the process, the pH is maintained with a 12% solution of ammonia water or ammonia.

Concentrated yeast biomass is combined and sent for drying and granulation in a drying apparatus.

The stock obtained after concentrating the yeast biomass in the second stage is sent for further treatment to the aeration tank and the water is returned to the grain crushing process, obtaining a practically closed water cycle in the process of obtaining high-protein yeast biomass from starch-containing grain raw materials.

Conducting a one-stage enzymatic hydrolysis of substandard grain raw materials without separating the grain shells with an amylolytic drug with thermostable alpha-amylase allows you to get fermentolizate with the content of dieth- and tetrasugars (maltose, maltotriosis and maltotetraosis) up to 80%. This contributes to the formation of intracellular true protein of more than 50-60% (up to 7075% of crude protein) and at the same time eliminates the need for additional stages: separation of the raw material into fractions, additional hydrolysis using other expensive enzyme preparations.

In the process of obtaining a high-protein feed additive from starch-containing grain raw materials, only two yeast strains are used, the use of which makes it possible to obtain yeast biomass with a crude protein content of at least 70%, which is 7% higher than in the prototype, while microbiological synthesis metabolites are used in the stock that allows you to use it again in the technological cycle and leads to a reduction in water consumption in the production of protein biomass of yeast.

Conducting a one-stage enzymatic hydrolysis, using fewer strains of yeast in the production process leads to a reduction in the number of technological operations and, accordingly, simplification of the technological scheme for the production of protein biomass of yeast.

Adding additional microelements and vitamins, such as cobalt and thiamine, to the nutrient medium makes it possible to obtain a high-protein yeast biomass with a high content of such amino acids as lysine, methionine and valine, the content of which is at least (%), respectively: lysine - 6.0; methionine - 2.1; valine - 3.0.

The combination of essential features results in a high-protein yeast biomass with a crude protein content of at least 70%, which contains essential amino acids (lysine, methionine, valine), the content of which is 1.4-3 times higher than in the prototype, vitamins of group B with the simultaneous deep processing of grain starch-containing raw materials, including substandard, with the receipt of the finished product - dietary fiber and autolysate of yeast biomass with a free amino acid content of more than 45%.

It should be understood that these and all examples cited in the application materials are not limiting and are provided only to illustrate the present invention.

Example 1

They took substandard (sprouted) grain of wheat in an amount of 72 kg with a moisture content of 13%, mixed with tap water in a ratio of 1: 3 and subjected to wet crushing on a rotary pulsation apparatus (RPA) ENA (Russia) once.

The resulting aqueous suspension of crushed raw materials in an amount of 288 L was subjected to enzymatic hydrolysis with an amylolytic preparation containing thermostable alpha-amylase, Termamyl SC DS from Novozymes (manufactured in Denmark). To do this, 89 ml of the indicated amylolytic preparation was introduced into the reactor with an aqueous suspension and enzymatic hydrolysis was carried out at a temperature of 90 ° C, pH 5.5-6.5 for 2 hours. Fermentolizate was obtained in an amount of 288.1 l with a total

- 7 033616 reducing substances 15.6%. After that, the grain shells were separated on a rotating sieve and received 57.6 kg with a moisture content of 92%.

After separation of the grain shells, the resulting fermentolizate in an amount of 115 L was sent for evaporation for 2 hours in a vacuum evaporator up to 60 L and with an ODS content of 30% for subsequent use in the process of growing yeast biomass as carbohydrate replenishment. The remaining half of the fermentolizate was used to prepare the main nutrient medium.

Inoculum of yeast Pichia guilliermondii VKPM Y-248 was prepared in an amount of 0.6 l on a nutrient medium containing ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) - 0.3%, magnesium sulfate (MgSO ₄ ) 0.05%, sodium chloride (NaCl) - 0.01%, calcium chloride (CaCl ₂ ) - 0.01%, yeast extract - 0.5%, thiamine (B1) - 50 μg / l, cobalt sulfate 7-water (CoSO ₄ -7H2O ) - 25 mg / l, fermentolizate (hydrolyzate) with an ODS content of 2%. The yeast culture Pichia guilliermondii VKPM Y-248 was washed with solid nutrient medium into a seed culture medium, poured into sterile rocking flasks containing 0.5 g of chalk, and cultivated on a rocking chair at a temperature of 40 ° C and a speed of 300 rpm in within 24 hours. Ready seed was combined and used for inoculating a working fermenter.

The cultivation of the yeast strain Pichia guilliermondii VKPM Y-248 was carried out in a fermenter with a working volume of 25 l, in which 18 l of the main nutrient medium was pre-sterilized.

The main nutrient medium:

(NH ₄ ) ₂ SO ₄ - 0.5%;

MgSO ₄ - 0.05%;

NaCl - 0.01%;

CaCl2 - 0.01%;

thiamine - 50 mcg / l;

CoSO ^ H ^ - 25 mg / l;

fermentolizate with a content of total reducing substances (ODS) - 6%.

The main cultivation process was carried out by the weaning-filling method at a temperature of 40 ° C, pH 6.5, air flow rate - 25 l / min, stirring speed - 500 rpm, the pH of the process was maintained by supplying 12% ammonia water.

After sowing the fermenter with 0.6 L Pichia guilliermondii VKPM Y-248 seed, biomass accumulation was carried out for 24 hours. Carbohydrate feed containing 30% ODS was given in the amount of 1.7 L after 8 hours with an increase in pH above 6.5 . After biomass accumulation up to 35 g / l, a partial discharge of the culture fluid was carried out and the main nutrient medium was added in a volume of 17 l. The process was also conducted with the supply of carbohydrate feed in the amount of 1.7 l with an increase in pH above 6.5. The number of weaning-topping operations was 5. The duration of the first weaning-topping operation was 14 hours, the next four - 12 hours each. The weaning operation - topping was carried out to achieve a concentration of biomass in the culture fluid of 45 g / l. The total cultivation process time was 86 hours, the amount of biomass obtained was 4.78 kg (by dry weight). The concentration of yeast biomass was carried out by separation of the culture fluid.

The obtained filtrate of the culture fluid in an amount of 110 l was combined with the runoff obtained after processing and squeezing the grain shells, and the resulting combined runoff was used in the second stage of production of high protein yeast biomass as the main nutrient medium for growing the Pichia guilliermondii VKPM Y-1858 yeast culture.

The cultivation of the yeast strain Pichia guilliermondii VKPM Y-1858 at the second stage of production of yeast biomass was carried out under conditions similar to the first stage, in a detachable-refill mode without filing a carbohydrate feed.

22.5 liters of pooled runoff was loaded into a 25 liter fermenter and 2 L inoculum was seeded from flasks with Pichia guilliermondii VKPM Y-1858 yeast culture. The accumulation of biomass took place over 16 hours, when the biomass concentration reached 6 g / l, the culture fluid was refluxed and culture medium (combined flow) was added in an amount of 17 l. After that, 5 weaning operations were carried out - topping up when the concentration of biomass in the culture fluid reached 8 g / l. The time of each operation was 8 hours. The total time of the cultivation process was 56 hours, the amount of biomass obtained was 0.85 kg (by dry weight).

The concentrated yeast biomass was combined with the biomass obtained in the first stage of production, after which drying and granulation were carried out and 6.2 kg of yeast biomass with a moisture content of 10% were obtained.

Biomass composition (in% by dry weight): crude protein - 72.8, crude fiber - 0.35, mass fraction of ash - 2.3, mass fraction of protein according to Barnstein - 60.9; amino acid composition of biomass: lysine - 6, methionine - 2.1, tryptophan - 0.7, arginine - 2.0, histidine - 1.8, leucine - 2.9, tyrosine - 2.12, alanine - 3.2% , threonine - 1.5, valine - 3.0, methionine + cystine - 3.1, glycine - 2.96, glutamic acid - 9.5, aspartic acid - 3.8; the content of vitamins (mg / g): thiamine (B ₁ ) - 5.3; riboflavin (B ₂ ) - 15.6; pantothenic acid (B ₃ ) - 39.5; choline (B ₄ ) - 1800; nicotinic acid (B ₅ ) - 39.4; pyridoxine (B ₆ ) - 18.7; cyanoba- 8 033616 lamin (B ₁₂ ) - 38.5. The total dissemination is 90-100 U / ml. The total conversion of carbohydrates to yeast biomass was at least 50%. The crude protein content is 10% higher than in the prototype, the lysine content is 40% higher, methionine 48%, valine 66%.

The amino acid composition of the product was determined by thin-layer chromatography and computer quantification after scanning chromatograms.

Example 2

Grain shells after enzymatic hydrolysis analogously to example 1 were separated on a rotating sieve. The resulting mass in the amount of 57.6 kg was placed in a reactor with a stirrer and was treated with a 31% solution of hydrogen peroxide in the amount of 2.88 l, creating a concentration of hydrogen peroxide in the solution of 1.5%. The processing of grain shells was carried out at a temperature of 70 ° C for two hours with vigorous stirring, then the mass was squeezed on a screw press to a solids content of 30% and dried in a vacuum dryer at a temperature of 40 ° C, after which it was crushed to particle sizes of 80 μm in diameter or 200 microns. Received 4.97 kg of the finished product with a moisture content of 5%.

A stock of 50 l after pressing the processed grain shells was sent to prepare a nutrient medium for the second stage of cultivation — the yeast strain Pichia guilliermondii VKPM Y-1858.

The resulting finished product - dietary fiber - can be used for veterinary and feed purposes for the prevention and treatment of gastrointestinal diseases of farm animals, including farm birds, as a detoxifying and sorbing agent; dietary fiber can be used as food and biologically active additives, the so-called biocorrectors, as they bind pathogenic microorganisms, their metabolic products, exogenous toxins, allergens, heavy metals, radioactive isotopes, ammonia, divalent cations and facilitate their excretion through the gastrointestinal tract intestinal tract.

Example 3

The yeast biomass after the separation stage, obtained analogously to example 1, in the amount of 20 l was sent to the reactor with a heating jacket with a working volume of 30 l. The proteolytic enzyme preparation Neutrase (manufactured by Novozymes A / S Denmark) was added to the yeast biomass at a rate of 10 U / g protein, the pH of the culture liquid was adjusted with a 10% sodium hydroxide solution to 5.6, and autolysis combined with fermentolysis at a temperature of 50-55 ° C for 6 hours. After that, the product was dried at a temperature of 120-125 ° C to a residual moisture content of 5% in the final product.

The resulting product, an autolysate of yeast biomass, contained more than 45% of water-soluble, easily digestible amino acids.

The composition of the yeast autolysate (in% on dry matter): crude protein - 72.8, crude fiber 0.35, amine nitrogen - 3.3; amino acid composition of biomass: lysine - 6, methionine - 2.1, tryptophan - 0.7, arginine - 2.0, histidine - 1.8, leucine - 2.9, tyrosine - 2.12, alanine - 3.2, threonine - 1.5, valine - 3.0, methionine + cystine - 3.1, glycine - 2.96.

As a result of autolysis, the protein molecule is divided into three-tetrapentapeptides by peptide bonds, which subsequently allows the product to be absorbed as quickly and efficiently as possible with less energy consumption of the body itself.

The above examples confirm that the proposed method for producing high protein yeast biomass allows to obtain a feed product of improved quality while reducing the number of operations of the production process of yeast biomass and simplifying the technological scheme of production compared to the prototype. At the same time, during the production process of yeast biomass, an additional product (dietary fiber) is obtained, which is used in veterinary medicine, which has an enterosorbing, detoxifying, antidiarrheal, antioxidant and lipid-lowering effect.

The obtained high-protein yeast biomass in terms of amino acid composition and crude protein content in the product approaches fishmeal and can be used as a substitute for fishmeal, which is considered the most valuable feed product, in the preparation of animal feed. The obtained yeast biomass, as well as yeast autolysates, can be used for the manufacture of a cosmetic product as well as a pharmaceutical one.

Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the gist of the present invention.

Claims (15)

CLAIM 1. A method of obtaining a high-protein biomass of yeast on a cultivation medium obtained from starch-containing grain raw materials, comprising the following stages: a) crushing starch-containing grain raw materials; b) carrying out high-temperature single-stage enzymatic hydrolysis of starch-containing - 9 033616 living grain raw materials obtained in stage a), without separation of the grain shells at a temperature of at least 90 ° C to obtain the fermentolizate; c) separating the grain shells from the fermentolizate; d) processing the grain shells obtained in stage c) with a solution of hydrogen peroxide at a temperature of not more than 75 ° C and extraction to obtain a drain; e) preparation of a culture medium based on the fermentolizate obtained in step c) with the addition of mineral salts, including cobalt (II), as well as thiamine; f) sequential cultivation, comprising the following stages: f.1) growing a strain of Pichia guilliermondii VKPM Y-248 on the culture medium obtained in stage e); f.2) separating the biomass of the strain Pichia guilliermondii VKPM Y-248 from the culture medium to obtain a filtrate of the culture fluid; f.3) growing the strain Pichia guilliermondii VKPM Y-1858 on a culture medium containing the filtrate of the culture fluid obtained in stage f.2), and the drain obtained in stage d); f.4) separating the biomass of the strain Pichia guilliermondii VKPM Y-1858 from the culture medium; g) combining the biomass of the strains obtained in steps f.2) and f.4) to produce the finished product. 2. The method according to claim 1, in which the crushing in stage a) is carried out by the wet method with a hydraulic module of 1: 3. 3. The method according to claim 1, in which the hydrolysis in stage b) is carried out by an enzyme preparation containing alpha-amylase. 4. The method according to claim 1, in which the hydrolysis in stage b) is carried out at a pH of 5.5-6.5. 5. The method according to claim 1, in which the culture medium obtained in stage e) contains the following components: (NH4) 2SO4 - 0.5%; MgSO ₄ - 0.05%; NaCl - 0.01%; CaCl ₂ - 0.01%; thiamine - 50 mcg / l; CoSO4-7H2O - 25 mg / L; fermentolizate to the content of total reducing substances (ODS) - 1-6%; water is the rest. 6. The method according to claim 1, in which at the stage f.1) the cultivation is carried out by the detachable-topping method and the topping is carried out by the cultivation medium obtained in the stage e). 7. The method according to claim 1, in which at stage f.3) the cultivation is carried out by the detachable-topping method and the topping is carried out with a culture medium containing the filtrate of the culture fluid obtained in stage f.2) and the stock obtained in stage d) . 8. The method according to claim 1, in which at stage f.1) the cultivation is carried out with the supply of carbohydrate feed. 9. The method of claim 8, in which the feed is carried out by a fermentolizate with a content of total reducing substances of 25-35%. 10. The method according to claim 1, in which at stage f) the pH of the medium is maintained at a predetermined level with a 12% solution of ammonia water or ammonia. 11. The method according to claim 1, in which to obtain the finished product, the biomass obtained in stage f.2) and f.4) is subjected to concentration, after which the biomass is combined, subjected to drying and optionally granulation. 12. The method according to claim 1, in which to obtain the finished product, the biomass obtained in stage f.2) and f.4) are combined and subjected to concentration, drying and optionally granulation. 13. The method according to claim 1, characterized in that to obtain the finished product, autolysis of the combined biomass of the strains obtained in stages f.2) and f.4) is additionally carried out, combined with the fermentolysis of a proteolytic enzyme, at a temperature of 50-55 ° C and pH 6.5 for 6 hours, while increasing the concentration of free amino acids to a content of at least 45%, and the autolysate is dried. 14. The method according to claim 1, characterized in that the grain shells obtained in stage d) are subjected to drying in a vacuum dryer at a temperature of 40 ° C and grinding to a size of not more than 80 microns or to a size of not more than 200 microns to obtain a finished feed and / or veterinary product. 15. A feed product containing a high-protein yeast biomass obtained according to any one of claims 1 to 13.