RU2704281C1 - Method for producing fodder microbiological protein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is a method for producing fodder microbiological protein comprising the preliminary preparation of a yeast culture Saccharomyces cerevisiae, which is added to the substrate in the form of extraction meal and water, cultivation of yeast biomass while stirring and aeration with subsequent drying of the obtained product, where the yeast culture is grown on the basis of the producer strain Saccharomyces cerevisiae Scm, is connected to the substrate in the form of a mixture containing in wt%: soybean meal – 30.0, rice flour – 60.0, rice husks – 10.0 and further – with water distilled, and cultivated at temperature of 32–34 °C for 10–18 hours at mixer rotation speed of 1,000–1,200 rpm and air supply of 2–4 l/min, characterized in that nutrient medium for cultivation of producer strain Saccharomyces cerevisiae Scm contains yeast extract – 10.0 %, peptone – 20.0 %, dextrose – 20.0 %, water distilled – up to 100 %, wherein 1 kg of mixture is 0.4–0.5 l of yeast and distilled water to 4 l and drying of the obtained product is carried out with thickness of 2–5 cm at temperature of 70–85 °C.

EFFECT: simple method with high protein content and balanced composition of amino acids.

4 cl, 3 tbl, 2 ex

Description

The invention relates to the field of biotechnology, microbiological, animal feed and processing industries and can be used to obtain biomass of fodder yeast by cultivating them on nutrient media based on waste processing of cereals or legumes for use as a feed additive in the diet of agricultural animals and poultry .

Fodder microbiological protein (fodder yeast) is a dry concentrated biomass of yeast cells specially grown for fodder for farm animals, poultry, fur animals, fish. Fodder yeast is rich in proteins and vitamins, and has high nutritional value. The microbial protein synthesized by yeast, in terms of digestibility and amino acid content, is superior to animal protein, increases the biological value of proteins of other feeds. Protein of fodder yeast is digested in animals by 95%. Sulfur and its compounds that are part of the composition are involved in the biological processes of the formation of amino acids. The yeast enzyme systems catalyze the processes of amino acid uptake and protein synthesis. Phosphorus and calcium in the yeast contribute to the normal development of the bone skeleton of farm animals. There are no contraindications to the use of fodder yeast. An overdose of fodder yeast does not cause side effects. The use of the product does not affect the timing of slaughter of animals and the use of milk.

Proteins are an essential part of feed diets. Animal life is inextricably linked to the formation and breakdown of proteins in the body. For full growth, for the production of milk, for a set of live weight, the animal must receive the necessary amount of protein in the diet. The most important criterion for feed quality is the amino acid composition of protein components. About a hundred amino acids are currently isolated and described. Amino acids in feed can be in the composition of proteins, and in a free state. Lysine is used to synthesize tissue proteins. Arginine promotes the synthesis of urea, is involved in the formation of seed producers, muscle proteins and insulin. Histidine is necessary for the formation of hemoglobin and adrenaline. Cystine activates insulin. Methionine is involved in the processes of fat metabolism, tryptophan - in the renewal of blood plasma proteins.

According to the content and ratio of essential amino acids, feed proteins are classified as full and inferior. If the diet contains only vegetable protein (grain, oilcake, hay, etc.), then such a diet is unlikely to be balanced by amino acids. There are more essential amino acids in animal proteins than in vegetable ones. The following amino acids are indispensable: arginine, valine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan, phenylalanine. Among the essential amino acids, especially important ones are identified, which are called critical. These are lysine, methionine + cystia, threonine and tryptophan. A deficiency, absence or imbalance of essential amino acids in animal diets is accompanied by a deterioration in protein use, metabolic disorders, and decreased productivity. Already a small protein deficiency in the diet slows down the metabolism, causes muscle weakness and a decrease in muscle volume. Enrichment of feed mixtures with yeast protein will increase the energy value of the feed and change the balance of amino acids in the right direction (V. Kosolapov. Problems of fodder production and ways to solve them at the present stage // Achievements of Science and Technology of the Agro-Industrial Complex. - 2010. - No. 11).

The accelerated development of livestock breeding involves a significant expansion of feed production and the creation of large-scale production based on technologies such as silage, yeast and premix development. In our country, the production of fodder yeast was started in the mid-1930s. Waste from agricultural production, such as straw, corn cobs, sawdust, was hydrolyzed with sulfuric acid, the hydrolysates were neutralized and used to grow Saccharomyces cerevisiae yeast (Kosolapov V.M., Trofimov I.A., Trofimova L.S., Yakovleva E.P. Feed production is the determining factor in Russian agriculture // Vestnik OrelGAU. 2012. No. 1).

Improving feed production requires biotechnological optimization of the feed protein producers and careful selection of the substrate for growing yeast to obtain a balanced amino acid composition of the finished feed mixture.

During the growth of biomass in a yeast cell, enzymatic protein synthesis occurs. Petroleum hydrocarbons (purified liquid paraffins), lower alcohols (ethanol and methanol), wood waste hydrolysates (sawdust, wood shavings, wood chips), agricultural waste hydrolysates (straw, seed husks, corncob) are used as raw materials (substrate) for nutrient media etc.), sulphite liquors of pulp and paper production, post-alcohol bards of hydrolysis and sulphite-alcohol production.

There are various methods for producing feed microbiological protein (KMB), including the hydrolysis or fermentolysis of waste products from processing cereals or legumes at a certain temperature, followed by growing cultures of protein-producing yeast (patent RU 2041946, op. 20.08.1995; patent RU 2042331, op. 27.08 .1995; patent RU 2333665, op. 20.08.2008; patent RU 2384612, op. 20.03.2010; patent RU 2437931, op. 27.12.2011; patent RU 2478701, op. 10.04.2013).

Known, for example, is a method for producing fodder microbiological protein, including pre-treatment of waste plant materials by hydrolysis and growing on the obtained hydrolyzate of yeast. Preliminary processing of the substrate is carried out by acid hydrolysis at a pH of 0.8-1.3, a temperature of 118-123 ° C for 30 minutes As a waste of plant materials, coffee slurry is used, and the yeast biomass is the Saccharomyces cerevisiae yeast biomass (patent RU 2393719, op. 10.07.2010).

The disadvantages of hydrolysis with mineral acids are the insufficient formation of simple carbohydrates for the subsequent fermentation process, due to the fact that during acid hydrolysis, not only starch is hydrolyzed to glucose, but glucose itself is destroyed and pentatom sugars are destroyed to furfural and hydroxymethyl furfural.

A known method for producing fodder microbiological protein, including grinding plant materials (soybeans), processing raw materials with enzymes to ensure the destruction of fiber of plant materials to glucose, introducing mineral salts into the obtained culture medium, introducing saccharomycetes into the culture medium to ensure yeast reproduction, yeast inactivation cells (pasteurization) and drying the resulting product (patent US 3958015, op. 05/18/1976). However, as a raw material in the known method, non-fat soybeans are used. Vegetable oil, which is found in soybeans, inhibits the action of enzymes, and can also act as an inhibitor of the development of yeast. In addition, soy contains a number of anti-nutritional substances, the neutralization of which requires heat treatment.

A known method of producing fodder microbiological protein, comprising crushing grain production waste, diluting the raw material with water (CB) and heating it to a temperature of 65-58 ^o C with a heating rate of 1.5 ° C / min in the presence of alkali or alkaline earth metal hypochlorite (0.2- 0.8% to SV) and ammonium sulfate (0.1% to SV), the mass is maintained at this temperature for 1 h, after which it is treated with amylosubtiline at 50 ° C for 1.5-2.5 h. The resulting substrate used for growing protein-producing yeast. The method allows to obtain a feed product with a high protein content (patent RU 2091492, op. September 27, 1997).

The disadvantages of the known methods, including the stages of hydrolysis or fermentolysis, are the complexity of the process, the duration, the complexity, as well as the high cost of proteolytic or amylolytic enzymes, which leads to higher prices and lower competitiveness of the finished product.

Modern protein enrichment technologies are based on the yeasting of more nutritious substrates. When yeast, protein is produced as part of concentrated feed during cooking.

As the closest analogue (prototype), a method for producing feed microbiological protein was adopted, including grinding sunflower meal, forming an aqueous suspension of ground sunflower meal, enzymatic hydrolysis of a suspension of sunflower meal to ensure the destruction of fiber meal to monosaccharides, introducing sugar culture yeast into the resulting nutrient medium and providing them reproduction, inactivation of yeast cells and concomitant microflora thermally and drying the resulting product. The mass fraction of crude protein (in terms of absolutely dry substance) is in the target product 33.5-39.0% (Patent RU 2561535, op. 08.28.2015).

The disadvantages of the closest analogue are the complexity of the process, the duration and high cost of the target product.

The problem to which the invention is directed, is the development of a simple and economical method for producing feed microbiological protein based on a strain of yeast Saccharomyces cerevisiae using waste from processing cereals and legumes.

The technical result that manifests itself in solving the problem is to simplify the method and reduce the cost of the target product, which has a high protein content and a balanced composition of amino acids.

The problem is solved in that in a method for producing a feed microbiological protein, including the preliminary preparation of a yeast culture Saccharomyces cerevisiae, which is added to the substrate in the form of meal and water, culturing the yeast biomass with stirring and aeration, followed by drying of the obtained product, the yeast culture is grown on the basis of the strain producer Saccharomyces cerevisiae Scm, combined with the substrate in the form of a mixture containing in wt.%: soybean meal - 30.0, rice flour - 60.0, rice husk - 10.0 and then with distilled water and cultivated at a temperature of 32-34 ° C for 10-18 hours at a speed of rotation of the mixer 1000-1200 rpm and an air supply of 2-4 l / min.

In addition, the nutrient medium for growing the producer strain Saccharomyces cerevisiae Scm contains yeast extract - 10.0%, peptone - 20.0%, dextrose - 20.0%, distilled water - up to 100%.

In addition, 0.4-0.5 liters of yeast and distilled water up to 4 liters are taken per 1 kg of the mixture.

In addition, the drying of the obtained product is carried out in layers 2-5 cm thick at a temperature of 70-85 ° C.

A comparative analysis of the set of essential features of the proposed technical solution and the set of essential features of the prototype and analogues indicates its compliance with the criterion of "novelty."

In this case, the distinguishing features of the claims solve the following functional tasks.

The sign “yeast culture is grown on the basis of the producer strain Saccharomyces cerevisiae Scm” allows to increase the protein content in the target product.

The strain of yeast Saccharomyces cerevisiae Scm has the following properties: accumulated biomass of more than 10 g / l; mass fraction of protein in biomass according to Barnstein> 50%; the resulting protein is not toxic; the producer strain is stored without loss of quality for more than 24 hours in a liquid medium at a temperature of 4-8 ° C, more than 10 days in a semi-solid medium at a temperature of 4-8 ° C and more than 60 days at a temperature below -70 ° C in a special environment with a cryopreservative (10% glycerol).

The signs "the substrate in the form of a mixture containing in wt.%: Soybean meal - 30.0, rice flour - 60.0, rice husk - 10.0" provide a balanced mixture of amino acids, and also allow you to get a complete fodder microbiological protein with low cost price.

The signs "yeast culture ... combine with the substrate ... and then with distilled water" and the signs of the second dependent claim provide the possibility of forming a culture medium.

The signs "cultivated at a temperature of 32-34 ° C for 10-18 hours at a speed of rotation of the mixer 1000-1200 rpm and an air supply of 2-4 l / min" describe the optimal conditions for growing yeast biomass.

The features of the first dependent claim describe the optimal composition of the nutrient medium for growing the producer strain Saccharomyces cerevisiae Scm.

The features of the third dependent claim describe the drying mode of the resulting product.

Thus, the claimed method allows to obtain easily digestible, balanced in amino acid composition of the target product with a high protein content.

The inventive method is carried out according to standard technology on standard equipment.

At the first stage, the strain is prepared for inoculation and its growth. For this, the yeast culture Saccharomyces cerevisiae Scm is transferred to a plate with agarized YPD medium and the plates are incubated for 18-36 hours at a temperature of 30 ° C without mixing. When the colonies reach a size of 2-3 mm, an individual colony is inoculated into a liquid YPD medium containing peptone - 20%, yeast extract - 10%, dextrose - 20%, distilled water - up to 100% to obtain a night culture. Grow for 18 hours on a shaker incubator at 30 ° C and 250 rpm.

Then the substrate is prepared in the form of a mixture containing in wt.%: Soybean meal 30.0, rice flour 60.0, rice husk 10.0.

The calculated amount of the mixture is added to the bioreactor, then the Saccharomyces cerevisiae Scm yeast strain is added, distilled water is added (0.4-0.5 L of yeast and distilled water to 4 L are taken per 1 kg of mixture) and cultivation is carried out for 10-18 hours at a temperature of 32-34 ° C, with a stirrer rotation speed of 1000-1200 rpm and an air supply (aeration) of 2-4 liters per minute.

At the end of cultivation, a suspension of yeast biomass is placed in pallets with a layer of 2-5 cm and dried at 70-85 ° C, with periodic stirring. Dried KMB is ground and packaged in a container.

The target product is a powder, flakes or granules from light yellow to grayish brown. The yield of the target product is 95-97%. Mass fraction of crude protein (in terms of absolutely dry substance) is 40-55%; Mass fraction of protein according to Barnstein (in terms of absolutely dry matter) is 50-55%; Mass fraction of moisture - 10-15%.

KMB is stored in a dry, dark place at a temperature of 0 to + 25 ° C. The shelf life of KMB is 1 year.

Description of the components of the feed mixture.

When processing rice at grain plants, in addition to the main product, rice flour, husk and grain waste are obtained in large quantities. The use of these wastes in fodder production is associated with some difficulties: rice flour rancid quickly, the husk has low nutritional properties. Meat torrents are considered highly nutritious feed products, since they contain particles of mealy core, fruit and seed membranes, hairs of grain and partially embryos, as well as a certain amount of crushed flower film. The meal contains a lot of starch and less fiber. The digestibility of nutrients in feed pellets is good not only in ruminants, but also in pigs and poultry. Wheat meal is digested by 86-90%, rye - by 76-83, barley - by 75-80%, oat - by 75-76, rice - by 65-70, pea - by 90-91 and buckwheat - by 70 -73%. About 60% of crude protein is contained in rice flour, which makes up 60% of the feed mixture used as a substrate.

Rice husk, which makes up 10% of the feed mixture, is rich in carbohydrates, vitamins (thiamine, riboflavin, etc.). Rice husk incorporates organic compounds (sugar, nitrogen, amino acids, organic acids) and inorganic substances (Ca, P, Mg, K, Na, Mn, Zn, Fe, Cu).

The use of soy protein in the feed mixture is an inexpensive and optimal solution for enriching the substrate with feed protein. Soya beans contain 33% crude protein. 1 kg of soybean contains 31.9 g of lysine. Compared to cereals, legumes have 2–3 times more crude protein and 3–5 times more lysine, the main limiting amino acid, in legumes. Soy protein is close to animal proteins in this indicator, as a result of which soy is an excellent component of feed for pigs and poultry. Table 1 presents the characteristics of the nutritional value of legumes.

Table 1

Nutritional indicators of cereals and legumes

Soybean meal, comprising 30% of the feed mixture, is the remnants of oil extraction production.

The amino acid content in the target product was determined by known methods (Methods of quality control of amino acids in pharmaceutical analysis. Development and registration of drugs, No. 11, May 2015). The obtained data on the content of amino acids in the target product (MBC) are shown in Table 2.

table 2

The amino acid content in the target product

Amino Acid (AK) Content, g / kg Associated AK Free AK Alanine 28.57 2.54 Glycine 7.12 0.61 Valine 18.3 1,50 Leucine 37.42 2.60 Isoleucine 5.20 0.48 Proline 26.65 0.44 Threonine 2,31 0.04 Methionine 0.44 <0.01 Phenylalanine 5.05 4.06 Histidine 9.23 0.36 Tyrosine 28.92 1.14 Tryptophan 0.94 0.19 Aspartic acid 0,03 <0.01 Lysine 0.51 0.10

From table 2 it is seen that the amino acids alanine, leucine, proline, tyrosine and valine are most represented in the composition of the protein (KMB).

Thus, the balance of the feed mixture by amino acids is carried out by yeasting the substrate with the yeast strain Saccharomyces cerevisiae Scm.

Based on the calculation of the composition of the substrate, the protein content in KMB is composed of soybean meal protein, rice flour protein and yeast protein produced by the Saccharomyces cerevisiae Scm strain. The crude protein content in KMB is 40.0 g / 100 g of yeast biomass. The exchange energy in yeast biomass is 220 kcal / 100 g, therefore, the yeast biomass adds 62.7 kcal / 100 g to the exchange energy of KMB

Table 3 presents data on the composition of the protein KMB.

Table 3

Characteristics of feedstock and feed microbiological protein

From table 3 it is seen that the exchange energy in KMB is 238.9 kcal / 100 g

Thus, the developed technology allows to obtain a complete fodder microbiological protein (KMB), suitable for feeding various farm animals. KMB is not toxic, does not contain toxic elements, has a pleasant characteristic odor and has a rather high feed value: protein content of more than 40%, metabolic energy content of about 240 kcal / 100 g.

The invention is illustrated by the following examples of specific performance.

Example 1

At the first stage, the strain was prepared for inoculation and the culture of the producer strain was grown on a liquid nutrient medium. For this purpose, the Saccharomyces cerevisiae Scm yeast culture was inoculated with a microbiological loop on a plate with agarized YPD medium. The plates were incubated for 18-36 hours at a temperature of 30 ° C in an atmosphere of ambient air on an incubator shaker without a mixing mode. Once the colonies reached a size of 2-3 mm, using a microbiological loop, an individual colony was inoculated into YPD liquid medium containing peptone - 20%, yeast extract - 10%, carbohydrate (dextrose or glucose) - 20%, distilled water - up to 100% for receiving night culture. They were grown for 18 hours on a shaker incubator at 30 ° C and 250 rpm.

A mixture was prepared containing soybean meal 30.0%, rice flour 60.0%, rice husk 10.0%, which is a powder with a particle size of not more than 1 mm. For this, the components of the mixture were mixed in the following proportion: soybean meal 300 g, rice flour 600 g, rice husk 100 g.

A substrate in the amount of 1 kg was added to a 5 liter bioreactor (BIOSTAT, Sartorius), 400 ml of the grown culture of the Saccharomyces cerevisiae Scm strain were added and distilled water was added to 4 liters. The cultivation was carried out automatically at a temperature of 32 ° C, for 10 hours at a mixer rotation speed of 1200 rpm and an air supply of 4 l / min.

At the end of the cultivation, a suspension of yeast biomass was placed in pallets with a layer of 2 cm and dried in an oven with periodic stirring at 70 ° C. The residual moisture content of the biomass was 10%. Dried KMB was crushed and packaged in containers. The weight of the preparation obtained after drying was ~ 950 grams.

Example 2

At the first stage, the strain was prepared for inoculation and the culture of the producer strain was grown analogously to example 1.

A substrate was prepared in the form of a mixture containing soybean meal 30.0%, rice flour 60.0%, and rice husk 10.0%.

A substrate of 1 kg, 500 ml of a culture of the Saccharomyces cerevisiae Scm yeast strain and distilled water up to 4 liters were added to a 5 liter bioreactor (BIOSTAT, Sartorius). The cultivation was carried out at a temperature of 34 ° C, for 18 hours at a mixer rotation speed of 1000 rpm and an air supply of 2 l / min.

At the end of cultivation, a suspension of yeast biomass was placed in pallets with a layer of 5 cm and dried in an oven at 85 ° C with periodic stirring. The residual biomass moisture was 12%. Dried KMB was ground into a uniform powder with a hammer mill and packaged in containers. The weight of the preparation obtained after drying was ~ 970 grams.

The developed method is simple and allows you to get a complete fodder microbiological protein (KMB) with a fairly low cost due to the use of grain waste, suitable for feeding various farm animals.

KMB is not toxic, does not contain toxic elements, has a pleasant characteristic odor and has a rather high feed value: protein content of more than 40%, metabolic energy content of about 240 kcal / 100 g.

Claims (1)

A method of obtaining a feed microbiological protein, including preliminary obtaining a yeast culture of Saccharomyces cerevisiae, which is added to the substrate in the form of meal and water, cultivating the yeast biomass with stirring and aeration, followed by drying of the obtained product, characterized in that the yeast culture is grown on the basis of the producer strain Saccharomyces cerevisiae Scm, combined with the substrate in the form of a mixture containing, wt.%: soybean meal - 30.0, rice flour - 60.0, rice husk - 10.0 and further - with distilled water, and cultivated at the same time a temperature of 32-34 ° C for 10-18 hours at a stirrer rotation speed of 1000-1200 rpm and an air supply of 2-4 l / min, that the nutrient medium for growing the producer strain Saccharomyces cerevisiae Scm contains yeast extract - 10.0 %, peptone - 20.0%, dextrose - 20.0%, distilled water - up to 100%, while 0.4-0.5 l of yeast is taken per 1 kg of the mixture and distilled water to 4 l and the resulting product is dried layers 2-5 cm thick at a temperature of 70-85 ° C.