RU2412612C1 - Method for production of "ferm km" probiotic fodder additive for domestic animals and birds - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: invention is intended for use in production of fodders. The method envisages production of liquid cultures of Bacillus subtilis VKPM V-8130, Bacillus subtilis VKPM V-2984, Bacillus subtilis VKPM V-4099 and Bacillus licheniformis VKPM V-4162 by way of their separate aerobic in-depth cultivation in nutrient media of a specified composition. The produced liquid cultures of Bacillus subtilis are mixed at a 6:6:1 ratio accordingly, applied onto a sterile mixture of beet cake and wheat bran preliminarily treated with a cellulolytic enzyme dissolved in a nutrient medium. PH and batch moisture content are brought to 7.5-8.0 and 43-48% accordingly; the mixture is thoroughly stirred and one proceeds with solid phase fermentation under restricted oxygen access conditions during 48 h at a temperature of 45-50°C and drying for moisture content to be 8-10%. Bacillus licheniformis VKPM V-4162 liquid culture is immediately mixed with beet cake and dried till moisture content is 8-9%. The dried products are mixed and crushed for a homogenous product production.

EFFECT: one provides for increased productivity of animals and birds due to fodder digestibility, stimulation of metabolic and immune processes in the organism and normalisation of intestinal microbiocenosis as well as livestock preservation enhancement.

3 cl, 2 tbl, 2 ex

Description

The invention relates to feed production, in particular to methods for the preparation of biologically active feed additives for mammals, birds, fish, which increase the efficiency of digestion, have a versatile effect on the animal organism and allow their use both for the treatment and prevention of gastrointestinal diseases, and for stimulating growth and increase animal productivity.

There is a known method of feeding farm animals, in which the waste of production of the Biosporin preparation, a probiotic from the association of Bacillus subtilis VKPM B-2335 and Bacillus licheniformis VKPM B-2336 bacilli, is added to the main diet. Waste is a centrate obtained after separation of cells, which is used in liquid form in animal diets daily during the feeding period or in courses of 14-21 days. The B. subtilis VKPM B-2335 and B. licheniformis VKPM B-2336 cells contained in the preparation help stabilize the normal intestinal microflora, displace pathogenic and conditionally pathogenic microorganisms, provide protection against a large group of intestinal viruses by stimulating local immunity, production of interferon, and increasing overall body resistance (1). It should be noted that obtaining probiotics consisting of an association of several types of bacteria requires control over the preservation of the necessary ratio of bacterial forms in the association to ensure stable indicators of the quality of the drug and its waste - the centrate.

A known method for producing animal feed from plant materials, including grinding plant materials and its enzymatic hydrolysis, which is carried out by introducing into the plant substrate a pre-prepared preparation based on the contents of the cecum of the common vole Microtus arvalis (2). However, the proposed association of bacteria cannot maintain a stable ratio of its constituents for a long time, which creates difficulties for obtaining a drug with desired properties.

A known method of obtaining a biologically active feed additive based on a bacterial strain B. subtilis VKPM B-8130 isolated from the cecum of the capercaillie. An additive is obtained by fermenting the bacteria in a liquid, nitrogen-free Ashby medium, with a cellulose-containing fine fraction substrate as a carbon source. Incubation is carried out for 5 to 72 hours under facultative anaerobic conditions, followed by drying of the target product at 60 ° C (3). But at the same time, there is no significant accumulation of biomass, which reduces the effectiveness of the drug.

The closest in technical essence and the achieved result is a method for producing a biologically active feed additive, which includes two stages of its production: deep aerobic cultivation of a probiotic cellulolytic bacillus - strain B. subtilis VKPM B-8130 to obtain a liquid culture (first stage), and subsequent solid-phase fermentation plant materials with difficult access of oxygen (second stage). The process is carried out in the presence of sodium bicarbonate (NaHCO ₃ ) to bind acetyl-CoA and neutralize acidic fermentation products, thereby using the Neuberg metabolic trap method for yeast fermentation. Sea buckthorn, dogrose, sunflower meal or cake, waste from flour milling, and cereal production are used as plant raw materials (4). The disadvantage is the use of a monoculture of bacteria, which limits the spectrum of the biological effect of the drug on the animal organism.

The technical result of the invention is to increase the productivity of animals and birds by normalizing the intestinal microbiocenosis, increasing the digestibility of feed, stimulating the metabolic and immune processes of the body. The use of the probiotic feed additive of KM Farms in the diets of farm animals and birds ensures their more intensive growth and development, increases feed conversion by 10% and increases the safety of the livestock.

To achieve a technical result when obtaining a probiotic feed additive, Farm KM for farm animals and poultry, separate deep cultivation is carried out: a cellulolytic culture of Bacillus subtilis VKPM B-8130 and Bacillus subtilis VKPM B-2984 on a nutrient medium containing, g / l:

molasses 24-26 corn gluten 14-16 corn extract 19-21 disubstituted potassium phosphate 1-3 sodium chloride 9-11 magnesium sulfate 0.4-0.6 water rest,

cellulolytic culture of Bacillus subtilis VKPM B-4099 on a nutrient medium containing, g / l:

molasses 14-16 corn gluten 9-11 corn extract 9-11 disubstituted ammonium phosphate 1-3 sodium chloride 9-12 magnesium sulfate 0.4-0.6 water rest,

and Bacillus licheniformis VKPM B-4162 on a nutrient medium containing, g / l:

molasses 24-26 corn extract 18-22 disubstituted potassium phosphate 1-3 sodium chloride 9-11 magnesium sulfate 0.4-0.6 water rest,

for 18-24 hours, then solid-phase fermentation is carried out, for which the obtained liquid cultures of Bacillus subtilis VKPM B-8130, Bacillus subtilis VKPM B-2984 and Bacillus subtilis VKPM B-4099 are mixed in a ratio of 6: 6: 1, respectively, applied to a sterile mixture of beet pulp and wheat bran, which is pre-treated with a cellulolytic enzyme dissolved in a nutrient medium containing 30 l of water: molasses - 255-265 g, potassium disubstituted phosphate - 98-102 g, magnesium sulfate - 25-26 g, yeast - 1.8-2.2 g, adjust the pH to 7.5-8.0, and the batch humidity to 43-48%, cm thoroughly mixed, solid-phase fermentation is carried out under conditions of limited oxygen access for 48 hours at a temperature of 45-50 ° C and dried at a temperature of 50 ° C to a moisture content of 8-10%, and the liquid culture of Bacillus licheniformis VKPM B-4162 is directly mixed with beet pulp and dried to a moisture content of 8-9%, the obtained dried products are stirred for 0.5 h and subjected to crushing to obtain a homogeneous product.

The composition of the probiotic supplement KM KM includes strains of Bacillus subtilis VKPM B-8130, Bacillus subtilis VKPM B-2984, Bacillus subtilis VKPM B-4099 and Bacillus licheniformis VKPM B-4162, which have unique antagonistic properties and efficiency in relation to almost 90% conditionally pathogenic intestinal bacteria. As a phytosubstrate for solid-phase fermentation, a mixture of dry beet pulp and wheat bran taken in a ratio of 4: 1 is used. For the treatment of phytosubstrate with a cellulolytic enzyme, celllovolidin or cellulox F with a cellulase content of at least 2000 U / g is used as a cellulolytic enzyme.

To increase the efficiency of feed use and stimulate the growth and productivity of animals and poultry, the biologically active additive Lactovit for animals and poultry manufactured by STC BIO LLC, Shebekino, Belgorod Region, is added to the obtained solid-phase fermentation product. according to TU 9291-002-54664067-2003. Lactovit contains cultures: Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacterium bifidum and / or Bifidobacterium longum and / or Bifidobacterium adolescentis, Lactococcus lactis subsp.cremoris or Lactococcus lactis subspobcuspus, lll. bulgaricus in the amount of 4 liters per load.

The following are examples of biologically active feed additives.

Example 1. The strains of Bacillus subtilis VKPM B-8130, Bacillus subtilis VKPM B-2984, Bacillus subtilis VKPM B-4099 and Bacillus licheniformis VKPM B-4162 are stored and grown in test tubes on solid nutrient media MPA or glucose-peptone medium composition, g / l:

glucose 20,0 peptone 4.0 yeast extract 1,5 disubstituted ammonium phosphate 1,5 magnesium sulfate 0.5 agar 2 tap water  up to 1 l

pH of the medium is 7.0 ± 0.5.

The medium is poured into test tubes (in fifty-millimeter tubes of approximately 20-25 ml of medium), sterilized at 124 ± 2 ° C for 40 minutes. Tubes with sterile medium after autoclaving are beveled to obtain mowed agar (jambs).

The sterile medium is seeded under aseptic conditions. Cultivation is carried out at 37-45 ° C for 24 hours. Reseeding on a solid medium is done once every 1-2 months. Culture tubes are transferred to a refrigerator and stored in a refrigerator at 4-6 ° C. In this way, cultures are stored and working jambs are prepared for preparing seed in flasks.

The cultivation of seed in flasks is carried out on a medium of the following composition, (g / l):

molasses fifteen corn gluten 10 yeast autolysate (ml / l) twenty magnesium sulfate 0.5 disubstituted potassium phosphate 2.0 tap water up to 1 l

pH 7.0 ± 0.5, on shakers at 190 rpm at 400 ° C for 36 hours

Obtaining liquid cultures of Bacillus subtilis and Bacillus licheniformis is carried out by separate deep cultivation of a pure microbiological culture in stainless steel fermenters, in sterile conditions, with aeration, mixing and constant temperature systems, on nutrient media presented in table 1.

Table 1 No. p / p The name of the crop The concentration of nutrients in the environment, g / l. Molasses Corn gluten Corn extract (NH) ₂ HPO ₄ K ₂ HPO NaCl MgSO ₄ Water up to 1 L one Bacillus subtilis VKPM B-8130 Bacillus subtilis VKPM B-2984 25 fifteen twenty - 2 10 0.5 2 Bacillus licheniformis VKPM B-4162 25 - twenty - 2 10 0.5 3 Bacillus subtilis VKPM B-4099 fifteen 10 10 2 - one one

The cultivation time of 20 hours

Then, solid phase fermentation is carried out, for which a fermentation mass is prepared. Dry beet pulp and wheat bran, taken in the amount of 200 kg, beet pulp and 5 kg, wheat bran, are used as a substrate. The products are thoroughly mixed for 0.5 hours. 30 liters of water are added to the substrate and the product is sterilized with constant stirring and a temperature of 100 ° C for 3.0 hours, for which sharp steam is supplied from the steam generator to the sterilization temperature. After the sterilization process is completed, the substrate is cooled to 50 ° C. Then, for 200 kg of cooled substrate, 30 l of pre-prepared nutrient medium is added, containing: molasses - 260 g (1.3 g per 1 kg of substrate), K ₂ HPO ₄ - 100 g (0.5 g per 1 kg of substrate), Mg SO ₄ - 26 g (0.13 g per 1 kg of substrate), yeast - 2.0 kg (10 g per 1 kg of substrate) and water - up to 30 liters. Before adding it to the substrate, celloviridin is added to the nutrient medium at a rate of 200 g per volume of medium — 1 g / kg of dry substrate with a cellulase content of at least 2000 U / g. The mixture is thoroughly mixed, kept at a temperature of 48 ° C for 2 hours, the pH of the mixture should not exceed 6.5. After the process of fermentolysis of the resulting mixture, a mixture of culture liquids Bacillus subtilis VKPM B-8130, Bacillus subtilis VKPM B-2984 of 30 L per charge and Bacillus subtilis VKPM B-4099 - 5 L per charge are added to it, the pH is adjusted to 8.0 and humidity kneading up to 45%. The mixture is thoroughly mixed, packaged in bags and sent to a fermentation chamber for solid phase fermentation at a temperature of 40 ° C for 2 days. After completion of the solid-phase fermentation process, drying is carried out at a temperature of 50 ° C with stirring to a moisture content of 10%.

At the same time, 65 l of Bacillus licheniformis VKPM B-4162 liquid culture containing at least 5.6 × 10 ⁸ CFU / g are applied to 200 kg of beet pulp, the mixture is thoroughly mixed and dried to a moisture content of not more than 9%.

The dried product after solid-phase fermentation is mixed in a mixer with a dry mixture of Bacillus licheniformis VKPM B-4162 with beet pulp in the ratio:

200 kg of dry product after solid-phase fermentation of phytosubstrate with a mixture of Bacillus subtilis strains and 25 kg of a dry mixture of Bacillus licheniformis VKPM B-4162 with beet pulp. The resulting mixture of products is stirred for 0.5 h, transferred to a two-stage crushing to obtain a homogeneous product with a particle size that meets the requirements of the feed industry products.

The finished product is packaged in bags of 20 kg according to NTD.

Example 2. Obtaining liquid cultures of Bacillus subtilis VKPM B-8130, Bacillus subtilis VKPM B-2984 and Bacillus subtilis VKPM B-4099, sterilization, substrate fermentolysis and mixing of all components is carried out analogously to example 1. Then, the resulting mixture of culture liquids Bacillus subtilis and sterile fermented substrate add biologically active additive Lactovit, manufactured by LLC "Scientific and Technical Center BIO" Shebekino, Belgorod region. according to TU 9291-002-54664067-2003 in the amount of 4 liters per load. The pH of the mixture is adjusted to 7.5 and the batch humidity is up to 43%. The mixture is thoroughly mixed, packaged in bags and sent to a fermentation chamber for solid phase fermentation at a temperature of 40 ° C for 2 days. After completion of the solid phase fermentation process, drying is carried out at a temperature of 50 ° C with stirring to a moisture content of 10%.

At the same time, 65 l of Bacillus licheniformis VKPM B-4162 liquid culture containing at least 5.6 × 10 ⁸ CFU / g are applied to 200 kg of beet pulp, the mixture is thoroughly mixed and dried to a moisture content of not more than 9%.

The dried product after solid-phase fermentation is mixed in a mixer with a dry mixture of Bacillus licheniformis VKPM B-4162 with beet pulp in the ratio:

200 kg of dry product after solid-phase fermentation of phytosubstrate with a mixture of Bacillus subtilis strains and 25 kg of a dry mixture of Bacillus licheniformis VKPM B-4162 with beet pulp. The resulting mixture of products is stirred for 0.5 h, transferred to a two-stage crushing to obtain a homogeneous product with a particle size that meets the requirements of the feed industry products.

The finished product is packaged in bags of 20 kg according to NTD.

Obtained by the proposed method, the probiotic feed additive of Farms KM for farm animals and poultry contains: cultures of Bacillus subtillis, Bacillus licheniformis, a complex of lactic acid bacteria and their metabolic products - a set of important enzymes - cellulase, endoglucanase, amylase, protease, lipase, organic acids, phytosorbents, biologically active substances, vitamins and amino acids. At the same time, microbiological cultures are in an immobilized form on a phytocarrier, which allows increasing their biological effectiveness and protecting bacteria from adverse environmental conditions. The combination of these factors ensures the destruction of fiber, proteins, polysaccharides, the neutralization of toxins, the suppression of pathogenic microorganisms in the animal body, providing an improvement in the secretory function of the digestive system, intestinal motility and blood circulation. The carrier of biologically active substances is phytosorbent - beet pulp and wheat bran.

Immobilization of microorganism cells on phytosorbent, their security, allows you to:

- microorganisms of the additive pass the acidic environment of the stomach and suppress pathogenic and conditionally pathogenic microorganisms in the intestine, stimulate the development of normoflora 5-7 times, in particular, ruminococci, lactobacilli;

- withstand the high temperature associated with the industrial production of granular feed (up to 105 ° C);

- maintain quality characteristics during long-term storage and in a wide temperature range.

Farm KM Probiotic Feed Additive is a yellow to brown bulk product. Compatible with components that are part of premixes, feed.

Recommended input rates for feed are presented in table 2.

The probiotic feed additive Farm KM is a new generation of enzyme-probiotic feed additive that provides increased productivity of animals and birds by increasing the digestibility of feed, stimulating the body's metabolic and immune processes.

Based on studies on the effectiveness of the use of the probiotic supplement Ferm KM for feeding animals and poultry, it was found that the use of this additive increases:

protein digestibility by 2.2-2.9%;

digestibility of fat by 0.55-1.4%;

the availability of lysine, methionine by 2.5-2.9%;

use of calcium, phosphorus by 5.6-3.5%;

increases the level of metabolic energy by 5-8%.

table 2 Application Application chart Result Chicken Broiler 1-20 Day 0.5 kg / t feed Normalization of microflora, stimulation of metabolic and immune processes, the rapid development of enzymatic and digestive systems, better feed eatability, an increase in average daily growth. Chicken broiler from day 20 - until slaughter 0.25 kg / ton of feed Chicken 1-20 day 0.5 kg / t feed Normalization of microflora, stimulation of metabolic and immune processes, the rapid development of enzymatic and digestive systems, better feed eatability. When transferring hens to the category of laying hens for 15 days and then for 2 months. 0.25 kg / ton of feed Stimulation of reproductive function and related consequences, reduction of embryonic mortality. Newborn calves 0.5 g / head day Normalization of microflora, stimulation of metabolic and immune processes, the rapid development of enzymatic and digestive systems, better feed eatability, increased average daily weight gain. Calves 2-6 months 1-2 g / head per day Calves from 6 months. 2-3 g / head per day (fattening period) Dairy cows 3-5 g / head per day Increased feed intake, improved metabolism, stimulates milk production. Sows 0.5 kg / t feed a month before farrowing and during lactation Bearing healthier piglets. Piglets from the beginning of feeding 1.0 kg / ton of cold-smoked for 2 months. Normalization of microflora, stimulation of metabolic and immune processes, rapid development of enzymatic and digestive systems, increased daily gain. Piglets at rearing 0.5 kg / tonne / fodder Fattening Pigs 0.25 kg / tonne / fodder

Bibliography

1. RU 2236147 C2, A23K 1/00, 09/20/2004.

2. RU 2153813 C1, A23K 1/165, 08/10/2000.

3. RU 2202224 C2, A23K 1/165, 04.20.2003.

4. RU 2346463 C2, A61K 1/165, 02.20.2009.

Claims (3)

1. A method of obtaining a probiotic feed additive for farm animals, which consists in the fact that they carry out separate aerobic deep cultivation: a cellulolytic culture of Bacillus subtilis VKPM B-8130 and Bacillus subtilis VKPM B-2984 on a nutrient medium containing, g / l:
molasses 24-26 corn gluten 14-16 corn extract 19-21 disubstituted potassium phosphate 1-3 sodium chloride 9-11 magnesium sulfate 0.4-0.6 water rest,
cellulolytic culture of Bacillus subtilis VKPM B-4099 on
nutrient medium containing, g / l:
molasses 14-16 corn gluten 9-11 corn extract 9-11 disubstituted ammonium phosphate 1-3 sodium chloride 9-12 magnesium sulfate 0.4-0.6 water rest,
and Bacillus licheniformis VKPM B-4162 on a nutrient medium containing, g / l:
molasses 24-26 corn extract 18-22 disubstituted potassium phosphate 1-3 sodium chloride 9-11 magnesium sulfate 0.4-0.6 water rest,
for 18-24 hours, then solid-phase fermentation is carried out, for which the obtained liquid cultures of Bacillus subtilis VKPM B-8130, Bacillus subtilis VKPM B-2984 and Bacillus subtilis VKPM B-4099 are mixed in a ratio of 6: 6: 1, respectively, applied to a sterile mixture of beet pulp and wheat bran, which is pre-treated with a cellulolytic enzyme dissolved in a nutrient medium containing 30 l of water: molasses - 255-265 g, potassium disubstituted phosphate - 98-102 g, magnesium sulfate - 25-26 g, yeast - 1.8-2.2 g, adjust the pH to 7.5-8.0, and kneading moisture to wet 43-48%, the mixture is thoroughly mixed, solid phase fermentation is carried out under conditions of limited oxygen supply for 48 hours at a temperature of 45-50 ° C and dried at a temperature of 50 ° C to a moisture content of 8-10%, and the Bacillus licheniformis liquid culture VKPM B -4162 is directly mixed with beet pulp and dried to a moisture content of 8-9%, the obtained dried products are mixed for 0.5 h and subjected to crushing to obtain a homogeneous product. 2. The method according to claim 1, characterized in that prior to the solid-phase cultivation, the biologically active additive Lactovit for animals and birds containing cultures: Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacterium bifidum and / or Bifidobacterium longum and additionally is introduced into the obtained Bacillus subtilis liquid cultures or Bifidobacterium adolescentis, Lactococcus lactis subsp. cremoris or Lactococcus lactis subsp.casei, Lactobacillus delbrueckii subsp. bulgaricus. 3. The method according to claim 1, characterized in that as cellulolytic enzyme use celloviridine or cellulox F with a cellulase content of at least 2000 u / g