JP7618246B2 - Livestock and fish feed protein additives - Google Patents

Description

GKPM-OBOLENSK B-8465 VKM B-3265D VKM B-3264D VKPM B-12549

The present invention is intended for use in livestock and fish farming, and in particular relates to a biological feed additive used in livestock and fish breeding in artificial reservoirs to improve productivity and natural resistance.

Currently, a decline in feed quality, including energy and protein value, is observed. Almost all types of feed used are low-protein, so that the amount of protein (especially for cattle) does not exceed 85 g instead of the 108 g prescribed by standard. This leads to low livestock productivity, high feed consumption per unit of production and high feed costs.

Researchers have focused on using plant ingredients, especially plant protein sources, due to their availability and low cost. The main source of protein is soybean meal. However, the natural environment in the country is not favorable for growing soybeans in sufficient quantities. Fishmeal is the basic source of protein traditionally for feeding fish. Nevertheless, the price of fishmeal is constantly increasing and fish production is increasing.

Those skilled in the art will need to look for other methods and alternative sources for producing feed protein.

The introduction of protein feed additives into the feed mixture can optimize the nutritional composition of the feed, improve bioavailability, and influence the growth rate of livestock and fish, as well as prevent diseases.

A patent search revealed the following sources:

Therefore, technologies have been developed for producing feed proteins from wastes of the alcohol (dried distillers grains with solubles) and flour milling (bran, flour) industries, as well as mixtures of distillers' spent grain and grain waste (RU 2159287; RU 2140449; USSR Inventor's Certificate No. 1595900; USSR Inventor's Certificate No. 1571061; USSR Inventor's Certificate No. 1532580; USSR Inventor's Certificate No. 1507787; RU 2054881; RU 2042713, etc.). In the above invention, the source culture is a Candida yeast-like fungus imperfecti. A drawback of the known microbial strain is that it cannot be actively grown in a long-term non-sterile culture process. Furthermore, the use of Candida yeast as the source culture requires complicating the production technology by forcing the introduction of a heat treatment step of the produced feed biomass to ensure that there are no live cells in the final product.

The prior art contains various protein-producing microbial strains (Russian Patent Applications Nos. 2244000, 2390554, and 2244001). A common defect of these strains is that the concentration of accumulated biomass at 2.5% lactic acid is insufficiently high, and as a result, the amount of protein that can be produced by cultivation is insufficient.

A promising method for producing wholesome protein feed products is to use protein-producing methanotropic bacteria. Under appropriate conditions, methanotropic bacteria exhibit vigorous processing of natural gas, rapid growth and accumulation of biomass that is rich in valuable proteins, vitamins, and other bioactive substances.

Producing proteins using methane has several advantages over liquid hydrocarbons: natural gas reserves are abundant, it has good transportability, and the final product can be produced without further removal of materials.

Russia has huge gas reserves, which according to some sources account for up to 40% of the world's reserves. In addition to bringing economic benefits, implementing microbiological protein production at Russian plants will contribute to the country's food security.

The prior art includes various feed protein producing microbial strains, including various species of methane oxidizing bacteria (methanotrophs), such as Pseudomonas methanica, Methylococcus capsulatus VKM B-2116, Methylocystis parvus VKM B-2129, Methylosinus sporium VKM B-2123, Methylosinus trichospohum VKM B-2131, Methylosinus trichospohum VKM B-2132, Methylosinus trichospohum VKM B-2133, Methylosinus trichospohum VKM B-2134, Methylosinus trichospohum VKM B-2135, Methylosinus trichospohum VKM B-2136, Methylosinus trichospohum VKM B-2137, Methylosinus trichospohum VKM B-2138, Methylosinus trichospohum VKM B-2139, Methylosinus trichospohum VKM B-2140, Methylosinus trichospohum VKM B-2141, Methylosinus trichospohum VKM B-2142, Methylosinus trichospohum VKM B-2143, Methylosinus trichospohum VKM B-2144, Methylosinus trichospohum VKM B-2145, Methylosinus trichospohum VKM B-2146, Methylosinus trichospohum VKM B-2147, Methylosinus trichospohum VKM B-2148, Methylosinus trichospohum VKM B-2149, Methylosinus trichospohum VKM B-2150, Methylosinus trichospohum VKM B-2117, Methylobacter acidophilus, Methylomonas rubra VSB-90, Methylococcus ChM-9, Methylococcus capsulatus VSB-874, Methylococcus minimus, Methylomonas methanica, and Methylomonas agile.

These are microbial synthetic products obtained by culturing methane-oxidizing bacteria using natural gas. The strains are characterized by different growth rates and biomass yields.

The prior art includes the Methylococcus capsulatus VSB-874 strain, which is a source of biomass production. This strain has been deposited at the museum of cultures of the VNIIgenetika Institute under collection number TsMPM V-1743 (USSR Inventor's Certificate No. 770200). The carbon source is both pure natural gas and methane in natural gas, and methanol. Products using this strain are available under the GAPRIN trademark. The disadvantages of this strain are poor productivity (8-10 g/lh), low raw protein content in the biomass (up to 70%), and unstable biomass yield (30-60 g/l).

The prior art includes an invention (RF2687137) related to biotechnology that can be used to produce microbial protein mass for feeding animals. A heterotrophic bacterial strain of Klebsiella pneumoniae 1-17 capable of using the co-oxidation products of methane homologues present in natural gas has been deposited at the State Collection of Pathogenic Microorganisms and Cell Cultures GKPM-OBOLENSK (accession number B-8465). This strain increases the productivity of methane-oxidizing bacteria.

The prior art includes inventions related to biotechnology that can be used to produce microbial protein mass for feeding animals. The heterotrophic bacterial strain Cupriavidus giraldii GBS-15-1, capable of using the co-oxidation products of methane homologs present in natural gas, has been deposited at the Russian National Collection of Microorganisms (G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences) (Accession No. VKM B-3265D). This strain increases the productivity of methane-oxidizing bacteria (RF2687135).

The prior art includes inventions related to the microbiology industry. Stenotrophomonas acidoaminophila GBS-15-2 heterotrophic bacterial strain is capable of using the co-oxidation products of methane homologues present in natural gas. Stenotrophomonas acidoaminophila heterotrophic bacterial strain is deposited at the Russian National Collection of Microorganisms (G.K.Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences) (Accession No. VKM B-3264D). The heterotrophic bacterial strain Stenotrophomonas acidoaminophila VKM B-3264D can be used to produce microbial protein mass (RF No. 2687136).

The closest analogue in terms of technical essence and claimed effect is the Methylococcus capsulatus GBS-15 methane-oxidizing bacteria strain. This strain is used to produce a protein and vitamin complex for balancing livestock feed. This strain has been deposited in the Russian State Collection of Industrial Microorganisms under the number VKPM V-12549. This strain allows the accumulation of biomass up to 32 g/l (RF No. 2613365). The minimum raw protein content in the biomass is 79%.

Russian Patent Application Publication No. 2159287 Russian Patent Application Publication No. 2140449 Russian Patent Application Publication No. 2054881 Russian Patent Application Publication No. 2042713 Russian Patent Application Publication No. 2244000 Russian Patent Application Publication No. 2390554 Russian Patent Application Publication No. 2244001

The technical objective is to expand the range of spent protein feed additives based on the methane-oxidizing bacterium Methylococcus capsulatus, resulting in higher technical feasibility, better cost-effectiveness of the process and a higher protein content in livestock and fish bred in artificial reservoirs.

The technical outcome of using the proposed feed additive is the creation of efficient additive production technology, improving additive performance by increasing livestock and fish productivity.

The technical result was achieved by using a protein feed additive for livestock and farmed fish, which contained the methane-oxidizing bacterial strain Methylococcus capsulatus (GBS-15) VKPM B-12549, which also contained the heterotrophic bacterial strain Cupriavidus gilardii (GBS-15-1) VKM B-3265D and/or the heterotrophic bacterial strain Stenotrophomonas acidoaminophila (GBS-15-2) VKM B-3264D and/or the heterotrophic bacterial strain Klebsiella pneumoniae 1-17 GKPM-OBOLENSK B-8465, in a uniform amount with the following component ratios by weight:
Methylococcus capsulatus VKPM B-12549
- Methane oxidizing strain 85:94
- Strain mixture 6:15

By feeding livestock with a diet containing a protein feed additive containing the proposed mixture of strains, weight gain due to muscle tissue growth is promoted. Meat quality is improved by reducing fat content in muscle tissue, reducing the subcutaneous fat layer, and increasing the loin area.

The present invention will enable increased productivity, survival and meat safety of livestock and fish, as well as production of meat with functional properties.

The claimed ingredient ratios of the feed additive allow the production of an easily digestible high protein additive containing heterotrophic bacteria. The protein feed additive for livestock and fish is produced by mixing the ingredients according to the claimed ratios, after which the product is granulated or subjected to extrusion as required. Table 1 shows the recipe for the proposed protein additive.

The following experiments were carried out using variations of the protein feed additive.
- Variant 1: a binary mixture of strains GBS-15 and GBS-15-2.
- Variant 2: a ternary mixture of strains GBS-15, GBS-15-1 and GBS-15-2.
- Variant 3: a quaternary mixture of strains GBS-15, GBS-15-1, GBS-15-2 and 1-17.
- Variant 4: GBS-15 variant base additive.

The experiment showed that the use of a feed additive mixture containing the proposed protein feed additive allows for a significant optimization of the nutrient composition of the feed and an improvement in bioavailability. Furthermore, the needs of fish, poultry and livestock for the necessary nutritional components are met, and the feed consumption per unit of production is reduced.

The source of the protein feed additive is the methanotroph Methylococcus capsulatus GBS-15. This strain was obtained in a long-term multi-stage selection process by culturing in a liquid mineral medium under aeration and mixing conditions in a natural gas atmosphere. Methylococcus capsulatus GBS-15 is cultivated in related cultures of heterotrophic relatives, such as Cupriavidus giraldii, Stenotrophomonas acidoaminophila, Klebsiella pneumoniae, etc., and utilizes the co-oxidation products of methane homologs present in natural gas and the main metabolic products of Methylococcus capsulatus GBS-15.

The resulting protein feed additive is a dried, inactivated biomass of methane-oxidizing bacteria and related heterotrophic bacteria.

Protein feed additives in the form of powder or granules are given in the required amount specified in the standards for use for preparing the feed mixture. Protein feed additives have high nutritional value and are safe for fish, livestock and poultry.

Methylococcus capsulatus GBS-15 and related bacteria are non-pathogenic, non-toxic, and safe.

The amount of protein in the protein feed additive is equivalent to that of animal protein (fish and meat and bone meal). This allows the protein feed additive to be widely used as a replacement for fish meal and soy meal in feed diets.

The biological value of the protein feed additive by chemical score is 65-70%. The limiting amino acid is methionine. The relative biological value with respect to casein in the Tetrahymena pyriformis test organism is 65-75%.

The nutritional properties of the protein feed additive were tested in animals for which the product was developed, including pigs, poultry, calves, sheep and fish. The animals were fed according to the adopted standards. The control diet contained pea flour as a protein source. The claimed amounts of the protein feed additive and the reference additive included 20% of the daily amount of digestible protein in the diet.

The essence of the present invention is illustrated through the following examples:
The reference diet used in all examples was GAPRIN.

Example 1. Tests on fish The feed additive was tested on carp fingerlings weighing 38-45 g under laboratory conditions in flow-through aquariums. In all tests, groups of 20 carp fingerlings were kept in 70 liter flow-through aquariums with a water temperature of 18-21°C and an oxygen content of 6.8-9 mg/l. The feeding course of the feed additive was 24 days.

After the feeding process was completed, a comparative analysis of the physiological status of the fish was performed using hematological parameters at each stage of the experiment. Calculations of absolute daily gain, relative gain, and average gain in mean body weight were all performed using conventional methods.

See Table 2 for test results.

The protein feed additive can be introduced into fish feed without any disturbance of the amino acids in the additive. The proposed strain mixture has high productivity levels, which contributes to increasing the feed consumption rate and weight gain rate by the fish.

Analysis of diets containing protein feed additives at different ages of fish showed that including this ingredient in the diet promotes the growth rate of fish without disturbing their physiological state and while reducing feed costs. Larval survival is also correlated with diet. The inclusion of protein feed additives in the experimental diets significantly increases the survival rate of carp to 93% compared to 63% for the reference diet.

Therefore, with the right balance of nutrients in fish feed, protein feed additives show similar results to other high protein ingredients.

Example 2. Tests in pigs The biological products were tested in the diet of pigs of different age groups and different classes.

Animals were fed according to the adopted standards. The control diet contained soybean flour as a protein source.

The test groups received biological additives in amounts up to 20% of the daily amount of digestible protein in the diet. At the end of each growth period, the weight of each individual animal was measured before the experiment. The physiological condition and health of the animals were normal throughout the experiment. No adverse effects were observed throughout the experiment. No differences were observed in health, feed consumption, and average daily weight gain values between the test and reference animals.

The level of protein feed additive in the test group was 20% of the daily value of digestible protein. The diet density was consistent with generally accepted standards for the age of the animals involved and was identical for the reference and test groups.

No deviations in the physiological status and health of the animals were observed throughout the experiment.

The use of protein feed additives in pig feed increased weight gain by 12% and survival rate of young pigs by 7.5% compared to the reference group without causing metabolic disorders, but no increase in incidence was observed in the test group.

The study results demonstrate the efficiency and safety of using protein feed additives in the diets of growing and fattening pigs.

Example 3. Poultry Trials A productivity trial was carried out on broiler poultry. The poultry in the test group were fed a diet containing a maximum of 5% protein feed additive instead of 50% animal protein. The birds in the reference group were fed a standard complete diet.

The main physiological parameters of the reference and test groups at the end of the experiment were as follows: survival rate of birds: test group 99.2%, reference group 98.8%. Egg laying intensity: test group 79.4%, reference group 72.4%.

Claims (1)

A protein feed additive for livestock and farmed fish , comprising the Methylococcus capsulatus VKPM V-12549 methane oxidizing bacteria strain,
The protein feed additive comprises 85-94% by weight of inactivated Methylococcus capsulatus VKPM V-12549 methane-oxidizing strain and the remainder a mixture of inactivated strains,
The mixture of strains is characterized in that it contains at least one of the heterotrophic bacterial strains Cupriavidus girardii VKM B-3265D, Stenotrophomonas acidoaminophila VKM B-3264D, and Klebsiella pneumoniae 1-17 GKPM-OBOLENSK V-8465 .