Strain of propionibacterium freudenreichii subsp shermanii-producer of feed protein

FIELD: medicine.

SUBSTANCE: strain of Propionibacterium freudenreichii subsp.shermanii Ac-103 is deposited into the Russian national Collection of microorganisms used in veterinary medicine and animal husbandry numbered VGNKI - 08/02/1957 DEP. This strain is a producer of feed protein.

EFFECT: invention enables to eliminate pollution at production of protein feed, to increase relative protein yield, to reduce energy costs at preparation of protein feed, to simplify medical equipment, to dispose wastes of manufactures using natural raw materials.

1 tbl, 9 ex

 

The invention relates to food industry, biotechnology, agriculture.

Known yeast Saccharomices cerevisiae race-1986 (Patent RU №2183666, C12F 3/10, publ. 2002) /1/.

Yeast this race is used for digestion of the grain mash in alcohol production. Their function is the processing of sugars in the wort into alcohol. The feed product is not the target product, its utilitarian function is not defined. He is the alcohol production waste, polluting the environment. Control over the quality and composition of the waste difficult to implement.

A known strain of the yeast Candida tropicalis BWA-C producing fodder protein (Patent RU №2042713, SR 1/100, publ. 20.06.2002 g) /2/.

The disadvantage of this known strain is use for feed protein of the yeast Candida, which are conditionally pathogenic microorganism that requires additional process of thermolysis and special equipment, a complex system of neutralization of effluents and air emissions from large volumes of used air. Sanitary and epidemiological rules SanPiN 2.3.2.1078-01 yeasts of the genus Candida included in the list of substances that have harmful effects on human health. The growth rate of cells known strain is low, which contributes to the production cycle which reduces the specific yield of the target product at the stage of biosynthesis. In addition, in the preparation of feed protein by the use of this famous strain of yeast that requires a large consumption of heat and energy for drying biomass and considerable expense (1.5 g/l) ammonium sulfate.

The use of bacteria as a producer of protein feed is more effective because the bacteria produce up to 75% protein by weight, while the yeast is not more than 60%. The use of a new strain of Propionibacterium freudenreichii subsp. shermanii 103/12 for the preparation of protein meal does not require air consumption and energy consumption per presentation, because this strain of propionic acid bacteria is gone anaerobic. The strain has a broad spectrum antimicrobial action, which eliminates the development of foreign microflora in the process of biosynthesis and therefore does not require special equipment for compliance with conditions of sterility. Opportunities for the recycling of various waste industries using natural raw materials, while increasing the biomass of a new strain with the purpose of preparation of protein feed solves environmental problems of enterprises.

Closest to the present invention is a strain of Propionibacterium freudenreichii subsp. shermanii 103/12 - producer of feed protein (Patent RU №2247149 C1, C12N 1/20, SR 21/00 // (12N 1/20, C12R 1/01) (publ. 27.02.2005,).

The disadvantage of this invention is not you who aka the concentration of biomass, accumulated in the presence of 1.5% propionic acid, and therefore, inadequate amounts of protein, which could be formed by culture.

The technical result achieved by the present invention, is the elimination of environmental pollution, the reduction of energy consumption in the preparation of feed protein, simplification of apparatus and equipment, disposal of waste products, using natural raw materials, increasing the biological value of feed protein by providing opportunities to enrich the intestinal microflora of animals living cells propionic acid bacteria.

This technical result is achieved by the fact that, as a producer of feed protein using the newly selected strain of Propionibacterium freudenreichii subsp. shermanii 103/27 deposited in the all-Russian State collection of strains of microorganisms used in veterinary medicine and animal husbandry, the registration number of a strain of Propionibacterium freudenreichii subsp. shermanii VGNKI-08.02.57.-DEPT (123022, Moscow, highway, and 5). The location of the strain collection is defined microorganisms wildebeest VNIIBT RAAS (111033, Moscow, street powered wheelchairs and scooters, dB).

Below are examples of implementation of the invention.

Example 1.

The strain of Propionibacterium freudenreichii subsp. shermanii 103/27 was obtained by multiple (at least 45) re-seeding of the cells of the initial strain Prpionibacterium freudenreichii subsp. shermanii Ac-103 on a dense nutrient medium of the following composition:

Yeast extract10.0 g
K2HPO41.0 g
Na2HPO4·2H2O3.0 g
Sodium lactate (70% solution)40,0 ml
CoSO41.0 mg
Agar20,0 g
Distilled water up to1000 ml

The content of free propionic acid in the medium was gradually increased from 0 to 3.0%. Cells were incubated on solid nutrient medium at 37°C for 48 hours Selection was carried out based on the number of colonies and acid-forming capacity, which was determined by titration. Was selected those colonies growing on solid culture medium is not inhibited in the presence of 3.0% propionic acid. After 45 transfers the strain reached genetic stability. The results of the selection of a new strain of Propionibacterium freudenreichii subsp. shermanii 103/27 by step breeding are shown in table 1, which shows that when the content in the environment is Rodionovo acid to 2.5% the cell growth of a new strain of propionic acid bacteria (unlike the original strain) is not inhibited and is characterized by a sufficiently large number of cells. At a higher content of propionic acid (3.0 per cent) there is a sharp decrease in the number of cells and the morphology of the cells changed.

The results obtained are presented in table 1.

Table 1
Comparison of the properties of the original and new strains of Propionibacterium freudenreichii subsp. shermanii
The name of the strainThe concentration of propionic acid %The number of cells in 1 ml culture fluid
Propionibacterium freudenreichii subsp. shermanii Ac-103 (original strain)03,0·108
0,55,0·106
1,01,0·102
Propionibacterium freudenreichii subsp. shermanii Ac-103/2703,0·1010
0,51,0·1010
1,0 5,0·108
2,01,0·106
3,02,0·103

Breeding strain of the largest colonies were perseval on liquid nutrient medium of the following composition:

Yeast extract10.0 g
KH2PO41.0 g
Na2HPO4·2H2O3.0 g
CoSO41.0 mg
Sodium lactate (70% solution)40,0 ml
Distilled water up to1000 ml

For long-term storage of cells of strain liofilizirovanny in separated milk and kept in the absence of oxygen. Cells of strain can also be stored in 10%sacharose-gelatin agar or in semi-solid medium under oil.

The strain is not zoopathogenic or phytopathogenic. It is not dangerous for other reasons.

In accordance with Bergey''s Manual of Determinative Bacteriology, 9th edition, 1994, Williams & Wilkins, USA in the divided strain identified as Propionibacterium freudenreichii subsp. shermanii 103/27.

Cultural and morphological characteristics of the strain still sticks by 0.5-1.5 mm, sometimes forming short curved chains, sometimes have the form of cocci. Gram-positive. Nesporoobrazuth. Aerotolerance. Fermented fructose, lactose, galactose, glucose, mannose. The main product of metabolism - propionic acid. In the cell wall of the main component is meso-DAN. The optimum pH of 7.0 to 7.2. The optimum temperature of 32°C.

Example 2.

The strain of Propionibacterium freudenreichii subsp. shermanii 103/27 was assessed by the ability to form a high content of protein by culturing it on a liquid nutrient medium composition specified in example 1, the results of measuring the amount formed in the culture fluid of biomass and crude protein.

The inoculate in the amount of ten volume percent was introduced into the flask with the liquid nutrient medium with a volume of 750 ml and were cultivated in a hospital if the following parameters: temperature 37°C, pH of 6.0. As the neutralizing agent used a 20%solution of sodium hydroxide. After 48 hours of cultivation was determined by the growth of bacteria and the number formed during the biosynthesis of biomass and crude protein in the culture fluid.

The results obtained are presented in table 2.

Table 2
Characterization of selected strains of Propionibacterium freudenreichii subsp. shermanii 103/27
Name of indicatorValue
The number of cells in 1 ml culture fluid1,5·109
The specific output from the substrate, wt.%96,0
Mass fraction of crude protein, %49,0

On the basis of obtained results it is concluded that the selected strain has a high productivity in relation to the target product - accumulation of biomass (initial content of bacterial cells was equal to 1.0·102), high specific output from the used substrate and the accumulation of crude protein to 49% vs. 25% of the original strain. The strain can be used on an industrial scale.

A new strain of Propionibacterium freudenreichii subsp. shermanii 103/27 allows to obtain protein food with high output when disposing of waste products: distillery stillage, spent grains, waste flour and starch production, waste grain and fruit raw materials.

Example 3.

In polisport the first bard contribute 1.1 mg/l CoSO 4and grown pure culture of Propionibacterium freudenreichii subsp. shermanii 103/27.

At the end of the fermentation process the resulting mass is divided into solid and liquid phase by filtration.

The solid phase is sent to drying at a temperature (80 to 90)°C.

Feedstuff obtained after drying of the solid phase contains 46,0% crude protein and has the following composition:

mass fraction of carbohydrates, % SV45,0
mass fraction of ash, % SV2,5
the total amino acid content, %44,0
of them
lysine + histidine2,15
arginine1,25
aspartic acid4,25
threonine2,0
serine2,08
glutamic acid14,84
methionine + cystine1,3
glycine 2,6
Proline3,14
phenylalanine + tyrosine2,65
alanine4,4
isoleucine + leucine2,53
mass fraction of protein Burstein, % SV37,1
mass fraction of fat, % SVthe 4.7
the total content of trace elements, mg/kg21800
of them
phosphorus5900
potassium3500
sodium500
calcium11600
magnesium1000
iron750
cobalta 4.9
the vitamin content, mg/kg
E (tocopherol)43,5
B1(thiamine)3,3
In2(Riboflavin)7,7
In3(Pantothenic acid)35,3
In4(choline)700
In5(nicotinic acid)26,8
In6(pyridoxine)8,2
In9(folic acid)18,0
In12(ciankobalamin)34,4
the exchange energy, MJthe 13.4
fodder units, kg1,39
Obtained after filtering the liquid phase content in g/l:
solidsthe 10.1
mass fraction of nitrogenous substances1,04
mass fraction of ash1,3
mass fraction of fat0,006
mass on the La carbohydrates 1,7
mass fraction of ammonia nitrogen0,04
the total amino acid content, %0,29
the total content of microelements1,05
the total content of vitamins1,03
content propionate, g/100 ml2,5

Received feed product contains living cells propionic acid bacteria, enriching the intestinal microflora of animals consuming food that increases its biological value.

Example 4.

Culture liquid after fermentation with Propionibacterium freudenreichii subsp. shermanii 103/27 in example 3 is subjected to pre-filtering. After filtration of the precipitate obtained by the humidity of 55-60%, which can be used as a ready wet food.

Example 5.

In milk serum solids content of 4-5% add 1.0 mg/l CoCl2is heated to 50°C, made of 20% vol. cells of a pure culture of Propionibacterium freudenreichii subsp. shermanii 103/27. The incubation is conducted for 8 hours at a temperature of 45°C and a pH of 6.0 to 6.5. After drying the obtained biomass get protein and vitamin food containing the th cells of propionic acid bacteria in the exponential growth phase.

Example 6.

Brewer diluted with tap water at a ratio of 1:1, set the pH level of 6.0 to 6.5, temperature 59-61°C and contribute to 1.4 units/g conditional starch α-amylase. The mixture is kept under these conditions for 55 min, and then 55 minutes at a temperature of 74-76°C. After cooling the mixture to 59-61°C give it to 0.4 units/g of α-amylase and maintained under these conditions for 35 minutes the Mixture is cooled to 45-50°C and contribute to 5.5 units/g conditional starch glucoamylase incubated at pH 5.0 to achieve the content of reducing substances 6%, then cooked fermentolizat spent grains impose 1.1 mg/l CoSO4and pure culture of Propionibacterium freudenreichii subsp. shermanii 103/27 in the amount of 10 vol.% Incubation is carried out at periodic stirring for 20 hours at a temperature of 50°C and a pH of 6.0 to 6.5.

The resulting biomass of propionic acid bacteria in the exponential growth phase, dried and get protein and vitamin food.

Cell viability in the finished dried protein stern remained.

The obtained protein-vitamin product contained protein to 49.9% in SV, amino acids 44,2% SV, carbohydrates 37,7% SV

Example 7.

Milling waste is crushed to pass at least 80% through a sieve with a mesh diameter of 1 mm is Prepared aqueous suspension of powdered milling waste by mixing them with water in the ratio 1:3. In th is prepared suspension is made of 20 units/g of dry matter of the pulp cellulolyticus enzyme β-glucanase and incubated the mixture at a temperature of (59-61)°C and a pH of 6.0 for 55 min, and then at 105°C for 25 minutes to reach the content of reducing substances 3-5%, then also add 8 units/g of cellulose β-glucanase and 5 units/g conditional starch glucoamylase. The mixture is maintained at a temperature of 45°C and pH 5.0 for 30 min before reaching the content of reducing substances 7-10%.

After processing in osaharennoe mass contribute 0.9 mg/l CoCl2and implement it on the incubation of microorganisms Propionibacterium freudenreichii subsp.shermanii 103/27 at a temperature of 45°C and a pH of 6.0 to 6.8. After 24 hours, the formed protein-vitamin product with a mass fraction of protein to 49.6% in SV, with the amino acid content of 44.1% for SV, the carbohydrate content of 37.7% SV

Example 8.

Starch waste is treated similarly raw grain and milling waste, excluding stage of grinding. When the preparation of a mixture of starch waste water, the amount of water is adjusted to obtain a slurry concentration of 14-16%.

On the environment carry out incubation Propionibacterium freudenreichii subsp. shermanii 103/27.

Get food, rich in biologically active substances and protein.

Example 9.

The crushed grain, milling waste or starch waste is used to make fermentolizate by treating them with aqueous suspensions of amylolytic enzymes. To do this, in aqueous suspension contribute to 1.4 units/g conditions the aqueous starch α-amylase, the mixture is kept for 55 min at a temperature of 59°C and pH 5.8, then 35 min at 95°C, after which the mixture is cooled to 45°C., introducing her to 0.4 units/g conditional starch α-amylase and 6.5 units/g conditional starch glucoamylase. Stand the mixture for 30 min at 45°C and pH 5.0 to achieve the content of reducing substances 7% with obtaining fermentolizate where incubated pure culture of Propionibacterium freudenreichii subsp. shermanii 103/27 with obtaining adequate protein and vitamin food.

The strain of Propionibacterium freudenreichii subsp. shermanii 103/27 producing protein feed deposited in the all-Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry.



 

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