Strain lactobacillus plantarum as producer of fodder protein

FIELD: biotechnology, microbiology, agriculture.

SUBSTANCE: the strain Lactobacillus plantarum 578/25 is obtained by method of step-by-step selection and selected by its ability to produce significant amount of crude protein and to accumulate the biomass. The strain is deposited in the VGNKI collection at number VGNKI-03.04.09.-DEP. Invention provides eliminating the pollution of environment in producing the protein fodder, to elevate the protein specific yield, to reduce energy consumptions in preparing protein fodder, to simplify and to accelerate the process of its preparing, to simplify apparatus equipment, to utilize waste in manufacturing using the natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

 

The invention relates to food industry, biotechnology, agriculture.

Known yeast Saccharomices cerevisiae race-1986 - (Patent RU №2183666, 12 F 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, 12 P 1/100, publ. 20.06.2002 g) /2/ (prototype).

The disadvantage of this known strain is use for feed protein of the yeast Candida, which are conventionally pathogenen a 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 of the n cycle and 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 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 of lactic acid bacteria.

This technical result is achieved by the fact that, as a producer of feed protein using the newly selected strain of Lactobacillus plantarum 578/25 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 Lactobacillus plantarum VGNKI - 03.04.09.-DEPT (123022, Moscow, highway, and 5). The location of the strain collection is defined microorganisms wildebeest VNIIBT (111033, Moscow, wsasocketa, dB).

The use of bacteria as a producer of protein feed is more effective because the bacteria produce up to 75% protein by weight, while drag and - no more than 60%. The use of a new strain of Lactobacillus plantarum 578/25 for the preparation of protein meal does not require air consumption and energy consumption per presentation, because this strain of lactic 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.

Below are examples of implementation of the invention.

Example 1. Lactobacillus plantarum 578/25 was obtained by multiple (at least 30) of the re-seeding of the cells of the initial strain of Lactobacillus plantarum In 578 on solid nutrient medium of the following composition:

Distilled water 600 ml

Meat water 400 ml

Yeast extract 5,0,

Sodium acetate in 5.0,

Glucose and 2.5,

Ammonium citrate, 2,5

To2NRA42,0,

MgSO4·7H2O 0,2,

MnO4·4H2O of 0.05,

Tween 80 1 ml

Agar 20,0,

The content of free lactic acid in the medium was gradually increased from 0 to 1.5%. Cells were incubated on nutrient dense is the ed at 37° C for 24 h Sampling was carried out according to 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 1.5% lactic acid. After 30 transfers the strain reached genetic stability. The results of the selection of a new strain of Lactobacillus plantarum 578/25 by step breeding are shown in table 1, suggests that the content in the environment of lactic acid to 2.0%, the cell growth of a new strain of lactic acid bacteria (unlike the original strain) is not inhibited and is characterized by a sufficiently large number of cells. At a higher content of lactic acid (2,5%) there is a sharp decrease in the number of cells and the morphology of the cells changed.

Table 1

Comparison of the properties of the original and new strains of Lactobacillus plantarum
The name of the strainThe concentration of lactic acid, %The number of cells in 1 ml culture fluid
Lactobacillus plantarum In 578 (original strain)01,5· 109
0,51,0· 107
1,01,0· 105
1,251,0· 0 3
Lactobacillus plantarum In 578/2502,0· 109
0,51,5· 109
1,01,0· 107
1,251,5· 106
1,51,0· 105
2,01,0· 104
2,50,5· 102

For breeding strain selected the largest colonies were perseval on liquid nutrient medium of the following composition:

Distilled water 600 ml

Meat water 400 ml

Yeast extract 5,0,

Sodium acetate in 5.0,

Glucose and 2.5,

Ammonium citrate, 2,5

K2HPO42,0,

MgSO4·7H2O 0,2,

MnO4·4H2O of 0.05,

Tween 80 1 ml

Agar 5,0,

For long-term storage of cells of strain lyophilizer in separated milk and store 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 selected strain is identified as Lactobacillus plantarum 578/25.

Cultural the-morfologicheski particular strain still sticks size (0,9-1,2)× (3-8 microns). Gram-positive. Nesporoobrazuth. Aerotolerance. Sprayway fructose, lactose, galactose, glucose (acid), maltose, mannitol, ribose, sucrose, cellobiose. Form DL-form of lactic acid. The optimum pH of 5.8-6.0. The optimum temperature 37° C.

Example 2.

Lactobacillus plantarum 578/25 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 five 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 6.0. As the neutralizing agent used a 20%solution of sodium hydroxide. After 24 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 Lactobacillus plantarum 578/25
Name of indicatorValue
The number of cells in 1 ml culture fluid0,8· 109
The specific output from the substrate, wt.%95,0
Mass fraction of crude protein, %49

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 Lactobacillus plantarum 578/25 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.

On distillery Barda grown pure culture of Lactobacillus plantarum 578/25.

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 (80-90)° C.

Feedstuff obtained after drying of the solid phase contains 45.9% of crude protein and has the following composition:

mass Dol is carbohydrates, % SV 44,2

mass fraction of ash, % 2,5 SV

the total amino acid content, % 43,4

of them

lysine+histidine 2,15

arginine 1,25

aspartic acid 4,25

threonine 2,0

serine 2,08

glutamic acid 14,84

methionine+cystine 1,3

glycine 2,6

Proline 3,14

phenylalanine+tyrosine 2,65

alanine 4,4

isoleucine+leucine 2,53

mass fraction of protein Burstein, % SV 37,1

mass fraction of fat, % SV 5,1

the total content of trace elements, mg/kg 21800

of them

phosphorus 5900

potassium 3500

sodium 500

calcium 11600

magnesium 1000

iron 750

cobalt 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, MJ 13,4

fodder units, kg 1,37

Obtained after filtering the liquid phase content in g/l:

dry matter 10,1

mass fraction of nitrogenous substances 1,04

mass fraction of ash 1,3

mass fraction of fat 0,006

mass fraction of carbohydrates 1,7

mass fraction of ammonia nitrogen 0,04

the total amino acid content, % 0,29

the total content of trace elements 1,05

the total content of vitamins 1,01

content is lactate (propionate), g/100 ml of 1.12

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

Example 4.

Culture liquid after fermentation with Lactobacillus plantarum 578/25 in example 3 is subjected to pre-filtering. After filtration of the precipitate obtained by the humidity of 50-60%, which can be used as a ready wet food.

Example 5.

In milk serum solids content of 4-5%, heated to 50° To contribute 20% vol. cells of a pure culture of Lactobacillus plantarum 578/25. The incubation is conducted for 3 hours at a temperature of 45° and a pH of 5.9 to 6.0. After drying the obtained biomass get protein and vitamin food containing living cells of lactic acid bacteria in the exponential growth phase.

Example 6.

Brewer diluted with tap water at a ratio of 1:1, set the pH at 5.8 to 6.0, temperature 59-61° and contribute to 1.4 u/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° give it to 0.4 u/g α -amylase and maintained under these conditions for 35 minutes the Mixture is cooled to 45-50° and contribute to 5.5 units/g conditional starch glucoamylase and maintain the pH 5.0 to achieve the content of reducing substances 6%, then in the prepared fermentolizat spent grains make a pure culture of Lactobacillus plantarum 578/25 in the amount of 5% by vol. Incubation is carried out at periodic stirring for 20 hours at a temperature of 50° and a pH of 5.9 to 6.0.

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

Cell viability in the finished dried protein stern preserved.

The obtained protein-vitamin product contained protein 49.6% SV, amino acids to 44.1% for 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 the prepared suspension is made of 20 u/g of dry matter of the pulp cellulolyticus enzyme β -glucanase and incubated the mixture at a temperature of (59-61)° C and pH 6.0 for 55 min, and then at a temperature of 105° C for 25 minutes to reach the content of reducing substances 3-5%, then also add to 8 IU/g cellulose β -glucanase and 5 u/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 which the processing osaharennoe mass used for incubation of the microorganisms Lactobacillus plantarum 578/25 at a temperature of 45° C and a pH of 5.9 to 6.0. 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.

Fruit pomace with a humidity of 50-60% is diluted with water in the ratio 1:2, the mixture is heated to a temperature of 45-50° With, in it add the cellulase in the amount of 1.8 u/l and the mixture was incubated for 3 h at a temperature of 50° before reaching the content of reducing substances 10-15% with obtaining fermentolizate. Seeded with a pure culture of microorganisms Lactobacillus plantarum 578/25, which contribute to the nutrient medium in the amount of 20% by volume environment. Incubation is carried out at periodic stirring at a temperature of 40° and a pH of 5.9 to 6.1 in 48 hours.

Example 9.

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 Lactobacillus plantarum 578/25.

Get food, rich in biologically active substances and protein.

Example 10.

The crushed grain, milling waste or starch waste is used to make fermentolizate by treating them with aqueous suspensions of the amylolytic farms is nami. To do this, in aqueous suspension contribute to 1.4 u/g conditional starch α -amylase, and the mixture is kept for 55 min at a temperature of 59° and a pH of 5.8, then 35 min at 95° C, after which the mixture is cooled to 45°C., introducing her to 0.4 u/g conditional starch α -amylase and 6.5 u/g conditional starch glucoamylase. Stand the mixture for 30 min at a temperature of 45° C and pH 5.0 to achieve the content of reducing substances 7% with obtaining fermentolizate where incubated pure culture of Lactobacillus plantarum 578/25 with obtaining adequate protein and vitamin food.

Lactobacillus plantarum VGNKI-03.04.09 - DEPT - producer of feed protein.



 

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