RU2244001C1 - Strain lactobacillus plantarum as producer of fodder protein - Google Patents

Abstract

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

Description

The invention relates to the food industry, biotechnology, agriculture.

Known yeast Saccharomices cerevisiae race U-1986 - (Patent RU No. 2183666, C 12 F 3/10, publ. 2002) / 1 /.

Yeast of this race is used to ferment grain wort in the alcohol industry. Their function is to convert wort sugars to alcohol. The feed product is not the target product, its utilitarian purpose is not defined. It is a waste of alcohol production, polluting the environment. It is difficult to control the quality and composition of this waste.

Known strain of yeast Candida tropicalis VSB-928K - producer of feed protein (Patent RU No. 2042713, C 12 P 1/100, publ. 06/20/2002) / 2 / (prototype).

The disadvantage of this known strain is the use of Candida yeast, which is a conditionally pathogenic microorganism, to obtain feed protein, which requires an additional thermolysis process and the availability of special equipment, a complex system for the treatment of wastewater and air emissions with significant volumes of air used. Sanitary and epidemiological rules SanPiN 2.3.2.1078-01 yeast of the genus Candida are included in the list of substances that have a harmful effect on human health. The cell growth rate of the known strain is low, which lengthens the production cycle and reduces the specific yield of the target product at the stage of biosynthesis. In addition, in the preparation of feed protein using this known strain of yeast, a large consumption of heat and energy for drying the biomass and a significant consumption (1.5 g / l) of ammonium sulfate are required.

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, the simplification of equipment, the disposal of waste products using natural raw materials, the increase in the biological value of feed protein by providing the ability to enrich the intestinal microflora of animals with living cells of lactic acid bacteria.

The specified technical result is achieved by the fact that the newly isolated strain of Lactobacillus plantarum 578/25, deposited in the All-Russian State collection of microorganism strains used in veterinary medicine and animal husbandry, is used as a producer of feed protein; registration number is the strain Lactobacillus plantarum (123022, Moscow, Zvenigorod highway, 5). The strain storage location is determined by the collection of microorganisms of GNU VNIIIPBT (111033, Moscow, Samokatnaya St., 4B).

The use of bacteria as a producer of protein feed is more effective, since bacteria form up to 75% of the protein by weight, while yeast - not more than 60%. The use of a new strain of Lactobacillus plantarum 578/25 for the preparation of protein feed does not require air consumption and energy consumption for its supply, since this strain of lactic acid bacteria is an anaerobic. The strain has a wide spectrum of antimicrobial activity, which excludes the development of extraneous microflora during biosynthesis and therefore, special equipment is not required to comply with sterility conditions. The possibility of recycling a variety of waste from industries using natural raw materials, while increasing the biomass of the new strain in order to prepare protein feed, solves the environmental problems of enterprises.

The following are examples of the implementation of the invention.

Example 1. The strain Lactobacillus plantarum 578/25 was obtained by repeated (at least 30) reseeding of cells of the original strain of Lactobacillus plantarum B 578 on a solid nutrient medium of the following composition:

Distilled water 600 ml.

Meat water 400 ml.

Yeast extract 5.0 g.

Sodium acetate 5.0 g.

Glucose 2.5 g.

Ammonium Citrate 2.5 g

K ₂ HPO ₄ 2.0 g

MgSO ₄ · 7H ₂ O 0.2 g.

MnO ₄ · 4H ₂ O 0.05 g.

Tween 80 1 ml.

Agar 20.0 g.

The content of free lactic acid in the medium was gradually increased from 0 to 1.5%. Cells were incubated in solid nutrient medium at 37 ° C for 24 hours. The selection was carried out according to the number of colonies and acid-forming ability, which was determined by titration. Those colonies were selected whose growth on solid nutrient medium was not inhibited in the presence of 1.5% lactic acid. After 30 passages, the strain reached genetic resistance. The results of the selection of a new strain of Lactobacillus plantarum 578/25 by stepwise selection are shown in Table 1, which indicates that when the content of lactic acid in the medium is up to 2.0%, the cell growth of the new strain of lactic acid bacteria (unlike the original strain) is not inhibited and characterized by a sufficiently large number of cells. With a higher content of lactic acid (2.5%), a sharp decrease in the number of cells occurs and the morphology of the cells changes.

Table 1
Comparison of the properties of the original and new strains of Lactobacillus plantarum The name of the strain The concentration of lactic acid,% The number of cells in 1 ml of culture fluid Lactobacillus plantarum B 578 (parent strain) 0 1.510 ⁹ 0.5 1,0 · 10 ⁷ 1,0 1,0 · 10 ⁵ 1.25 1,0 · 10 ³ Lactobacillus plantarum B 578/25 0 2.010 ⁹ 0.5 1.510 ⁹ 1,0 1,0 · 10 ⁷ 1.25 1.5 · 10 ⁶ 1,5 1,0 · 10 ⁵ 2.0 1,0 · 10 ⁴ 2,5 0.5 · 10 ²

To propagate the strain, the selected largest colonies were subcultured onto a liquid nutrient medium of the following composition:

Distilled water 600 ml.

Meat water 400 ml.

Yeast extract 5.0 g.

Sodium acetate 5.0 g.

Glucose 2.5 g.

Ammonium Citrate 2.5 g

K ₂ HPO ₄ 2.0 g

MgSO ₄ · 7H ₂ O 0.2 g.

MnO ₄ · 4H ₂ O 0.05 g.

Tween 80 1 ml.

Agar 5.0 g.

For long-term storage, the cells of the strain are lyophilized in separated milk and stored in the absence of oxygen. The cells of the strain can also be stored on 10% sucrose-gelatin agar or in a semi-liquid medium under oil.

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

According to the Bergey’s Manual of Determinative Bacteriology, 9th edition, 1994, Williams & Wilkins, USA, the isolated strain is identified as Lactobacillus plantarum 578/25.

Cultural and morphological features of the strain: fixed rods of size (0.9-1.2) × (3-8) microns. Gram-positive. Non-spore forming. Aerial Tolerance. Fermentation of fructose, lactose, galactose, glucose (acid), maltose, mannitol, ribose, sucrose, cellobiose. Forms DL-forms of lactic acid. The optimum pH is 5.8-6.0. The optimum temperature is 37 ° C.

Example 2

The strain Lactobacillus plantarum 578/25 was evaluated by its ability to form a high protein content by culturing it on a liquid nutrient medium of the composition described in example 1, by measuring the amount of biomass and crude protein formed in the culture fluid.

An inoculum in an amount of five volume percent was introduced into flasks with a 750 ml liquid nutrient medium and cultivation was carried out under stationary conditions, subject to the following parameters: temperature 37 ° C, pH 6.0. A 20% sodium hydroxide solution was used as a neutralizing agent. After 24 hours of cultivation, bacterial growth and the amount of biomass and crude protein formed in the biosynthesis in the culture fluid were determined.

The results are presented in table 2.

table 2
Characterization of the selected strain of Lactobacillus plantarum 578/25 Name of indicator Indicator value The number of cells in 1 ml of culture fluid 0.810 ⁹ The specific yield of the substrate, wt.% 95.0 Mass fraction of crude protein,% 49

Based on the results obtained, it was concluded that the selected strain has high productivity in relation to the target product - biomass accumulation (the initial content of bacterial cells was 1.0 · 10 ² ), a high specific yield from the substrate used and the accumulation of crude protein up to 49% against 25% in the original strain. The strain can be used on an industrial scale.

A new strain of Lactobacillus plantarum 578/25 allows you to get protein feed with a high yield when disposing of industrial waste: post-alcohol stillage, beer grains, waste from flour and starch production, waste from processing grain and fruit raw materials.

Example 3

A pure culture of Lactobacillus plantarum 578/25 is grown on the post-alcohol bard.

At the end of the fermentation process, the resulting mass is separated into solid and liquid phases by filtration.

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

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

mass fraction of carbohydrates,% d.v. 44,2

mass fraction of ash,% d.v. 2,5

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 according to Barshteyn,% on d.v. 37.1

mass fraction of fat,% d.v. 5.1

total micronutrient content, mg / kg 21800

of them

phosphorus 5900

potassium 3500

sodium 500

calcium 11600

magnesium 1000

iron 750

cobalt 4.9

the content of vitamins, mg / kg

E (tocopherol) 43.5

B ₁ (thiamine) 3.3

B ₂ (riboflavin) 7.7

B ₃ (pantothenic acid) 35.3

B ₄ (choline) 700

B ₅ (nicotinic acid) 26.8

B ₆ (pyridoxine) 8.2

B ₉ (folic acid) 18.0

B ₁₂ (cyancobalamin) 34.4

exchange energy, MJ 13.4

feed units, kg 1.37

The liquid phase obtained after filtration with a content in g / l:

solids 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

total amino acid content,% 0.29

total micronutrient content 1.05

total vitamins 1.01

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

The resulting feed product contains living cells of lactic acid bacteria that enrich the intestinal microflora of animals that consume food, which leads to an increase in its biological value.

Example 4

The culture fluid after fermentation using Lactobacillus plantarum 578/25 according to example 3 is subjected to preliminary filtration. After filtration, a precipitate with a moisture content of 50-60% is obtained, which can be used as a finished wet feed.

Example 5

In whey with a solids content of 4-5%, heated to 50 ° C, make 20% vol. cells of a pure culture of Lactobacillus plantarum 578/25. Incubation is carried out for 3 hours at a temperature of 45 ° C and a pH of 5.9-6.0. After drying the obtained biomass, a protein-vitamin feed is obtained containing live cells of lactic acid bacteria in the exponential growth phase.

Example 6

The beer pellet is diluted with tap water in a ratio of 1: 1, the pH is set at 5.8-6.0, the temperature is 59-61 ° C and 1.4 u / g of conditional starch of α-amylase are added. The mixture is kept under these conditions for 55 minutes and then 55 minutes at a temperature of 74-76 ° C. After the mixture has cooled to 59-61 ° C, 0.4 u / g of α-amylase are introduced into it and kept under these conditions. for 35 minutes The mixture is cooled to 45-50 ° C and 5.5 u / g of conditional glucoamylase starch are added and kept at pH 5.0 until a reducing substance content of 6% is reached, after which a pure culture of Lactobacillus plantarum 578/25 is introduced into the prepared the amount of 5% vol. Incubation is carried out with periodic stirring for 20 hours at a temperature of 50 ° C and a pH of 5.9-6.0.

The resulting biomass of lactic acid bacteria in the exponential growth phase is dried and a protein-vitamin feed is obtained.

Cell viability in the finished dried protein feed was preserved.

The resulting protein-vitamin product contained protein 49.6% by d.v., amino acids 44.1% by d.v., carbohydrates 37.7% by d.v.

Example 7

Flour waste is crushed to a passage of at least 80% through a sieve with cells with a diameter of 1 mm. An aqueous suspension of crushed flour wastes is prepared by mixing them with water in a ratio of 1: 3. 20 u / g of dry matter of cellulolytic β-glucanase cellulolytic enzyme are added to the prepared suspension and the prepared mixture is kept at a temperature of (59-61) ° С and pH 6.0 for 55 minutes, and then at a temperature of 105 ° С for 25 minutes to achieve a content of reducing substances of 3-5%, after which 8 units / g of β-glucanase cellulose and 5 units / g of conditional glucoamylase starch are additionally added. The prepared mixture is maintained at a temperature of 45 ° C and pH 5.0 for 30 minutes until reaching a content of reducing substances of 7-10%.

After processing, the saccharified mass is used to incubate Lactobacillus plantarum 578/25 microorganisms on it at a temperature of 45 ° C and a pH of 5.9-6.0. After 24 hours, a protein-vitamin product is formed with a mass fraction of protein up to 49.6% d.v., with an amino acid content of 44.1% d.v., a carbohydrate content of 37.7% d.v.

Example 8

Fruit squeezes with a humidity of 50-60% are diluted with water in a ratio of 1: 2, the mixture is heated to a temperature of 45-50 ° C, cellulase is added in an amount of 1.8 u / l and the resulting mixture is kept for 3 hours at a temperature of 50 ° C to achieve a content of reducing substances of 10-15% to obtain a fermentolizate. Inoculated with a pure culture of microorganisms Lactobacillus plantarum 578/25, which is introduced into the nutrient medium in an amount of 20% by volume of the medium. Incubation is carried out with periodic stirring at a temperature of 40 ° C and a pH of 5.9-6.1 for 48 hours.

Example 9

Starch waste is treated similarly to grain raw materials and flour milling waste, excluding the grinding stage. When preparing the mixture - starch waste - water, the amount of water is regulated so as to obtain a suspension with a concentration of 14-16%.

The resulting medium is incubated with Lactobacillus plantarum 578/25.

Get food rich in biologically active substances and protein.

Example 10

Crushed grain, milling waste or starchy waste is used to prepare fermentolysates by treating their aqueous suspensions with amylolytic enzymes. For this, 1.4 u / g of conditional starch of α-amylase are added to the aqueous suspension, the mixture is incubated for 55 minutes at a temperature of 59 ° C and a pH of 5.8, then 35 minutes at 95 ° C, after which the mixture is cooled to 45 ° C. 0.4 u / g of conditional starch of α-amylase and 6.5 u / g of conditional starch of glucoamylase are introduced into it. The prepared mixture is kept for 30 minutes at a temperature of 45 ° C and pH 5.0 until a reducing substance content of 7% is obtained to obtain an enzymeolysate, on which a pure culture of Lactobacillus plantarum 578/25 is incubated to obtain a complete protein-vitamin feed.

Claims (1)

Strain Lactobacillus plantarum VGNKI-04.04.09 - DEPT - producer of feed protein.