Reproduction of E.coli in nutrient media and its factors. Nutrient medium for the cultivation of bacteria of the genus pseudomonas Density of the prepared medium

The reproduction of E. coli under standard conditions can be described by a curve, which represents the dependence of the number of cells E.coli in suspension from the time of growth (see figure in the presentation). Number of cells E.coli in suspension is equivalent to the optical density of an E. coli culture measured at 600 nm (“suspension turbidity”). Reproduction curve E.coli includes 4 main phases: (initial phase, lag-phase), exponential phase (exponential, log-phase), stationary phase (stationary phase) and death phase. During the initial growth phase, the increase in cellular biomass occurs very slowly. This is due to the initially small number of cells. When the optical density of the suspension reaches approximately 0.1, the growth of E. coli enters the exponential phase - the rapid growth phase. It was found that during the exponential phase, the optical density of the culture doubles on average every 30 minutes. When the optical density of the suspension becomes approximately 1.5, due to the large number of cells and small amount of nutrients in the medium, growth E.coli slows down significantly and enters the stationary phase. And finally, an excessively large number of cells in the suspension, almost complete depletion of the nutrient medium and a high concentration of bacterial metabolites in it lead to the death of bacterial cells, their lysis and a decrease in the density of the bacterial culture (death phase).

Standard growth conditions E.coli in the suspension are the following parameters: LB-broth nutrient medium, temperature 37 °C, intensive stirring (at least 150 rpm) and sufficient aeration. It should be noted that the growth E.coli possible not only in suspension, but also on so-called solid nutrient media containing agar. In this case, intensive mixing is not required.

Nutrient media for growthE.coli .

Liquid culture media. As mentioned above, the most widely used growth medium is E.coli in suspension is LB-broth medium (Luria Bertani medium, lysogeny broth). This highly nutritious medium and its main components are tryptone or peptone, yeast extract and NaCl. Tryptone (peptone) are products of casein hydrolysis under the action of trypsin (pepsin) and serve as sources of amino acids and peptides. Yeast extract is a source of carbon, vitamins (including group B), as well as minerals such as magnesium, sulfur, and calcium ions; and NaCl provides bacterial cells with Na + ions to implement effective transport and maintain osmotic balance. To prepare LB medium, dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl (or 25 g ready-made mixture) in 1 liter of water, autoclave and cool. It should be noted that the growth of various strains E.coli in LB medium occurs at different rates and some strains are very sensitive to high NaCl content. It is known that NaCl at 10% concentration can have an inhibitory effect on the growth of some strains of E. coli. In this regard, protocols were developed for medium-salt LB medium (5 g NaCl/L LB) and low-salt LB medium (0.5 g NaCl/L) with identical peptone and yeast extract contents. The low-salt version of LB medium is also used if an antibiotic sensitive to high salt concentrations is to be added to the growth medium. The pH of the LB medium without additional adjustment is approximately 7.0 – 7.2.

Normally, to achieve the stationary phase, grow E.coli should be 14-18 hours, however, sometimes situations arise when it is necessary to grow E.coli for several days (for example, to produce large amounts of biomass during protein expression). In this case, the use of LB medium is not effective, since there is a depletion of nutrients and a decrease in the pH of the medium caused by a high concentration of metabolites, which initiates cell death and inhibits their growth. In such cases, for growth E.coli use other media containing additional nutrients and buffer systems to maintain pH. To increase nutrients, most of these media contain two or three times the amount of tryptone and yeast extract. In addition, some media include: glycerol or glucose as additional carbon sources (TB, SOC media), a phosphate buffer system or CaCO 3, which prevent acidification of the medium and maintain its pH in the stationary growth phase E.coli(TB environment), as well as Mg 2+ and K + salts (2*YT, SOB, SOC environments).

For growth E.coli Liquid culture media typically use pre-sterilized or alcohol-treated conical glass flasks or 50 ml plastic Falcon tubes. Biomass expansion E.coli for subsequent isolation of plasmid DNA, it is usually carried out overnight, i.e., an “overnight” culture is obtained. To do this, a small amount of bacteria pre-transformed with a plasmid is placed in a flask or plastic tube with a medium in the presence of the required antibiotic and the culture is grown at 37°C and 150 rpm for 14–18 hours. The finished “overnight” culture is a turbid suspension of cells in the medium. To implement some tasks (for example, obtaining competent cells or expressing recombinant proteins), it is necessary to grow cells to certain OD600 values ​​(usually 0.3 – 0.8). In such cases, it is necessary to monitor the optical density of the growing crop so as not to miss the moment of achieving the desired optical density.

Solid nutrient media. Solid nutrient medium is used when it is necessary to obtain single colonies E.coli. This is usually required when cells are transformed with a heterogeneous mixture of plasmid DNA (for example, in the case of a ligase mixture) and it is necessary to find a colony containing the correct variant of the plasmid. Typically in the case of solid growth media E.coli use disposable plastic cups with a diameter of 90 mm. The most common solid culture medium is 1.5% agar prepared on LB (LB agar). To prepare 1 liter of such a medium, 15 g of agar, 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl (or 25 g of the prepared LB mixture) must be dissolved in 1 liter of distilled water and autoclaved. The agar should be cooled to a temperature of 50°C - 60°C, the required antibiotic should be added to it, poured into cups with approximately 10 ml per cup and left to harden under a laminar next to the burner, with the lid slightly opened. Once the condensation has evaporated, the cup can be used to inoculate bacteria.

Temperature. With decreasing temperature the increase E.coli slows down, but in some cases E. coli is grown at lower temperatures for several days, such as when cooked from a culture E.coli competent cells or upon expression of recombinant proteins.

Stirring and aeration. To ensure sufficient aeration, grow E.coli in suspension it is necessary with intense stirring (at least 150 rpm), and the vessel (flask or plastic test tube) must be filled with the medium to a maximum of 1/10 of the total volume. To improve aeration, the lids of the tubes used for the growth of the bacterial culture are closed loosely, and foil is used to close the flasks. In some cases, flasks with special internal blades - “bumpers” are used. When stirring in such flasks, the surface of contact between the medium and air increases noticeably due to the splashing of the liquid. The more bumpers in the flask, the stronger the splashing of the liquid and the higher the aeration. In case of growth E.coli on a solid nutrient medium, aeration occurs due to the fact that some space is provided between the walls of the bacterial dish and the lid for air penetration.

Antibiotics As mentioned above, most plasmids contain an antibiotic resistance gene, in the presence of which there is a selection of cells containing the plasmid from cells that do not contain it. The most widely used antibiotics for selection are ampicillin (working concentration - 100 μg/ml), kanamycin (working concentration - 30 μg/ml), and chloramphenicol (working concentration - 25-30 μg/ml). Antibiotics are usually dissolved in ethanol or water (each antibiotic has its own solubility conditions), thousand-fold stock solutions are prepared and stored at -20°C. Before adding antibiotics to LB agar, it is necessary to cool it to 50-60°C, since antibiotics are easily destroyed at high temperatures. Some antibiotics, such as ampicillin, are light-sensitive, so it is advisable to grow bacteria in their presence in the dark.

These media are used for cultivating and storing recombinant strains Escherichia coli, as well as for the usual cultivation of not very fastidious microorganisms.

Compound**:

** The composition has been verified and adjusted to meet the required parameters

Preparation:

Mix 35.0 g of M557 powder or 20.0 g of M575 powder or 40.0 g of M1151 powder or 25.0 g of M1245 powder in 1000 ml of distilled water. Heat to completely dissolve the particles. Sterilize by autoclaving at 1.1 atm (121°C) for 15 minutes. Pour into appropriate containers.

Principle and evaluation of the result:

Agar and Luria broth are prepared according to the Lennox recipe (1) for the cultivation and storage of recombinant strains Escherichia coli. The remaining media (2) are slightly different in that they have double the concentration of sodium chloride. These media contain sufficient nutrients for the growth of recombinant strains. Such strains are usually derivatives Escherichia coli K12, deficient in the synthesis of vitamin B. Because of this and other auxotrophy markers obtained during mutagenesis, recombinant strains may lose the ability to grow on media of depleted composition.

Casein hydrolyzate ensures the presence of peptides in the medium, yeast extract - B vitamins, sodium chloride - sodium ions for membrane transport and maintaining optimal osmotic pressure.

Quality control:

Powder appearance:

Homogeneous free-flowing light yellow powder.

Density of the finished medium:

A medium is formed corresponding in density to a 1.5% agar gel (M557 or M1151).

Color and transparency of the finished medium:

The medium is yellow or amber in color, transparent or slightly opalescent if a gel forms in test tubes or Petri dishes.

Acidity of the environment:

At 25°C, aqueous solutions of M557 (3.5% w/v) or M575 (2.0% w/v) have a pH of 7.0 ± 0.2, and aqueous solutions of M1151 (4.0% w/v) or M1245 (2.5% w/v) have a pH of 7.5 ± 0.2.

Topic: Protein isolation and purificationpkb5

experimental part

Strains and media.

The strain used in this work E. coliB.L.21(DE3) pLysS, with plasmid pET32a:Pkb5 (Fig), containing the catalytic domain of the Bifidobacterium longum protein kinase pkb5.

For growing cells E. coli LB and TB nutrient media were used.

Table 1 - Nutrient media used for cultivation E. coli

The media were sterilized in an autoclave at an excess pressure of 0.8 atm for 40 minutes.

To maintain the strain E. coliB.L.21(DE3) pLysS, containing the pET32a:Pkb5 plasmid, monoclonal sieving was carried out on Petri dishes with LB agar medium with the addition of chloramphenicol (30 μg/ml) and ampicillin (150 μg/ml).

To generate cell biomass E. coliB.L.21(DE3) pLysS, containing the pET32a:Pkb5 plasmid, were grown in flasks on TV medium with the addition of chloramphenicol and ampicillin.

At the first stage, night crops were grown in TV medium. At the second stage, 1.5 overnight cultures were added to flasks with 150 ml of fresh medium. Growing was carried out under aerated conditions at 250 rpm and a temperature of 37 0 C until the optical density OD 600 = 0.6 (~ 2 hours), then expression was induced by adding IPTG (isopropyl - β-D-thiogalactoside) to a final concentration of 0.5 mm. Next, cultivation was carried out at 28 0 C for 18 hours, after which the biomass was pelleted by centrifugation for 10 minutes at 6,000 rpm, washed with 1M Tris-HCl (pH 7.6-8.0) and frozen at -20 0 C .

Rice. Scheme of plasmid pET32a construction: Pkb5 .

Preparation of lysateE .With oli

Thawed cells E.Witholi at a temperature of 4 0 C. Thawed cells were washed from the growth medium with 20 volumes (18 ml) of 1 M Tris-HCl, pH 7.8. The cells were pelleted by centrifugation at 7500 rpm for 10 min at a temperature of 4 0 C. The weight of the sediment was determined to be 1.61 g on an analytical balance. The sediments were resuspended in 10 volumes of lysis buffer containing 300 mM KCl, 50 mM KH 2 PO 4 , 5 mM Imidazole, 6 M urea inhibitor cocktail (complete EDTA-free, Roche Diagnostics Gmbh, Germany). Cells were lysed using an ultrasonic disintegrator SONICS VIBRA CELL 3 times for 59 seconds with an interval of 15 seconds at an amplitude of 40% and a temperature of 4 0 C. To sediment cell wall fragments, the lysate was centrifuged at 10,000 rpm for 20 minutes at a temperature of 4 0 C The supernatant was collected and filtered through a filter (0.22 µm pore diameter, Merck Millipore Ltd, Germany) immediately before application to the column.

Metal affinity chromatography

The metal affinity chromatography method is based on the non-covalent interaction of the 6 histidine fragment of the recombinant protein (His-Tag) with nickel ions (Ni 2+), forming coordination bonds with nitroacetic acid residues.

Purification was carried out on a Bio Logic LP Model 2110 Fraction Collector (Bio Rad, USA) using a 1 ml Bio-Scale Mini Profinity IMAC Cartridges column.

Protein purification was carried out according to the Profinite IMAC Resins method manual (Bio Rad, USA).

Before starting work, the system was washed with distilled water, a Bio-Scale Mini Profinity IMAC Cartridges column was installed, and the column was washed with distilled water at a rate of 2 ml/min. The column was equilibrated with 8 volumes of lysis buffer at a rate of 2 ml/min, lysate was applied in a volume of 15 ml at a rate of 0.5 ml/min, washed with 15 volumes of lysis buffer at a rate of 2 ml/min, then washed with 15- 3 volumes of wash buffer at a rate of 2 ml/min, elution was carried out with 15 volumes of elution buffer at a rate of 2 ml/min. Fractions were collected in 2 ml volumes. Fractions corresponding to the eluted protein were collected separately.

At the end of the work, the column was washed with 2 ml of distilled water at a rate of 2 ml/min and regenerated with 5 volumes of 6 M guanidine-HCl and then with distilled water. The used column was stored in 20% ethanol at 4 0 C.

Buffer solution composition

Determination of protein amount

Protein amounts were determined using the Bradford method (Bradford M.M., 1976).

Using a PD-303 digital spectrophotometer, the optical density at a wavelength of 595 nm was determined for fractions 31 and 32 and the amount of protein was determined from the graph (Fig.) and the concentration was calculated.

Paint composition: 100 mg CBB (Coomassie Brilliant Blue G-250), 95% C 2 H 5 OH, 85% H 3 PO 4

Rice. Graph for determining the amount of test sample using the Breedford method

Staged dialysis

The dialysis tubes used in the work were SnakeSkin Pleated Dialysis Tubing (Thermo scientific), pore diameter 3,500 MVCO.

Selected fractions were dialyzed against a buffer solution containing 50 mM Tris-HCl, 5 mM β-mercaptoethanol, 10% glycerol, 3 M urea at a temperature of 4 0 C for 2.5 hours with constant stirring. Next, the dialysis buffer was replaced with a dialysis buffer containing 50 mM Tris-HCl, 5 mM β-mercaptoethanol, 10% glycerol, 1.5 M urea, 2 mM MgCl2. Left overnight with constant stirring at 4 0 C.

Electrophoretic separation of proteins in denaturing SDS-polyacrylamide gel

Electrophoresis was carried out according to the Laemmli method (Laemmli U. K., 1970) using a Mini proteam sistem. A 12% polyacrylamide gel in a buffer containing 0.375 M Tris-HCl pH 8.8, 0.1% SDS was used as a working gel; a 3% polyacrylamide gel containing 0.125 M Tris-HCl pH 6.8 was used as a forming gel. 0.1% SDS.

The electrode buffer was 0.025 M Tris, 0.192 M glycine, 0.1% SDS.

Before application, samples were heated in a buffer containing 0.0625 M Tris-HCl pH 6.8, 2.3% SDS, 5% β-mercaptoethanol, 10% glycerol and 0.001% bromine phenol blue. Electrophoresis was carried out at a constant voltage of 200 volts. At the end of electrophoresis, PAGE was fixed for 30 min in a solution containing 50% C2H5OH and 10% CH3COOH. Next, staining was carried out in a solution containing 0.2% SBB g-250, 40% C 2 H 5 OH, 8% CH 3 COOH with heating. PAGE was washed in 7% CH3COOH.

Concentration

To further measure the activity of the pkb 5 catalytic domain, the eluted protein was concentrated using Amicon Ultra Centrifugal Filters, pore diameter 50,000 NMWL (Merck Millipore Ltd).

Concentration of the protein sample was carried out using Eppendorf 5430 R centrifugation at 7500 rpm for 1 hour 20 minutes at a temperature of 4 0 C.

Activity check

Determination of autophosphorylation of pkb 5 (recombinant protein) was carried out using the Kinase-Glo r Plus Luminiscent Kinase Assey (Promega V3772), used to measure the extent of phosphorylation by the level of ATP remaining during the reaction.

Using a Biomek 3000 Beckman Coulter© automated workstation, 15 μl of pkb 5 solution (5 μg and 1 μg in the reaction mixture) in reaction buffer (15 mM HEPES pH 7.4; 20 mM NaCl, 10 mM MgCl 2 , 0.5 mM EDTA, 0.02% Tween-20, 10 mg/ml BSA) and 15 μl reaction buffer (control without protein kinase).

20 μM ATP (15 μl) was added to each well, and the contents of the wells were mixed. Incubate at room temperature for 30 min, covering the plate with a lid to prevent evaporation.

At the end of the enzymatic reaction, 30 μl of the reagent for determining luminescence was added to each well, the plate was incubated for 40 minutes at room temperature, and luminescence was measured on a Beckman Coulter© DTX 880 Multimode Detector.

The discussion of the results

At the stage of metal affinity chromatography, it was determined from the chromatogram that the protein pkb 5 under study was contained in fractions 31 and 32.

Fig. Chromatogram of the isolation of the pkb 5 catalytic domain

Fractions 31 and 32 were combined because they contained the protein under study.

Since the catalytic domain of pkb5 was isolated under denaturing conditions, it was necessary to refold this protein for further study.

In the next step, the pooled fractions were dialyzed to refold pkb5 from denaturing conditions to native conditions.

At the next stage, to check the optimal conditions for chromatographic purification of pkb5 of various fractions, electrophoretic separation of proteins was carried out in a denaturing SDS-polyacrylamide gel.

Rice. Electropherogram obtained as a result of purification of pkb5; a) 1- lysate, 2- fractions 13-20, 3- fractions 26-27, 4- fraction 31, 5- test sample after dialysis, 6- marker proteins (Prestained Protein Molecular Weight Marker, 20-120 kDa); b) 1- lysate, 2- fractions 13-20, 3- fractions 26-27, 4- fraction 31, 5- marker proteins (Prestained Protein Marker, Broad Range, 7-175 kDa), 6- test sample after dialysis

The table (..) indicates the final amount of proteins applied to electrophoresis.

Table(..)

At the next stage after stepwise dialysis, the concentration of the studied protein pkb 5 was increased for further measurement of activity.

The phosphotransferase activity of the catalytic domain of pkb5 was previously tested. Employees of the laboratory of genetics of microorganisms senior researcher, Ph.D. Mavletova D. A. and junior researcher Mironcheva T. A. IOGen im. N.I. Vavilova RAS carried out autophosphorylation of the catalytic domain of pkb5 using [γ-32P]-ATP (Fig.).

A) b)

Rice. a) Electropherogram of pkb5 autophosphorylation. b) Autoradiogram of pkb5 autophosphorylation

Activity check

The Kinase-Glo reagent uses the residual amount of ATP as a substrate for Ultra-Glo TM Luciferase, catalyzing the monooxygenation of luciferin to produce a photon of light (Figure 1). Protein kinase activity is inversely proportional to the intensity of the luminescence signal.

Rice. 1. Kinase-Glo® Assey Reaction Scheme

Based on the luminescent data, a graph was constructed of the dependence of the percentage of autophosphorylation on the amount of protein kinase pkb 5 ( rice.).

Rice. Percent autophosphorylation of pkb 5

Owners of patent RU 2303061:

The invention relates to biotechnology and can be used for cultivating Pseudomonas bacteria. The nutrient medium contains as a source of amino acids the autolysate of spent yeast of the 7-8th generation, K 2 HPO 4, MgSO 4 × 7H 2 O and tap water. The invention allows to increase the yield of biomass of bacteria of the genus Pseudomonas. 2 tables

The nutrient medium can be used in biotechnology for cultivating bacteria of the genus Pseudomonas in order to obtain biological products based on these microorganisms that have antagonistic activity against fungal phytopathogens.

There are known strains of bacteria of the genus Pseudomonas that have antagonistic activity towards fungal phytopathogens and are used to obtain drugs against wheat diseases caused by fungal phytopathogens.

The nutrient medium LB is known, which is one of the main ones for the cultivation of bacteria of the genus Pseudomonas - producers of substances with fungicidal activity and consisting, g/l:

Peptone - 10

Yeast extract - 5

Sodium chloride - 10

Tap water up to 1 liter.

The disadvantage of the nutrient medium is its high cost, since it contains such an expensive component as peptone (approximately 700 rubles/kg).

Known nutrient medium King B, adopted as a prototype, is also used for cultivating bacteria of the genus Pseudomonas. It includes peptone, glycerin, various mineral salts, g/l:

Peptone - 20

Glycerin - 10

K 2 HPO 4 - 1.5

MgSO 4 7H 2 O - 1.5

Distilled water up to 1 liter.

Its disadvantage is also the high cost associated with the presence in its composition of such an expensive ingredient as peptone.

In the industrial production of biological products based on bacteria of the genus Pseudomonas, intended for use in plant growing, the high cost of the nutrient medium will inevitably lead to an increase in the price of the target product.

The technical objective of the proposed invention is to reduce the cost of the nutrient medium for cultivating bacteria of the genus Pseudomonas and increase the yield of biomass.

The stated technical task of reducing the cost of a nutrient medium for cultivating bacteria of the genus Pseudomonas and increasing the yield of biomass is solved by using a nutrient medium of the following composition, g/l:

Autolysate of spent brewer's yeast 7-8 generations (dry matter) - 26.8

K 2 HPO 4 - 1.5

MgSO 4 7H 2 O - 1.5

Tap water up to 1 liter.

Spent brewer's yeast of the 7th-8th generation is not used in beer production, since the cells of such yeast are no longer capable of performing their main function during the fermentation process; they also lose their ability to reproduce. The number of dead cells in such a generation reaches 89%, with weak vital activity - up to 10%. Currently, spent brewer's yeast does not find qualified use and, as a rule, is discharged into the sewer. At the same time, spent brewer's yeast contains significant quantities of vitamins B, PP and E, as well as all essential amino acids.

Microbial cultures during their cultivation are highly sensitive to the composition of the nutrient medium. With the successful selection of all components in terms of qualitative and quantitative composition, in particular the set of amino acids, the medium ensures fairly rapid growth and development of the population of microorganisms and is considered balanced. In cellular protein, individual amino acids account for 1-5% of the total protein, from which the amount of amino acids required as growth factors can be roughly estimated. The exception is glutamic acid and glutamine, which quantitatively play a large role in amino acid metabolism and the content of which in the medium should significantly exceed the content of other amino acids. Table 1 shows the amino acid composition determined by us of spent brewer's yeast from various beer manufacturers, as well as a sample of commercial peptone intended for use in some nutrient media in microbiology.

As follows from the data in Table 1, spent brewer's yeast contains glutamic acid in a significant amount, and its content in percentage terms is higher than in peptone. It should also be noted that the content of all essential amino acids is higher than that of peptone, which indicates a more balanced amino acid composition of the proposed component of the nutrient medium.

The proposed nutrient medium is prepared as follows.

To obtain autolysate, spent brewer's yeast of the 7th-8th generation is subjected to heat treatment at 100°C for an hour.

Mineral components are added to the yeast autolysate, the volume of the resulting mixture is adjusted to 1 liter with tap water and autoclaved. Inoculum is added (40 ml of Pseudomonas bacterial culture). The strain is grown for 48 hours at 28-30°C with constant aeration. Then the titer in the culture liquid is determined by a known dilution method.

Example 1. A nutrient medium of the following composition is prepared. To 26.8 g of yeast autolysate add 1.5 g of K 2 HPO 4 and 1.5 g of MgSO 4 · 7H 2 O, bring the volume of the resulting mixture to 1 liter with tap water and autoclave. Then 40 ml of the bacterial culture Pseudomonas aureofaciens IB 51 is inoculated into the nutrient medium. Fermentation is carried out for 48 hours at 28-30°C with constant aeration. The titer of the culture liquid at the end of fermentation is 5·10 11 CFU/ml.

Under similar conditions, the strain Pseudomonas aureofaciens IB 51 is cultivated on the known King V medium. The number of microorganisms at the end of the cultivation process was 3·10 10 CFU/ml.

Example 2. The nutrient medium is prepared as described above according to example 1 and 40 ml of Pseudomonas putida IB 17 culture is inoculated. Fermentation is carried out in a similar way according to example 1. The titer of the culture liquid at the end of fermentation is 5·10 11 CFU/ml.

Under similar conditions, the strain Pseudomonas putida IB 17 is cultivated on the known King V medium. The number of microorganisms at the end of the cultivation process was 6·10 10 CFU/ml.

Table 2 shows the results of cultivating bacteria of the genus Pseudomonas on King B medium (according to the prototype) and the proposed medium. As can be seen, the highest titer of bacteria of the genus Pseudomonas is maintained in samples where the nutrient medium contains, instead of peptone and glycerol, the autolysate of spent brewer's yeast of the 7th-8th generation.

Example 3. Testing the preservation of the antagonistic activity of the Pseudomonas aureofaciens strain IB 51 grown on the proposed medium.

A suspension of fungal phytopathogen spores ( Bipolaris sorokiniana). Then, bacteria of the Pseudomonas aureofaciens strain IB 51, grown on the proposed medium, are planted by injection on the same plates. The dishes are incubated at 28°C for 3 days.

Accounting for antagonistic activity is carried out by the average diameter of the zone of absence or suppression of growth of the test fungus around the colony of the strain. For Bipolaris sorokiniana it was 55 mm.

Under similar conditions, the antagonistic activity of the Pseudomonas aureofaciens strain IB 51, cultivated on the well-known King V medium, is taken into account. The average diameter of the fungal growth suppression zone was 55 mm.

Example 4. Checking the preservation and taking into account the antagonistic activity of the Pseudomonas putida strain IB 17, grown on the proposed medium, is carried out in the manner described above according to example 3. The average diameter of the zone of suppression of the growth of the test fungus was 22 mm.

Under similar conditions, the antagonistic activity of the Pseudomonas putida strain IB 17, cultivated on the well-known King V medium, is taken into account. The average diameter of the fungal growth suppression zone was 22 mm.

The results are shown in Table 2 and indicate the preservation of the antagonistic effect of strains of bacteria of the genus Pseudomonas grown on the proposed medium on the test culture of the phytopathogenic fungus Bipolaris sorokiniana.

Thus, the use of the proposed nutrient medium containing autolysate of spent brewer's yeast of the 7th-8th generation as a source of amino acids for the cultivation of bacteria of the genus Pseudomonas, which have antagonistic activity towards fungal phytopathogens, can significantly reduce the cost of biological products based on these microorganisms and increase the yield of biomass .

Literature

1. RF patent 2203945, 7 С12N 1/20, А01N 63/00 // (С12N 1/20, С12R 1:38). A strain of bacteria Pseudomonas aureofaciens to obtain a drug against wheat diseases caused by fungal phytopathogens. / Loginov O.N., Sveshnikova E.V., Silishchev N.N., Melentyev A.I., Galimzyanova N.F., Boyko T.F.; declared 08/17/2001; publ. 05/10/2003. Bulletin 13.

2. RF patent 2213774, 7 С12N 1/20 // (С12N 1/20, С12R 1:40). A strain of Pseudomonas putida bacteria to obtain a drug against wheat diseases caused by fungal phytopathogens. / Loginov O.N., Sveshnikova E.V., Silishchev N.N., Melentyev A.I., Galimzyanova N.F., Boyko T.F., Isaev R.F.; declared 03/25/2002; publ. 10.10.2003. Bulletin 28.

3. RF patent 2130265, 6 A01N 63/00 // (A01C 1/00). A method of combating plant pathogens. / Dashkevich V.S., Dashkevich N.Yu., Ashmarina L.F., Shusharo A.I., declared 12/29/97; publ. 05.20.99. Bulletin 14.

4. King E.O., Ward M.K., Raney D.E. Two simple media for the demonstration of pyocyanin and fluorescin. //J.Lab. Clin. Med. - 1954. - V.44. - P.301-307.

5. Smirnov V.V., Kiprianova E.A. Bacteria of the genus Pseudomonas. Kyiv, “Naukova Dumka”, 1990. - P.222.

6. Gurevich Yu.L. Stability and regulation of reproduction in microbial populations. - Novosibirsk, Science, 1984. - P.28-29.

7. Manakov M.N., Pobedimsky D.G. Theoretical foundations of microbiological production. - M.: Agropromizdat, 1990. - P. 180-181.

8. Perth S.J. Basics of cultivation of microorganisms and cells. M.: Mir, 1978. - pp. 147-148.

9. Tepper E.Z., Shilnikova V.K., Pereverzeva G.I. Workshop on microbiology. - M.: Bustard, 2004. - 256 p.

Table 1
Amino acid composition of peptone and spent yeast from various breweries
	Spent yeast (Ufa, Efes)	Spent yeast (Sterlitamak, Shikhan-Heineken)	Spent yeast (Novotroitsk, PIT)	peptone
Threonine	5,56%	5,45%	5,25%	2,09%
Valin	4,67%	4,34%	5,34%	2,22%
Methionine	1,83%	1,85%	2,00%	0,96%
Isoleucine	5,22%	4,50%	5,30%	1,81%
Leucine	6,93%	6,31%	7,03%	2,98%
Tyrosine	4,28%	3,72%	4,44%	0,78%
Phenylalanine	4,97%	4,86%	5,37%	2,34%
Lysine	11,07%	10,57%	11,71%	4,85%
Cystine	1,24%	1,72%	1,55%	0,23%
Serin	5,35%	5,86%	5,87%	3,50%
Glutamic acid	15,20%	13,70%	13,40%	11,35%
Proline	5,40%	6,23%	4,87%	14,46%
Glycine	5,78%	5,33%	5,36%	27,33%
Alanin	8,37%	7,42%	7,80%	8,97%
Histidine	3,34%	2,00%	3,47%	0,75%
Arginine	0,79%	6,54%	1,09%	9,52%
Aspartic acid	10,00%	9,60%	10,15%	5,86%

Nutrient medium for the cultivation of bacteria of the genus Pseudomonas used for the production of antifungal drugs, containing a source of amino acids, K 2 HPO 4, MgSO 4 ·7H 2 O and water, characterized in that as a source of amino acids it contains autolysate of spent brewer's yeast of the 7-8th generation , and water - tap water with the following ratio of components, g/l:

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