Method for obtaining actinobacillus pleuropneumonia apxi or apxiii toxins in liquid culture media supplemented by air enriched with carbon dioxide

FIELD: biotechnologies.

SUBSTANCE: invention relates to method for obtaining of RTX-toxins Apxl or ApxIII by culturing of Actinobacillus pleuropneumoniae bacteria in liquid culture media. Characterised method consists in the following: during exponential growth phase of bacteria and production of RTX-toxins air passes through the medium, carbon dioxide content in air is above normal atmospheric level and is up to 10 % vol.

EFFECT: invention allows increasing Apxl or ApxIII toxins output, this may be used during vaccines production.

7 cl, 4 tbl

 

The present invention relates to a method for RTX toxins ApxI or ApxIII by culturing bacteria Actinobacillus pleuropneumoniae in a liquid culture medium.

Pleuropneumonia of swine, the main respiratory disease of pigs, is distributed worldwide and causes severe economic losses in pig production due to lightning deaths, ill pigs and delays in sales because of chronically infected animals. The aetiological agent is Actinobacillus pleuropneumoniae. It is transmitted primarily through direct contact between animals, and the infection leads to disease, ranging from lightning to chronic. The disease mainly is an infection of the respiratory tract, with clinical signs of high fever, severe respiratory distress, cough and anorexia. Onset rapid, and morbidity and mortality is high. One way to control infection by bacteria Actinobacillus pleuropneumoniae (hereinafter also referred to as "ADR") is a program of vaccination. These programs were used fried bacteria, but aware of their severe side effects. Currently widely used in subunit vaccines based toxins ARR.

The RDA produces the so-called RTX-toxins (RTX hereafter which includes the repeat in toxin). The presence of these RTX-toxins contribute to the pathogenic nature of this bacteria. RTX-toxins have been discussed in detail previously and described in the literature. As is well known, not all serotypes of APP produce all RTX-toxins. For example, serotypes 1, 5, 9 and 11 produce ApxI and ApxII. Serotypes 2, 3, 4, 6 and 8 produce ApxII and ApxIII. Serotype 10 produces only ApxI, and serotypes 7 and 12 produce only ApxII. Current commercially available vaccines against the RDA is based on the toxins ApxI, ApxII and ApxIII. Recently it was discovered that all serotypes of APP produce the fourth RTX-toxin, currently called ApxIV (see EP 0875574).

It is widely known how to get RTX-toxins ApxI or ApxIII through cultivation of Actinobacillus pleuropneumoniae in the liquid culture medium. In particular, earlier in the EP 0453024 describes a method for ApxI (see "example 2", paragraph 2 "Purification and characterization of hemolysin", subsection "Methods") or ApxIII (see "example 4", paragraph 2 "Purification and characterization of macrophage toxin RDAs (Mat)", subsection "Methods"). It is noted that used ApxI should be marked "HLY", while ApxIII, usually meant "Mat" (see Frey et al. in the journal “J Gen Environ.”, August 1993; 139(8): 1723-8). The environment must support the growth of bacteria ARR. It is well known how to construct an environment that ensures the growth of bacteria. Klas is practical culture medium was originally developed by Eagle, Ham and others in 1950-60, They found that the environment that satisfies the basic needs of growth, must contain inorganic salts, a nitrogen source (for example, in the form of nitrogen-containing compounds, such as peptides or proteins), the source of carbon and vitamins. Environment mainly sautereau to prevent them from either acidification or alkalization. This basic recipe is available a large number of different compositions. For example, to provide the amino acids you can choose components of animal origin, but you can also choose chemically defined amino acids. Other connections are also possible large number of variants. Actually, to make an environment that ensures the growth of bacteria, is relatively easy. However, to optimize growth and/or receiving metabolites may take some time to develop, particularly if the preferred environment, which does not contain serum or other animal ingredients. Strategies to improve fermentation environment, however, well known in this field and are described in detail in the literature (see, for example, a review article Kennedy and Krouse in journal of Industrial Microbiology & Biotechnology (1999) 23, 456-475). This optimization is part of routine experiments in the laboratory of fermentation. In the case of coltivirus is of the RDA part of an environment essentially amounts to NAD (nicotinamide adenine dinucleotide), because the bacterium RDAs is a NAD-dependent. In the absence of NAD environment will not support the growth of bacteria Actinobacillus pleuropneumoniae and therefore can not be considered as a liquid medium to support the growth of the RDA from the point of view of this application and the attached claims. A liquid medium to support growth of bacteria or components for making such media are commercially available from numerous companies such as Sigma Aldrich, Quest International, Oxoid, Becton Dickinson, Pharmacia, VGD Inc, Mediatech, Invitrogen, Marcor, Irvin Scientific, etc.

Despite the fact that in the prior art suggests ways of receiving RTX toxins ApxI and ApxIII by culturing the RDA, it is desirable to improve the yield. To date, attempts to improve the yield, were mainly aimed at the production speed of toxins in the stationary phase of the cultivation of the RDA because it is known that the maximum production RTX-toxins observed in high-density cells, i.e. at the end of the exponential phase of growth (see, for example, Microbial Pathogenesis 37 (2004) 29-33). These attempts have not led to a substantial improvement of the full output. Unexpectedly, however, the applicant has found that when passing through a medium of air during the phase of production (i.e. during the phase of growth and/or stationary phase bacteria RDAs) of these toxins Actinobacillus pleuropneumoniae, if sod is neigh in the air of carbon dioxide above the normal atmospheric level, products RTX-toxins is significantly increased. Actually, generally known about the use of elevated levels of carbon dioxide in the process of cultivation of bacterial colonies on plates (see, for example, U.S. patent 6019984: EXAMPLES of Bacterial strains and growth conditions"). However, this refers to the cultivation of colonies of bacteria, and the bacteria are then used for inoculation of the fermenter. At this stage only the growth of bacteria, but not products RTX-toxins (at least on the material level). As soon as the bacteria of the RDA transferred into liquid medium for rearing to a high density of cells, suitable for receiving RTX-toxin, in the prior art it is proposed to dispense with the increased level of carbon dioxide. This, of course, is consistent with the assumption of the prior art mentioned herein above that the maximum production of the Architect in the fermenters occurs only when a high density of cells, i.e. at the end of the exponential phase of growth. At this stage, the cell growth was over and generally has no value, and thus, the carbon dioxide previously thought unimportant. Moreover, bacteria RDAs themselves produce carbon dioxide during the formation of RTX-toxins. Thus, I believe that purposeful adding to the atmosphere of carbon dioxide even suppresses products is July toxin. These facts explain why carbon dioxide has never been considered as a stimulating factor to obtain the RTX-toxin. The reason, however, why carbon dioxide stimulates the production of ApxI and ApxIII, not clear, especially considering that the carbon dioxide does not have a positive impact on the level of production RTX-toxin ApxII.

It is noted that there are a large number of techniques to pass air through a medium. A widely used approach is to pass air through the device, which allows air to enter anywhere in the environment (i.e. under the surface of the medium) in the form of bubbles. Such devices may have a single nozzle or a large number of nozzles, depending, among other things, on whether the desire to establish (approximate) situation of equilibrium in the environment, and if so, how quickly this balance should be achieved. In any case, the transmission of air through the environment contrary to the use of free air space above the liquid based mostly just on diffusion. It was found that this technique leads to inadequate results. "Air" in the context of the present invention means a gaseous environment containing one or more gaseous components that are normally present in atmospheric air, such as oxygen, nitrogen, diox the d carbon helium, neon, argon, xenon, radon, etc. "the Normal atmospheric level of carbon dioxide" is 0.04% of the volume of CO2the total volume of air.

In one embodiment, the implementation of air is passed during the exponential phase of bacterial growth of Actinobacillus pleuropneumoniae. The exponential growth phase in contrast to what has been described in the prior art, is a phase, which is part of all phases of production RTX-toxins (along with the stationary phase). Unexpectedly, the applicant has found that the transmission of carbon dioxide during the exponential phase of growth led to a very significant stimulation of production RTX-toxin, so that even at the end of this phase in the fermenter is economically significant quantities of toxin. Therefore, in this embodiment, it is proposed to choose the end of the fermentation at the end of the exponential growth phase or early stationary phase. An important advantage is that it can reduce a significant production time, and that the number of lipopolysaccharides in the final product may be reduced.

In another embodiment, where the environment sautereau (i.e. add a substance that minimizes the change in the acidity of a solution when added to a solution of acid or base), it sautereau using bicarbonate ie salt containing ions HCO3-). When using bicarbonate buffer found that the effect of decreasing pH, the inherent excess of carbon dioxide, can be very effectively neutralized. Obviously, using such a buffer, for example, sodium bicarbonate or bicarbonate buffer with another alkali metal in the environment will almost immediately reached (approximate) equilibrium state.

In another embodiment, air is passed through a medium at a constant flow. In fact, can be designed many different ways of passing the gas through a medium. One of them is a pulsating stream of air having an extremely high content of carbon dioxide (up to, for example, 90%). However, the authors found that very good results can be obtained with constant flow. With this constant flow of air can be used moderate levels of carbon dioxide. This creates the advantage that the buffer will be better able to maintain the pH value around the equilibrium value at any time. It is noted that a constant flow does not necessarily mean that the addition of carbon dioxide is generally not interrupted at some point of time. For example, a short break flow in the cultivation process does not exclude the fact that before and the pic is E. this break the flow constant. In one embodiment, the implementation of air is passed continuously during the exponential growth phase of the bacteria Actinobacillus pleuropneumoniae, i.e. during the exponential phase of growth, the flow must not be interrupted.

In one embodiment, the implementation of the carbon dioxide content of up to 10 vol.%. In this embodiment, the maximum volumetric concentration of carbon dioxide in the air is 10%. Above this level it is possible that the buffer will not be able to balance all the time at high speed. This can adversely affect the output of the RTX-toxin. In a preferred embodiment, the carbon dioxide content is 5 vol.%. When the content of carbon dioxide were obtained good results, but also from an economic point of view this is the preferred amounts of carbon dioxide, because this mixture is commercially available and very cheap.

In one of the embodiments in which the RTX-toxin is a ApxI, the culture medium contains borogluconate calcium. In fact, it is widely known that the transcriptional activity of the ApxI operon is enhanced when added to growth medium calcium (see Environ Pathogenesis 37 (2004) 29-33). They found certain advantages when using borogluconate (2,3-dihydroxy-3-[2-hydroxy-5-(hydroxymethyl)-1,3,2-dioxanone the EN-4-yl]propanoate) for the formation of a complex with calcium ions. First, it is shown that common problem precipitiously of calcium salts during the process, in particular filters, which tend to clog, can be prevented or at least significantly reduced. Next it is shown that it is possible to get ApxI at a level that is significantly higher compared to the level achieved by the methods of the prior art which use other complexing agents such as EDTA. Obviously, using this specific complexing agent, so that the medium contains a complex of calcium-borogluconate (i.e. 2,3-dihydroxy-3-[2-hydroxy-5-(hydroxymethyl)-1,3,2-dioxaborolan-4-yl] propanoate calcium, also known as D-gluconic acid, cyclic 4,5-ester with boric acid, calcium salt of 2:1), can be prevented significant precipitation of calcium ions with other negative ions, at the same time, calcium ions remain able to enhance the transcriptional activity of the ApxI operon of the bacterium Actinobacillus pleuropneumoniae.

Although it is not essential to the present invention, the medium may not contain components of animal origin. The lack of many methods of the prior art is that they are based on the use of media containing components of animal origin, such as Colombian culture among the and. Other components of animal origin referred to in the prior art, are, for example, a modified Colombian culture medium or cardio-cerebral infusion environment. As is well known, the use of components of animal origin has some significant drawbacks. First, the chemical composition can vary significantly between batches. In addition, supplements of animal origin may be contaminated by infectious agents. The most dangerous is the presence of prions that cause TSE in humans or animals. You can simply choose the environment that does not contain animal ingredients (often referred to as "ACF"). Component of animal origin" in this sense means any component that is present as such in the animal (e.g., blood or protein) or is of such a component (for example, a modified serum obtained from the blood, or amino acids derived from protein). The applicant, however, found that the production efficiency of the ApxI significantly lower when using these environments ACF compared with media containing components of animal origin, even if the concentration of calcium is adequate. Not referring to theory, it is possible that when using serum samples the EMA precipitation of the calcium salt is less sharp due to the presence of agents, which form soluble complexes with calcium ions. In any case, when using borogluconate for the formation of a complex with calcium ions can be obtained a significant increase in the yield of ApxI, suddenly getting the output, which is even higher than the yield obtained using the accepted medium containing serum.

MATERIALS AND METHODS

Bacterial strain and environment

The studies were carried out using a strain of Actinobacillus pleuropneumoniae, produce ApxI, serotype 10, later in this document called the RDA 10, and the strain producing ApxII and ApxIII, namely the strain of serotype 2, later in this document called the RDA 2. In all cases, work seed these strains were recovered using a Cup with a base Columbia blood agar (BAB) (production company Becton, Dickinson, USA). Used liquid medium consisted of either Colombian culture medium (production company Becton, Dickinson USA), or the environment, does not contain animal ingredients (referred to as "ACF"). Last Wednesday was held as buffer mixture To2NRA4(14.6 g/l) and NaH2PO4(3.6 g/l) and, in addition, NaNO3(0.2 g/l), 50% glucose solution (10 ml), yeast extract (15 g/l) (production company Becton, Dickinson), trace elements (for example, 2.5 ml of a solution SL-10 specified in the guidance Handbook of Microbiological Media, 3rdrdition, Ronald Atlas, CRCpress, 2004), and 10 mm solution of amino acids (containing all 20 amino acids, except tryptophan). Alternative tested environment, which does not contain components of animal origin (called "ACF-alt")contain cysteine·HCl (0.1 g/l), NaNO3(0.5 g/l), KCl (0.1 g/l), trace elements (see above), 50% glucose solution (10 ml) and 10 mm solution of amino acids (see above), HEPES buffer (6 g/l; for example, the production company Sigma Aldrich) and yeast extract (10 g/l).

These have been used in preculture and fermentation. Nicotinamide adenine dinucleotide (0,01%) used preculture and fermentation. All the media were sterilized by filtration with a pore diameter of 0.22 μm. Before using fermentation environment was heated at 85°C for one minute.

CULTIVATION

Preculture

Working seed materials strains RDAs were sown in a Cup with Columbia agar VAV, and incubated for approximately 24 hours at 37°C. Several colonies were selected for inoculation vessels with a volume of 500 ml containing 75 ml Colombian medium. The vessels were incubated for approximately 6 hours at 37°C with shaking for education preculture. Using these preculture carried out several fermentati.

Culturing in the fermenter SIXFORS

In the fermenter SIXFORS (production company Infors AG, Switzerland), containing at listello 400 ml of culture medium, added as inoculum about 20 ml of preculture. The cultivation temperature is 37°C, pH 7,2±0,1 (by adding 4 n sodium hydroxide solution or a 4 n solution of acetic acid) and the aeration is carried out at 50% Rho2. Culturing in the fermenter was stopped after about 24 hours.

Culturing in the fermenter patch BIOSTAT

In fermenters patch BIOSTAT C (production company B. Braun Biotech, Germany), which contained 10 l of medium, as inoculum was added 500 ml of preculture ARR. Used the same parameters as in the fermenters SIXFORS. However, the carbon dioxide level is increased, maintaining a constant air flow of 1 vvm (volume of gas to volume of medium per minute) for the gas mixture air/CO295/5 about/about. This leads to the aeration parameters with RDF2about 5%. Culturing in the fermenter was stopped at the end of the exponential phase after approximately 8.5 hours.

Cultivation in scale pilot plant

Experiments on a pilot plant was carried out in sterilized fermenter with a volume of 100 L. the Axis of the stirrer was equipped with three of a six-bladed turbine agitators Rushton. The fermenter was filled with 75 liters of environment and inoculable 3 liters of preculture. The temperature was maintained at 37°C, at pH of 7.3 with automatic pH controller using a 20% (weight/volume) solution of NaOH and 8 N. the solution of the UKS the Noah acid. Standard cultivation was carried out at an elevated pressure of CO2in free space (500 mbar), and the stirrer was rotated at a constant speed of 250 Rev/min oxygen Pressure is not controlled. For cultivation experiments with spray CO2the medium was supplemented with NaHCO3to a final concentration of 10 mm and aeronavali constant air flow of 0.25 vvm air enriched 0,014 vvm CO2. This leads to the pCO2about 5%. In the case of strain ARR 10, the medium was supplemented with 25 mm solution of CaCl2and the aeration was carried out at 50% Rho2.

Analysis

At the end of each experiment, samples of the cultures of bacterial cells were aseptically taken from the fermenter to determine the optical density at 648 nm and level ApxI, ApxII and/or ApxIII (ELISA; units per ml) and optional level LPS (analysis LAL; units×105per ml).

RESULTS

The first experiment was conducted at the scale of the experimental setup with the strain of RDA 2 to study the effect of carbon dioxide passed through the environment (experiments with "sprayed" CO2). Used environment was a Colombian environment. The results are presented in table 1. The results of the two experiments (exp 1 and exp 2).

Table 1
The method of cultivationTime (h)OD648ApxIIApxIIILPS
Standard, exp 1121,35441300,36
Standard, exp 2111,47771460,32
Spraying CO2, exp 151,42522420,34
Spraying CO2, exp 25to 1.86382870,09

From these results it becomes clear that the products ApxIII can be almost doubled by passing carbon dioxide through the medium at a level above the normal atmospheric pressure. On the level of ApxII was not observed absolute increase. The concentration of LPS in the late exponential phase of growth (coalsales the end of each experiment, table 1) did not differ greatly. But because the level ApxIII twice, the number of LPS at a dose ApxIII in the final product will be a maximum of about half the number of LPS of the product obtained with the standard options.

The second experiment was also carried out at the scale of the experimental setup, but now with the strain ARR 10, to study the effect of carbon dioxide passed through the environment (experiments with "spray" CO2), to obtain ApxI. Used environment was a Colombian environment. The results are presented in table 2. Again the results of two experiments (exp 1 and exp 2).

Table 2
The method of cultivationTime (h)OD648ApxILPS
Standard, exp 1122,995204,10
Standard, exp 2113,116436,10
Sprayed WITH2, exp 16is 3.08 10743,36
Sprayed WITH2, exp 252,9917745,48

As is clear from table 2, in respect of ApxI can be obtained comparable results.

In the third experiment studied the effect of carbon dioxide passed through the environment using the environment ACF described in this document above. The first cultivation was carried out in a fermenter SIXFORS in the absence of additional carbon dioxide can flow through the environment. The second cultivation was carried out in a fermenter patch BIOSTAT C in the presence of additional carbon dioxide passed through the environment, as specified in this document above. Due to the fact that in the patch BIOSTAT fermenter conditions can be controlled a little better, it was expected that the output of the toxin Architect will (all parameters equal) about 2 times higher (maximum 3 times)than in the fermenter SIXFORS. The results are presented in table 3.

/tr>
Table 3
The method of cultivationTime (h)OD660ApxIII
ACF, without requiring the use of CO274,001003
ACF, spray CO24,753,134575

Because the level ApxIII in the patch BIOSTAT fermenter about 4 1/2 times above, we can conclude that when using the environment ACF carbon dioxide passed through the environment, also has a positive effect on the production of Apx.

In the fourth experiment, carried out in a fermenter SIXFORS strain ARR 10, the authors studied whether it is possible to add calcium in the form borogluconate complex (instead of calcium, not formed complex). The concentration of borogluconate varied between 40, 50 and 70 mm. The cultivation was stopped at the end of the exponential phase of growth. The results are presented in table 4.

Table 4
The method of cultivationApxI
ACF-alt, without added calcium, 5% CO20
ACF-alt, added 40 mm Sa-borogluconate, 5% CO2520
ACF-alt is objavlen 50 mm CA-borogluconate, 5% CO2357
ACF-alt, added 70 mm Sa-borogluconate, 5% CO2222

Based on the results, we can conclude that to obtain satisfactory levels of ApxI calcium may be present in the form of a complex calcium-borogluconate. The advantage of this is that the precipitates of calcium is no longer influenced in a negative way on the progress of the processing environment (in particular for the filtration process). Apparently, a higher concentration of borogluconate adversely affect the output.

1. The method of obtaining RTX toxins ApxI or ApxIII by culturing bacteria Actinobacillus pleuropneumoniae in a liquid culture medium, which ensures the growth of bacteria, differentthe,during the exponential phase of bacterial growth and production of RTX-toxins through the environment, preventing pollution, and carbon dioxide content in the air is higher than the normal atmospheric level and up to 10 vol.%.

2. The method according to claim 1, in which the environment sautereau.

3. The method according to claim 2, characterized in that environment sautereau using bicarbonate.

4. The method according to claim 1, characterizedthe,that air is passed through a medium at a constant flow.

5. The method according to claim 4, characterizedthe,that air is passed continuously over the former is nenciarini phase growth of bacteria Actinobacillus pleuropneumoniae.

6. The method according to any of the preceding items, characterizedthe,the carbon dioxide content is 5%vol.

7. The method according to claim 1, in which the RTX-toxin is a ApxI differentthe fact that the culture medium contains borogluconate calcium.



 

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16 cl, 2 dwg, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: method includes a stage of yeast cultivation, transformed by a vector containing a DNA sequence, determined by the formula X-B-Y-A, coding the precursor of insulin glargine, where X is a sequence of leader peptide, containing at least one amino acid. B is a B1-B30 sequence of amino acids of B-chain of the insulin glargine molecule. Y is a linker peptide containing at least two amino acids. A is an A1-A21 sequence of amino acids of an A-chain of a molecule insulin glargine, a stage of extraction of an expressing precursor of insulin glargine, a stage of crystallisation of the extracted precursor of insulin glargine, a stage of completion of fermentative conversion of insulin glargine precursor crystals at pH from 8 to 10 in presence of tripsin or tripsin-like ferment and water soluble organic dissolvents at the ratio from 40% to 60% of the reaction mix with formation of insulin glargine, containing at least one related admixture. Then the stage of insulin glargine treatment by reverse phase highly efficient liquid chromatography is carried out on a chromatographic matrix, using a polar organic buffer dissolvent in a water phase, containing a buffer based on organic acid, in which the matrix is first balanced with 10% acetonitrile in 250 mM of acetic acid with further elution of insulin glargine in the specified acetonitrile. Then the matrix is again balanced with 10% acetonitrile in the buffer on the basis of organic acid in concentration from 20 mM to 200 mM at pH from 3 to 8.5 with subsequent elution of insulin glargine in the specified acetonitrile, and further repeatedly the matrix is balanced with 6% ethanol in the buffer on the basis of organic acid in concentration from 10 mM to 50 mM with subsequent elution of the specified insulin glargine in the specified ethanol. Further the treated insulin glargine is deposited by means of addition of the buffer on the basis of citric acid and zinc chloride at pH from 6 to 8.

EFFECT: invention makes it possible to produce insulin glargine with high purity and low content of glycolised admixtures.

12 cl, 9 dwg, 8 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to genetically modified bacteria Salmonella enterica, which contain at least one operon pgI from Campylobacter jejuni or its functional derivative and refers to presentation of at least one N-glycan from Campylobacter jejuni or derivative of this N-glycan on their cell surface. In bacteria one or several genes for biosynthesis of bacillozamine are inactivated by mutation and/or partial or complete deletion of genes pgID, E, F, G. Invention provides method for obtainment of genetically modified bacteria Salmonella enterica. Invention is directed to application of modified bacteria in pharmacological compositions of medical and veterinary purpose and methods for treatment and/or prevention of infections Campylobacter and, not obligatory, Salmonella.

EFFECT: invention provides safety and efficiency of treatment and prevention of infections, which affect people and animals, namely livestock and fowl.

18 cl, 4 dwg, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbiology and biotechnology. Disclosed is a consortium of Lactobacillus rhamnosus VKM B-2726D and Lactobacillus plantarum VKM B-2725D strains.

EFFECT: consortium is used to produce a bacterial preparation and a direct administration ferment for producing fermented milk and fermented beet juice as functional nutrition products.

15 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Disclosed are Bacillus subtilis DSM 19467, Bacillus subtilis DSM 19489 and Bacillus subtilis DSM 19466 strains which produce high levels of phytase. An animal feed composition is obtained based on any of said strains coupled with other suitable additives. Also disclosed is a method of feeding animals, which involves serving the composition to animals along with other feed ingredients.

EFFECT: invention provides a probiotic effect and increases assimilation and use of nutrients in animal feed.

8 cl, 3 dwg, 2 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: strain Lactococcus casei VKPM V-8730 is obtained at available growth-supporting media and is used as starter material in cultured milk products production.

EFFECT: invention allows reducing the time of technical process and increasing cultured product shelf life.

1 ex

FIELD: biotechnologies.

SUBSTANCE: strain Enterococcus durans VKPM B-8731 is obtained at available growth-supporting medium and is used as starter material in cultured milk products production.

EFFECT: invention allows reducing final acidity of cultured milk product, reducing the time of technical process and increasing cultured product shelf life.

1 ex

FIELD: biotechnologies.

SUBSTANCE: invention pertains to the use of L. casei ssp. paracasei CNCM 1-1518 strain for giving antimycotic properties to fermented dairy product. Inhibiting of mould development of ascomycete class in the said product is made by dairy substrate fermentation in presence of this strain for, at least, 5 hours. Thallome diameter after 7 days of incubation amounts 8.8±1 mm.

EFFECT: increase of antimycotic activity in fermented dairy product.

6 cl, 3 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: group of inventions refers to microbiology and biotechnology. Proposed strains Lactobacillus crispatus "ВКМ" B-2727D, Lactobacillus gasseri "ВКМ" B-2728D and Lactobacillus plantarum "ВКМ" B-2731D have antagonistic activity in relation to pathogenic and semi-pathogenic microorganisms. Besides, the invention proposes a consortium based on the above strains for production of a bacterial preparation, a biologically active additive, and direct inoculation for obtaining fermented milk product of functional nutrition.

EFFECT: consortium has higher antagonistic activity against pathogenic and semi-pathogenic microorganisms in relation to individual strains of lactobacilli, use of a consortium will allow enlarging the range of devices intended for prophylaxis and treatment of urogenital infections of females.

4 cl, 1 dwg, 7 tbl, 12 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant bacteria strain Escherichia coli All-Russian collection of industrial microorganisms B-11271 - producer of hydrolase of esters of alpha-amino acids from Xanthomonas rubrilineans All-Russian collection of industrial microorganisms B-9915 is built, which is obtained by transformation of strain-recipient Escherichia coli BL21(DE3) of plasmid DNA corresponding to nucleotide sequence SEQ ID NO 1 containing gene aehR of hydrolase of alpha-amino acids esters from Xanthomonas rubrilineans All-Russian collection of industrial microorganisms B-9915, which is not modified on 5'-end with the sequence coding a polyhistidin tail and being under control of promoter T7, as well as resistance gene to kanamycin Kan.

EFFECT: invention allows obtaining hydrolase of esters of alpha-amino acids with high efficiency degree.

2 cl, 3 dwg, 1 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: exometabolites extracted from Phaeodactylum tricornutum sea microalgae by ethyl acetate extraction of culture fluid obtained during growth of Phaeodactylum tricornutum sea microalgae with further removal of a solvent from ethyl acetate extract and by drying of ethyl acetate extract till constant weight are used as Yersinia pseudotuberculosis growth stimulators. If required, additional cleaning of ethyl acetate is performed.

EFFECT: invention allows enlarging a range of Yersinia pseudotuberculosis growth stimulators.

2 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: method involves preparation of bacterial suspension, separation of the obtained biomass of bacteria by centrifugation, dilution of the obtained biomass in physiological solution, preparation of monolayer of CaCo-2 cells, introduction of bacterial culture, cultivation of cells, flushing with physical solution, removal of monolayer with bacterial cells and counting of the amount of bacterial cells related to 1000 cells of CaCo-2; with that, bacteria refer to highly-adhesive ones if the number of bonded cells is 1010 to 3000, to average-adhesive ones of 210 to 1000, and to low-adhesive ones of 0 to 200.

EFFECT: improvement of the method.

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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