RU2530552C1 - STRAIN Bacillus lentus - PRODUCER OF BACTERIOCIN-LIKE SUBSTANCE OF ANTIMICROBIAL ACTION AND METHOD OF OBTAINING BACTERIOCIN-LIKE SUBSTANCE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain Bacillus lentus "GKPM-Obolensk" B-7150 - producer of bacteriocin-like substance of broad spectrum of antimicrobial action is proposed, having molecular weight according to data of SDS-PAGE of about 4 kDa. Also the method of obtaining bacteriocin-like substance is provided, comprising submerged cultivation of Bacillus lentus "GKPM-Obolensk" B-7150 at a temperature 29-30°C in carbohydrate-yeast medium based on inorganic source of amino nitrogen with a limit of oxygen and carbohydrate. Isolation of bacteriocin-like substance from acellular fluid is carried out using dichloromethane or silica gel type adsorbent, and from the surface of cells - using ethanol. Then the fractions are combined and dried.

EFFECT: strain has antagonistic activity against coliform and other gram-negative bacteria and some species of gram-positive microorganisms.

2 cl, 3 dwg, 5 tbl, 7 ex

Description

The invention relates to the field of microbiology, biotechnology, in particular to the production of a bacillary strain as a producer of an antimicrobial substance, which can be used as a growth-promoting additive in poultry feed instead of antibiotics for use in veterinary medicine and agriculture.

BACKGROUND

Species of Bacillus as part of the probiotics of the so-called “live drugs” and food additives approved for human and animal consumption include only certain types of bacilli B. subtilis, B. lichenformis, B. coagulans, B. toyoi (cereus), B. natto (subtilis), B. clausii (lentus), B. polyfermentans, and B. cereus [Hoa NT, Baccigalupi L., Huxham A. etal., 2000; Lee KH, Jun KD, Kim WS et al., 2001; Pinchuk IV, Bressollier P., Verneuil B. et al., 2001; Sanders ME, Morelli L., Tompkins T., 2003]. The bacteria Bacillus lentus (better known as Bacillus clausii in the English literature) are part of the non-pathogenic transit microflora of the intestines of humans and animals, but have their own characteristic differences [Urdaci, Maria C., Bressollier, Ph., Pinchuk, I, 2004; Senesi S., Celandroni F., Tavanti A. et al., 2001]. Bacteria of this species are known to be alkaline tolerant and are known producers of highly alkaline protease [Denizci A.A., Kazan D., Abeln ECA, and Erarslan A., 2004]. Studies have shown that B. clausii is safe and effective in the treatment of respiratory infections, acute otitis media in children, and also have a number of positive effects on the human body, especially on the immune system [Marseglia GL, Tosca M., Cirillo I., et al., 2007]. The spectrum of antimicrobial activity of most probiotic strains of bacilli is limited to gram-positive types of microorganisms. This, for example, applies to four strains of B.clausii (lentus), O / C, N / R, SIN and T (Enterogermina ^® preparation), B.subtilis 3, B.licheniformis 31 (Biosporine preparation), B.cereus IP 5832 (Bactisubtil preparation), B.cereus NT (Biosubtyl preparation), B.cereus DM-423 (Cereobiogen preparation) and reference B.subtilis 168 [Hoa NT, Baccigalupi L., Huxham A. et al., 2000; Sanders ME, Morelli L., Tompkins T., 2003]. None of them had activity against gram-negative bacteria. Therefore, the search for microorganisms with a wide spectrum of antimicrobial activity is relevant for their use both as part of a new generation of probiotic preparations and as producers of bacteriocin-like substances (BPS; in the BLlS English literature - bacteriocin-like inhibitory substances) - a possible alternative to antibiotics. This would facilitate the development of new antibiotic-resistant microorganisms.

Antagonistically active strains of bacilli that are part of probiotic preparations are widely known: B.subtilis 534 - probiotic Sporo-bacterin patent SU 1708350, B.subtilis 3H - probiotic Baktis-porin patent RU 2067616. Basically, all of them act on gram-positive bacteria, which is their main drawback, as well as probiotic preparations based on them.

Known strains of B. clausii in pharmaceutical compositions that offer adsorption in different ratios to a solid matrix [US-A-4935353, WO-A-9949877, GB-A-1061894].

Known strain B. clausii (lentus) NG121 producer of a new bacteriocin active against Listeria and Staphylococcus aureus [Sharma N, Kapoor G, Neopaney B]. The authors propose using this bacteriocin as a bioconservative of food products.

A known method of producing a peptide with antibacterial activity using strain B. clausii (patent application FR No. 200990312262 from 2009-12-17). However, the preparation of this bacteriocin is active only against gram-positive bacteria - S. aureus, Enterococcus faecium, Micrococcus sp, Lactococcus lactis, Clostridium difficile, Clostridium perfringens, Listeria monocytogenes, and the method for its preparation is laborious, multi-stage and expensive.

The objective of the invention is to obtain a new strain producing a bacteriocin-like substance with a wide spectrum of antimicrobial activity directed against gram-positive and gram-negative microorganisms, including Listeria spp., Clostridium spp., Bacillus anthracis, Salmonella spp., Escherichia spp., Campylobacterobacterppact. and simplification of the method of obtaining it.

The problem is solved by the fact that the proposed strain of Bacillus lentus B-7150 producing a bacteriocin-like substance having a molecular weight according to SDS-PAG electrophoresis of about 4 kDa, a wide spectrum of antimicrobial activity directed against gram-positive and gram-negative microorganisms, including Listeria spp., Clostridium s. , Bacillus anthracis, Salmonella spp., Escherichia spp., Campylobacter spp., Acinetobacter spp.

The task is also solved by the fact that the proposed method for producing the bacteriocin-like substance Bacillus, including deep cultivation of the producer at a temperature of 29-30 ° C, separation of the culture fluid, and the cultivation of the strain of Bacillus lentus B-7150 is carried out in a carbohydrate-yeast medium based on an inorganic source of amine nitrogen with a limit on oxygen and carbon, and the isolation of a bacteriocin-like substance from a cell-free liquid is carried out using a non-polar solvent of dichloromethane or an adsorbent of the type silica gel, and from the cell surface using a polar ethanol solvent, followed by drying and combining fractions to increase the spectrum of antimicrobial activity.

The strain of Bacillus lentus isolated from an aqueous extract of wheat straw.

The strain Bacillus lentus was deposited in the State collection of microorganisms and cell cultures "GKPM-Obolensk." The strain is assigned a registration number: B-7150. Depositor: Federal State Institution of Science "State Scientific Center for Applied Microbiology and Biotechnology" of the Federal Service for Supervision of Consumer Rights Protection and Human Well-being (FBUN SSC PMB).

Cultural and morphological characteristics of the strain.

The strain Bacillus lentus GKPM-Obolensk B-7150 propagates in general nutrient media (timing agar, MPA, potato agar) at an optimal temperature (30 ± 1) ° C and pH 7 ± 2. The colonies are whitish, light cream, with a slight bulge, dense consistency, opaque with a diameter of 23 mm and an uneven edge, smell pleasantly. Gram-positive strain cells have the form of long (up to 10 μm) thin rods with a diameter of 0.50.8 μm and form short chains. With age, the length of the cells shortens (up to 2.54 microns). Ellipsoidal spores are located closer to the center in slightly inflated sporangia.

Physiological and biochemical characteristics of the strain.

The strain of Bacillus lentus ferments glucose, fructose, lactose, sucrose, mannose, mannitol, ribose, salicin, maltose, cellobiose, trehalose without gas formation. Hydrolyzes gelatin, casein, starch, esculin and glycogen. Does not hydrolyze twins (20, 40, 60, 80) and hippurate. Indole and hydrogen sulfide does not form. Reduces nitrates and nitrites. Produces catalase and proteolytic enzymes. In the process of growth, it accumulates antimicrobial substances that are released into the environment and are sorbed on the cells.

Recognition. The strain was identified as Bacillus lentus using API 50 CHB strips (Bio-Merieux, France) with% ID = 99.0; T = 0.85 (very good identification by V4.0)

Storage. The strain of Bacillus lentus support for the method of periodic reseeding on nutrient media for general purposes (timing agar, MPA, potato agar) and growing at a temperature of (30 ± 1) ° C. The number of passages is not more than 5. Long-term storage is carried out by lyophilization using a solution of lactose (7%) and polyglucin (2%) as a protective medium. To do this, for 48 h, the biomass of vegetative cells grown on the above agarized media is washed out of the cups with a lactose-polyglucin protective medium to achieve an optical density in suspension of at least 1.0 billion cells / ml according to the standard turbidity L.I. Tarasevich. A stabilized suspension of the cell mass is poured into glass vials or ampoules with a layer of up to 10 mm, frozen at minus 50 ° C and lyophilized for 20-24 hours. The vials are closed and sealed, the ampoules are sealed. Containers with lyophilized cells are stored in a household refrigerator at a temperature (4-8 ° C).

A distinctive feature of the strain of Bacillus lentus is its antagonistic activity against coliform and other gram-negative bacteria and some types of gram-positive microorganisms, which is caused by the production under certain cultivation conditions of a practically valuable metabolite - a bacteriocin-like substance (BPS) with a molecular weight according to SDS-PAAG (SDS-PAAG) ) electrophoresis for proteins of about 4 kDa. This makes it possible to use the Bacillus lentus strain as a producer of antimicrobial substances that replace the feed antibiotic, and living cells as a component of the probiotic preparation.

The accumulation of BPS by Bacillus lentus strain in practically significant quantities occurs under strictly defined conditions of deep cultivation - in a nutrient medium based on an inorganic source of amine nitrogen with an oxygen and carbohydrate limit at a temperature of 29-30 ° C for 24 hours.

The strain of Bacillus lentus and its metabolites according to the present invention is used for the following purposes.

As a producer of a bacteriocin-like substance (the conditional name is “BPS Lentocin PS1”) of a wide spectrum of antimicrobial action;

Antagonistically active cell mass, which is a waste when producing BPS as a component of a probiotic composition, as well as an additive to poultry feed;

“BPS Lentocin PS1” as an additive to poultry feed instead of antibiotics.

The invention is as follows

1. The strain Bacillus lentus is isolated from an aqueous extract of wheat straw, which is the habitat of several species of bacilli: Bacillus subtilis, Bacillus circulans, Bacillus macerans, Bacillus cereus, Paenibacillus polymyxa, etc. Selection of antagonistically active isolates is carried out by the presence of growth inhibition microorganisms, usually signs Gram-positive and gram-negative bacteria strains. It differs from these microorganisms in speed, temperature optimum of growth, morphology of cells and spores, biochemical and antagonistic properties. The size, location and shape of the spores is close to Bacillus subtilis, but differs in the greater length of vegetative cells. According to the temperature optimum of growth, it is close to Bacillus circulans, Bacillus macerans, but differs in the location and morphology of the spores. By production of antimicrobial metabolites during deep cultivation, it is close to Paenibacillus polymyxa and Bacillus circulans, but with a wider spectrum, affecting some gram-positive bacteria. The strain of Bacillus lentus grows in general-purpose broth media: timing broth, meat-peptone broth (BCH), Hottinger broth and others, however, a noticeable accumulation of the bactericidal substance occurs in relatively poor media, which contain salt instead of a source of animal nitrogen. The deep cultivation of the strain of Bacillus lentus is carried out in broth with yeast extract as a source of vitamins, with tartrate or ammonium sulfate as a source of nitrogen, with glucose or sucrose as a source of carbon and energy, as well as sodium, magnesium and phosphorus salts with a neutral pH level at at a temperature of 30 ° C, stirring for 48 hours. At the end of the cultivation, the culture fluid (QL) and its components obtained by centrifugation — the supernatant and cells, are applied in 10 μl onto freshly sown lawns of Escheri strains chia coli and Listeria monocytogenes in Petri dishes with timing agar. After 6 hours of cultivation at 37 ° C, zones of suppression of test microorganisms are formed around the applied samples: at least 10 mm from QOL and cells and at least 8 mm from cell-free supernatant.

2. The preparation of the bacteriocin-like substance “BPS Lentocin PS1” for experimental study and at the same time seed for hardware cultivation of the Bacillus lentus strain is carried out in 750 ml rocking flasks with 100 ml of the following medium (%) culture medium: yeast extract - 0.5; glucose - 0.7; potassium phosphate 2-substituted 7-water (K ₂ HPO ₄ ) - 0.3-0.5; ammonium tartrate - 0.6; sodium chloride - 0.1; magnesium sulfate - 0.01. The resulting nutrient solution was adjusted to a value of 7.2 - 7.5 pH units by the additional introduction of K ₂ HPO ₄ and sterilized by autoclaving (110 ° C, 30 min). A flask with 100 ml of medium is inoculated with 1-5 ml of a suspension of 48 hours of cell mass obtained by incubation of the strain on GRM agar, followed by washing with saline. Crops are grown in a rocking chair at a temperature of 29-30 ° C and 120-130 rpm. Growth time 24-36 hours

The culture fluid (QL) is separated by centrifugation (8000 rpm, 20 min). The supernatant is collected in a separate container, an equal in volume (1: 1) anhydrous non-polar solvent dichloromethane (methylene chloride) is added to it. The mixture is homogenized using a mixer equipped with a metal stirrer. Mixing time 15 minutes.

The resulting emulsion is poured into metal glasses and centrifuged (5000 rpm, 10 minutes). A film is formed at the phase boundary, which is easily removed from the centrifuge cup after draining by decantation of the upper aqueous and lower phases - dichloromethane (DCM).

 The figure 1 shows a microscopic image of a concentrated emulsion of the type "water in oil" in the drug "crushed drop" (Uv. 1200x. 1 - microdrops of the aqueous fraction. 2 - solvent).

To remove DCM, an interphase film containing about 10% of the aqueous fraction is placed in a glass cup and kept in a dry heat oven at a temperature of 60 ° C until the solvent smell disappears completely. The remaining precipitate is a crude concentrate of the bacteriocin-like substance “BPS Lentocin PS1”. Its indicators for antimicrobial activity are given in table 1. The figure 2 presents the results of protein SDS-PAGE electrophoresis of a BPS Bacillus lentus sample obtained from a cell-free filtrate using dichloromethane. Judging by the standard set of Page ruler tags, B lentus BPS has a molecular weight of about 4 kDa.

It can then be purified by reprecipitation in a solvent system and by liquid chromatography for subsequent study and use.

Table 1. The results of the evaluation of the antimicrobial activity of samples of BPS B lentus Impressions
tel Effect on Gy (-) bacteria, including: Effect on Gy (+) bacteria, including: E.coli MP Salm. enteriditis E.coli R3 L.monocyt. Cl.perfrin. B.anthraces Values 6400 AU * / ml 3200 AU * / ml 6400 AU * / ml 3200 AU * / ml 1600 AU * / ml 1600 AU * / ml Activity is expressed in arbitration units (AE) - the maximum dilution of a substance with a pronounced effect.

3. The preparation of the bacteriocin-like substance “BPS Lentocin PS1” for rough cleaning and the cell mass of the strain of Bacillus lentus is carried out in a 10 L fermenter (BioFlo 110 NBS, USA) with 5.0 L of culture medium according to paragraph 2. Fermentation is carried out at a temperature of 30 ° C, s aeration. The stirrer speed is automatically controlled through the CASCADE system according to the concentration of dissolved oxygen (10%). The process is carried out without pH adjustment. With strong foaming, up to 3 ml of antifoam is sterilized into the fermenter.

Table 2. Technological map of the cultivation of B.lentus strain and properties of enzymes Growth hour pH T, ° C Oboro
you, min ^-1 Aera
cts, l / min OP ₅₄₆ PO ₂ ,% AA to E.col i BK, CFU / ml 0 7.22 29.3 fifty 1,0 0.213 80.6 (10%) 0 1,5.10 ⁵ 2 7.03 30,0 263 1,0 0,303 1.3 0 2.2.10 ⁵ four 6.67 30,0 444 1,0 0.735 5,0 0 6 6.16 30,0 255 1,0 1.26 4.0 0 7.7.10 ⁶ 8 6.49 30,0 415 1,0 1.55 9.0 0 10 6.77 30,0 487 1,0 2.25 10.0 + 12 6.99 30,0 474 1,0 2.08 11.2 + 3.4.10 ⁶ fourteen 7.10 30,0 417 1,0 1,56 9.2 +

16 7.19 30,0 302 1,0 1.41 12.1 + eighteen 7.29 30,0 247 1,0 1,1 9.9 + twenty 7.35 30,0 189 1,0 0.96 9.9 + 1.5.10 ⁶ 22 7.40 30,0 135 1,0 0.98 9.8 + 24 7.42 30,0 88 1,0 0.92 9.7 + 1.4. 10 ⁶ OP - optical density; AA - antimicrobial activity against E. coli; BK - biological concentration (number of living cells).

The culture fluid is separated by ultrafiltration in an UPL-0.6 apparatus (NPK Biotest, Kirishi, RF) on hollow fibers of the AR VPU type with a cut-off by molecular weight of 15 kDa. To do this, QL is supplied to the hydraulic system of the installation, the injection pump is turned on, the working pressure (0.15 ± 0.03) MPa is set. The filtrate (permeate) in the process of membrane separation accumulates on the outside of the hollow fibers and merges into one container, and the cell mass concentrate held inside the fibers is then collected in another container. The cell mass concentrate is precipitated by centrifugation (8000 rpm), and the resulting supernatant is added to a container with filtrate for subsequent extraction of BPS.

A stabilizer solution is added to the cell mass sediment (two volume parts of the stabilizer are applied to one weight part of the precipitate: lactose - 7%, polyglucin - 2%), the sediment is resuspended, poured into containers (bottles, baking sheets) for subsequent lyophilization and use as a dry probiotic component or feed additives. Freezing is carried out in a low-temperature freezer NZ 250/75 ("Frigera", Czechoslovakia) at a temperature of (-50) ° C for 3 hours, lyophilization in a BT-4k installation ("Virtis", USA) for 20-24 hours until residual humidity 3-5%. Lyophilized biomass is collected, ground to a powder state, weighed, poured into an airtight container, from which a sample is taken to determine the number of living cells by serial dilutions with culture on nutrient agar. After the container with the lyophilizate is marked, activity data are applied, placed in the refrigerator until subsequent use.

To collect BPS by adsorption, silica gel, dried to a constant weight, is slowly added (10%, weight / volume) to the filtrate volume; for column chromatography, the particle size is 100/250 μm, mixing the liquid. The mixture was left for 1.5-2 hours to precipitate silica gel. The liquid is decanted without affecting the precipitate, and the precipitate is washed three times with distilled water. The volume of water at each washing is equal to the volume of the starting filtrate. Ethyl alcohol (or another polar solvent) is added to the precipitate washed with water for desorption (elution) of BPS from it in an amount sufficient to cover silica gel and left for at least 1 h with periodic agitation by circularly pumping the tank. The eluant is separated from the silica gel precipitate by filtration or centrifugation. To remove alcohol, the solution is dehydrated to dryness by convective or vacuum drying in a dry heat oven or on a rotary evaporator, respectively, at a temperature of 65-70 ° C. The obtained precipitate, which is a crude concentrate of BPS "Lentocin PS1", is crushed to powder, collected in an airtight container, weighed.

To control activity, 10-100 mg of powder is taken, diluted in 100-1000 μl of water. A series of successive double dilutions is prepared from the resulting solution, from which 10 μl are taken and applied to freshly planted lawns of test microorganisms. By the presence of a zone of inhibition of the growth of microorganisms around the spot of the applied sample and taking into account its dilution, the presence and value of the antimicrobial activity of a coarse sample of BPS "Lentocin PS1" are judged. If it is necessary to obtain a purified preparation, a coarse BPS sample is reprecipitated in a solvent system and liquid chromatography.

The characterized LPS sample “Lentocin PS1” and the lyophilized biomass of the strain cells can be used respectively as the basis for an antimicrobial biological product and a bacillary probiotic.

4. Obtaining BPS "Lentocin PS1" coarse purification in 10 l of fermenter (NBS mod. 511, USA) with 6.5 l of culture medium of the following composition (g / l): yeast extract - 5; glucose - 6.5 (by the final value); ammonium sulfate - 5, potassium phosphate 2-substituted 7-aqueous - 3.5; potassium chloride - 4. The medium in a volume of 6.2 l is poured into the fermenter without glucose and phosphate (pH 6.5), concentrated solutions of which are prepared separately. The phosphate solution is introduced into the medium immediately in full (pH 7.3), and the glucose solution is fractional after the fermenter is seeded. The fermenter is inoculated with 300 ml of 24 hours QL (see paragraph 1). Fermentation is carried out for 20 hours at a temperature of 28-30 ° C, stirring 120-400 rpm, aeration and maintaining the pH in the range of 6.2-7.2 using a 20% KOH solution (table 3).

Table 3. Technological map of the cultivation of B.lentus strain and properties of enzymes Growth hour pH Oboro
you, min ^-1 O ₂ , L / min OP + 20% KOH + 50% C ₆ H ₁₂ O ₆ AA to E.S. AA to L.m. BK, CFU / ml 0 7.7 120 1,0 0,045 14 ml 0 0 2.2.10 ⁶ 2 7.7 120 1,0 0,049 22 ml 2.6.10 ⁶ four 7.65 120 1,0 0,068 14 ml 0 0 6 7.45 200 1,0 0.116 0 0 1.0.10 ⁷ 8 7.2 250 1,0 0.260 0 5 10 6.7 300 1,0 0.576 0 6 12 6.8 400 1,0 1.44 17 ml 20 ml 0 6 2,3.10 ⁶ fourteen 6.7 350 1,0 4.92 ~ 24 ml 20 ml 2 6 16 6.3 / 6.6 300 <1.0 2.22 ~ 24 ml 5 6 eighteen 6.7 300 <1.0 2.27 ~ 15 ml 7 7 twenty 6.8 300 <1.0 2.17 7 7 1.7.10 ⁶ Total: ~ 100 ml 90 ml OP - optical density; AA - antimicrobial activity (⌀, mm); BK - biological concentration (number of living cells).

QOL is separated by ultrafiltration in a UPL-0.6 apparatus on hollow fibers according to paragraph 3, separately collecting the filtrate and cell mass concentrate. In order to save the reagent used further in the isolation of BPS, the filtrate is concentrated approximately 10 times on a Heidolph rotary vacuum evaporator, model Laborota 4000 (Germany), at a water bath temperature of 60-70 ° C. A suspension of cell mass is precipitated by centrifugation (8000 rpm), and the resulting supernatant is combined with a concentrated filtrate.

The combined solution of the filtrate and supernatant is treated with an equal volume of anhydrous dichloromethane (DCM) by intensively homogenizing the mixture for 15 minutes using a mixer. Figure 3 reflects the picture of the change in the appearance of the liquid in the direction of obtaining a stable emulsion (3.1 - ingredients before mixing: the filtrate is below, the solvent is above; 3.2 - the final emulsion).

The emulsion obtained is poured into metal glasses, centrifuged (5000 rpm, 10 minutes), and the interphase film (IFP) formed at the phase boundary containing the BPS aqueous fraction and the remains of DCM is collected in a glass cup or other suitable container with a developed surface. To evaporate the solvent, the container with the IFP sample is kept in a dry heat oven at a temperature of 60 ° C until the smell disappears completely. The precipitate remaining after drying is collected, crushed to a powder state, weighed and placed in a sealed container, designated as “BPS IFP”.

The sediment of the cell mass is resuspended directly in a centrifuge beaker with a small amount of water (1: 1). A cell mass suspension (KM) is treated with ethanol (1: 2) by stirring for 30 minutes. The alcohol fraction is separated from the precipitate by centrifugation. To remove alcohol, the supernatant is subjected to convective or vacuum drying in a dry heat oven or on a rotary evaporator, respectively, at a temperature of 65-70 ° C. A dry sample is collected, ground into powder, weighed and placed in an airtight container, designated as “BPS KM”.

To control the activity, 10-100 mg of the obtained BPS powders (IFP, KM) are taken, diluted in 100-1000 μl of water. A series of serial two-fold dilutions are prepared from the resulting solutions, from which 10 μl are taken and applied to freshly planted lawns of test microorganisms. By the presence of a zone of inhibition of the growth of microorganisms around the spot of the applied sample, taking into account its dilution, the antimicrobial activity of coarse samples of BPS “Lentocin PS1” obtained from QL filtrate and the cell surface of the producer strain is judged. If necessary, coarse BPS samples can be chromatographically purified.

The characterized samples serve as components of the BPS "Lentocin PS1" antimicrobial biological product.

5. The preparation of untreated Lentocin PS1 BPS and cell mass is carried out in a 10 L fermenter (NBS mod. 511, USA; or BioFlo 110 NBS, USA; or 3U, RF or similar) with 6.0 ± 1.0 L of culture medium (g / l): yeast extract - 5; glucose - 7 (by the final value); ammonium tartrate - 7, potassium phosphate 2-substituted 7-water - 3.5; sodium chloride - 1 according to paragraph 4. QOL is separated by ultrafiltration in a UPL-0.6 apparatus on hollow fibers according to paragraph 3, separately collecting the filtrate and concentrated cell mass suspension.

A suspension of cell mass is precipitated by centrifugation (8000 rpm), the resulting supernatant is added to the filtrate. A stabilizer solution is added to the cell mass sediment (two volume parts of the stabilizer are applied to one weight part of the precipitate: lactose - 7%, polyglucin - 2%), the sediment is resuspended, poured into containers (bottles, baking sheets) for subsequent lyophilization according to paragraph 3 and use as dry component of probiotic or feed supplement.

To the combined acellular fluid (filtrate and supernatant), a portion of a portion of polyvinylpyrrolidone (povidone) or sodium chloride is added in portions to a final dry solids value of 4-5% (weight: volume) with stirring until dissolved. The solution is heated to boiling or autoclaved at 0.5 atm (110 ° C, 10 min) to remove extraneous microflora, cooled and dried by spray drying at the Anhydro installation (Denmark). Drying mode: temperature at the inlet to the drying chamber 135 ± 5 ° C, at the outlet - 65 ± 5 ° C. The dry concentrate is collected in an airtight container, weighed and characterized by antimicrobial activity, which should not be lower than 10,000 AU / g according to E. coli.

The crude bacteriocin-like substance “Lentocin-PS1” obtained in this way is used as an additive to poultry feed instead of an antibiotic.

6. The use of dry cell mass of the strain B.lentus as an additive in food. The lyophilized crushed B.lentus cell mass obtained in paragraphs 2 and 4 is added to the feed of 30 day old broiler poultry in an amount of 0.1% (weight: weight), mixed thoroughly. Birds are divided into two groups - experimental and control. The experimental group was given food with a cell mass of B.lentus, the control group was given the usual food of type PK-6 in the same amount. The content of birds is outdoor for six days with a control of live weight, with periodic analysis of the microflora of fecal mass, and after slaughter, attention is drawn to the appearance of internal organs, to their norm or pathology.

The results of applying the cell mass of the B.lentus strain to feed 30-day-old broiler chickens are shown in Table 4.

Table 4. The effect of adding dry biomass of B.lentus strain to chick feed Group Oso
hit pieces Live weight of a bird, g Adding
ka by weight, g Microfloor changes
fish Organ pathology Elementary Finite Experienced 5 890 ± 12 1035 ± 65 145 Gr (+) cocci BUT* The control
naya 5 935 ± 5 1045 ± 55 110 BUT BUT* (*) BUT - not detected; Gr (+) cocci - there was a tendency to increase them

7. The use of dry untreated BPS "Lentocin PS1" strain B.lentus as an additive to feed instead of an antibiotic. 900 g BPS "Lentocin PS1" strain B.lentus was added per 1 ton of feed for broilers produced by LLC "PROVIMI" (Moscow). We used Ross-308 broiler chickens (males) from three groups: experimental, control with antibiotic (K + A) and control without antibiotic (K). There are 80 chickens in each group, the fattening period is 35 days, and weighing is weekly. After slaughter, the microflora of the blind processes was examined.

The main results on the use of BPS "Lentocin PS1" as a feed additive against the background of control options for feeding chickens are shown in table 5.

Table 5. The data of a large-scale experiment on the use of dry untreated BPS "Lentocin PS1" in the feed of Ross-308 broiler chickens Indicator Groups of chickens, indicator values Indicator changes to ... experienced control + A the control control + A control Live weight (g): - source (0) 46.3 ± 0.1 46.3 ± 0.2 46.3 ± 0.2 1,000 1,000 - for 21 days 762 ± 9.2 714 ± 9.1 693 ± 9.1 1,067 1,099 - on day 35 1808 ± 34.7 1517 ± 34.6 1504 ± 34.5 1,192 1,202 Growth (g / day): - 0-35 days 50.7 42.0 41.7 1,207 1,216 Microbes per 1 g of feces, including: - general aerobes 7.6 10 ⁶ 4.1. 10 ⁶ 5.9.10 ⁶ 1.85 1,288 - E.faecium 1.3. 10 ⁶ 3,5.10 ⁵ 4.4.10 ⁵ 3.71 2.95 - Lactobacillus spp. 1,5.10 ⁷ 1.4.10 ⁷ 4.8.10 ⁷ 1,07 0.31

Claims (2)

1. The strain Bacillus lentus is a producer of a bacteriocin-like substance having a molecular weight according to SDS-PAGE of about 4 kDa, a wide spectrum of antimicrobial activity against gram-positive and gram-negative microorganisms, deposited in the collection of microorganisms "GKPM - Obolensk" of the Federal Budget Institution of Science, State Scientific and Research Center for Applied Microbiology and biotechnology, collection number B-7150. 2. A method of obtaining a bacteriocin-like substance Bacillus, including deep cultivation of the producer, separation of the culture fluid, characterized in that the producer uses the Bacillus lentus strain "GKPM - Obolensk" B-7150, which is cultivated at a temperature of 29-30 ° C in carbohydrate-yeast medium based on an inorganic source of amine nitrogen with a limit on oxygen and carbohydrate, and the isolation of a bacteriocin-like substance from a cell-free liquid is carried out using a non-polar solvent of dichloromethane or adsorbed a type of silica, and from the cell surface - using a polar solvent of ethanol, followed by drying and combining fractions.

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