RU2627164C1 - Bacterium strains lactobacillus rhamnosus, with a wide spectrum of antagonistic activity relating to pathogenic and conditionally pathogenic microorganisms - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Lactobacillus rhamnoses 1 ds, which has antagonistic activity against pathogenic and opportunistic microorganisms, including against yeast fungi of the genus Candida, deposited in the All-Russian Collection of Industrial Microorganisms under the registration number VKPM V-12597 and can be used in the production of probiotic bacterial preparations, biologically active food additives, fermented and unfermented food products.

EFFECT: improved strain properties.

1 dwg, 7 tbl, 2 ex

Description

The invention relates to biotechnology, food and medical industry, and can be used in the manufacture of preparations containing live bacteria, biologically active food additives, fermented milk and fermented non-fermented foods.

Currently, an urgent and important task is the development of new antimicrobial preparations of probiotics containing new, non-toxic, bioavailable strains of microorganisms that can be used in the form of mono- and mixed cultures as part of probiotic preparations and food products. Probiotic cultures differ in spectrum and in strength, adhesiveness, sensitivity to antimicrobials, resistance to bile, digestive enzymes, growth characteristics on nutrient media, the ability to ferment milk, etc.

When treating various acute intestinal infections and with dysbiosis of various etiologies, the probiotic preparation Lactobacterin, based on the Lactobacillus plantarum 8A-P3 strain, is widely used [Zh. microbiol. 2004. No1. P. 84-92.] However, in the treatment of acute intestinal diseases, this drug is not effective enough.

A number of probiotic strains of Lactobacillus salivarius are known (RU 2302458). These strains are isolated from resected and washed human gastrointestinal tract and have antagonism with L.innocua, L.fermentum KLD, P. fluorescens, E. coli V 157. The strains are characterized by sensitivity to a number of antibacterial drugs (ampicillin, amoxacillin, ceftaxime, ceftriaxone, ciprofloxacin, cefradine, rifampicin, chloramphenicol), resistant to physiological concentrations of hydrochloric acid and bile, have the ability to adhere to gastrointestinal epithelial cells. However, the spectrum of their antagonistic activity does not include yeast-like fungi of the genus Candida, which is relevant today.

The objective of the invention is the expansion of the range of strains with antagonistic activity against pathogenic and opportunistic microorganisms.

The problem is solved in that a Lactobacillus fermentum strain of 11 stars is obtained, characterized by high antagonistic activity against pathogenic and conditionally pathogenic microorganisms, including against yeast fungi of the genus Candida, as well as the ability to adhere to epithelial cells of the oral mucosa .

The 11 star strain Lactobacillus fermentum was isolated from the oral fluid of a man aged 38 years. Tver, who was clinically healthy at the time of the examination, did not have a history of infectious and somatic diseases of the gastrointestinal tract and other organ systems. The strain was obtained in October 2007 by isolating a pure culture as a result of the cultivation of the test material and plating on optimal nutrient media (MRS agar, lactoagar, MRS broth). The cultivation was carried out without the use of genetic modification methods at a temperature of 37 ° C for 24 hours in a desiccator with a candle at a high content of CO ₂ on the basis of the bacteriological laboratory of the Department of Microbiology and Virology with an immunology course at the SBEI HPE of the Tver State Medical University of the Russian Ministry of Health.

The resulting strain was deposited in the All-Russian collection of industrial microorganisms of the Federal State Unitary Enterprise GosNIIgenetika (Moscow, 1st Dorozhny pr., 1) and has a registration number VKPM B-12597.

The resulting strain is characterized by the following features.

Cultural and morphological characters.

Under microscopy, gram-positive non-spore-forming rods are short and long with rounded ends, some are arranged in a chain, 2-5 microns in size. Cell sizes vary with growth on various nutrient media in the range of 0.5-1.2 × 1.0-10 microns.

When growing on MRS agar (Difco ™ Lactobacilli MRS Agar, BD) and lactoagar (State Scientific Center for Applied Microbiology and Biotechnology Obolensk) at 37 ± 1 ° C under anaerobic conditions using a microaerostat and GasPak + system, in a desiccator with a candle at increased content of CO ₂ , and aerobically for 24-48 hours, S-shaped colonies of 1-2 mm, convex, smooth, light yellow or white with smooth edges.

When grown on MRS broth (Difco ™ Lactobacilli MRS Broth, BD) at a temperature of 37 ± 1 ° C for 24-48 hours under aerobic conditions, growth is in the form of turbidity of the broth and a white-gray precipitate at the bottom.

Composition of MRS Agar (Difco ™ Lactobacilli MRS Asar, BD)

Approximate Formula Per Liter

Proteose Peptone No. 3 10,0 g Beef extract 10,0 g Yeast extract 5.0 g Dextrose 20,0 g Polysorbate 80 1,0 g Ammonium citrate 2.0 g Sodium acetate 5.0 g Magnesium Sulfate 0,1g Manganese sulfate 0.05 g Dipotassium phosphate 2.0 g Agar 15.0 g

Composition of MRS broth (Difco ™ Lactobacilli MRS Broth, BD):

Approximate Formula Per Liter

Proteose Peptone No. 3 10,0 g Beef extract 10,0 g Yeast extract 5.0 g Dextrose 20,0 g Polysorbate 80 1,0 g Ammonium citrate 2.0 g Sodium acetate 5.0 g Magnesium Sulfate 0,1g Manganese sulfate 0.05 g Dipotassium phosphate 2.0 g Distilled water 1000,0 ml

On protein media (MPA, MPB, milk), there is no growth (sometimes very poor development is observed). Starch does not hydrolyze. Gelatin does not thin.

Physiological and biochemical characteristics

Mesophile, growth is possible in the range of 20-40 ° C, the optimum temperature for growth is 37 ± 1 ° C.

Optional anaerobic. In case of deep fermentation on a liquid nutrient medium, the optimal aeration-mixing mode is 1 volume of air / 1 volume of the nutrient medium, which corresponds to a sulfite number of 1.

Optimal for growth pH = 6.5 ± 0.2.

Attitude to carbohydrate sources.

Grows well on: Glycerol + (48 h), D-Arabinose + (48 h), L-Arabinose + (24 h), D-Ribose +, D-Xylose + (48 h), D-Galactose +, D-Glucose +, D-Fructose +, D-Mannose +, L-Sorbose + (48 h), L-Rhamnose +, Inositol + (48 h), D-Mannotol +, D-Sorbitol +, Methyl-bD-Mannopyranoside + (48 h), Methyl-bD-Glucopyranoside +, N-AcetylGlucosamine +, Amygdaline +, Arbutine +, Esculine citrate Fe +, Salicine +, D-Cellobiose +, D-Maltose +, D-Lactose +, D-Melibiose +, D-Saccharose +, D-Trehalose +, Inuline + (48 h), D-Melezitose +, D-Ridonose +, Gentiobiose +, D-Turanose +, D-Lyxose + (48 h), D-Tgatose +, K Gluconate +.

He grows on: Erythritol -, L-Xylose -, D-Adonitol -, Methyl-in D-Xylopyranoside -, Dulcitol -, Glycogene -, Xylitol -, D-Fucose -, L-Fucose -, D-Arabitol L-Arabitol - K 2-Cetogluconate K 5-Cetogluconate -.

From alcohols, it metabolizes: D-sorbitol, D-mannitol, glycyrin, inositol.

Of the alcohols does not absorb: erythritol, D-adonitol, dulcitol, xylitol, D and L-arabitol.

Cleaves: K-gluconate.

Does not break down: K 2-ketogluconate, K 5-ketogluconate, glycogen

Does not absorb citrate, cellulose, ethanol, butanol.

Guard milk after 12-14 hours.

The studied strain is phenotypically apatogenic, i.e. it does not have hemolytic, proteolytic, leticinase, hyaluronidase, antilizocymal, DNA and RNA activity.

Antagonistic activity.

The study of antagonistic activity was carried out by the method described in (Blinkova L.P. Obtaining the tomicide of a new bacterial preparation and the study of its biological activity. Abstract of thesis. Doctor of Biological Sciences. M., - 1986. - P. 25) on the test strains of Staphylococcus aureus ATCC 25923, Candida albicans ATCC 885-653, Pseudomonas aeruginosa ATCC 27853, Shigella sonnei I phase 941, Bacillus subtillis 534, Escherichia coli 25922. Klebsiella pneumoniae K ₁ 5054, through S. 24 typhim hours of incubation at 37 ° C in a thermostat according to the size of the zone of lack of growth of the test culture. The degree of antagonism was determined by the following criteria: 10 mm or less - low, 10-20 mm - medium, 21 mm or more - high.

It was shown that the studied strain inhibits the growth of pathogenic and conditionally pathogenic microorganisms: S.aureus, B.subtilis, E.coli, Sh.sonnei I phases, P.aeruginosa, Klebsiella pneumoniae, S. typhimurium 415 and yeast fungi C.albicans ( table 1).

Attitude to antibacterial drugs

The ratio of the 11-star Lactobacillus fermentum strain to antimicrobial agents (qualitatively) was studied in accordance with the methods recommended by the Research Center for Pharmacotherapy (NICF)

www.nicf.spb.ru

Studies have shown that the claimed strain is sensitive to benzylpenicillin, ampicillin, vancomycin, gentamicin, streptomycin, erythromycin, tetracycline, doxycycline, rifampicin, levomycetin, meropenem, linezolid, intermediate sensitivity to furadonin, and leviculinucin ciprofrof.

The 11 star Lactobacillus fermentum culture is stored for a long time in cryo-tags on trypticase soy broth (TSB) (TSB: Difco Laboratories, Detroit, Mich.) With 15% glycerol in a freezer at -80 ° C for 5 years or -20 ° C up to 3-6 months, and also in lyophilized form for more than 10 years.

The invention is illustrated by the following examples.

Example 1. The cultivation of a strain of Lactobacillus fermentum 11 stars in the laboratory.

The 11 star strain Lactobacillus fermentum was grown in flasks on liquid MRS broth (Difco ™ Lactobacilli MRS Broth) at a temperature of 36 ± 1 ° C for 24-48 hours under aerobic conditions. The volume of the nutrient medium is 500 ml. Sowing a loop of one isolated colony. After 24 and 48 hours there is an increase in the form of turbidity of the broth and the precipitation of a white-gray precipitate at the bottom. To determine the number of viable lactobacilli, the grown bacteria are triturated on an isotonic sodium chloride solution in steps of 10 and then plated from different dilutions of 0.3 ml per dense MRS or lactoagar medium. After 24 hours of cultivation at 36 ± 1 ° C under aerobic conditions, the colonies are counted and the number of viable microorganisms - colony forming units (CFU) in 1 ml of liquid nutrient medium (MRS broth) is calculated. As a result of the determination, it was found that after 24 hours of cultivation, the number of lactobacilli grown was 4⋅10 ⁸ -1.7⋅10 ⁹ CFU / ml. After 48 cultivations, 1.3-2⋅10 ⁹ CFU / ml.

Example 2. The study of the properties of the strain Lactobacillus fermentum 11 stars.

Acid-forming ability.

The activity of acid formation is determined by the titrometric method (Liannaya AM, Goncharova GI, Vysotsky V.V. Morphology of bifidobacteria at the light-optical and electron-microscopic level and their biological properties // Microbiology. - 1979. No. 6. - P. 54-58. ) Measurements are carried out for 72 hours using culture media without glucose and with the addition of glucose and sampling the culture fluid every 24 hours. The result is expressed in degrees Turner (T ° is a value expressing the amount of 0.1 N alkali, which went to the titration of 100 ml of the test sample).

The results obtained (Table 3) show that during the first day there was an acidification of the cultivation medium without glucose (acidity 14 ° T). Subsequently, a gradual increase in acidity occurs, which amounted to 46 ° T by 2 days and 54 ° T by 3 days. After glucose was added to the medium as a nutrient substrate, the following data were obtained: by the end of the first day, the acidity was 36 ° T, by the end of the second day the acidity significantly increased to 88 ° T, and by the third day to 102 ° T.

The ability to adhesion.

The degree of adhesion of microorganisms is determined using the average adhesion index (SPA) according to the method of V. Brilis. (Brilis V.I., Brilene T.A., Lenzner H.P. et al. Method for Studying the Adhesive Process // Lab. Business. - 1986. - No. 4. - P. 210-212) on human red blood cells O (I ) of the group Rh +.

Studies show that the adhesion degree of the strain of Lactobacillus fermentum 11 sv is 5.72 ± 2.21, which is a high indicator.

The ability to autoaggregate.

Autoaggregation of lactobacilli is determined by the method of A. Andreu et al. (Andreu A., Stapleton, A., Fennell, C., Hillier, S., and Stamm, E. Hemagglutination, adherence and surface properties of vaginal Lactobacillus species. J. Infect. Dis - 1995 - 171: 1237-1243) . Autoaggregation is assessed as the ability to form clusters (aggregates) within 2 minutes. Studies show that the Lactobacillus fermentum strain of 11 stars has a high rate of autoaggregation.

Ability to surface hydrophobicity

The surface hydrophobicity of lactobacilli is studied by the salt aggregation test (TCA) (Andreu A., Stapleton, A., Fennell, C., Hillier, S., and Stamm, E. Hemagglutination, adherence and surface properties of vaginal Lactobacillus species. J. Infect . Dis - 1995 - 171: 1237-1243). Studies show that the Lactobacillus fermentum strain of 11 stars has a high surface hydrophobicity (TCA <0.9 mol / L).

The ability to coaggregate.

The ability of lactobacilli to coaggregate is determined by G. Reid et al. (Reid G., Mac Groarty, J., Chow, A., Bruce, A., Eisen, A., and Costerton, W. Coaggregation of urogenital bacteria in vitro and in vivo. Curr. Microbiol. 1990, 20: 47 -52) The following pathogenic and opportunistic strains of Staphylococcus aureus 209, Candida albicans 531, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25922 are used as test cultures for this technique; Salmonella typhimurium 11, Shigella flexneri 26, Bacillus subtilis 534. The coaggregation test is considered positive if the studied culture forms aggregates with another type of microorganism. Studies show that the strain L.fermentum 11 stars. coaggregates only with Bacillus subtilis.

Sensitivity to hydrochloric acid and bile.

The sensitivity of the strain to hydrochloric acid and bile, which largely determines its ability to persistence in the digestive tract, is determined by the change in the optical density (OD) of the daily broth culture, depending on the concentration of hydrochloric acid or bile (Casey PG, Casey GD, Gardiner GE et al . (2004) Isolation and characterization of anti-Salmonella lactic acid bacteria from the porcine gastrointestinal tract. Lett Appl Microbiol 39: 431-438). Measurements are carried out on a KFK-3 spectrophotometer at a wavelength of 590 μm or 600 μm, respectively. As a control, a pure daily broth culture is used.

The results show that the minimum inhibitory concentration (MIC) of hydrochloric acid is 0.95% (Table 4), and bile in various concentrations (Table 5) inhibits the growth of the broth culture of the 11 star Lactobacillus fermentum strain at a bile concentration of 20%.

Determination of gas signaling molecules.

The analysis of gas signal molecules is carried out by gas chromatography using a Crystallux 400M gas chromatograph equipped with a thermal conductivity detector and a flame ionization detector connected in series, which provides simultaneous analysis of combustible and non-combustible components. Separation of the gas mixture is carried out on a three-meter column filled with polymer MN270, fractions 100-125 microns. The analysis is carried out in temperature programming mode for 25 minutes: 40 ° C (4 minutes) - 12 ° C (up to 250 ° C) - 250 ° C (3 minutes). Pure gases were used as a reference for the calibration curves. The sample volume of 1 ml, all concentrations were recalculated from the current analysis conditions to standard conditions. It was found that Lactobacillus fermentum 11 stars emits in a small amount of CO ₂ and CO, the production of methane, propane and ethane was not determined (Table 6).

Ammonia production was determined by the photometric method using the Nessler reagent according to GOST 33045-2014. Quantitative chemical analysis of water. Method for measuring the mass concentration of ammonium, potassium, sodium, lithium, magnesium, strontium, barium and calcium cations in drinking, natural (including mineral) and waste water samples by capillary electrophoresis using the “Drops” capillary electrophoresis system. PND F 14.1: 2: 4.167-2000. Moscow 2000. Measurements were performed on the basis of the Tver Oblast Hygiene and Epidemiology Center using the KAPEL 105 apparatus. When calculating the results, the concentration of ammonia in the control sterile MPC broth was taken into account. The difference in the broth with and without lactobacilli is presented in the table (table. 7).

The results show that Lactobacillus fermentum 11 stars emits ammonia, and during repeated measurements its amount remained practically unchanged.

The claimed solution is illustrated by the following figures.

FIG. 1 - sensitivity of the strain Lactobacillus fermentum 11 stars to hydrochloric acid.

Thus, the claimed strain of Lactobacillus fermentum 11 star In KPM B-12597, has a high antagonistic activity against pathogenic and conditionally pathogenic microorganisms: S.aureus ATCC 25923, B.subtilis 534, E.coli ATCC 25922, Sh.sonnei I phase 941, P. aeruginosa ATCC 27853, Klebsiella pneumoniae K ₁ 5054, S. typhimurium 415, and yeast fungi C. albicans ATCC 885-653. Acid production, expressed in degrees Turner, ranges from 14 to 56 ° T in a medium without glucose and from 36 to 102 ° T when glucose is added to a nutrient medium. L. fermentum 11 stars is sensitive to benzylpenicillin, ampicillin, vancomycin, gentamicin, streptomycin, erythromycin, tetracycline, doxycycline, rifampicin, levomycin, meropenem, linezolid. The studied strain is phenotypically apatogenic, i.e. It does not have hemolytic, proteolytic, leticinase, hyaluronidase, antilysocyme, DNA and RNA activity. L. fermentum 11 stars has high adhesion (5.72 ± 2.21) and surface hydrophobicity (<0.9 mol / L), high autoaggregation and coaggregation (coaggregates only with Bacillus subtilis). The minimum inhibitory concentration of hydrochloric acid for L. fermentum 11 stars is 0.95%. Bile only in a concentration of 20% inhibits the reproduction and growth of the broth culture of the strain Lactobacillus fermentum 11 star The production of gas signaling molecules was revealed: ammonia, carbon monoxide and carbon dioxide.

The strain is high-tech, on artificial nutrient media quickly accumulates biomass with a high concentration of lactobacilli.

All this allows us to offer a strain of Lactobacillus fermentum 11 star VKPM B-12597 for use in the production of probiotic bacterial preparations, biologically active food additives, fermented milk fermented and non-fermented foods, starter cultures providing a probiotic effect and normalization of human body microbiocenoses, including gastrointestinal and genitourinary tracts, skin and mucous membranes.

Claims (1)

The bacterial strain Lactobacillus fermentum VKPM B-12597, which has a wide spectrum of antagonistic activity against pathogenic and conditionally pathogenic microorganisms.

Images