Lactobacillus rhamnosus bacteria strain having wide range of antagonistic activity with respect to pathogens and opportunistic pathogens

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Obtained is a Lactobacillus rhamnosus 38k bacteria strain, having antagonistic activity with respect to pathogens and opportunistic pathogens, including Candida yeast fungi, average adhesion and self-aggregation properties, high surface hydrophobicity, average coaggregation, is deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under registration number VKPM V11155.

EFFECT: strain can be used in production of probiotic bacterial preparations, biologically active food additives, fermented milk and non-fermented food products.

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

At present, the urgent and important task is the development of new antimicrobial drugs probiotics containing new, non-toxic, bioavailable strains of microorganisms which can be in the form of mono - and mixed cultures to use in the composition of probiotic preparations and food products. Probiotic cultures differ in range and potency, adhesion, sensitivity to antimicrobial agents, resistance to the action of bile, digestive enzymes, growth on nutrient media, the ability to skachivat milk etc.

When different aftercare of acute intestinal infections and with the dysbacteriosis of various etiologies widely used probiotic preparation Lactobacterin, based on the strain Lactobacillus plantarum 8A-P3 [Ukr. microbiol. 2004. No. 1. P.84-92] However, in the treatment of acute intestinal diseases, this drug is not effective enough.

Known for a number of probiotic strains Lactobacillus salivarius (EN 2302458). These strains were isolated from resected and washed the digestive tract is the ne and are antagonistic towards L.innocua, L.fermentum KLD, P.fluorescens, E.coli V 157. Strains are characterized by their sensitivity to a number of antimicrobial agents (ampicillin, amoxicillin, cefixim, Ceftriaxone, ciprofloxacin, cefradine, rifampicin, chloramphenicol), resistant to physiological concentrations of hydrochloric acid and bile, have the ability to adhesives to the epithelial cells of the gastrointestinal tract. However, the range of their antagonistic activity does not include yeast-like fungi of the genus Candida, which is relevant for today.

Object of the invention is to widen the range of strains with antagonistic activity.

The problem is solved by the fact that the resulting strain Lactobacillus rhamnosus C with high antagonistic activity against pathogenic and conditionally pathogenic microorganisms, including in relation to yeast Candida.

Lactobacillus rhamnosus C isolated from faeces of healthy young men aged 17 years, who at the time of examination was clinically healthy, had no history of infectious and somatic diseases of the gastrointestinal tract and other organ systems, by separating the pure culture by rascaroli investigated material and sowing at the optimum nutrient medium (MRS agar, lacto-agar MRS broth). The cultivation was carried out without the use of genetic methods option is the ratification at 37°C for 24 hours in a desiccator with a candle with a higher content of CO 2on the basis of the bacteriological laboratory of the Department of Microbiology, Virology and immunology GBOU VPO Tver state medical Academy of the Ministry of health and social development of Russia.

The resulting strain deposited in Russian national collection of industrial microorganisms FSUE gosniigenetika, Moscow (1st Road Ave, 1) and has the registration number VKPM B-11155.

The resulting strain is characterized by the following features.

Cultural morphological traits

When using the microscope, the cells are fixed, large sticks of the correct form with rounded ends, single, surrounded by a capsule. In young culture there are cells in pairs.

The size of the cells varies during growth on different nutrient media in the range of 0.5 to 1.2×1.0 to 10 μm. Gram-positive.

When grown on MRS agar (Difco™ Lactobacilli MRS Agar) and lacto-agar (fsri State research center for applied Microbiology and biotechnology goblins) at 37±1°C under anaerobic conditions using microbiostatic and GasPak system+in a desiccator with a candle with a higher content of CO2and aerobic mode within 24-48 hours of the colony S-shape of 1-2 mm, convex, smooth, white with smooth edges.

When grown in MRS broth (Difco™ Lactobacilli MRS Broth) at a temperature of 37±1°C for 24-48 hours in aerobic conditions, the increase in turbidity Boo is gone and loss of white-gray sediment at the bottom.

On protein environments (MPA, MPB, milk) no growth (sometimes there is very little development). Starch is not hydrolyses. The gelatin liquefies.

Physiological and biochemical characteristics

The species is mesophilous, growth is possible in the range of 20-40°C, the optimal growth temperature of 37±1°C.

Optional gone anaerobic. In submerged fermentation liquid nutrient medium optimum aeration-mixing 1 volume of air/volume 1 nutrient medium, which corresponds to sulfite number 1.

For optimal growth pH=6,5±0,2.

The sources of carbohydrates.

Grows well in: D-glucose, D-sucrose, D-fructose, D-maltose, D-galactose, D-ribose, D-mannose, L-rhamnose, D-lactose, D-cellobiose, N-acetylglucosamine, methyl-D-glycopyranoside, citrate esculin iron, salicin, arbutin, amygdalin, D-trehalose, D-melezitose, gentiobiose, D-turanose, D-tagatose.

Not growing on: methyl-D-mannopyranoside, D - and L-xylose, D-arabinose, L-arabinose, methyl-D-xylopyranoside, L-sorbose, D-melibiose, inulin, D-raffinose, amidone, D-lyxose, D - and L-fucose, D - and L-arabitol.

From alcohols learns: D-sorbitol, D-mannitol, glycerin, Inositol, lactate.

Of the alcohols is not absorbed: aritra, D-adonitol, dulcitol, xylitol.

Splits: K-gluconate.

Not splits: 2-ketogluconic, 5-ketogluconic, glycogen.

Not absorbed citrate, cellulose, ethanol, butanol.

curtailing milk milk 12-14 hours.

The investigated strain phenotypic pathogeny, i.e. does not have hemolytic, proteolytic, medicinskoi, hyaluronidases, the antilysocyme, DNA-Noah and RNA-tion activity.

Antagonistic activity

Study the antagonistic activity was performed by the method described in (Blinkova L.P. Getting tomized new bacterial preparation and study of its biological activity. Abstract. dis. Dr. Biol. Sciences. M., 1986. S) on the test strains Staphylococcus aureus was ATSS 25923, Candida albicans was ATSS 885-653, Pseudomonas aeruginosa was ATSS 27853, Shigella sonnei phase I 941, Bacillus subtillis 534, Escherichia coli 25922. Klebsiella pneumoniae K15054. Analysis was performed 24-48 hours incubation at 37°C in thermostat largest zone of no growth of the test cultures. The degree of antagonism was determined by the following criteria: 10 mm or less is low, 10-20 mm - 21 mm - high.

It is shown that the investigated strain inhibits the growth of pathogenic and conditionally pathogenic microorganisms: S.aureus, B.subtilis, E.coli, Sh.sonnei phase I, P.aeruginosa, Klebsiella pneumoniae and yeast-like fungi .albicans (table 1).

The ratio of strain L.rhamnosus C to antimicrobial agents (kachestvennno) was studied in accordance with the methods recommended by the research centre for pharmacotherapy (NICF) www.nicf.spb.ru

Studies have shown that the claimed strain has sensitivity to ampicillin, streptomycin is, tetracycline, doxycycline, rifampin, Meropenem, linezolid, as well as intermediate sensitivity to erythromycin (table 2).

Lactobacillus rhamnosus C permanently stored in cryovials at trypticase-soy broth (TSB) (TSB: Difco Laboratories, Detroit, Mich.) with 15% glycerol in a freezer at a temperature of -80°C to 5 years or -20°C up to 3-6 months, and in dried form for more than 10 years. The claimed solution is illustrated by the following figures.

Figure 1 the sensitivity of the strain Lactobacillus rhamnosus K for the hydrochloric acid.

Figure 2 sensitivity of the strain Lactobacillus rhamnosus C to bile.

The invention is illustrated by the following examples.

Example 1. Cultivation of Lactobacillus rhamnosus C in the laboratory.

Lactobacillus rhamnosus C grown in flasks in liquid nutrient medium MRS broth (Difco™ Lactobacilli MRS Broth) at a temperature of 36±1°C for 24-48 hours under aerobic conditions. The volume of nutrient medium 500 ml Inoculation loop of single isolated colony. After 24 and 48 hours of growth in the form of turbidity of the broth and the loss of white-gray sediment at the bottom. To determine the number of viable lactobacilli are restframe grown bacteria on istoricheskom solution of sodium chloride in increments of 10, and successive planting of different dilutions of 0.3 ml in a dense medium MRS or lactogen. After 24 hours of cultivation at 36±1°C in AE the conditions hold for one count colonies and calculate the number of viable microorganisms colony forming units (CFU) in 1 ml of liquid nutrient medium (MRS broth). By understanding it is established that after 24 hours of cultivation, the number of grown lactobacilli was 4·108-1,7·109CFU/ml After 48 cultivation - 1,3-2·109CFU/ml

Example 2. The study of the properties of Lactobacillus rhamnosus C.

Acid-forming capability.

The activity of acid generating define out by a titrimetric method (Lanna A.M., Goncharova GI, Vysotsky CENTURIES Morphology of bifidobacteria in optical and electron microscopic level and their biological properties // Microbiology. - 1979. No. 6. - P.54-58). The measurements were carried out within 72 hours, using a nutrient medium without glucose and with the addition of glucose and taking samples of the culture fluid every 24 hours. The results are expressed in degrees Turner (T°) is a value expressing the amount of 0.1 N alkali, followed by titration of 100 ml of sample).

The results obtained (table 3) show that during the first day was the acidification of the environment of cultivation without glucose (pH 30°T). In the subsequent gradual increase in acidity, which amounted to 2 days 34°T to 3 days 40°T. After adding on Wednesday glucose as a nutrient substrate following data were obtained: by the end of the first day of the acidity with the set of 30°T, by the end of the second day the acidity increased to 34°T, to the third day to 36°T.

The ability to adhesion

The degree of adhesion of microorganisms determined using an average adhesion (SPA) according to the method of Briles VI (Briles VI, Brylane T.A., Lenzner HP and other techniques for studying adhesion process // lab. case. - 1986. No. 4. - S-212) on human erythrocytes On (I) the group is Rh+.

Studies show that the degree of adhesion of strain L.rhamnosus C is 2.5±1.3, which is the average of.

The ability to autoaggregate

The self-aggregation of lactobacilli define method A.Andreu et al. (Andreu, A., Stapleton, A., Fennell, C., Hillier, S., and E. Stamm Hemagglutination, adherence and surface properties of vaginal Lactobacillus species. J. Infect. Dis. - 1995. - 171: 1237-1243). The self-aggregation is assessed as the ability to form clusters (aggregates) for 2 minutes. Studies show that the strain L.rhamnosus C has the highest rate of autoaggregation.

The ability to surface hydrophobicity

Surface hydrophobicity of lactobacilli study test salt aggregation (TCA) (Andreu, A., Stapleton, A., Fennell, S., Hillier, S., and E. Stamm Hemagglutination, adherence and surface properties of vaginal Lactobacillus species. J. Infect. Dis. - 1995. - 171: 1237-1243). Studies show that the strain L.rhamnosus C has an average surface hydrophobicity (TCA 1.5 mol/l).

The ability to coaggregation

The ability of lactobacilli to coaggregation determined by the method of G. Reid et al. (eid G., Mac Groarty J., Chow, A., Bruce, A., Eisen, A., and W. Costerton Coaggregation of urogenital bacteria in vitro and in vivo. Curr. Environ. 1990, 20: 47-52). As test crops for this method use the following pathogenic and conditionally-pathogenic strains of Staphylococcus aureus 209, Candida albicans 531, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25922; Salmonella typhimurium 11, Shigella flexneri 26, Bacillus subtilis 534. Test coaggregation is considered positive if the studied culture forms aggregates with other species of microorganisms. Studies show that the strain L.rhamnosus C corregiruet with Staphylococcus aureus, Escherichia coli, Candida albicans, Pseudomonas aeruginosa, Bacillus subtilis.

Sensitivity to hydrochloric acid and bile

The strain sensitivity for the hydrochloric acid and bile, which largely determines its ability to persistently in the digestive tract, determined by the change in optical density (OD) 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 Environ 39: 431-438). The measurements were carried out on a spectrophotometer KLF-3 at a wavelength of 590 μm or 600 μm, respectively. As a control, use clean daily broth culture.

The results show that the minimum inhibitory concentration (MIC) of hydrochloric acid is 1.25% (table 4 and figure 1), and bile in different concentrations (table 5, figure 2) not only inhibits, kostimulirovat reproduction and growth in broth culture of strain L.rhamnosus K, the most pronounced effect does the concentration of bile 10%.

Thus, the claimed strain L.rhamnosus C VKPM B-11155 high antagonistic activity against pathogenic and conditionally pathogenic microorganisms: S.aureus was ATSS 25923, B.subtilis 534, E.coli was ATSS 25922, Sh.sonnei phase I 941, P.aeruginosa ADS 27853, Klebsiella pneumoniae K15054, and yeast-like fungi C.albicans ADS 885-653. Kislotoproduccia, expressed in degrees Turner, ranges from 30 to 40°T in medium without glucose and from 30 to 36°T adding glucose to the culture medium. L.rhamnosus C has sensitivity to ampicillin, streptomycin, erythromycin, tetracycline, doxycycline, rifampin, Meropenem, linezolid. The investigated strain phenotypic pathogeny, i.e. does not have hemolytic, proteolytic, medicinskoi, hyaluronidases, the antilysocyme, DNA-Noah and RNA-tion activity. L.rhamnosus C has an average adhesion (2,5±1,3) and surface hydrophobicity (1.5 mol/l), high performance auto-aggregated and coaggregation (coaggregation with Staphylococcus aureus, Escherichia coli, Candida albicans, Pseudomonas aeruginosa, Bacillus subtilis). Minimum inhibitory concentration of hydrochloric acid for L.rhamnosus C is 1.25%. Bile in various concentrations stimulates reproduction and growth in broth culture of strain L.rhamnosus C.

The strain of high-tech artificial Pete is a recreational environments quickly accumulates biomass with a high concentration of lactobacilli

All this allows us to offer strain L.rhamnosus C VKPM B-11155. for use in the production of probiotic bacterial preparations, biologically active additives to food, fermented milk fermented and non-fermented food starters, providing probiotic effect and normalization of microbiocenosis of the human body, including the gastrointestinal and urogenital tracts, skin and mucous coats.

Relation to antibacterial drugs

Table 2
The sensitivity of the strain Lactobacillus rhamnosus C to antibiotics
№ p/pName antibioticThe degree of sensitivity
1.Penicillin
2.AmpicillinH
3.Vancomycin
4.Gentamicin
5.StreptomycinH
6.ErythromycinP
7.TetracyclineH
8.DoxycyclineH
9.Ciprofloxacin
10.Levofloxacin
11.RifampicinH
12.Chloramphenicol
13.Furadonin
14.MeropenemH
15.LinezolidH
Note: H - sensitive, N - intermediate, U - stable.

Table 3
Kislotoproduktsiya the ability of Lactobacillus rhamnosus C
Strain1 day, T°2 nights) (T°3 day, T°
L.rhamnosus Cwithout adding glucose
303440
with the addition of glucose
303436

Table 4
The sensitivity of the strain Lactobacillus rhamnosus K for the hydrochloric acid
The concentration of HCL, %ControlMICK
52,51,25of 0.6250,31250,15625
OP0,0090,012 0,0110,0290,0250,0790,0951,25

Table 5
The sensitivity of the strain Lactobacillus rhamnosus C to bile
The concentration of bile, %Control
201052,51,25of 0.625
OP0,1960,4960,297is 0.1020,40,0910,092

The bacterial strain Lactobacillus rhamnosus VKPM B-11155 with a broad spectrum of antagonistic activity against pathogenic and conditionally pathogenic microorganisms.



 

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