RU2482177C1 - Lactobacillus plantarum BACTERIA STRAIN HAVING WIDE RANGE OF ANTAGONISTIC ACTIVITY WITH RESPECT TO PATHOGENS AND OPPORTUNISTIC PATHOGENS - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and biotechnology and can be used in production of probiotic bacterial preparations, biologically active food additives, fermented milk and non-fermented food products. The Lactobacillus plantarum VKPM V-11154 strain has high antagonistic activity with respect to pathogens and opportunistic pathogens, including Candida yeast fungi. The strain is phenotypically apathogenic, has average adhesion and self-aggregation properties, high surface hydrophobicity, average coaggregation; hydrochloric acid has minimum inhibiting concentration of 1.25%; the bile stimulates propagation and growth of the culture.

EFFECT: invention enables faster accumulation of biomass with high concentration of lactic bacteria on artificial nutrient media.

2 dwg, 5 tbl

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 [Journal. 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 (RU 2302458) are known. 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 nomenclature of strains with antagonistic activity.

The problem is solved by the fact that the obtained strain of Lactobacillus plantarum 36k, characterized by high antagonistic activity against pathogenic and opportunistic microorganisms, including against yeast of the genus Candida.

The strain Lactobacillus plantarum 36k was isolated from the feces of a healthy girl at the age of 20, 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, by isolating a pure culture by planting the test material and plating on optimal nutrient media (MRS agar, lacto-agar, 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, Virology and Immunology, GBOU VPO Tver State Medical Academy of the Ministry of Health and Social Development of Russia.

The resulting strain was deposited in the All-Russian collection of industrial microorganisms FSUE GosNIIgenetika (Moscow, 1st Dorozhny pr., 1) and has a registration number VKPM B-11154.

The resulting strain is characterized by the following features:

Cultural and morphological features

During microscopy, the cells are fixed, short and long large sticks of regular shape with rounded ends, single, surrounded by a capsule. In young culture, cells are found in pairs, some are arranged in a chain.

Cell sizes vary with growth on various nutrient media in the range of 0.5-1.2 × 1.0-10 microns. Gram-positive.

When growing on MRS agar (Difco ™ Lactobacilli MRS Agar) and lacto-agar (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 CO ₂ content, and aerobically for 24-48 hours, S-shaped colonies of 1-2 mm, convex, smooth, gray-yellow or white with smooth edges.

When grown on MRS broth (Difco ™ Lactobacilli MRS Broth) 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.

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 nutrient medium, which corresponds to a sulfite number of 1.

Optimal for pH growth of 6.5 ± 0.2.

Attitude towards carbohydrate sources

It grows well on: D-melibiosis, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-sucrose, D-maltose, D-lactose, D-cellobiose, N-acetylglucosamine, methyl - D-mannopyranoside, esculin citrate, iron, salicium, arbutin, amygdalin, D-trehalase, D-melesitosis, gentiobiosis, D-raffinose.

Does not grow on: methyl - D-glycopyranoside, L-ramnose, D- and L-xylose, D-arabinose, L-arabinose, methyl-D-xylopyranoside, L-sorbose, inulin, amidone, D-lyxose, D- and L-fucose, D-turanose, D- and L-arabitole, D-tgatose.

From alcohols, it metabolizes: glycyrin, D-mannitol, lactate.

Of the alcohols does not absorb: D-sorbitol, erythritol, D-adonitol, dulcitol, inositol, xylitol.

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, antilysocyme, 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. Dr. 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. C in the 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 phase I, P.aeruginosa, Klebsiella pneumoniae and yeast-like fungi C.albicans (Table 1).

Attitude to antibacterial drugs

The ratio of the strain Lactobacillus plantarum 36k to antimicrobial agents (qualitatively) was carried out 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 ampicillin, gentamicin, streptomycin, tetracycline, rifampicin, meropenem, linezolid, as well as intermediate sensitivity to erythromycia, doxycycline, furadonin (table 2).

The Lactobacillus plantarum 36k culture is stored for a long time in cryovials in trypticase-soy broth (TSB) (TSB: Difco Laboratories, Detroit, Mich.) With 15% glycerol in a freezer at -80 ° C for 5 years or -20 ° C to 3- 6 months, and also in a lyophilized form for more than 10 years.

The claimed solution is illustrated by the following figures:

Figure 1 The sensitivity of the strain Lactobacillus plantarum 36k to hydrochloric acid.

Figure 2 The sensitivity of the strain Lactobacillus plantarum 36k to bile.

The invention is illustrated by the following examples:

Example 1. Cultivation of a strain of Lactobacillus plantarum 36k in the laboratory

The strain Lactobacillus plantarum 36k is 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 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 with an isatonic sodium chloride solution in steps of 10 and subsequent plating 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 2.7 · 10 ⁸ -5 · 10 ⁸ CFU / ml. After 48 cultivations, 5 · 10 ⁸ -1.3 · 10 ⁹ CFU / ml.

Example 2. The study of the properties of the strain Lactobacillus plantarum 36k

Acid-forming ability.

The activity of acid formation is determined by the titrometric method (Liannaya AM, Goncharova GI, Vysotsky VV Morphology of bifidobacteria at the optical and electron microscopic level and their biological properties // Microbiology. - 1979. No. 6. - P.54-58. ) The measurements are carried out for 72 hours, using nutrient-free 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 the 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, the cultivation medium was acidified without glucose (acidity 32 ° T). Subsequently, there was a gradual increase in acidity, which amounted to 36 ° T by 2 days and 38 ° 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, acidification of the medium was observed (acidity - 32 ° T), by the second day, acidity increased to 34 ° T, by the third day - to 40 ° T.

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 degree of adhesion of the strain L.plantarum 36k is 2.3 ± 1.2, which is an average.

Ability to autoaggregate

Autoaggregation of lactobacilli is determined by 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 strain L.plantarum 36k has an average 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 strain L.plantarum 36k has a high surface hydrophobicity (TCA 0.8 mol / l).

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. plantarum 36k coagulates with Staphylococcus aureus, Escherichia coli, Candida albicans, 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. Pure daily broth culture was used as a control.

The results show that the minimum inhibitory concentration (MIC) of hydrochloric acid is 1.25% (Table 4 and Figure 1), and bile in various concentrations (Table 5, Figure 2) stimulates the propagation and growth of the broth culture of strain L. plantarum 36k.

Thus, the inventive strain L.plantarum 36k VKPM B-11154 is characterized by high antagonistic activity against pathogenic and opportunistic 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, and yeast-like fungi C. albicans ATCC 885-653. Acid production, expressed in degrees Turner, ranges from 32 to 38 ° T in a medium without glucose and from 32 to 40 ° T when glucose is added to a nutrient medium. L.plantarum 36k is sensitive to ampicillin, gentamicin, streptomycin, tetracycline, rifampicin, meropenem, linezolid, as well as intermediate sensitivity to erythromycin, doxycycline, furadonin. The studied strain is phenotypically apatogenic, i.e. It does not have hemolytic, proteolytic, leticinase, hyaluronidase, antilysocyme, DNA and RNA activity. L. plantarum 36к has average adhesion (2.3 ± 1.2) and autoaggregation, high surface hydrophobicity (0.8 mol / L) and coaggregation (coaggregates with Staphylococcus aureus, Escherichia coli, Candida albicans, Bacillus subtilis). The minimum inhibitory concentration of hydrochloric acid for L.plantarum 36k is 1.25%. Bile in various concentrations stimulates the reproduction and growth of the broth culture of strain L.plantarum 36k.

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

All this allows us to offer the strain L.plantarum 36k VKPM B-11154 for use in the production of probiotic bacterial preparations, biologically active food additives, fermented milk and fermented non-fermented foods, starter cultures providing a probiotic effect and normalization of human microbiocenoses, including gastrointestinal and genitourinary tracts, skin and mucous membranes.

Table 1 The spectrum of antagonistic activity of the strain Lactobacillus plantarum 36k Zones of growth inhibition of test cultures in mm S.aureus ATCC 25923 B.subtili s534 E.coli ATCC 25922 Sh.sonnei I phase 941 P.aeruginosa ATCC 27853 Klebsiella pneumoniae K ₁ 5054 C.albicans ATCC 885-653 thirty 24 38 27 40 22 25 table 2 The sensitivity of the strain Lactobacillus plantarum 36k to antibacterial drugs. No. p / p Name of antibiotic Degree of sensitivity one. Benzylpenicillin At 2. Ampicillin H 3. Vancomycin At four. Gentamicin H 5. Streptomycin H 6. Erythromycin P 7. Tetracycline H 8. Doxycycline P 9. Ciprofloxacin At 10. Levofloxacin At eleven. Rifampicin H 12. Chloramphenicol At 13. Furadonin P fourteen. Meropenem H fifteen. Linezolid H * note: H - sensitive, P - intermediate, U - stable Table 3 Acid-producing ability of the strain Lactobacillus plantarum 36k Strain 1 day T ° 2 days T ° 3 days T ° L.plantarum 36k no glucose added 32 36 38 with glucose 32 34 40

Table 4 The sensitivity of the strain Lactobacillus plantarum 36k to hydrochloric acid The concentration of HCl,% The control MIC 5 2,5 1.25 0.625 0.3125 0.15625 OP 0.011 0.009 0.014 0,038 0,031 0.1 0.127 1.25

Table 5 The sensitivity of the strain Lactobacillus plantarum 36k to bile The concentration of bile,% The control twenty 10 5 2,5 7.25 0.625 OP 0.253 0.605 0.506 0.475 0.103 0,092 0,087

Claims (1)

The bacterial strain Lactobacillus plantarum VKPM B-11154, which has a wide spectrum of antagonistic activity against pathogenic and conditionally pathogenic microorganisms.

Images