RU2375439C1 - STRAIN OF BASIDIUM FUNGUS Fomitopsis officinalis, WHICH EXPRESSES ANTIBACTERIAL ACTIVITY IN RELATION TO BACTERIA Yersinia pseudotuberculosis - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain of basidium fungus Fomitopsis officinalis KKBG 24 is described, which expresses bactericide activity in relation to bacteria Yersinia pseudotuberculosis.

EFFECT: invention may be used as base for production of biological preparations and medical preparations having antibiotic activity in relation to bacteria Y pseudotuberculosis, which are pathogenic for animals and humans.

1 dwg, 2 tbl, 3 ex

Description

The invention relates to biotechnology, is a strain of the basidiomycete fungus Fomitopsis officinalis, which can be used as the basis for biologics and drugs with antibacterial activity against pathogenic bacteria for humans and animals Yersinia pseudotuberculosis.

The basidiomycete Fomitopsis officinalis has been actively used in folk and official medicine for several millennia. It is distinguished by pronounced physiological activity, exhibits a sedative, hemostatic effect. The mushroom is best known in medicine in the countries of the Asia-Pacific region, especially in Japan (Kjosiev P.A. Complete reference book of medicinal plants. M., 2002. 992 p.).

Recently, the fungus began to be studied as a producer of biologically active substances. Carotenoids, sterols, unsaturated fatty acids, agaricin acid, bioflavonoids, B, P, E, A vitamins, and essential oils were isolated from the fruit body of F.officinalis. This will expand the range of medicinal and technical raw materials to obtain biologically active components used as the basis for the production of biological products with different biological activity (RF patent No. 2257222, A61K 35/84, publ. 2005.07.27).

It is known that different types of basidiomycetes have different biological activity. So, Trametes versicolor (L.Ex Fr.) Lloyd or multicolored tinder has antitumor and antibacterial activity, Ganoderna applanatum (Pers ex Wallr.) Pat, or flat tinder has antiviral, antibacterial and antidiabetic properties. Fomitopsis officinalis or medicinal tinder, is known as a fungus that has bactericidal activity against the causative agent of tuberculosis and in the perfume industry as an antiperspirant.

We studied the antimicrobial effect of water extracts from the mycelium of wood-destroying basidiomycetes, including Fomitopsis offlcinalis against staphylococcal bacteria of various species (Staphylococcus aureus, S.caprae, S.warneri, S.cophnii). However, the results of the study showed that Fomitopsis officinalis does not possess biological activity against staphylococcal bacteria (Kovaleva G.K., Gromovy T.I. Prospects for creating new biotechnologies based on Siberian strains of basidiomycetes // Collection of abstracts of the Russian school-conference "Genetics of microorganisms and biotechnology ". Moscow - Pushchino-on-Oka, 2006. S.135-136).

The antibacterial properties of isolates of the basidiomycete fungus Ganoderna spp. against Escherihia coli, Candida albicans and Trichophyton mentagrophytes (Smania Junior A., Smania EP, Della Monache F., Pizzolatti MG, Delia Monache G. Derivatization does not influence antimicrobial and antifungal activities of applanoxidic asids and sterols from Ganoderma spp. // Z .Naturforsch [C] .2006. 61 (1-2): 31-34).

The antibacterial properties of isolates from the fruiting bodies of 23 species of basidiomycetes against gram-negative bacteria (Escherihia coli, Pseudomonas aeruginosa) and gram-positive bacteria (Staphilociccus aureus, Bacillus subtilis, Micrococcus flavus) are known. (A1-Fatimi M., Wurster M., Kreisel H., Lindequist U. Antimicrobial Cytotoxic and antioxidant activity of selected basidiomycetes from Yemen. // Die Pharmazie. 2005 Oct; 60 (10): 776-780).

However, among these 23 species of basidiomycetes, Fomitopsis officinalis is not indicated.

Known antibacterial activity of the mycelium of the basidiomycete Laetiporus sulphureus grown deeply (in a liquid medium) against a wide range of gram-positive and gram-negative bacteria. (Ershova E.Yu., Tikhonova O.V., Lurie L.M., Efremenkova O.V., Komzolkina O.V., Dudnik Yu.V. Antimicrobial activity of strains of Laetiporus sulphureus under conditions of deep cultivation // Antibiotics and chemotherapy M., 2003. T. 48. To 1. S.18-22).

However, with respect to Yersinia pseudotuberculosis, the antibacterial activity of Laetiporus sulphureus mycelium was not determined.

The antimicrobial action of the mycelium and the culture fluid of the wood-destroying fungus Coriolus pubescens, as well as its isolates on Staphylococcus aureus, Escherihia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis (Chkhenkeli V.A. : Fr.) Quel. // Abstract of thesis ... Doctors of Biological Sciences. Irkutsk, 2006, 48 p).

The biological activity of carbon dioxide extract of the fruit body Fomitopsis officinalis is known (Sharikov AM, Oorzhak U.S., Peryanova O.V., Ushanova V.M., Neshumaev D.A. Bactericidal activity of metabolites of the fungus Fomitopsis officinalis against opportunistic bacteria // Mushrooms in natural and man-made systems. Proceedings of the international conference. St. Petersburg, 2005. T 2. S.304-307). However, with respect to Yersinia pseudotuberculosis, activity of carbon dioxide extract of the fetal body Fomitopsis officinalis was not detected.

The antibacterial activity of the cultured mycelium Fomitopsis officinalis against any bacteria, including the causative agent of Far Eastern scarlet fever (pseudotuberculosis) Yersinia pseudotuberculosis, has not been previously studied and it was unambiguously assumed that Fomitopsis officinalis would not have such activity against puberism

To date, the biological activity of Fomitopsis officinalis against bacteria Yersinia pseudotuberculosis is not known to the applicant.

The objective of the invention is to obtain funds with an effective antibacterial effect against Yersinia pseudotuberculosis, the causative agent of pseudotuberculosis in humans and animals.

The technical result of the claimed invention is the isolation of a strain having an antibacterial effect against Yersinia pseudotuberculosis from the fruit body of the basidiomycete Fomitopsis officinalis fungus.

The strain Fomitopsis officinalis was isolated by tissue from the fruiting body of the basidiomycete fungus Fomitopsis officinalis (Vill .: Fr.) Bond. et Sing (larch fungus, larch sponge), found on daur larch (Larix dahurica (Rupr.) Rupr.) in the Bastak Nature Reserve (Jewish Autonomous Region).

The strain is stored in the collection of cultures of basidiomycetes of the Biological and Soil Institute of the Far Eastern Branch of the Russian Academy of Sciences with the KKBG24 full measure.

The strain Fomitopsis officinalis KKBG 24 is characterized by the following cultural-morphological and physiological-biochemical characteristics.

Cultural and morphological signs:

On wort agar and agarized glucose-mineral medium, it forms rounded, irregularly shaped colonies with a wavy edge, with white fluffy mycelium. Radial seals extend from the center of the colony.

In a liquid glucose-mineral medium, it forms round, regular shapes, of various diameters, cream-colored colonies with a dense center and star-like outgrowths over the entire surface.

Physiological and biochemical signs:

Aerobe. The temperature range is 20-28 ° C. The optimum growth temperature is 25-26 ° C.

Optimum pH 5.5-5.8.

The strain assimilates various sources of carbon and nitrogen, but prefers media containing wort and / or glucose.

The strain is stored on an agar medium or wort agar in test tubes at a temperature of 5-6 ° C. For long-term storage, lyophilization, cryopreservation can be used.

The invention is illustrated by the following examples:

Example 1. The strain Fomitopsis officinalis KKBG 24 isolated tissue method from the fruiting body of the basidiomycete Fomitopsis officinalis. The mycelium of the strain is grown on a Petri dish by plating a piece of fruiting body on an agarized glucose-mineral medium of the following composition: glucose - 20.0 g, NH ₄ NO ₃ - 3.5 g, KCl-0.5 g, K ₂ PO ₄ - 1, 0 g, MgSO ₄ - 0.5 g, wort (15 ° Balling) - 115.0 ml, water - up to 1 l, agar - 20.0 g, pH of the medium - 5.6.

Cultivation is carried out at 25 ° C in a darkened place for 7 days. The diameter of the colony reaches 15 cm.

Example 2. The strain Fomitopsis officinalis KKBG 24 isolated tissue method from the fruiting body of the basidiomycetes Fomitopsis officinalis. The mycelium of the Fomitopsis officinalis strain KKBG 24 is grown on Petri dishes on an agarized glucose-mineral medium of the following composition: glucose - 10.0 g, NH ₄ NO ₃ - 3.5 g, KCl - 0.5 g, K ₂ PO ₄ - 1.0 g MgSO ₄ - 0.5 g, wort (15 ° Balling) - 115.0 ml, water - up to 1 l, agar - 20.0 g of pH -5.5. Cultivation is carried out at 21 ° C in a darkened place for 7 days. The diameter of the colony reaches 10 cm.

Example 3. The strain Fomitopsis officinalis KKBG 24 isolated tissue method from the fruiting body of the basidiomycete. Fomitopsis officinalis. The mycelium of the Fomitopsis officinalis strain KKBG 24 is grown on Petri dishes on an agarized glucose-mineral medium of the following composition: glucose - 30.0 g, NH ₄ NO ₃ - 3.5 g, KCl - 0.5 g, K ₂ PO ₄ - 1.0 g MgSO ₄ - 0.5 g, wort (15 ° Balling) - 115.0 ml, water - up to 1 l, agar - 20.0 g, pH of the medium - 5.8.

Cultivation is carried out at 22 ° C in a darkened place for 7 days. The diameter of the colony reaches 10 cm.

The new strain can be used for the industrial production of agents with antibacterial activity against Yersinia pseudotuberculosis, the causative agent of pseudotuberculosis in humans and animals.

The determination of the antibacterial activity of the Fomitopsis officinalis strain KKBG 24 was carried out against a number of pathogenic and conditionally pathogenic bacteria.

As test organisms, 4 strains of the following bacteria were used: Yersinia pseudotuberculosis pcs. 282, 512, 907, 3515H, Escherichia coli, Pseudomonas putida, Ps. fluorescence, Staphylococcus aureus, Listeria monocytogenes pcs. A, P, K, 10CN, Salmonella typhimurium, obtained from the Museum of the All-Russian Center for Yersiniosis and Pseudotuberculosis (Research Institute of Epidemiology and Microbiology SB RAMS, Vladivostok) and from the All-Russian State Control Institute of Veterinary Medicines (Moscow).

For analysis, the grown Fomitopsis officinalis KKBG 24 strain was taken in the form of agar blocks cut from agar medium with growing mycelium.

Determination of antibacterial activity

On a Petri dish with AGV medium (a standard medium for determining the sensitivity of microbes to antibiotics), a Yersinia pseudotuberculosis culture is seeded on a lawn onto which a standard block of seed Fomitopsis officinalis KKBG 24 (grown according to Example 1) is cut 5 mm in diameter, cut out from the growth zone of the colony of the fungal strain on wort agar. Cups are incubated in a thermostat at 37 ° C for a day. As a result, zones of lack of bacterial growth appear around the mycelial blocks, which indicates that the Fomitopsis officinalis strain KKBG 24 exhibits bactericidal (antibacterial) properties against bacteria of the species Y. pseudotuberculosis.

The experiment was carried out in four replicates. The results of bactericidal activity are shown in table 1 and shown in the drawing.

At the same time, the antibacterial activity of the Fomitopsis officinalis strain KKBG 24 against bacteria of the species Escherichia coli, Pseudomonas putida, Ps.fluorescence, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium was studied.

It was found that the F.officinalis strain KKBG 24 does not have bactericidal activity against bacteria Escherichia coli, Pseudomonas putida, Ps. fluorescence, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, but it was reliably established that in relation to different strains of Yersuberia Pseudosis F.officinalis KKBG 24 has bactericidal activity.

Table 1 Bactericidal activity of the strain Fomitopsis officinalis KKBG 24 against bacteria Yersinia pseudotuberculosis Bacterial strains The diameter of the zones of absence of bacterial growth, (mm) repetition one 2 3 four Y. pseudotuberculosis strain 282 23.0 24.0 17.0 14.0 Y. pseudotuberculosis strain 907 20,0 20,0 13.0 15.0 Y. pseudotuberculosis strain 512 20,0 18.0 14.0 14.0 Y. pseudotuberculosis strain 3515H 19.0 16,0 12.0 12.0

The results are comparable with the action of known antimicrobial agents, and in some cases the antibacterial activity of the claimed strain of F. officinalis KKBG 24 exceeds the effect of those.

table 2 The generally accepted diameters of growth retardation zones when determining the sensitivity of microorganisms to antimicrobials (AGV medium) Antimicrobial agents The diameter of the zones of sensitivity of microorganisms (mm) Ampicillin ≥14 Azlocilin ≥16 Cefazolin ≥19 Cefoperazone ≥19 Ceftazidime ≥18 Doxycycline ≥20 Gentamicin ≥16 Amikacin ≥17 Piperacillin ≥18

As can be seen from tables 1 and 2, the antibacterial activity of the claimed strain F.officinalis KKBG 24 according to example 1 is higher than the antibacterial ability of the most powerful antimicrobial drug doxycycline.

Thus, the advantage of the claimed strain F.officinalis KKBG 24 is its high antibacterial properties compared with known antimicrobials and selectivity, expressed in the active suppression of the growth of Y. pseudotuberculosis, without affecting the other microflora.

Claims (1)

The strain of the basidiomycete Fomitopsis officinalis KKBG 24, showing bactericidal activity against bacteria Yersinia pseudotuberculosis.