CS212526B1 - Method of preparation of the antibacterial and antifungicide antibiotic from the head mushroom - Google Patents

Description

It is an object of the present invention to provide a method of preparing an antibacterial and an antifungal antibiotic from a capped stalk fungus.

To date, mucidine, prepared from a submersible culture of the fungus Oudemansiella mucida (U.S. Patent 136,492), is known from antifungal antibiotics from hats.

Mucidine (Mucidermin SPOFA) is applied topically, for example in the form of a spray or an ointment. Antibacterial antibiotics, obtained by fermentation via the capillary stalk fungi, are not yet used in clinical practice.

The method of preparation of the antibacterial and antifungal antibiotic according to the invention is based on the fact that the mycelial culture of the Agrocybe aegerita (Brig.) Sing ,, stem, Ag ae I strain, is cultivated by deep cultivation at 24 ° C (- 4 °) for 14 to 24 days in a liquid nutrient medium containing assimilable carbon and nitrogen sources of mineral nutrient salts and growth agents, then the antibiotic-containing culture fluid is filtered off.

The organism that produces the novel antibiotic of the invention is Agrocybe aegerita (Brig.) Sing. (Basidiomycetes) from the order of Agaricales. It is a thermophilous wood edible fungus (M. Moser: Rohrlinge und Blatterpilze, Kleine Kryptogamenflora IIb / 2 Basidiomyzeten ”, Gustav Fischer Verlag Stuttgart, New York, 1978, p. 286) Her synonym is Pholiota cylindracea (DC ex Fr.). The appearance and occurrence of natural fruiting bodies is described by Pilate (A. Pilate: The Key to Determining Our Boletus Mushrooms, Brázda, 1951, p. 356) and Hennig (Michael Hennig: Handbuch fiir Pilzfreunde, IV. Blatterpilze-Dunkelblater, VEB Gustav Fischer Verlag Jene, 1967, p. 230).

Pure culture of Agrocybe eegerita was obtained by 1971 isolation by the explanter method from fresh fruiting bodies from nature. Ag ae I strain Agrocybe aegerita is stored in the collection of cultures of basidiomycetes of the Department of Experimental Mycology of the Institute of Experimental Medicine of the Czech Academy of Sciences, part of the Czechoslovak Collection of Organic Organism.

The mycelium is maintained on the 8 ° Ball sweetener ager. in test tubes. Mycelial colony is cotton wool, dense, yellow-white, without pigmentation into nutrient egar. The hyphae fibers are 3 to 5 Aun thick, with no vegetative spores. Growth rate is moderate (most mycelium fungi grow slowly), Petri dish of 0 cm diameter, inoculated with dicaryotic culture, will grow with mycelium within 10 days. Sexuality of this kind is governed by a tetrapolar incompatibility mechanism (Esser and Kuenen: Genetic der Pilze. Springer Verlag Berlin-Heidelberg-New York, 1967). The dicaryotic culture grows a little faster than the monocaryotic and has buckles on the hyphae, characteristic of most stalk fungus cultures.

The monocaryotic culture (from 1 spore) is macroscopically similar to the dicaryotic, but has a simple septum on the hyphae. Agrocybe aegerita meets the prerequisites for breeding because it forms a fruiting body under suitable conditions in the laboratory, has a well-sprouted basidiospore and a relatively short development cycle (Esser, Semerdzhieva, Downloaded: Genetische Untersuchungen an dem Basidiomyzeten Agrocybe aegerita, Theoret. Appl. (1974). The mycelium grown by the deep cultivation method on a shaking machine in liquid soil in a flask is the ball of that, later the balls disintegrate. Submerged culture growth is slower than that of penicillia or actinomycetes.

The broth must contain a source of assimilable carbon and organic or inorganic nitrogen, mineral salts, traces of metals and growth agents. From complex soils, the fungus grows best on sweeteners with a concentration of 8 ° Ball. at an initial pH of 5.5-7.0, from synthetic soils on MS-soil (glucose 30 g, lutamic acid 2.5 g, glycine 0.1 g, ammonium tartrate 0.5 g, potassium phosphate primary 1.0 g, crystalline magnesium sulphate 0,5 g, calcium chloride 0,25 g, 1 ml of trace element solution Fe, Zn, B, Mn, Cu, Na, KJ, aneurin 100, µg, distilled water 1000 ml, pH 5, 8).

The antibiotic produced by the submersible culture of said stalk fungus has not yet been isolated in a chemically pure state. The antibiotic contained in the culture filtrate is extracellular, water-soluble. It is effective against both gram-positive and gram-negative bacteria and fungicides. Table 1 shows the antibiotic effect of an antibiotic of the invention (contained in the culture filtrate) on some pathogenic and conditional pathogenic microorganisms.

Table 1

Antibiotic effect of Agrocybe aegerita culture filtrate on some pathogenic a conditionally pathogenic microorganisms type pathogenic microorganism number sledov. pathos. strains antibiotic effect (zone diameter in mm) determined by diffusion titration (5 mm hot plate) dripped 0.1 ml of filtrate bactericidal bal íteriostaticl gram + Pneumococcus 15 Dec 8 to 28 coca Staphyloooccus aureus 7 8 to 10 Streptococcus faecalis 23 8 to 13 Streptocoocus hemolyt. AND 8 , 0 to 15 15 Dec to 30 Streptococcus hemolyt. (B) 16 12 to 26 19 Dec to 34 gram - Aeromonas 2 16 to 26 sticks Enterobacter agglomeratus 1 12 Enterobacter cloaceae 2 8 to 12 Enterobacter hafniae 2 8 to 10

continued table 1 yeast type

pathogenic microorganism number sledov. patog. strains antibiotic effect (zone diameter in mm) determined by diffusion titration (5 mm plate stack) drip 0.1 ml of filtrate bactericidal bacteriostatic Escherichia coli 18 8 to 8 10 to 36 Klebsiella pneumoniae 2 8 to 10 Proteus mirabilis Próteus morgani Próteus rettgeri , 0 1 2 11 to 26 12 to 25 10 Proteus vulgaris Providentia Pseudomonas aeruginosa 6 1 5 16 8 to 20 10 to 18 Salmonella 2 8 to 12 Serratia Yersinia 1 2 30 14 to 20 Candida albicans 8 fungicidní 8 to 12 Candida pseudotropicalis 1 4 18 8 to 1 0

From tab. Figure 1 shows that Gram-positive cocci are most effective against β haemolytic streptococci (inhibition zones up to 34 mm), Gram-negative cocci against Aeromonas, Escherichia coli, Proteus mirabilis and P. rettgeri and Serratia (Inhibition zones 25 to 36 mm); on the Candida yeast, the antibiotic has a fungicidal effect.

The antibiotic activity of the culture filtrate (lyophilisate) was determined by plate diffusion microbiological titration. It was not found in any part of Agrocybe aegerita. Stationary cultures show weak activity against gram-positive bacteria.

The following is an example of a method of preparing an antibiotic of the invention.

Example 1

An Erlenmeyer flask (and 300 ml) containing 100 ml of liquid 8 ° Ball was placed in a rotary shaker. sweets (pH 5.5). The flasks were inoculated with 10 to 20 wires of well grown Ag and e I (20 days) mycelial culture from the sloping agar in a test tube and sealed with a cotton plug. After 20 days of shaking, the culture filtrate was separated from the mycelium and tested by plate titration for antibacterial and antifungal activity with Baccillus subtilis, Escherichia coli and Candida pseudotropicalis strains using streptomycin and fungieidine as a reference standard. The activity effect (0.1 ml filtrate sample) corresponded to an antibacterial activity of about 100 µl streptomycin and an antifungal activity of about 500 µl fungieidine. Dry mycelium weight 22 mg / ml.

Example 2

Cultivation on a rotary shaker as in Example 1, but on synthetic soil (MS-soil: its composition indicated earlier). The culture grows slightly weaker (maximum 15 mg / ml dry weight of mycelium) and slower (maximum antibiotic production after 28 days of culture). The culture filtrate also exhibits antibiotic activity against gram-positive, gram-negative bacteria and yeast. The concentration of the antibiotic is slightly lower than that of Example 1.

Figure 1 is a graphical representation of antibiotic activity on Bacillus subtilis, Escherichia coli, and Candida pseudotropicalis microorganisms over 28 days of submersive culture of the starting strain Ag and e I on an 8 ° Ball. sweet soil. The figure shows, for example, that the greatest antibacterial effect against Escherichia coli is after 24 days of culture of the parent strain, where the diameter of the inhibition zone is about 24 mm. The curves in the figure show:

- ^α —– 'gram + bacteria (Bacillus subtilis) —δ — ú— gram - bacteria (Escherichia coli) —o — o- yeast (Candida pseudotropicelis)

--------- dry mycelium weight

Claims (1)

SUBJECT OF THE INVENTION A method for the preparation of an antibacterial and antifungal antibiotic from a capillary stalk fungus characterized in that the mycelial culture of the capillary stalk fungus Agrocy be aegerita, strain Ag and e I, is cultivated by a deep cultivation method at 24 ° C ± 4 ° on a sweetened liquid medium at pH 5.5 to 7, for a period of 4 to 24 days, after which the culture liquid containing the antibiotic is filtered off.