PL242259B1 - Composition and method for preparing a composition for use in the treatment of bee foulbrood - Google Patents

Abstract

The subject of the application is a composition for the treatment of bee foulbrood, a brood disease of the honeybee Apis mellifera caused by spore-forming bacteria Paenibacillus larvae, prepared on the basis of a dry extract in the form of a powder obtained from the fruiting bodies of the polyporous fungus Tyromyces fissilis, which is mixed directly with water in a proportion of 1 :20. For the treatment of bee foulbrood, it is used after mixing with sugar syrup in the proportion of 1:10 or prophylactically in the proportion of 1:20.

Description

Description of the invention

The subject of the invention is a composition and a method for obtaining a composition containing a dry methanol extract in the form of a powder from the fruiting bodies of the polyporoidal fungus Tyromyces fissilis for use in the treatment of bee foulbrood, a brood disease of the honey bee Apis mellifera.

Malignant foulbrood (American Foulbrood AFB) is one of the most dangerous diseases of bees in the world. It is caused by Gram-positive bacteria Paenibacillus larvae, which produce spores in the form of endospores with exceptional resistance. They belong to the group of aerobic (aerobic) bacteria, but they are also able to grow in anaerobic conditions (relative anaerobes). Paenibacillus larvae infect and kill larvae and pupae (bee brood), while adult bees are immune to them. Bees act as carriers of endospores, which they feed to the larvae. The development of the disease and death can be caused by the penetration of only thirty-five bacterial spores into the body of the larva, and only ten spores are enough to infect the youngest larvae. As a result, foulbroodbrood is one of the most contagious diseases known to man. The endospores of P. larvae show a unique viability. In ambient conditions, they remain active for over 35 years, and the temperature of 100 ° C kills them only after 5 days. The habitat of infection is the remains of dead foulbrood larvae, each of which contains many millions of endospores. Worker bees cleaning the comb cells from the remnants of dead larvae spread endospores around the hive and contaminate food - honey and bee bread. Laboratory measurement of the amount of P. larvae spores in 1 g of honey is used to diagnose the possibility of occurrence of foulbrood in a bee colony. This number can be relatively high and amount to as much as 15,000 spores in 1 g. Spores are a potential source of infection, which is especially revealed in unfavorable living conditions for bees, when weakened larvae begin to die. The severity of the disease occurs in the summer, usually in the second half of summer.

In Poland, bee foulbrood is a disease combated ex officio. The scale of the potential threat is evidenced by the results of 5-year epidemiological studies (Skubida et al., 2014), which found a significant spread of P. larvae bacteria in 38% of the 4090 apiaries examined. The worst situation was found in the Małopolskie and Warmińsko-Mazurskie voivodeships: 71 and 58% of infected apiaries, respectively, in the remaining voivodships this rate ranged from 25 to 48%. The above test results are confirmed by the data of the Chief Veterinary Inspector https://www.wetgiw.gov.pl/publikacje/biuletyn-stan-chorob-zakaznych-zwierzat. From year to year, a systematic increase in foulbrood outbreaks can be observed. And so in 2017 there were 137, in 2018-170, and in 2019 already 260 (GIW). It is significant that in 2020 the first four outbreaks of foulbrood were found in March (GIW). This means that P. larvae bacteria were present in the winter food in large numbers, and the extremely unfavorable weather conditions in the early spring of 2020 led to the onset of clinical symptoms of the disease.

In most countries, the fight against foulbrood involves the destruction of infected bee colonies by sulphurizing and burning the bees along with the contents of the hive and associated equipment. It is possible to thermally decontaminate hives and equipment with a flame (Buczek, 2011). In Poland, it is allowed to treat sick families by resettlement to a disinfected hive under the supervision of a veterinarian. It is mandatory to destroy all old brood combs and hive equipment (see Beekeeping, No. 5, 2011). For a long period of time, antibiotics (tetracycline, virginiamycin, flavomycin, erythromycin and others) or sulfonamides were commonly used to treat sick bee colonies with American foulbrood. However, the side effects of particularly long-term use of antibiotics and polysulfamides for bees and humans (contaminated bee products) led to the ban on their use in the European Union (Regulation EEC 2377/90 with subsequent amendments).

Currently, there are no pharmacological agents approved for the treatment of patients with American foulbrood in bee colonies that could replace the banned antibiotics and polysulfamides. Attempts to breed bee lines resistant to foulbrood also did not give satisfactory results. Meanwhile, the above-mentioned data from the General Inspector of Inspection clearly show that the threat of this disease of bees is constantly growing. This is due to the overlapping of a number of unfavorable factors. These include problems with varroosis, viral diseases, a new variety of nosemosis - nosema ceranae, or disorders of the bee colony caused by the massive use of chemicals, such as neonicotinoids. The problem related to the threat of malignant foulbrood to bee colonies is global and affects all countries where bees are kept.

Substances of natural origin are sought in the treatment of bee foulbrood. Essential oils from various plants and propolis were tested. Most of these substances have relatively weak activity against P. larvae. The value of the minimum concentration inhibiting the development of P. larvae bacteria (MIC) ranges from 250 to 400 μg/mL), however, even at these concentrations, they are toxic to bees, e.g. thymol. Some of the authors of this application are also the authors of the patent application number P.422063 of June 29, 2017, in which a non-polar extract (hexane or obtained as a result of extraction with supercritical carbon dioxide) from thin birch twigs was used to treat bee foulbrood. Own research allowed to optimize the composition of the composition obtained on the basis of a non-polar extract from thin birch twigs and determine its minimum MIC inhibitory concentration at the level of 1.562 mg/ml. Verification of the effectiveness of the above composition directly in bee colonies allowed to conclude that it is well tolerated by adult bees and can be used especially for prophylaxis or supportive treatment after resettlement of bee colonies. Unfortunately, research conducted at the National Veterinary Institute in the Laboratory of Bee Diseases showed that at therapeutic doses (above the MIC) the composition prepared on the basis of extracts from thin birch twigs is toxic to bee larvae.

The subject of the invention is a composition and a method for obtaining a composition containing a dry methanol extract in the form of a powder from the fruiting bodies of the polyporoidal fungus Tyromyces fissilis for use in the treatment of bee foulbrood, a brood disease of the honey bee Apis mellifera.

The essence of the invention is a composition for use in the treatment of bee foulbrood, a brood disease of the honey bee Apis mellifera caused by spore-forming bacteria Paenibacillus larvae. It contains a dry methanol extract in the form of a powder obtained from the fruiting bodies of the polyporous fungus Tyromyces fissilis, mixed with water in a ratio of 1:20.

The composition is used in the treatment of bee foulbrood, honey bee brood disease Apis mellifera caused by spore-forming bacteria Paenibacillus larvae when mixed with sugar syrup in a ratio of 1:10, and in the prevention of bee foulbrood when mixed with sugar syrup in a ratio of 1:20.

According to the subsequent stages of preparation of the composition: the fruiting bodies of Tyromyces fissilis are crushed, flooded with methanol, macerated and mixed, then filtered to separate the extract from the macerate, and then evaporated from methanol in a vacuum evaporator at a temperature below 50°C and pressure p < 20 kPa (200 mbar), and the dry extract thus obtained is mixed with water.

Research initiated in 2017 and related to the search for substances of natural origin drew the team's attention to polyporoid (arboreal) fungi. At that time, quite accidentally conducted research showed that some mushroom extracts have a much stronger bactericidal effect than extracts from birch twigs. In 2019, extensive research was carried out on the antibacterial properties of extracts obtained from arboreal fungi, consisting of three stages.

The first stage consisted in verifying the qualitative antibacterial activity of fungal preparations (compositions) against Paenibacillus larvae bacteria representing two genotypes, i.e. Eric I and Eric II (two reference strains and four wild strains) at different concentrations of extracts. These were typical screening tests (screening) of 125 extracts obtained from polyporoidal fungi collected at the Forest Department in Hajnówka. These extracts were dry powders obtained after evaporation of methanol from mushroom macerates in a vacuum dryer. In the initial phase of the research, it was shown that preparations in water solutions at a concentration of 5% showed the highest activity. At higher extract concentrations, there was no significant increase in the bacterial inactivation radius. Therefore, a 5% concentration of the dry formulation in aqueous solutions was used in all studies. The qualitative reading of the antibacterial activity against P. larvae was carried out analogously to the reading of the antibiograms. The diameters of the obtained circles of inhibited growth of P. larvae were measured using a ruler in millimetres. The measurement was carried out in three repetitions, from which the average value was taken, provided that the difference was not more than 2 mm. Where the measurements differed by more than 2 mm

PL 242259 Β1 the test was repeated to verify the result. The result of this stage of research was the indication of fungal species whose extracts have a bactericidal effect against P. Iarvae. Out of 125 mushroom extracts, 6 were selected that showed a particularly strong bactericidal effect against P. Iarvae. Table 1 presents the results of the studies described above for 6 selected species of polyporoid fungi.

Table 1. Antibacterial activity of selected 6 fungal preparations with the highest activity against P. larvae strains (qualitative test).

IN no Composition number and fungus species with common and Latin names The trophy of inhibition of P. hundred for different strains of atvae [mm] ERIC1 ER1C II P. larvac LMG 9820 P. Iarvae WILD 1 P.larvat WILD 5 P. CCUG 48973 larva P. larvat WILD 3 P. Iarvat WILD 7 1. Pine bullfinch Hcterohasidion annosum 20 21 19 22 23 21 2. G36 Tyromyces fissilis nasty ragweed 20 18 19 21 21 20 3. G40 Oligoporus guttulatus 20 20 19 21 20 20 4. G52 Sparassis crispa pine tree 13 13 16 14 14 15 5. G73 Purple earthworm Chondrostercum purpurcum 10 10 <10 14 13 15 6. G124 Larch officinalis Fomitopsis officinalis 15 <10 12 17 17 16

In the second stage, the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of extracts from six selected species of fungi were determined, which are presented in Table 2. The results of the MIC and MBC tests show that the concentration values differ significantly for different species of fungi and different strains of P. Iarvae. The reference strains P. Iarvae LMG 9820 (Erie I) and CCUG 48973 (Erie II) turned out to be the most resistant to the tested compositions. The strongest bactericidal effect was shown by the composition prepared on the basis of the extract marked with the symbol G36 and obtained from the fruiting bodies of Tyromyces fissilis, and it was selected for the third stage of the research. The research described above was carried out at the Applied Microbiology Laboratory of the University of Bialystok.

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Table 2. Qualitative results of antibacterial activity of six selected fungal preparations against P. larvae strains

b no Composition number and fungus species with common and Latin names Bracing type Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values against P. larvac (pg/mL) ERIC I ERIC II P. Iarvac LMG 9820 P.larvae WILD 1 P. larvac WILD 5 P. larvac CCUG 48973 P. Iarvae WILD 3 P. larvae WILD 7 1. G11 Pine rootstock Hcterobasidion annosum MIC 4882.81 76.29 76.29 4.77 1.19 1.19 MBC 19531.25 305.17 305.17 19.07 4,769 4,769 2. G36 Golden Porcupine Tyromyces fissilis MIC 305.18 1.19 0.30 4.77 1.19 0.30 MBC 1220.7 4.77 1.192 19.07 4.77 1.19 3. G40 Oligoporus guttulatus MIC 19531.25 4882.81 4882.81 305.18 19.07 0.30 MBC 78125.0 19531.25 19531.25 1220.7 76.29 1.19 4. G52 Sparassis crispa pine tree MIC doesn't work 4882.81 4882.81 19531.25 19531.25 4882.81 MBC doesn't work 19531.25 19531.25 78125.0 78125.0 19531.25 5. G73 Chondrosterc purpureum MIC doesn't work 78125 78125 78125 78125 78125 MBC doesn't work 312500.0 312500.0 312500.0 312500.0 312500.0 6. G124 larch officinalis. Fomitopsis officinalis MIC MBC doesn't work nothing works 5000 doesn't work 5000 nothing works 5000 doesn't work 5000 nothing works 5000 nothing works

b

The research related to stage III was carried out at the Department of Bee Diseases of the National Veterinary Institute in Puławy. It consisted in in vivo verification of the effect of a selected preparation - a composition prepared on the basis of a methanol extract from Tyromyces fissilis (a 5% water solution of a dry methanol extract) on bee larvae infected with P. Iarvae spores of Erie I and Erie II genotypes. The main objective of the study was to evaluate the bactericidal properties of the preparation prepared on the basis of a mushroom extract in vivo conditions, i.e. after administration to bee larvae infected with Paenibacillus larvae bacteria.

The research was carried out based on the guidelines of The COLOSS BEEBOOK, volume I "Standard methods for Apis mellifera research", chapters: "Standard methods for artificial rearing of Apis mellifera larvae"; “Standard methods for toxicology research in Apis mellifera”; volume II, "Standard methods for Apis mellifera pest and pathogen research", chapter "Standard methods for American foulbrood research" and research procedures of the Department of Bee Diseases NIV-PIB. One-day-old larvae of Apis mellifera carnica bees from clinically healthy bee colonies stationed in the PIWet-NRI apiary in Puławy were used for laboratory tests. To obtain day-old larvae, four days before the start of each series of laboratory tests, in a selected colony, the queen bee was placed on an empty comb in a single-frame insulator. After the patch was turned red by the queen and the three-day period of the egg stage had elapsed, the patches with one-day-old larvae were transferred to the laboratory for rearing under laboratory conditions.

The evaluation of the antibacterial effect of the fungal preparation was carried out on bee larvae infected with field strains of P. Iarvae of the ERIC I and ERIC II genotypes found in domestic apiaries. To determine the dose of P. Iarvae bacteria, with which the larvae were infected, a series of ten-fold dilutions of the initial suspension of ERIC I and ERIC II bacteria (from ^10'1 to 10 ^-7 ) was prepared and inoculated onto solid growth media. Based on the number of bacterial colonies obtained in Petri dishes, the titre of P. Iarvae bacteria of the ERIC I and ERIC II genotypes in the initial suspensions was calculated. The ERIC I inoculum used to infect the larvae contained 3.0 χ 10 ⁵ spores/ml, and the ERIC II inoculum contained 9.0 χ 10 ⁵ spores/ml. Day-old bee larvae were infected with a single dose of the inoculum added to the first portion of the royal jelly. Infected bee larvae

PL 242259 Β1 with bacteria of ERIC I genotype consumed 1.0 x 10 ³ spores/larva with food, while bee larvae infected with bacteria of ERIC II genotype consumed 2.0 χ 10 ³ spores/larva.

In order to achieve the aim of the study, experimental groups of 30 bee larvae were created. The test was performed in 3 repetitions:

• group A1 - bee larvae infected with P. Iarvae bacteria, ERIC I genotype, fed with royal jelly without the addition of a fungal composition (untreated);

• group A2 - bee larvae infected with P. Iarvae, genotype ERIC I fed with the addition of 0.025% of the fungal composition;

• group A3 - bee larvae infected with P. Iarvae, genotype ERIC I fed with 0.05% with the addition of a fungal composition;

• group B1 - bee larvae infected with P. Iarvae bacteria, genotype ERIC II, fed with royal jelly without the addition of a fungal composition (untreated);

• group B2 - bee larvae infected with P. Iarvae, genotype ERIC II, fed with 0.025% of the fungal composition;

• group B3 - bee larvae infected with P. Iarvae, genotype ERIC II, fed with the addition of 0.05% of the fungal composition;

• group K1 - bee larvae not infected with P. Iarvae, fed with royal jelly without the addition of a mushroom composition;

• group K2 - bee larvae not infected with P. Iarvae, fed with the addition of 0.025% of the mushroom composition;

• group K3 - bee larvae not infected with P. Iarvae, fed with the addition of 0.05% of the mushroom composition.

Observations of the larvae (development of infection, mortality) were carried out daily throughout the duration of each test series (11 days). The condition of each larva was assessed individually using a stereoscopic microscope.

The results of the effect of the mushroom extract on the development of P.larvae infection and the survival of bee larvae obtained during the study (three repetitions in total) are presented in Table 3. number of larvae tested (n=90).

Table 3. Results of studies on the effect of the fungal composition in invivo conditions in the form of the percentage of mortality of larvae infected with P. Iarvae spores of the Erie I and Erie Π genotypes.

Experience day A1 infected with ERKI, untreated A2 infected with ERIC I, treated with 0.025% extract A3 infected with ERIC 1, treated with 0.05% extract B1 infected with ERIC II, untreated B2 infected with ERIC II, treated with 0.025% extract B3 infected with ERIC II, treated with 0.05% extract KI uninfected, fed milk without extract K2 uninfected, fed with 0.025% extract K3 uninfected fed with 0.05% extract Feeding 1 0 .... 0 0 0 0 0 0 2 0 , 0 0 0 0 0 0 5 3 0 7 0 13 13 0 15 '7 4 10 thirty 43 35 33 19 5 15 7 5 42 80 65B 97 67 33 5 15 10 6 42 80 80 100 67 50 5 15 15 7 58 80 90 100 67 50 10 25 16 8 61 90 100 100 67 50 10 27 16 9 74 90 100 100 67 50 10 27 18 10 100 93 100 100 67 50 10 28 18 11 100 97 100 100 67 50 10 28 18

The lowest mortality of bee larvae was found in the control group (K1) of larvae not infected with P. larvae bacteria, fed with royal jelly without the addition of a mushroom preparation, in which the pupal stage reached 90% of larvae. In groups of uninfected larvae fed milk with the addition of mushroom extract (K2, K3), mortality was slightly higher, and the pupal stage was reached by 72% of larvae fed with food containing 0.025% extract and 82% of larvae fed with food containing 0.05% extract. Although in the groups of larvae receiving the addition of the fungal preparation, a lower survival of the larvae was found, it is difficult to state unequivocally that it was the result of the toxic effect of the preparation on the larvae. These doubts are raised by the higher survival rate of larvae in the group of larvae fed milk with a higher concentration of mushroom extract.

The course of infection with P. larvae, assessed on the basis of observations of larvae infected with ERIC I and ERIC, but not treated with the mushroom extract (groups A1, B1), was consistent with the development of American foulbrood described by the World Organization for Animal Health (OIE). In the group infected with the ERIC I strain, most of the larvae died before reaching the prepupal stage (61%), and the rest before pupae. In the case of larvae infected with the ERIC II strain, characterized by greater virulence, all larvae died on the fourth and fifth day after infection, i.e. during the uncapped brood phase in vivo.

Administration of ERIC I infected larvae (groups A2, A3) with a fungal preparation at a concentration of 0.025% and 0.05% did not inhibit the death of larvae resulting from the development of infection. The death of the larvae was even much faster compared to the infected but untreated larvae (A1). Although in the group of larvae treated with the mushroom extract at a concentration of 0.025%, 3% of the larvae that reached the pupal stage did not develop infection, but in the group treated with the 0.05% extract, the mortality of the larvae was 100%, as in the group of untreated larvae. However, 3% of the larvae infected with the lethal dose of P. larvae survived infection with the Eric I genotype. It can be concluded that the administered dose of the composition was too low. This is confirmed by the results of the MIC and MBC tests presented in Table 2.

The bactericidal effect of the mushroom extract was unequivocally confirmed in groups of larvae infected with the Eric II bacterial strain (group B2, B3). The development of infection was inhibited in 33% of the larvae treated with the 0.025% extract, while in the group of larvae fed with the 0.05% extract, 50% of the infected larvae reached the pupal stage. In the group infected with Eric II but not treated (B1), 100% of the larvae died due to the development of the infection. It is significant that the critical period of larval development occurs between the 3rd and 5th day of life. It should be noted that the Eric II bacterial strains are generally considered to be "more virulent".

The test results presented above allow to conclude about the bactericidal effect of the fungal composition prepared on the basis of Tyromyces fissilis extract on P. larvae bacteria, and at least on its selective effect on Eric II genotypes.

The chemical composition of the dry methanol extract from Tyromyces fissilis (after alcohol evaporation) was determined using the GC-MS technique after prior silylation of the samples. The identification of the composition was based on the following databases: Wiley Registry: Mass Spectral Library, 11th Edition; NIST 11 and based on Isidorow W. "GC-MS of Biologically and Environmentally Significant Organic Compounds: TMS Derivatives, 2020, John Wiley & Sons, ISBN 978-1-11961134-9". Figure 1 shows the chromatogram obtained from the GC-MS analysis, and table 4 shows the chemical composition of the extract. The research was carried out in the chemical laboratory of the Institute of Forest Sciences, Faculty of Civil and Environmental Sciences.

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Figure 1. Chromatogram from GC-MS analysis of a methanolic extract of Tyromyces fissilis

The analysis of the chemical composition shows that the identified substances responsible for the bactericidal effect of the extract are the group of hydroxy acids and dicarboxylic acids, the content of which can be estimated at about 9%. Unfortunately, it was not possible to identify as much as 28.95% of the substances that were observed in the chromatograms despite the updated database. This is quite a characteristic problem in the identification of secondary metabolites in fungal species that are relatively unknown.

PL 242259 B1

Table 4. Chemical composition of the methanolic extract of Tyromyces fissilis determined using GC-MS, after prior silylation of the samples.

A group of chemical compounds CAS number (minutes) Contents (%) Hydroxy acids and dicarboxylic acids, including: 9.06 Lactic acid, dl-TMS 17596-96-2 12.052 0.06 Dimethyl 2-hydroxysuccinate, TMS 5S590-73-3 20.652 0.56 Succinic acid, di-TMS 40309-57-7 23.070 0.61 Fumaric acid, di-TMS 17962-03-7 24,500 1.38 Malic acid, tri-TMS 107241-82-7 30.810 6.26 Citric acid, tetra-TMS 14330-97-3 43.183 0.18 Carbohydrates, including: 52.35 Rhamnose, tetra-TMS 19127-15-2 36.531 2.10 Xylitol, penta-TMS 14199-72-5 39.594 0.23 Ribitol, penta-TMS 32381-53-6 40.067 39.28 Methyl α-glucopyranoside, tetra-TMS 2641-79-4 45.302 0.07 α-D-Gyucopyranose, penta-TMS 3327-61-5 45.954 4.34 Mannitol, hexa-TMS 14317-07-8 47.101 0.34 Galactitol, hexa-TMS 18919-39-6 47,597 0.13 β-Glucopyranose, penta-TMS 2775-90-3 48.882 0.65 myo-lnozitol, hexa-TMS 2582-79-8 51.826 0.13 Fatty acids and fatty acid esters, including; 5.07 Methyl palmitate 112-39-0 45.582 0.81 Palmitic acid, TMS 55520-89-3 49,498 0.10 Methyl linoleate 112-63-0 50,783 2.62 Methyl oleate 112-62-9 50,979 1.29 Linoleic acid, TMS 56259-07-5 54.328 0.17 Oleic acid, TMS 21556-26-3 54.495 0.10 Amino acids, including: 0.62 Alanine, N,O-di-TMS 2899-44-7 13,811 0.12 Valina, N,O-di-TMS 7364-44-5 18,846 0.07 Serine, N,O,O-tri-TMS 64625-17-8 25.459 0.07 Pyroglutamic acid, N,O-di-TMS 30274-77-2 31,560 0.37 Other chemicals, including: 3.94 Methyl phosphate, di-TMS 18291-81-1 17.190 0.33 1-Octanol, TMS 14246-16-3 17/421 0.37 Phosphoric acid, tri-TMS 10497-05-9 21.575 0.64 Glycerol, tri-TMS 6787-10-6 21,779 2.60 Unidentified chemicals 28.95

Tyromyces fissilis is not a common species in Poland. Therefore, at the Institute of Forest Sciences of the Bialystok University of Technology, in cooperation with the Jagiellonian University, attempts were made to cultivate this fungus on a solid substrate and in hydroponic cultivation. The results of research on Złotoporek niemitego Tyromyces fissilis breeding are very promising. Breeding will allow to obtain an unlimited resource of raw material in the form of fruiting bodies or micelles (hydroponic cultivation) for use in the production of a veterinary medicine for the treatment of malignant foulbrood.

Claims (4)

1. A composition comprising a dry methanol extract in the form of a powder from the fruiting bodies of the polyporoidal fungus Tyromyces fissilis, mixed with water in a ratio of 1:20 for use in the treatment of bee foulbrood, a brood disease of the honey bee Apis mellifera caused by spore-forming bacteria Paenibacillus larvae. 2. The composition for use according to claim A method according to claim 1, characterized in that it is mixed with sugar syrup in a ratio of 1:10. 3. The composition for use according to claim A method according to claim 1, characterized in that it is mixed with sugar syrup in a ratio of 1:20. 4. A method for obtaining a composition as defined in claim 1. The fruiting bodies of Tyromyces fissilis are crushed, flooded with methanol, macerated and mixed, then filtered to separate the extract from the macerate, and then evaporated from methanol in a vacuum evaporator at a temperature below 50°C and a pressure p < 20 kPa (200 mbar), and the dry extract thus obtained is mixed with water.