WO2023287313A1 - The method of obtaining a fortified mycelium of fomitopsis officinalis, active substances contained in it, compositions comprising it and health-promoting use thereof - Google Patents

Abstract

The object of the invention is a method of obtaining a fortified mycelium of Fomitopsis officinalis, in which a small-scale pre-culture is carried out, in which the liquid medium according to Oddoux is inoculated with biomass derived from in vitro cultures of F. officinalis on a solid medium according to Oddoux, and in the next step the main culture is carried out in bioreactors with air-lift system, in which the liquid medium according to Oddoux is supplemented with Mg, Zn salts, preferably their salts containing CI^- and SO₄ ^2- ions. The invention also relates to the fortified mycelium produced by this method, compositions comprising them, uses thereof, a dietary supplement and biologically active substances for the human body present in such a mycelium, and health-promoting uses thereof.

Description

The method of obtaining a fortified mycelium of Fomitopsis officinalis, active substances contained in it, compositions comprising it and health-promoting use thereof

TECHNICAL FIELD

The object of the invention is a method of obtaining a fortified mycelium of Fomitopsis officinalis, in which a small-scale pre-culture is carried out, in which the liquid medium according to Oddoux is inoculated with biomass derived from cultures of F. officinalis on a solid medium, and in the next step the main, large-scale (industrial) culture is carried out, in which the liquid medium according to Oddoux is supplemented with Mg, Zn salts, preferably their salts containing Cl^" and SO₄ ^2-. The invention also relates to the fortified mycelium produced by this method, compositions comprising it, biologically active substances contained in the fortified mycelium, and prophylactic and therapeutic uses thereof.

STATE OF THE ART

Fomitopsis officinalis (quinine conk) is a arboreal mushroom that lives on alive or dead trunks having documented medicinal, i.a. anti-inflammatory, antimicrobial, immunostimulating, antioxidant, anti-inflammatory and anti-tumor effects. F. officinalis is a bitter tasting medicinal species. F. officinalis is used in traditional Asian and European medicine, in the treatment of various diseases and conditions, including excessive sweating, respiratory diseases, gastrointestinal diseases, dizziness, rheumatism, tuberculosis and as an anti-tumor raw material.

The agarikon obtained from quinine conk was also used outside the natural areas of occurrence of the species. In F. officinalis there is a different content of biologically active substances in fruiting bodies, what has fundamental consequences for the possible medicinal preparations obtained from various parts of this organism and for obtaining it, and also has an impact on the management of mycelium and its harvest.

The properties of the raw material are determined by the unique composition of biologically active compounds contained in it, such as: triterpenoids, polysaccharides, organic acids, coumarins, flavonoids. The effect of agaricin (a citric acid derivative) present in quinine conk was determined to reduce the secretion of sweat and gastric glands and, in higher doses, to stimulate peristalsis in the gastrointestinal tract.

F. officinalis fruiting bodies are characterized by the ability to synthesize metabolites with immunostimulating and antioxidant effect (such as, e.g., phenolic and indolic compounds) used in medical, pharmaceutical or cosmetic preparations. The defined polysaccharides contained in F. officinalis fruiting bodies strengthen the immune system, as well as have antiviral and antibacterial properties. F. officinalis fruiting bodies are a valuable source of vitamins and are characterized by the ability to accumulate bioelements.

F. officinalis is a species which is dying out and in danger of extinction. Therefore, at present F. officinalis fruiting bodies are rarely used in medicine, and in Poland, due to legal protection, they cannot be obtained from the natural state.

Due to the wide medical demand for F. officinalis and almost complete lack of available natural raw material, possibilities to optimize the conditions for the culturing and composition of fruiting bodies and/or mycelium, especially for large- and full-scale culturing, allowing to obtain a standardized raw material with an enriched composition, allowing for the production of dietary supplements and pharmaceutical preparations are being sought.

There are no known methods of culturing of F. officinalis in liquid media in order to obtain the maximum increase of its biomass and obtain increased amounts of the desired active substances, as well as to optimize the production of mycelial biomass with improved health-promoting properties.

DISCLOSURE OF THE INVENTION

In the view of the described state of the art, the object of the present invention is to overcome the indicated disadvantages and to provide an efficient method of obtaining F. officinalis mycelium in in vitro conditions, with improved biological properties to provide essential macro- and microelements and biologically relevant organic compounds, which will allow the restoration of this medicinal wood fungus as ingredient of medicaments, dietary supplements, functional food or a source of active ingredients for pharmaceutical and cosmetic formulations.

The aim of the study was to obtain an efficient, repeatable, standardized method of F. officinalis culturing, which simultaneously will provide mycelium with better health-promoting properties by enriching it with bioelements important for the human body and their salts containing Mg, Zn, S, Cl. The aim was also to obtain mycelium with an increased content of biologically active organic compounds (such as phenylalanine, phenolic compounds with antioxidant activity (e.g., gallic acid, p-hydroxybenzoic acid), indolic compounds: 5-hydroxy-L-tryptophan, L-tryptophan, tr/ptamine, melatonin, serotonin - important in the prophylaxis of depression and pain).

Unexpectedly, it was found that it is possible to efficiently culture F. officinalis in a liquid medium enriched with selected and appropriately chosen salts containing Zn, Mg, Cl, S - especially MgS04-7H₂O, MgCl₂-6H₂O, ZnSO₄7H₂O, ZnCl₂, preferably ZnSO₄-7H₂O and/or MgCl₂-6H₂O , what leads to the highest increase in biomass and allows for standardization of the culture, to obtain the biomass of mycelium in cultures on an industrial scale. Unexpectedly, it was found that a mycelium fortified with Zn, Mg, S and Cl in the form of inorganic salts: sulfate(VI) and chloride salts is obtained in this way, due to the possibility of supplying macro- and microelements important for the human body (Mg andZn, S and Cl), but also organic compounds produced by the mycelium, such as phenylalanine, phenolic compounds with antioxidant activity (e.g., gallic acid, p- hydroxybenzoic acid), indolic compounds with important biological properties, such as 5-hydroxy- L-tryptophan, L-tryptophan, tryptamine, melatonin, serotonin. Unexpectedly, it was found that these compounds are produced in significant amounts in the mycelium fortified in the described method of culturing of F. officinalis, but they are also biologically available to the human body, as they are released in significant amounts from the obtained mycelium to the digestive juices.

The mycelium grown by the method according to the invention on a medium enriched with selected salts, comprising Zn, Mg, Cl, S in specific concentrations and forms, produces more biologically active and health-relevant compounds; it is also an excellent source for supplementing macro- and microelements Zn, Mg, Cl, S (their high bioavailability to the human body has also been demonstrated).

The invention relates to the method of efficient preparation of fortified mycelium of Fomitopsis officinalis in liquid in vitro cultures, including the steps, in which a) a small-scale pre-culture is carried out, in which the liquid medium according to Oddoux with the pH: 5.8±0.5 is inoculated with the biomass of the derived pure culture of F. officinalis, and the mycelium is cultured for a period of 10-16 days, more preferably the mycelium is grown for 14 days, at the temperature of 20-30 °C, more preferably at the temperature of 25+2 °C, with shaking in the range of 110-190 rpm, more preferably with shaking about 140 rpm; b) a large-scale, main culture is carried out, in which the liquid medium according to Oddoux, with the pH: 5.8±0.5, is supplemented with at least one Mg and/or Zn salt, selected from MgSO₄.7H₂O in the range of 1580-2400 mg/L, MgCI₂-6H₂O in the range of 2670-4000 mg/L_; ZnSO₄-7H₂O in the range of 80-120 mg/L, ZnCI₂ in the range of 32-48 mg/L or a combination thereof; the supplemented liquid medium is inoculated with the F. officinalis mycelium biomass prepared in step a) and the mycelia are grown at the temperature of 20-30 °C, more preferably at the temperature of 25±2 °C, for a period of 8-12 days, more preferably for 10 days, in bioreactors with centrifugal, bottom-up and uniform aeration providing simultaneous mixing of the culture to produce the Mg and/or Zn fortified mycelium of Fomitopsis officinalis.

Preferably the method in step b) of the main culture is carried out in a medium supplemented with:

MgSO₄-7H₂O in the range of 1780-2180 mg/L, more preferably 1880-2070 mg/L, most preferably about 1980 mg/L, and/or MgCI₂-6H₂O in the range of 3000-3675 mg/L, more preferably 3270-3500 mg/L, most preferably about 3340 mg/L, and/or ZnSO₄-7H₂O in the range of 90-110 mg/L, preferably 95-105mg/L, most preferably about 100 mg/L, and/or ZnCI₂ in the range of 36-44 mg/L, preferably 38-42 mg/L, most preferably 40 mg/L, or a mixture thereof.

Preferably the method in the step b) of the main culture is carried out on a medium supplemented with ZnSO₄-7H₂O and/or MgCI₂-6H₂O.

Preferably, the method in the step a) of the pre-culture and/or the step b) of the main culture is carried out by providing a photoperiod: 14-18 h light, preferably 16 h light, at 700-1100 lx, preferably 900 lx and with 6-10 h without light, preferably 8 h without light.

In the preferred method, the mycelium form the pre-culture from the step a) is homogenized before being added to the main culture in the step b).

In the preferred method, in the main culture the step b), the supply of air with oxygen to the bioreactor and the discharge of air with carbon dioxide are carried out through a system of antibacterial filters.

The invention also relates to the fortified mycelium of Fomitopsis officinalis produced by the method of efficient preparation according to the invention, which mycelium of Fomitopsis officinalis is fortified with Mg and/or Zn.

The invention also relates to a composition, which comprises the Mg and/or Zn fortified mycelium of F. officinalis produced by the method of efficient preparation according to the invention, and a carrier acceptable for food and/or pharmaceutical and/or veterinary purposes. The composition is preferably in the form of a powder, granules, paste, drink, functional drink, suspension, tablet, capsule, freeze-dried mycelium.

The composition is preferably in the form of a functional food and/or dietary supplement, preferably it is intended for supplying and/or supplementation of the body in diseases and conditions, in which the fortified mycelium of Fomitopsis officinalis acts as an agent with antioxidant, anti-aging, anti-inflammatory, anti-depressant, anti-proliferative, anti-tumor effect.

The invention further relates to a pharmaceutical composition comprising the fortified mycelium of Fomitopsis officinalis produced by the method of efficient preparation of the fortified mycelium of Fomitopsis officinalis in in vitro liquid cultures according to the invention, for use as a medicament in the treatment of tumors, depression, inflammation, signs of aging; as a medicament with antioxidant, anti-inflammatory, anti-aging and immunomodulating activity; as a medicament with antibacterial, antifungal and antiviral activity; as a medicament for supplementing deficiencies of at least one of Mg, Zn, S, Cl, preferably for supplementing deficiencies of Mg and Zn.

The pharmaceutical composition is preferably intended for use in the treatment and prevention of tumor growth, selected from lung cancer, prostate cancer and melanoma.

The composition is preferably intended to supply and/or supplement the organism with phenylalanine, phenolic compounds, gallic acid, p-hydroxybenzoic acid, 5-hydroxy-L-tryptophan, tryptophan, tryptamine, melatonin, serotonin and 6-CH₃-D,L-tryptophan.

The invention also relates to the use of the fortified mycelium of Fomitopsis officinalis produced by the efficient preparation according to the invention in the biotechnology, cosmetic, food and dietary supplement industries.

In a preferred application, the fortified mycelium of Fomitopsis officinalis is used as a dietary supplement and/or functional food to supplement the body with Mg and Zn.

The invention also relates to the use of a fortified mycelium of Fomitopsis officinalis produced by the method of efficient preparation according to the invention for the treatment and prevention of tumor growth, preferably selected from lung cancer, prostate cancer and melanoma.

Another object of the invention is a dietary supplement intended for the supplementation of Mg and Zn deficiencies, which contains the fortified mycelium of Fomitopsis officinalis produced by the method of efficient preparation according to the invention, wherein the dietary supplement comprises the Mg and/or Zn fortified mycelium of F. officinalis in an amount of 0.01% to 100% by weight.

DETAILED DESCRIPTION OF THE INVENTION

The mycelium of F. officinalis, as well as the preparations and compositions comprising it, produced by the disclosed culturing method, have a beneficial effect on health due to the synergistic action of the active substances present in it, while maintaining the safety of its use. The obtained mycelium shows nutritional, anti-inflammatory, regenerative and antioxidant properties, which is why it will be successfully used as a diet substitute, functional food, product for the treatment and prevention of specific diseases, as an active ingredient of cosmetics or ingredients of balanced food for animals and humans.

The recommended daily dose of Mg for an adult human is about 400 mg and Zn is about 20 mg. One of the richest food products in Mg are pumpkin seeds, the consumption of 100 g of seeds supplements 130% of the body's daily magnesium requirement (550 mg Mg/100 d.w.). On the other hand, oysters have the highest Zn content (61 mg Zn/100 dry weight). It was found that 100 g of F. officinalis mycelium biomass, obtained by the method of in vitro culture according to the invention in a liquid medium supplemented with ZnSO₄-7H₂O and MgCl₂-6H₂O, provides as much as 200% of the daily demand of an adult human body for Mg and about 400% of the human body's demand for Zn (Tab. 3).

In the presented studies, it was shown that zinc and magnesium salts increase biomass growth compared to the control (not-enriched material). The liquid culture of F. officinalis mycellium in a medium enriched with Zn and Mg salts causes the accumulation of these bioelements, which makes it a valuable source for their supply to the human and/or animal body. The mycelium grown in this way has more biologically active compounds, including phenolic and indolic compounds than the mycelium grown without such supplements. Unexpectedly, it was also found that more active substances, bioelements and biologically active compounds are extracted from the F. officinalis mycelium enriched with Zn and Mg salts to the digestive juices in the artificial gastrointestinal tract model.

Ultimately, based on the conducted research, it was determined that the optimal compounds enhancing the biological activity of F. officinalis mycelium, including immunostimulating, antitumor, antioxidant, anti-aging, anti-inflammatory and anti-depressant activity, resulting from the organic compounds present in it, as well as from the activity of Zn, Mg, are MgSO₄-7H₂O, MgCI₂-6H₂O, ZnSO₄.7H₂,O ZnCI₂ salts and combinations thereof, more preferably ZnSO₄-7H₂O and/or MgCI₂-6H₂O.

It will be evident for a person skilled in the art that in one embodiment the mycelium may be grown on one of the selected salts MgSO₄.7H₂O, MgCI₂.6H₂O, ZnSO₄.7H₂,O ZnCI₂ more preferably ZnSO₄-7H₂O and/or MgCI₂-6H₂O, and then different mycelia from different cultures may be combined in specific proportions to obtain a raw material with a specific final composition and desired properties. It will be evident for a person skilled in the art that in another embodiment the mycelium will be grown on a combination of several of the above-mentioned salts, specifically, it will be supplemented with ZnSO₄.7H₂O and MgCI₂.6H₂O to obtain the final composition enriched in Mg, Zn, S and Cl from a single culture.

The anti-tumor and antioxidant properties of medicinal fungi are related, i.a., to the ability to accumulate significant amounts of zinc and magnesium in their fruiting bodies. So far, the possibilities of accumulation of Zn and Mg in a large-scale liquid culture of the mycelium of F. officinalis, as well as their subsequent bioavailability to the organism, were unknown.

Zinc is most abundant in the human central nervous system, especially in the cerebral cortex and the hippocampus. Zinc has functions related to nerve conduction, moreover, zinc has been shown to play an important role in the physiology and pathology of the skin, demonstrating antiinflammatory and regenerative effects. It is necessary for the production of insulin.

Magnesium, which has an anti-depressant effect similar to zinc, is a cofactor of respiratory enzymes, it is responsible for the main functioning of skeletal muscles and cooperates with iron. The contents of these bioelements determined in the mycelium of F. officinalis obtained in the method of production according to the invention allow for their very efficient supplementation to the human body compared to other natural sources.

Chlorine is a valuable macroelement, necessary for the production of digestive juices in the stomach and participates in the gas exchange process, i.e. the transport of CO₂ from the body to the lungs.

Sulfur is a component of cysteine and methionine (an exogenous amino acid), which are the basic component of proteins (responsible for their tertiary structure), including enzymes, and are part of compounds that build cartilage tissue.

Therapeutically important, non-hallucinogenic derivatives of indole found in F. officinalis include L-tryptophan, 5-hydroxy-L-tryptophan, melatonin, serotonin and tryptamine. These compounds act as neurotransmitters or their precursors. They affect the daily cycle by regulating appetite, mood and body temperature. They are responsible for the clotting processes, immune response and cell regeneration. In addition, they exhibit antioxidant (reduction of lipid peroxidation), antitumor, cardioprotective and analgesic properties. All the tested compounds were present in the fortified mycelium of F. officinalis in greater amounts than in the fruiting bodies of this species. 5- hydroxy-L-tryptophan is also a direct precursor of serotonin. Taken with food, it easily crosses the blood-brain barrier in the central nervous system, where it is converted to serotonin (serotonin does not cross the blood-brain barrier). In some countries (GB, USA, CA) it is used as a dietary supplement with anti-depressant, appetite-reducing and hypnotic effect. This compound is well absorbed after oral administration, about 70% of the dose enters into the bloodstream. It has been shown that the administration of 5-hydroxy-L-tryptophan in therapy can be effective in treating conditions such as depression, fibromyalgia, chronic headache and insomnia. It has been shown that the addition of Mg and Zn salts increases the content of these neurotransmitters.

The fortified mycelium from the in vitro culture according to the invention has a significant content of phenolic compounds. Phenolic compounds have a strong antioxidant effect - eating foods rich in phenols prevents cancer, type II diabetes and cardiovascular diseases (CVD). Much stronger antioxidant activity (%DPPH) of the mycelium obtained from fortified in vitro cultures results from the proven higher content of indole and phenolic compounds.

Phenylalanine is an amino acid that is characteristic for animal meat, it is needed for the correct structure of proteins in the human body, but it is also a precursor of neurotransmitters. The mycelium of F. officinalis obtained from liquid cultures fortified with Mg and/or Zn salts according to the invention is a comparable source of this compound with animal meat, and therefore - a good alternative for vegetarians. It was found that the phenylalanine in liquid cultures of F. officinalis fortified with Mg and/or Zn salts, according to the invention, is produced at a level from 13 to 46 times higher than in non-fortified mycelia, and that the bioavailability of phenylalanine demonstrated by the analysis of the release from mycelium in gastric and intestinal juices is up to 23 higher for all variants of the mycelium fortified with Mg and/or Zn salts, or even 29 times higher for ZnSO₄x7H₂O.

Therefore, the mycelium of F. officinalis obtained from liquid cultures fortified with salts shows increased health-promoting properties for the age, diseases and conditions, it is a good source for supplementing deficiencies of macro- and microelements such as Mg, Zn, as well as S and Cl. BRIEF DESCRIPTION OF FIGURES

Exemplary implementations of the invention are presented in the figures, in which:

Fig.l Shows a picture of an in vitro culture of F. officinalis on a solid medium, 3 days after initiation. Fig. 2 Shows a liquid culture of F. officinalis in a 10 L bioreactor with centrifugal aeration, providing simultaneous mixing.

Fig.3 The amount of biomass [g/L of the medium] obtained in in vitro cultures of F. officinalis.

Fig.4 Content of a) metals: Mg, Zn and b) Cl-, SO₄ ^2- ions in fruiting bodies, mycelium and mycelium enriched with inorganic salts [mg/100 g of dry weight] obtained for F. officinalis (F.o).

METHODS OF CARRYING OUT THE INVENTION

In the following examples, unless it was otherwise indicated, manufacturers' instructions for specific materials and methods were followed. The following examples were presented merely to illustrate the invention and to clarify its various aspects, but are not intended to be limitative, and should not be equated with all its scope, which is defined in the appended claims. Publications cited in the description, and the references given therein, are also incorporated herein as references.

EXAMPLES Example 1: Derivation of pure cultures of Fomitopsis officinalis mycelium

The starting material for the derivation of mycelium cultures were fruiting bodies of F. officinalis, obtained from a natural site in the Swietokrzyskie Voivodeship, Poland.

Fragments of the hymenial layer of the fruiting bodies, with an area of about 2 mm², were used to initiate in vitro cultures of F. officinalis. Explants were degreased with 70% ethyl alcohol, sterile redistilled water, transferred to BD Sabouraud Agar with Chloramphenicol solid medium (per 1 L of water: pancreatic hydrolysate of casein 5.0 g; pepsin hydrolysate of animal tissue 5.0 g, glucose 40.0 g, agar 15.0 g), and incubated at 22 °C for 14 days (Fig. 1). The derived stationary cultures allowed for obtaining a pure F. officinalis mycelium. Example 2 Liquid cultures of the mycelium of F. officinalis

Liquid cultures were carried out in the basal medium according to Oddoux and its modifications. After all the components were combined, the medium was made up with water to the required volume and the pH was adjusted to 5.8±0.5 with 1 M NaOH or 1 M HCI. Tab, 1. Composition of the basal medium according to Oddoux.

a) Pre-cultures with shaking

To the modified liquid medium according to Oddoux, 0.1 g of F. officinalis biomass obtained from 2-week in vitro cultures on the solid medium from Example 1 was transferred to 0.5 L flasks with 250 mL of the liquid medium, and shaken at 140 rpm for 14 days.

Initial and specific in vitro cultures on a liquid medium were carried out on the medium according to Oddoux pH: 5.8±0.5, (with optional supplements) in optimized physicochemical conditions: 25±2 °C, photoperiod: 16 h light 900 lx and 8 h without light. b ) Main cultures in bioreactors with mixing and centrifugal aeration (of the air-lift system type)

To determine the optimal conditions for the most effective growth off. officinalis biomass, which at the same time will ensure that the obtained mycelium cultured on an industrial scale shows the best properties for the production of biologically active compounds and their bioavailability for the human body, about 100 ml of mycelium from liquid cultures in 0.5 L flasks were transferred to 10 L bioreactors with liquid medium according to Oddoux (control), and with the same substrate but with different additives (see c). Cultures were carried out in self-designed bioreactors to cultivate F. officinalis on a large scale with aeration carried out in a bottom-up, uniform and centrifugal manner, which ensured not only the supply of air (oxygen), but gentle mixing that did not destroy or cut the hyphae of the growing mycelium.

In a preferred embodiment, 250 mL of the pre-culture was homogenized before the addition to the main culture, which additionally increased the amount of finally produced biomass and uniformed the growth rate of the culture, and finally increased the culture yield/L

The air was supplied to 10 L of bioreactors through a system of antibacterial filters. The applied aeration system with simultaneous mixing, and thus increasing the access of mycelium to nutrients, also ensured the removal of carbon dioxide, through antibacterial filters, as a byproduct of fungal metabolism. The use of centrifugal bottom-up aeration, which simultaneously ensures mixing of the biomass instead of mechanical mixing e.g. by paddles, prevents damage to the mycelium and ensures stable culture conditions to optimize mycelium growth (Fig. 2). After 10 days of incubation in the bioreactor, the resulting mycelium was separated from the medium and subjected to microscopic examination and confirmation of the purity of the cultured species by sequencing the fungal ITS region of the ribosomal RNA gene amplified with ITS1 and ITS4 universal primers by PCR as described in White et al. (1990). The remaining material was subjected to various tests and determinations, or was subjected to lyophilization (Freezone 4.5 freeze dryer, Labconco; temperature: -40 °C) for further determinations and tests. c) Fortification of the mycelium with bioelements to optimize the conditions of large-scale culture and to obtain mycelium with improved health-promoting properties

The research was aimed at determining which inorganic salt added to the medium most effectively influenced the growth of F. officinalis biomass and the degree of accumulation of supplemented bioelements, and enhanced the metabolism of beneficial organic compounds, especially those with health-promoting properties.

To optimize the conditions of large-scale industrial culture and increase the efficiency of culturing of F. officinalis mycelium with the simultaneous obtaining of mycelium with improved health- promoting properties, the basic liquid media according to Oddoux in cultures in 10 L bioreactors were modified and enriched with bioelements, looking for a solution that would provide the synergy effect, i.e. at the same time, it will allow to obtain mycelium with an increased amount of bioelements important for the human body (fortified mycelium), as well as ensure optimized growth of F. officinalis biomass and induce the metabolism of beneficial organic compounds produced by mycelium with health-promoting properties. At the same time, they searched for such additives that would be safe for the mycelium and for organisms consuming such mycelium, that would affect the mycelium in such a way that their bioavailability from the mycelium in the human digestive tract would be at least good, preferably better than from mycelium grown in conditions without the addition of these bioelements.

For this purpose, the culture media in 10 L bioreactors were supplemented with the addition of various organic and inorganic salts separately and with a mixture containing iron, copper, zinc and magnesium. Due to the observed beneficial effect on the growth of biomass, zinc and magnesium salts were selected for further research. These elements are additionally beneficial in supplementing the human body. It was found that copper, and especially iron, inhibited the growth of mycelium. Tab. 2. List of the selected medium additives

Example 3 Determination of the growth of F. officinalis mycelium biomass in various large-scale liquid cultures

In the first step, the increase in biomass (Fig. 3) in the main cultures in 10 L bioreactors in a liquid medium according to Oddoux supplemented with various salts and their combinations - see Tab. 2. By analyzing the effectiveness of biomass growth, it was shown that for each of the in vitro cultures carried out on an enriched medium, a greater growth of mycelium was obtained than for the control - a culture carried out on the standard medium according to Oddoux (2.8 g/L of the medium). Due to the increase in the growth of F. officinalis biomass by nearly 1 g/L of the medium, sulfate(VI) (ZnSO₄x7H₂O, MgSO₄><7H₂O) and chloride(ZnCl₂, MgCI₂x6H₂O) salts were selected.

Effective, that is a two-fold increase in biomass was obtained for in vitro cultures carried out on a medium enriched by the addition of sulfate(VI) salts, respectively 5.5 g/L of the medium in the case of the addition of MgSO_4’7H₂O salt, and 5.6 g/L of the medium for the addition of ZnSO₄-7H₂O salt. In turn, the amount of biomass obtained for the in vitro culture of F. officinalis with the addition of chloride salts was about 40% lower than the sulfate ones - 3.2 g/L of the medium for the addition of ZnCI₂ and 3.6 g/L of the medium for the addition of MgCI₂-6H₂O, but still higher than with the unenriched medium.

Example 4. Comparative analysis of biomass content of fruiting body, mycelium and extracts for different cultures of F. officinalis

Analyses of F. officinalis fruiting bodies were carried out on fruiting bodies harvested from the natural state with the consent of the Minister of the Environment, Poland.

Analysis of inorganic compounds a) Analysis of metals in fruiting bodies, mycelium and extracts from in vitro cultures of F. officinalis

Tests were carried out to determine the concentration of selected bioelements - Mg and Zn present in the fruiting bodies of F. officinalis and in the mycelium obtained in in vitro culture conditions on the initial liquid medium according to Oddoux (control) and the same medium enriched with inorganic sulfate(VI) and chloride salts.

The obtained biomass from in vitro culture of F. officinalis was divided into two parts. The first was subjected to a wet mineralization process in a closed system in the Magnum (ERTEC II) device. For this purpose, 6 mL of concentrated 65% nitric acid and 2 mL of a 30% perhydrol solution were used. All was transferred to teflon vessels and subjected to mineralization according to an optimized program. The obtained colorless solution was evaporated in quartz evaporators on a heating plate at the temperature of 120 °C. The residue was quantitatively transferred to 10 mL flasks and made up to the volume with 4 times distilled water.

The other part of the material was weighed in an amount of 0.5 g in three independent replications and extracted into artificial digestive juices (Example 5). The extraction was carried out in three steps. In the first step, the material was transferred to 100 mL Erlenmeyer flasks and wetted with 3 mL of artificial saliva solution, and then 20 mL of gastric juice solution was added. The process was carried out for 60 minutes. The extracts thus obtained were filtered using filter paper and syringes. The biomass remaining on the filter was transferred back to the flasks and extracted with 20 mL of intestinal juice. The extraction process in this medium was carried out for a period of 150 minutes. Then the filtration procedure was repeated. All extraction steps were carried out in the Gastroel-2014 apparatus, which was designed to test the release of substances into artificial digestive juices.

In the mineralized fruiting bodies and mycelium from in vitro cultures of F. officinalis, as well as in the obtained extracts, the concentrations of Mg and Zn were determined using the method of atomic absorption spectrometry (flame technique - FAAS). An atomic absorption spectrometer from Thermo Scientific (iCE 3500 - UK) was used for the performed measurements. Each prepared analytical sample was tested in three independent replications, and the obtained test results were presented as mean values supplemented with standard deviation.

Based on of the analysis of the content of Mg and Zn, it was found that the concentration of these elements is the lowest in fruiting bodies of the species F. officinalis (Mg - 132.13 mg/100 g dry weight (d.w.) and Zn - 15.3 mg/100 g d.w.) and their mycelia obtained on in unenriched substrates according to Oddoux (Mg - 98.84 mg/100 g d.w., Zn - 12.6 mg/100 g d.w.) (Fig. 4 a). b) Analysis of sulfate and chloride ions

OG and SO₄ ^2- ions were analyzed by spectrophotometry on the Spectroquant Nova 60 spectrophotometer (Merck KGaA, Darmstadt, Germany). Validated tests were used to analyze SO₄ ^2- (Cat. No. 101812, Merck KGaA, Darmstadt, Germany) and Cl- (Cat. No. 114897, Merck KGaA, Darmstadt, Germany). Spectrophotometric analyses were performed in quartz cuvettes. The intestinal juice samples were filtered using a 0.45 pm membrane (Merck KGaA, Darmstadt, Germany). The results of the analysis of the content of Cl- and SO₄ ^2- anions are presented as mean values from three independent measurements and are summarized in Tab. 3. The independent result was corrected for the amount of background (the content of individual anions present in the control sample - digestive juice solution). In fruiting bodies of F. officinalis, sulfate(VI) ions were determined in the amount of 429 mg/100 g d.w. In turn, the content of chloride ions in fruiting bodies was 3 times higher and amounted to 1904 mg/100 g d.w. The addition of inorganic sulfate salt (VI) to the substrates caused a significant increase in the amount of SO₄ ^2- anion in the tested biomass. The highest concentrations of SO₄ ^2- were found in the case of magnesium sulfate(VI) salts of 1733 mg/100 g of d.w. However, the analysis of Cl- ions showed that only the addition of a salt containing chloride ions in the form of ZnCI₂ to the culture medium increases the amount of this ion in the mycelial biomass (1750 mg/100 g d.w.). (Fig. 4 b).

Taking into account the form of the compound in which the above-mentioned elements were added to the liquid culture medium, it was shown that the highest amounts of magnesium were determined in the biomass of F. officinalis grown on a medium enriched with inorganic magnesium salt - magnesium(ll) sulfate at a concentration of 3340 mg/L of the medium. The amount was 1261.2 mg/100 g d.w. and was about 25% higher compared to the amount of magnesium in mycelium from in vitro cultures grown on media enriched with magnesium chloride (951.7 mg/100 g d.w.). The same trend was shown for the analysis of zinc content. The highest concentration of zinc was determined in the biomass of F. officinalis obtained in a medium also enriched in inorganic zinc salt -zinc sulfate at the concentration of 100 mg/L of the medium. The determined amount of zinc was 182.8 mg/100 g d.w., respectively, and it was also higher than the determined amount of this element in the mycelium from in vitro cultures carried out on media enriched in zinc chloride (136.4 mg/100 g d.w.). By analyzing the amounts of Mg and Zn determined in the control biomass (Fig. 4 a) in relation to these elements in the biomass of in vitro cultures obtained on enriched media, it was shown that the in vitro cultivated mycelium of F. officinalis efficiently accumulates metals from the medium in which it grows. Which means that not only fruiting bodies, but also fortified mycelium from a liquid in vitro culture can be used as a natural carrier of bioelements and minerals and, as a result, constitute their natural source as a dietary supplement of Mg and Zn, as well as S and Cl, or a natural functional food with health-promoting properties provided by the mycelium.

Based on of the conducted analyzes, the salts that were selected as an additive to the medium for further research were ZnSO₄-7H₂O in the amount of 100 mg/L and MgCl₂-6H₂O in the amount of 3340 mg/L, because the mycelium showed a good accumulation of Zn and Mg, which have known health-promoting properties for the human body, as well as showed a positive effect on increasing the production of mycelial biomass.

Analysis of organic compounds c) Preparation of extracts to analyse the content of phenolic compounds, phenylalanine and indolic compounds

These extracts were obtained from freeze-dried fungal material (5 g), which was homogenized in an agate mortar and then extracted five times with 100 ml of methanol by means of ultrasound at 49 kHz for 30 minutes (Sonic-2, Polsonic, Poland). The extracts obtained were evaporated to dryness. The dry residue was dissolved in 10 ml of HPLC purified methanol. Each of the test samples was filtered through syringe filters (Millex, Millipore Corporation, USA). The obtained F. officinalis extracts were analyzed using a Hitachi HPLC chromatograph (pump: L-7100; column: Purospher^® RP-18 (4x200 mm, 5 pm), with an isocratic elution program for lovastatin and a gradient for phenolic acids (UJ CM). d) Analysis of phenolic acids and L-phenylalanine

The analysis was performed using the Reverse High Performance Liquid Chromatography (RP- HPLC with DAD detection) method using a VWR Hitachi-Merc HPLC apparatus with the following specifications: L-2200 autosampler, L-2130 pump, RP-18e LiChrospher column (4 mm ^c 250 mm, 5 pm), thermostat at 25 °C, L - 2350 thermostat, L - 2455 diode detector operating in the UV wavelength range of 200-400 nm. The components used in the mobile phase were: solvent A: methanol/0.5% acetic acid 1:4 (v/v) and solvent B: methanol. The gradient was set to: 100:0 time 0-25 min; 70:30 time 35 min; 50:50 time 45 min; 0:100 time 50-55 min; 100:0 time 57-67 min. The comparison of the UV spectra and the retention times in relation to the standard compounds made it possible to identify phenolic compounds. The quantitative analysis was performed using a calibration curve assuming the initial linearity of the size of the area under the peak in relation to the concentration of the standard used. The obtained results of determining the content of phenolic compounds and L-phenylalnine in fruiting bodies, mycelium cultures and extracts from digestive juices were expressed in mg/100 g dry weight. e) Analysis of indolic compounds

The analysis of the content of indolic compounds in the previously prepared extracts was carried out using the RP-HPLC method with UV detection. The extracts were dissolved quantitatively in a 1.5 m solvent system (methanol/water/ammonium acetate 15:14:1 v/v/v) and separated on a Hitachi RP-HPLC with UV detection (Merck, Japan) equipped with an L-pump -7100. A Purospher^® RP-18 column (4 mm ^c 200 mm, 5 pm) was kept at 25 °C, while the UV detector was operated at a wavelength of l = 280 nm. The liquid phase used was a mixture of methanol/water/ammonium acetate (15: 14: 1 v/v/v) and the flow rate was determined to be 1 mL/min. Qualitative and quantitative analysis of indolic compounds was performed analogously to phenolic compounds and L-phenylalanine. f) Antioxidant properties

The antioxidant activity was determined using a l,l-diphenyl-2-picrylhydrazyl (DPPH) radical (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 2 g of fungal material was extracted with 8 mL of methanol. From this methanol extract, 0.1 mL was added to 4.9 mL of 0.1 mM DPPH solution dissolved in 100% methanol. In the case of digestive juices, 0.1 mL of artificial juice was added without dissolving. The reaction mixtures were incubated for 20 min in the dark at 22 °C. The absorbance at 517 nm was then measured with a UV-VIS Helios Beta spectrophotometer (Thermo Fisher Scientific Inc., Waltham, USA). The reduction of DPPH radicals was calculated using the following equation: AA (%) = [(AO - A1)/A0] · 100, where AA is the antioxidant activity expressed in%, AO is the absorbance of the blank/blank sample and A1 is the absorbance of the tested concentration (for a description of the DPPH test, see also reference no. 29).

The antioxidant activity showed a diversified antioxidant potential of the studied F. officinalis extracts. In the case of mycelia supplemented with salts, the bioelements used in the experiment showed their higher antioxidant activity compared to the extracts from the control (mycelium without the addition of salts) and from fruiting bodies. The highest antioxidant activity was found in the mycelium supplemented with ZnSCU. In studies of the antioxidant potential of fungal material extracts for gastric and intestinal juices, it was shown that the substances contained in fruiting bodies and reducing the DPPH radical are not digested and act on a similar level in the obtained digestive juice extracts. For mycelium supplemented with zinc after extraction into gastric juice, greater antioxidant activity was determined compared to unenriched mycelium (control) and mycelia supplemented with magnesium.

The results of the analyses are presented in Tab.4.

Example 5 Determination of the occurrence and bioavailability of elements, ions and organic compounds from fruiting bodies and mycelium of F. officinalis from various culture conditions

The actual bioavailability of elements, salts and organic compounds for the human body from the obtained from fruiting bodies and fortified mycelium and control mycelium, including the bioavailability of Mg and Zn from the obtained bioelements enriched mycelium obtained in 10 L bioreactors by the method described in Example 2, was also determined.

For this purpose, studies of the release of these compounds were carried out under conditions simylating the human gastrointestinal tract (temperature 37 °C and peristaltic movements) into gastric juice and intestinal juice in the Gastroel-2014 apparatus (see also Example 4). The analysis of the content of Mg and Zn bioelements in extracts obtained by etching in artificial digestive juices was carried out using the method of atomic absorption spectrometry (AAS) and the AAS3500 spectrometer (ThermoScientific). The use of the ASA method, which is characterized by high sensitivity of determinations, accuracy and precision, combined with appropriate preparation - i.e. mineralization of analytical samples (microwave mineralizer, ERTEC), made it possible to conduct a reliable analysis of their composition.

The research was carried out in artificial digestive juices, which were prepared according to current literature data, in accordance with the following recipe: Artificial saliva: 100 mL KhhPCU with concentration 25 mmol/L, 100 mL KHCO3 100 mL, 100 mL MgCI₂ 1.5 mmol/L, Na2HPC>424 mmol/L, 150 mmol/L, 6 mL citric acid 25 mmol/L, 100 mL CaCh 15 mmol/L made up to 1 L with distilled water.

Artificial gastric juice: 2.0 g of NaCI and 3.2 g of pepsin were dissolved in four times distilled water, then 80 mL of 1 mol/L HCI acid was added, made up to 1 L with distilled water.

Artificial intestinal juice: 120 mg of bile salts, 8.4 g of NaHCOs and 20 mg of pancreatic extract were dissolved and made up to 1 L with distilled water. a) Bioavailability of elements and ions

Based on of the obtained results, it was found that after the extraction of fruiting bodies of F. officinalis in artificial digestive juices, the determined amounts of Mg and Zn elements are much lower than in the biomass obtained in liquid culture in 10 L bioreactors on Oddoux medium alone and with appropriate supplements of bioelements (Tab. 3).

Tab. 3. The analyzed content of Mg and Zn and Cl- and SO4^2- ions [mg/100 g of dry biomass] in fruiting bodies, in vitro cultures and in vitro cultures of the F. officinalis species enriched with the addition of zinc and magnesium salts after extraction to artificial digestive juices.

N=6; values in the same row marked with different letters are significantly different (p <0.05); n.d. not determined It has been shown that the most effective extraction of bioelements takes place through artificial gastric juice. The amount of zinc determined after extraction into gastric juice from biomass obtained on a ZnSO₄-enriched medium was 96.8 mg/100 g d.w., while enriched with ZnCI₂ - 62.2 mg/100 g d.w. In the case of extraction in intestinal juice, these values were lower and amounted to, respectively, 29.2 mg/100 g of dry weight in the case of the addition of ZnSCU to the medium and 15.8 mg/100 g of dry weight, for the addition of ZnCI2. For mycelium enriched with MgS04, 823 mg/100 g D.W. was extracted into the gastric juice, and 92 mg/100 g d.w. for intest Inal juice. In turn, in the case of mycelia with MgCI₂ in the gastric juice, Zn was determined at the level of 633 mg/100 g d.w., while in the intestinal juice this level was lower and amounted to 76 mg/100 g d.w., respectively. In all the discussed cases, the amount of extracted bioelements (Mg and Zn) for artificial digestive juices was higher for the biomass obtained on culture media enriched with inorganic Mg and Zn salts. The greatest amounts of the released elements tested were found in the case of the addition of sulfate(VI) salts. Each time the amounts of extracted Zn and Mg for artificial gastric juice were higher than those determined in the tested solution of artificial intestinal juice. The obtained results confirm that the biomass from in vitro cultures of F. officinalis enriched with the addition of Zn and Mg salts to the medium is a rich source of these elements and allows to cover the daily needs of the human body for these bioelements and they will be effectively released from the mycelium in the human digestive tract. 100 g of the culture according to the invention on a medium supplemented with MgSO₄-7H₂O and ZnSOWFhO provides 200% of the daily requirement of an adult human body for Mg and about 400% for Zn.

By analyzing the amounts of anions extracted into artificial digestive juices, it was found that the most effective extraction of anions was into artificial gastric juice (Tab. 3). The highest concentration of Cl- ions was determined in the case of extraction from the biomass obtained on the medium with the addition of ZnCI₂ - 1000 mg/100 g of dry weight, while the smallest amount of Cl- ions was extracted from the biomass carried out on the medium with the addition of MgCl₂-6H₂O - 133 mg/100 g d.w. In the case of SO₄ ^2- ion, the smallest amounts of this ion were determined in gastric juice, into which natural fruiting bodies were extracted (332 mg/100 g d.w.) and biomass from in vitro cultures carried out on media without the addition of salt (371 mg/100 g d.w.). The best result was observed with the addition of MgSO₄-7H₂O salt to the culture media. In the extract for gastric juice from biomass obtained on the medium with the addition of this salt, the highest content of SO₄ ^2- was determined, which was - 1322 mg/100 g d.w. Considering the daily requirement of the human body for Cl- (1850-2300 mg) and SO₄ ^2-ions (460 mg), it was found that the biomass obtained from the in vitro culture of F. officinalis allows for the complete coverage of the daily requirement of the human body for SO₄ ^2- ions (Tab. 3). The above confirms that the biomass obtained from the mycelia fortified according to the invention is a rich source of not only bioelements, but also SO₄ ^2" and Cl- ions, and therefore it will supplement the demand for elements such as: Mg, Zn, S and Cl. b) Occurrence and bioavailability of organic compounds

Organic bioactive compounds (i.a. indolic compounds, lovastatin, phenolic acids) were determined in methanol extracts (for preparation see Example 4).

During the analyzes, the presence of various organic compounds in fruiting bodies, mycelium and mycelium fortified with bioelements and grown in accordance with Example 2 was determined for the presence of both phenolic compounds: gallic acid and p-hydroxybenzoic acid as well as L- phenylalanine and indolic compounds: 5-hydroxy-L- tryptophan, L-tryptophan, tryptamine, melatonin, serotonin and 6-CH₃-D, L-tryptophan (Tab. 4). The DPPH test was performed as described in Example 4.

Based on the analysis of phenolic compounds, the presence of gallic acid in methanol extracts was confirmed in concentrations ranging from 1.3 mg/100 g d.w. (MgSO₄-7H₂O) to 5.6 mg/100 g d.w. (MgCl₂-6H₂O) for the mycelium grown in the Oddoux medium and in fruiting bodies at the level of 2.1-2.2 mg/100 g d.w. The amounts of this compound in the control mycelium and the mycelium enriched with Zn salts were determined in gastric juice extracts in the amount between 1.3-2.0 mg/100 g d.w., while no gallic acid was found in intestinal juice extracts. Another marked phenolic compound was MgCI₂-SH₂O p-hydroxybenzoic acid, which in the mycelium contained about 0.003 mg/100 g of d.w., and in the mycelium obtained on a medium enriched in MgCI₂ 0.1 mg/100 g d.w. The highest amount of p-hydroxybenzoic acid was determined in the mycelium enriched with chlorides, especially ZnCI₂, in the extract 0.4 mg/100 g d.w. and gastric juice 0.23 mg/100 g d.w.

In the case of L-phenylalanine analyses, its content was determined in fruiting bodies, mycelium and mycelium from a culture with a substrate enriched with magnesium sulfate(VI) and chloride and zinc sulfate(VI) and chloride (Tab. 4). The analysis of the content by the RP-HPLC method revealed the presence of L-phenylalanine in the case of methanol extracts, as well as those obtained after extraction in artificial gastric and intestinal juices under conditions imitating the human gastrointestinal tract. The highest amount of L-phenylalanine was found in F. officinalis mycelium extracts obtained in vitro in a medium enriched with Zn salts (502 mg/100 g d.w. for ZnSO₄.7H₂O and 794 mg/100 g d.w. for ZnCI₂). The analysis of extracts of biomass enriched with ZnSCh-ZH₂O to artificial digestive juices showed the release of the largest amounts of L-phenylalanine in gastric juice - 331 mg/100 g d.w., while in intestinal juice - 59.5 mg/100 g d.w. The enrichment of the substrate with Mg salts also increased the content of L-phenylalanine compared to fruiting bodies. L- phenylalanine is a common component of medicinal fungi and is responsible for their health- promoting effect, it is an exogenous amino acid, a precursor of neurotransmitters (adrenaline, serotonin and a building component of proteins). The enrichment of the medium for growing F. officinalis mycelium with the addition of inorganic Mg and Zn salts significantly increased the content of L-phenylalanine in the fortified mycelia, compared to fruiting bodies and unforced mycelium. The highest amounts of L-phenylalanine were determined after adding zinc compounds, and this effect was confirmed after extraction into artificial digestive juices under conditions mimicking those prevailing in the human gastrointestinal tract. Unexpectedly, it was found that in order to increase the amount of L-phenylalanine, the mycelium of F. officinalis should be grown with the addition of inorganic salts of Mg and Zn, selected from MgSO₄-7H₂O, MgCI₂-SfhO, ZnSCVZH₂O, ZnCI₂, preferably ZnSO₄-7H₂O and/or MgCI₂-6H₂O. Phenolic compounds show a strong antioxidant effect, protecting cells against free radica Is. The gallic acid present in medicinal fungi additionally has anti-inflammatory, antibacterial, antifungal and antiviral effects. The addition of inorganic salts to the growth medium of F. officinalis did not adversely affect the content of gallic acid and p-hydroxybenzoic acid in mycelial extracts. Contrary to Mg salt, fortification with Zn salts, especially ZnCI₂, favorably translated into the presence of the above compounds in gastric juices determined by RP-HPLC method.

Based on of the analysis of indolic compounds in the tested extracts, the presence of: 5-hydroxy-L- tryptophan, L-tryptophan, tryptamine, melatonin, serotonin and 6-CH3-D, L-tryptophan was found

(Tab. 4).

The highest amount of 5-OH-L-tryptophan was determined in F. officinalis mycelium obtained in Oddoux medium (518 mg/100 g dry weight). Interestingly, this compound was not determined in the fruiting bodies extract. The addition of Mg and Zn to the media decreased the amount of 5-OH-L- tryptophan in the extracts of such cultures, and a higher level of this compound was detected in the samples with the addition of chlorides, especially MgCI₂-6H₂O (291 mg/100 g d.w.). A lower content of 5-OH-L-tryptophan was found in extracts containing the addition of sulfate salts, with the lowest result for ZnSO₄-7H₂O (117 mg/100 g d.w.). However, the highest amount of 5-hydroxy-L-tryptophan extracted into the gastric juice (177 mg/100 g d.w.) was demonstrated for this additive, with a significantly lower content in the control mycelium not enriched with salt, amounting to 69.4 mg/100 g d.w. In the case of the remaining samples, the mean higher results were again found in the extracts for gastric juices for the CT salt. In the case of intestinal juice extracts, the highest content of 5- hydroxy-L-tryptophan was achieved by the addition of MgSO₄-7H₂O (60.9 mg/100 g d.w.), only in this case the content of this indole compound was higher than in the gastric juice extract. The smallest amount was determined for the mycelium on the Oddoux medium (9.5 mg/100 g d.w.) without the addition of Mg and Zn salts, the results for the remaining salt-enriched extracts ranged from 28-41 mg/100 g d.w.

L-tryptophan content was the highest for fruiting bodies of 70 mg/100 g d.w. In the case of gastric juice extracts, 1.6 mg/100 g d.w. was obtained for mycelia with the addition of MgSO₄-7H₂O and slightly higher for ZnSO₄-7H₂O, (i.e. 3.9 mg/100 g d.w.), in which the content of L-tryptophan in intestinal juices was also determined to be lower by more than a half.

In the case of the remaining analyzed indolic compounds, single determinations for methanolic extracts of mycelium and fruiting bodies were obtained, however, the presence of tryptamine, melatonin, serotonin and 6-CH3-D, L-tryptophan was not detected in the extracts of digestive juices: gastric and intestinal. It should be assumed that they have been efficiently digested and metabolized. Tryptamine in an amount of 4.1 mg/100 g d.w. was determined only in the extract of mycelium grown in a medium enriched with MgCI₂. Serotonin, in turn, only in the mycelium with the addition of MgSCU (46.9 mg/100 g d.w.) and ZnCI₂ (74 mg/100 g d.w.). The enrichment of the substrate is the main factor that influences the presence of these indolic compounds in the analyzed biomass.

Among the tested indolic compounds, the most interesting results were obtained for the serotonin precursors, i.e. 5-hydroxy-L-tryptophan and L-tryptophan. The enriched cultures reduced the 5- hydroxy-L-tryptophan content compared to the control mycelium grown without enrichment. Interestingly, however, the addition of Mg and Zn chlorides as well as Mg sulfate led to an increase in the content of 5-hydroxy-L-tryptophan in gastric and intestinal juices compared to the control. No 5-hydroxy-L-tryptophan was detected in F. officinalis fruiting bodies extract.

L-tryptophan content was the highest in the fruiting bodies extract, i.e. almost ten times higher than in the mycelium grown without enrichment. The remaining extracts of the enriched mycelium showed no or very little L-tryptophan. The salt additions significantly lowered the amount of the compound in the culture process. Thus, inverse proportions of L-tryptophan and 5-hydroxy-L- tryptophan were observed in the mycelium and fruiting bodies of F. officinalis. Since L-tryptophan is metabolized to 5-hydroxy-L-tryptophan, it is possible that in the mycelium this process is more intense in the mycelium of in vitro cultures than in the fruiting bodies of the fungi.

Serotonin was determined only in mycelium enriched with magnesium sulfate(IV) and zinc chloride. Tryptamine, in turn, only in the extract of mycelium grown with the addition of magnesium chloride.

The antioxidant activity determined by the DPPH radical reduction method showed different antioxidant potential of the examined F. officinalis extracts. In the case of mycelia supplemented with salts, the bioelements used in the experiment showed their higher antioxidant activity compared to the extracts from the control (mycelium without salt addition) and from fruiting bodies (Tab. 4). The highest antioxidant activity was found in the mycelium supplemented with ZnSCU. In studies of the antioxidant potential of fungi material extracts for gastric and intestinal juices, it was shown that the substances contained in fruiting bodies and reducing the DPPH radical are not digested and act on a similar level in the obtained digestive juice extracts. For the mycelium supplemented with Zn after extraction into gastric juice, higher antioxidant activity was determined compared to the control and the mycelium supplemented with Mg.

Example 6: Anti-proliferative properties of the fortified mycelium of F. officinalis a) Cell cultures In the study the A549 lung cancer cell line (ATCC^®), the DU 145 prostate cancer cell line (ATCC^®), and A375 melanoma cell lines (ATCC^®) were used. Cells were grown in culture flasks under standard conditions of temperature (37 °C) and CO2 concentration (5%). A549 and A375 cell lines were grown in DMEM medium (Gibco) supplemented with 10% FBS (Gibco) and antibiotics (Lonza); while DU 145 was grown in DMEM-F12 medium (Gibco) supplemented with 10% FBS (Gibco) and antibiotics (Lonza). b) Viability of tumor cells in the presence of extracts from the fortified liquid in vitro cultures of F. officinalis

Cells were seeded in 96-well plates at a density of 5000 cells/well. After 24 hours, cells were incubated with extracts of fortified and unforced F. officinalis mycelium dissolved in DMSO (dry methanol extracts prepared as in Example 4 dissolved in DMSO were used) and the plates were mixed gently.

After 48 hours of incubation, an MTT solution (5 mg/mL) was added to the medium. After another 3 hours, when black formazan crystals appeared at the bottom of the wells, the medium was removed and the formazan was dissolved in DMSO. Absorbance was read on a multi-well reader (Spectra Max iD3, Molecular Devices) at 570 nm. Viability was determined by dividing the absorbance of the experimental wells by the absorbance of the control wells (xl00%). Three separate replications of the experiment were performed. DMSO had no significant effect on viability. IC50 was calculated using GraphPad Prism 7.01.

The extracts showed various effects on the viability of cancer cells. The most pronounced effect was shown by methanol extracts from mycelium MgSO₄-7H₂O and ZnSOrTFhO, active against DU 145 prostate cancer cell lines. In A549 and A375 they showed different, but still the strongest activity. The effect of the mycelial extracts was an IC50 value between 35.54 pg/mL and 83.23 pg/mL.

Thus, the anti-tumor properties of the MgSOWFhO and ZnSOWFhO fortified mycelium was shown to be stronger than for the control, during the culture according to the invention.

Example 7: Preparation of fortified mycelia compositions in the form of a sachet with powder

Two parallel, separate cultures of F. officinalis mycelium (as in Example 2) were performed on medium with ZnSO₄-7H₂O (100 mg/L) and a second culture with MgCI₂-SFhO (3340 mg/L). The obtained mycelium from both the cultures was freeze-dried and ground to a powder. a) The powder was combined in a 1:1 w/w ratio and 2 g of the obtained mixture were placed in sachets. b) In another embodiment, maltodextrin is used as the carrier. The resulting powder was combined in a 1:1 w/w ratio, and placed 2 g in a sachet. c) In another embodiment, 4 g of mycelial powder, 4 g of powdered fiber, 1 g of L-ascorbic acid (vitamin C) in powder form, and 1 g of maltodextrin powdered were weighed and mixed. 2 g of the mixture obtained were placed in sachets.

The contents of the sachets, prepared according to a-c, showed the stability of the quantitative and qualitative composition after being stored at room temperature for one year.

Summary:

The present invention provides an optimized method of obtaining the mycelium of F. officinalis in in vitro conditions, with improved health-promoting properties, which allows for the large-scale production of health-enhancing mycelium, thereby allowing the restoration of this medicinal wood fungus as a component of medicaments, supplements and its use as functional food in the daily functioning of the human body. The presented research proved that enrichment of the liquid culture medium according to Oddoux for large-scale industrial culturing with magnesium and zinc salts, especially MgSO₄-7H₂O, MgCI₂-6H₂O, ZnSO₄-7H₂O, ZnCI₂, preferably ZnSO₄-7H₂O and/or MgCI₂-6H₂O, affects the accumulation of bioelements, macro- and microelements valuable for the human diet, and increases the production of substances with, among others, antioxidant, anti-aging, antiinflammatory and anti-depressant effect, thus enhancing the natural, health-promoting properties of F. officinalis.

Ultimately, based on the conducted research, it was established that the optimal compounds enhancing the biological activity of F. officinalis mycelium, including antioxidant, anti-inflammatory, anti-depressant and anti-tumor effects, especially against lung cancer, prostate cancer and melanoma, also resulting from the activity of Zn, Mg, Cl, S supplementation and delivery of organic compounds produced in the mycelium are MgSO₄-7H₂O, MgCI₂-6H₂O, ZnSO₄-7H₂O, ZnCI₂, salts, preferably ZnSO₄-7H₂O and/or MgCI₂-6H₂O.

The presented studies showed that the MgSO₄-7H₂O, MgCI₂-6H₂O, ZnSO₄-7H₂O, ZnCI₂, preferably ZnSO₄-7H₂O and/or MgCI₂-6H₂O, increase biomass growth compared to the control. The mycelium of F. officinalis in a medium enriched with salts of MgSO₄-7H₂O, MgCI₂-6H₂O, ZnSO₄-7H₂O, ZnCI₂, preferably ZnSO₄-7H₂O and/or MgCI₂-6H₂O causes accumulation of Mg, Zn, Cl and S, which makes it a valuable source for their delivery to the human and/or animal body, and the mycelium grown in this way has more biologically active compounds, including phenylalanine, phenolic compounds (gallic acid, p-hydroxybenzoic acid) with antioxidant activity, indolic compounds such as 5-OH-L- tryptophan, L- tryptophan, tryptamine, melatonin, serotonin than mycelium grown w/ithout such supplements. Unexpectedly, it was also found that more active substances, macro- and microelements and biologically active compounds are extracted into digestive juices in the artificial gastrointestinal model from the mycelium of F. officinalis enriched with the above-mentioned Zn and Mg salts during culture than in the control. F. officinalis mycelium grown on media enriched with MgSO₄-7H₂O, MgCI₂-6H₂O, ZnSO₄-7H₂O, ZnCI₂, preferably ZnSO₄-7H₂O and/or MgCI₂-6H₂0, exhibits anti-tumor, inhibitory properties against lung cancer, prostate cancer and melanoma cell lines, hence it should be considered that the mycelium fortified with salts will show improved properties for the prophylaxis, prevention and support of the treatment of human and animal tumors.

The present invention allows for the production of preparations comprising the fortified mycelium of F. officinalis for the production of medicaments and dietary supplements intended to supplement Mg, Zn, S and Cl deficiencies, simultaneously providing anti-tumor, immunostimulatory, antioxidant properties, stabilizing the nervous system, acting as anti-depressants, acting on the processes of acceleration of skin regeneration, affecting the proper functioning of skeletal muscles and preventing muscle contractions, stabilizing insulin production and preventing type II diabetes, providing neurotransmitters and/or their precursors, regulating the circadian cycle and circadian rhythm of the human body, regulating appetite, improving mood, regulating blood coagulation processes, enhancing the immune response, having cardioprotective properties, showing analgesic properties, preventing depression, treating headaches and insomnia. The preparation and/or composition containing the fortified mycelium of F. officinalis according to the invention is a multi-component dietary supplement providing Mg, Zn, preferably also S, Cl, but also provides exogenous amino acids, including phenylalanine, thus being a suitable meat substitute for vegetarians and vegans, and has an immunostimulating effect on the immune system, e.g., through the b-glucans contained in the mycelium.

LITERATURE

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Claims

1. An efficient method of obtaining the fortified mycelium of Fomitopsis officinalis in liquid in vitro cultures, characterized in that it includes the steps, in which a) a small-scale pre-culture is carried out, in which the liquid medium according to Oddoux with the pH: 5.8±0.5, is inoculated with the biomass of the derived pure culture of F. officinalis and the mycelium is cultured for a period of 10-16 days, more preferably the mycelium is grown for 14 days, at the temperature of 20-30 °C, more preferably at the temperature of 25±2 °C, with shaking in the range of 110-190 rpm, more preferably with shaking at 140 rpm; b) the main, large-scale culture is carried out, in which the liquid medium according to Oddoux, pH: 5.8±0.5, is supplemented with at least one Mg and/or Zn salt, selected from MgSO₄-7H₂O in the range of 1580-2400 mg/L, MgCl₂-6H₂O in the range of 2670-4000 mg/L, ZnSO₄-7H₂O in the range of 80-120 mg/L, ZnCI₂ in the range of 32-48 mg/L or a combination thereof,_, the supplemented liquid medium is inoculated with the F. officinalis mycelium biomass prepared in step a) and the mycelia are grown at the temperature of 20-30 °C, more preferably at the temperature of 25±2 °C, for a period of 8-12 days, more preferably for 10 days, in bioreactors with centrifugal, bottom-up and uniform aeration providing simultaneous mixing of the culture to produce the Mg and/or Zn fortified mycelium of Fomitopsis officinalis.

2. The method according to claim 1, characterized in that the step b) of the main culture is carried out in a medium supplemented with:

MgSO₄-7H₂O in the range of 1780-2180 mg/L, more preferably 1880-2070 mg/L, most preferably 1980 mg/L, and/or

MgCl₂-6H₂O in the range of 3000-3675 mg/L, more preferably 3270- 3500 mg/L, most preferably 3340 mg/L, and/or

ZnSO₄-7H₂O in the range of 90-110 mg/L, preferably 95-105mg/L, most preferably 100 mg/L, and/or

ZnCl₂ in the range of 36-44 mg/L, preferably 38-42 mg/L, most preferably 40 mg/L_, or a mixture thereof.

3. The method according to claim 2, characterized in that the step b) of the main culture is carried out in a medium supplemented with SO₄.7H₂O and/or MgCl₂-6H₂O.

4. The method according to claims 1-3, characterized in that the step a) of the pre-culture and/or the step b) of the main culture is carried out by providing a photoperiod: 14-18 h light, preferably 16 h light, at 700-1100 lx, preferably 900 lx and from 6-10 h without light, preferably 8 h without light.

5. The method according to claims 1-4, characterized in that the mycelium from the pre-culture from the step a) is homogenized before being added to the main culture in the step b).

6. The method according to claims 1-5, characterized in that in the main culture step b), the supply of air with oxygen to the bioreactor and the discharge of air with carbon dioxide are carried out through a system of antibacterial filters.

7. A fortified mycelium of Fomitopsis officinalis produced by the method as defined in claims from 1 to 6, wherein the mycelium of Fomitopsis officinalis is fortified with Mg and/or Zn.

8. A composition, characterized in that it comprises a Mg and/or Zn fortified mycelium of F. officinalis defined in claim 7 and a carrier acceptable for food and/or pharmaceutical and/or veterinary purposes.

9. The composition according to claim 8, characterized in that it is in the form of a powder, granules, paste, drink, functional drink, suspension, tablet, capsule, freeze-dried mycelium.

10. The composition according to claims 8-9, characterized in that it is preferably in the form of a functional food and/or dietary supplement, preferably it is intended for supplying and/or supplementation of the body in diseases and conditions, in which the fortified mycelium of Fomitopsis officinalis acts as an agent with antioxidant, anti-aging, anti-inflammatory, antidepressant, anti-proliferative, anti-tumor effect.

11. The composition according to claims 9-10, characterized in that it is preferably intended to supply and/or supplement the organism with phenylalanine, phenolic compounds, gallic acid, p- hydroxybenzoic acid, 5-hydroxy-L-tryptophan, tryptophan, tryptamine, melatonin, serotonin and 6- CH₃-D, L-tryptophan.

12. A pharmaceutical composition characterized in that it comprises the Mg and/or Zn fortified mycelium of f. officinalis as defined in claim 7 and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition containing the fortified mycelium of Fomitopsis officinalis as defined in claim 7 for use as a medicament for the treatment of tumors, depression, inflammation, signs of aging; a medicament with antioxidant, anti-inflammatory, anti-aging and immunomodulating effect; a medicament with antibacterial, antifungal and antiviral effect; a medicament for supplementing deficiencies of at least one of Mg, Zn, S, Cl, preferably for supplementing deficiencies of Mg and Zn.

14. A pharmaceutical composition for use according to claim 13 characterized in that it is used as a medicament for the treatment and prevention of tumor growth, selected from lung cancer, prostate cancer and melanoma.

15. The use of the fortified mycelium of Fomitopsis officinalis as defined in claim 7 in the biotechnological, cosmetic, food and dietary supplement industries.

16. The use according to claim 15, characterized in that the fortified mycelium of Fomitopsis officinalis is used as a dietary supplement and/or functional food for supplementing the body with Mg and/or Zn.

17. The use of the fortified mycelium of Fomitopsis officinalis as defined in claim 7 for the treatment and prevention of tumor growth, preferably selected from lung cancer, prostate cancer and melanoma.

18. A dietary supplement containing the fortified mycelium of Fomitopsis officinalis characterized in that it contains the Mg and/or Zn fortified mycelium of F. officinalis as defined in claim 7 in an amount of 0.01% to 100%, wherein the dietary supplement is intended for supplementing deficiencies of Mg and Zn.

19. The use of the fortified mycelium of Fomitopsis officinalis as defined in claim 7 as a dietary supplement intended for supplementing deficiencies of Mg and Zn.

20. The use of the fortified mycelium of Fomitopsis officinalis as defined in claim 7 as a dietary supplement intended for supplementing deficiencies of phenylalanine, phenolic compounds, gallic acid, p-hydroxybenzoic acid, 5-hydroxy-L-tryptophan, tryptophan, tryptamine, melatonin, serotonin and 6-CH₃-D, L-tryptophan in an individual.