Liquid culture medium with enriched formulation for the purpose of creating liquid spawn and developing Psilocybe medicinal mushroom culture with optimal production capability of the medicinal substance psilocybin

Abstract

Liquid culture medium with an enriched formulation for the purpose of creating liquid spawn and developing the cultivation of Psilocybe medicinal mushrooms with the ability to optimally produce the medicinal substance psilocybin has industrial, manufacturing, pharmaceutical and research applications. Considering the importance of medicinal mushrooms belonging to the genus Psilocybe in the production of the active medicinal substance psilocybin, in the present invention, the production of mushroom biomass with high efficiency has been made possible by designing a liquid culture medium with an optimally defined composition of amino acids, sugars and mineral compounds for the propagation of mycelium and the production of liquid mushroom spawn.

Description

Description of the invention Title of the invention Liquid culture medium with enriched formulation for the purpose of creating liquid spawn and developing the cultivation of medicinal Psilocybe mushrooms with the ability to optimally produce the medicinal substance psilocybin liquid culture medium with enriched formulation for creating liquid spawn and developing cultivation of Psilocybe mushrooms with the ability to optimally produce the medicinal substance psilocybin Technical background of the relevant invention This invention relates to the production of a liquid culture medium with suitable physical and chemical properties for cultivating psilocybe mushrooms, which is based on amino acids and sugar compounds and can be replaced with conventional solid culture medium (cereal grains) for spawn production. This invention relates to the field of producing medicinal products based on medicinal mushrooms. Technical problem and stating the objectives of the invention Mental health disorders account for 7.4% of the total burden of disease. It is estimated that 1 billion people worldwide suffer from a mental health disorder, which, with depression and anxiety being the two most common disorders, costs the global economy $1 trillion annually. The COVID-19 pandemic has had a significant impact on mental health and well-being. Looking ahead, the increased demand for mental health care after the pandemic is over indicates an urgent need to strengthen clinical capacity by integrating new and cost-effective approaches into existing systems. Studies have shown that among hospitalized COVID-19 patients, approximately 96% of patients suffer from symptoms of posttraumatic stress disorder. Studies among intensive care unit (ICU) patients with COVID-19 show high rates of posttraumatic stress disorder (PTSD), depression, and anxiety (30–40%) that persist for months after discharge. The COVID-19 pandemic has significantly changed lives in ways that exacerbate mental health problems due to increased grief, social isolation, economic and housing instability, and reduced access to critical services associated with quarantine. In addition, available data on the effects of COVID-19 on substance use patterns indicate an increase in alcohol and other substance use in response to stress and negative emotions. The mental health problems that will be left behind by COVID-19 require innovative solutions to address the rising rates of PTSD, depression, anxiety, addiction, and other psychiatric disorders. There is significant and growing evidence of the therapeutic potential of the drug psilocybin for the treatment of PTSD, depression, anxiety, and substance use disorders. Given the importance of this drug in the treatment of psychiatric disorders, it is important to produce it with the highest efficiency and quality, and in a cost-effective manner. Today, the production of psilocybe mushrooms, with their high medicinal properties for promoting mental health, has been the focus of attention and emphasis by many executive and research centers, including the World Health Organization (WHO). The existence of valuable medicinal compounds psilocin, psilocybine, and psilocetin, as well as the demonstration of their functional properties on humans in numerous clinical studies, has led to them receiving more and more attention from pharmaceutical companies. A total of 12 genera of mushrooms contain psilocybin, with 115 species discovered in the genus Psilocybe alone. These include P. azurescense, P. semilanceata, P. baeocystis, P. bohemica, and P. cubensis, all of which can produce psilocybin. One of the factors that is important in the production of the active pharmaceutical ingredient psilocybin and other pharmaceutical compounds from medicinal mushrooms is the quantitative and qualitative characteristics of the medicinal mushroom that produces it. The quantitative and qualitative characteristics of medicinal mushrooms are influenced by various factors such as: species, strain, characteristics of the mycelium and fruiting body, physicochemical characteristics of the culture medium, and their genetic characteristics. One of the most important non-genetic factors affecting the quantitative and qualitative characteristics of medicinal mushrooms is the characteristics of the culture medium. Direct mushroom cultivation is dependent on mycelium. The physical and chemical properties of the culture medium must be appropriate for the type of mycelium organ. Important environmental factors affecting the growth of mushroom mycelium include nutrients (carbon and nitrogen sources), pH of the culture medium, oxygen concentration, and humidity. Spawn is an intermediate nutrient medium that can maintain mushroom mycelium for a long time. Spawn production requires a suitable nutrient medium in which the mushroom mycelium can grow under controlled environmental conditions. Spawn quality is one of the most important factors affecting the final product in the production of mushrooms and their metabolites. Cereal grains are generally used in the production of psilocybe mushroom species spawn, the most important of which is wheat. Despite the widespread use of cereal grains and solid culture media in spawn production, contamination of spawn is one of the most important problems during mushroom cultivation. Cereal grains are a rich source of carbohydrates and are therefore susceptible to contamination, especially with pathogenic species of the fungus Trichoderma.This pathogen is capable of spreading on a large scale and grows rapidly in a suitable environment, often causing heavy damage and many losses in the final products of medicinal mushrooms. Another major disadvantage of using cereal grains for spawn production is the change in nutrient composition, water content, osmotic process and even pH of cereal grains during spawn formation. Therefore, one cannot expect a controlled and uniform combination of culture conditions during the spawn growth process. Therefore, it is important to create a culture environment that consists of defined food sources, its conditions are controllable and leads to high-yield biomass production. The main goal of the present invention is to create a liquid culture medium with an optimally defined combination of carbon and nitrogen sources, in order to achieve maximum production of psilocybe mushroom mycelium and develop liquid spawn with the ability to optimally produce mushrooms and increase the production efficiency of psilocybin. Other objectives of this invention include: Reducing the production costs of Psilocybe medicinal mushrooms by eliminating the spawn production stage in a solid culture medium containing grains and increasing the distribution of mycelium by using liquid spawn through complete and uniform penetration and spread of mycelium in the mushroom fruiting culture medium and mass production of mushroom fruiting organs in a practical and cost-effective manner are other objectives of the present invention. A description of the state of the prior art and the history of developments related to the claimed invention. Mushroom growth requires environments containing a high carbohydrate source, a rich nitrogen source, in the pH range of 5 to 6, and a temperature range of 15 to 37 degrees Celsius. There are two general types of culture media for growing mushrooms: natural and synthetic. Natural media consist of natural substrates such as grass or woody stems, seeds, leaves, cornmeal, wheat germ, and oatmeal, etc. Natural media are usually easy to prepare, but have an undefined and unknown composition. Such as: Corn agar, potato dextrose agar, V-8 agar. On the other hand, synthetic culture media contain materials with a known composition. These types of culture media contain specific amounts of carbohydrates, nitrogen and vitamin sources and Czapek-Dox, glucose-asparagine and Neurosporacrassa minimal culture media fall into this category which are used for specific cultivation of fungi. The general media commonly used for cultivation of various fungi is Sabouraud dextrose agar (SDA) which is nutritionally poor with an acidic pH (5.6). Cereal seeds are generally used to prepare the spawn of psilocybe species. Currently, in various studies and inventions, wheat, millet, and rye seeds are used to cultivate this medicinal mushroom species. In the study by Klára Gotvaldová et al., the mushroom spores were first grown in a dextrose agar medium, and then, to form the spawn, the mycelium was inoculated into a sterilized hydrated wheat substrate in glass jars and incubated at 28°C, and then the desired spawn was transferred to a fruiting culture medium. In the patent (US3183172A), to prepare mushroom spawn, a compost consisting of fermented wheat straw and a mixture of corn leaves and corn stalks or wild grass stems was poured into clay containers and the compost was inoculated with mycelium from initial cultures on agar and incubated for approximately two weeks at 24-27°C. In traditional and old methods, mycelium is grown in solid culture medium and the separated spores are added to the culture medium and after going through the growth stages, the fungal mycelium develops and fungal colonies are created in the culture medium medium. Recently, liquid culture medium is used for wider colonization of mycelium. In this method, the fungal mycelium grows and develops in liquid culture medium and in bottles or glass vials (Friel et al., 2000). The mycelium expanded in liquid culture medium is used as an intermediate culture medium for inoculation into the grain culture medium to prepare spawn, then the spawn expanded in the grain medium is transferred to the culture medium to produce fruiting bodies. These culture media often contain malt extract or potato extract along with other sugar compounds and some salts with undefined compounds that contain undefined amounts of carbohydrates, nitrogen and vitamin sources. In none of the studies has a defined liquid culture medium been used to prepare spawn and transfer it to the culture medium to produce fruiting bodies. Liquid spawn is easy to control, disperse, and store because it is liquid. In addition, the use of liquid spawn allows the design of an automatic system for transferring liquid spawn to the fruiting medium, which can minimize contamination and enable automatic inoculation for industrial-scale production. Liquid spawn is more evenly dispersed in the medium than solid spawn, which is why a high volume of mushroom biomass can be achieved in the cultivation and breeding of psilocybe medicinal mushroom species. One of the most important prerequisites for the growth of mushrooms in liquid cultures is defined and controllable nutritional and environmental conditions. Liquid mushroom cultivation has the advantage of producing large amounts of mycelium in a short period of time and at any time of the year. In addition, since the pH, type of food and concentration of nutrients in the defined liquid culture medium can be controlled, the final mushroom product is produced in a quality and uniform manner. On the other hand, since industrial-scale spawn production requires expensive equipment, liquid spawn can be available and cost-effective for mushroom producers and related pharmaceutical industries. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention How to make a liquid culture medium with an enriched formulation with the appropriate ratio of carbon and nitrogen sources for mycelium propagation and mushroom liquid spawn production To prepare one liter of liquid culture medium with enriched formulation, the necessary compounds including amino acids and sugars are dissolved in 500 ml of autoclaved deionized water in the amounts mentioned in Table 1 in the map file, without increasing the temperature. Then, 50 microliters of 1M calcium chloride, 250 microliters of thiamine hydrochloride, 5 microliters of ferrous sulfate, and 500 microliters of 1M magnesium sulfate are added to the above solution and the volume is brought to one liter with autoclaved deionized water. After preparing the culture medium, appropriate antibiotics should be selected from the group of classical beta-lactam antibiotics (penicillin, e.g. ampicillin, amoxicillin, carbenicillin), subgroups of beta-lactam antibiotics such as cephalosporins (e.g. cefotaxime), tetracyclines, sulfonamides, macrolides, quinolones, aminoglycosides, nitrofurans, or any other group of antibiotics that are effective against gram-negative or gram-positive bacteria and added to the culture medium. The resulting culture medium is passed through a 0.2 micron filter after preparation. Propagation of mycelium and preparation of mushroom liquid spawn Liquid spawn is a solution enriched with mushroom mycelium. The liquid spawn solution can be inoculated into the culture medium to produce mushroom fruiting bodies and also serves as a nutrient-rich culture medium to maintain the mycelium for a long time. To produce liquid spawn from psilocybe mushrooms, first the Psilocybe mushroom spores are transferred to an 8 cm Petri dish containing fresh commercial PDA (Potato-Dextros-Agar) solid culture medium. The agar culture medium containing the mushroom spores is kept at 25°C in the dark for 14 days until the spores transform into mycelium and grow well in it. The germinated mycelium is then transferred to a liquid culture medium with an enriched formulation and cultivated for 14 days at 25-28°C, specifically at 27°C, until the mycelium covers the entire liquid culture medium. Also, to facilitate the distribution of nutrients and gas exchange, the culture medium is shaken constantly at 30 rpm. Alternatively, the mycelium culture can be carried out in a bioreactor that allows for a high surface area to liquid volume ratio or gas exchange. Double layer culture for mushroom fruiting body production First, the ingredients necessary for preparing the culture medium include: compost, dead sphagnum moss tissues, perlite and are autoclaved at 121 degrees for 99 minutes. Then, the fruiting boxes are filled halfway with compost and perlite in a ratio of 5 to 1, then liquid spawn is spread on its surface and mixed completely with the culture medium, and compost is poured on it again and its surface is covered with dead sphagnum moss tissues. Then, the culture boxes are placed in a dark room with 90-100% humidity for 14 days. When pinning begins, the culture medium is exposed to light with a wavelength of 460 nm and 100% humidity at 8-hour intervals. Before the radial grooves open, the highest amount of psilocybin is present in the mushroom, so the mushroom is harvested at this time. Comparison of mushroom biomass production and psilocybin production using grain spawn and liquid spawn In order to confirm the effect of the enriched formulation liquid culture medium on increasing mushroom biomass production and psilocybin production, two common methods for preparing psilocybin mushroom spawn were compared with the method in the present invention. In the first method, the mycelium of the medicinal mushroom psilocybin was colonized in cereal spawn and then transferred to the fruiting medium. In the second method, the potato dextrose intermediate liquid culture medium was used to expand the mycelium, and then the propagated mycelium was colonized in the cereal culture medium to produce spawn, and then the cereal spawn was inoculated into the fruiting medium. In the third method, the germinated mycelium in an agar plate was inoculated into the enriched formulation liquid culture medium to produce liquid spawn, and then the liquid spawn was inoculated into the fruiting medium, and the amount of mushroom biomass and the amount of psilocybin production in these three methods were compared with each other. The results indicated a significant effect (p<0.05) of the enriched liquid culture medium on the growth of mushroom mycelium in spawn production, increase in mushroom biomass, and production of psilocybin.Based on the results and observations presented in Figure 1 in the map file, the liquid culture medium with enriched formulation for spawn production shows significant results in increasing mushroom biomass and psilocybin production. Explanation of shapes, maps and diagrams Table 1: Elements and components of liquid culture medium with enriched formulation for producing liquid spawn of Psilocybe mushrooms Figure 1: Comparison of the effects of different spawn culture media on mushroom biomass and psilocybin production. Figure 2: The steps for cultivating and growing psilocybe medicinal mushrooms using liquid spawn can be seen in the form of a flowchart and image in the map file. A clear and precise statement of the advantages of the claimed invention over prior inventions. The present invention is a rich and efficient culture medium for producing liquid spawn of psilocybe mushroom species, which can be used in a continuous culture cycle. The mycelium colonized in the culture medium introduced in the present invention can be continuously produced in laboratory and industrial conditions and directly transferred to create new cultures for the production of fruiting bodies, thereby enabling mass and cost-effective production of mushrooms. Cultivation of mycelium of various species of psilocybe mushrooms. It is carried out in enriched liquid culture medium. The volume and composition of the liquid medium in the continuous mycelium cultivation method and liquid spawn production can be carried out in any suitable culture vessel capable of maintaining the desired amount of liquid culture medium. The volume of liquid culture medium in the mycelium cultivation system of the present invention is preferably 200-400 ml on a laboratory scale, but can easily be increased to several liters or several thousand liters on an industrial scale. The use of the liquid culture medium defined in the present invention for mycelium colonization and liquid spawn formation leads to reduced mushroom production costs, quick and easy spawn production, less space required for production, rapid colonization, easy inoculation, and easy integration and distribution in the culture medium for mushroom fruiting. The mycelium colonized in the enriched liquid culture medium of the present invention is capable of growing, multiplying, and maintaining for two years at ambient temperature and sterile conditions, and can be used whenever needed to create highly efficient fruiting bodies. The liquid culture medium with the optimally defined composition, in the present invention, leads to improved mushroom growth performance and shortens the minimum time required for the production of mushroom mycelium into fruiting bodies. The mycelium grown in the enriched liquid culture medium successfully colonizes and produces the compounds psilocybin and psilocin, which were successfully identified using HPLC analysis. In this process, a high yield of psilocybin is produced from the psilocybe mushroom. Description of at least one implementation method for implementing the invention To use the enriched liquid culture medium for producing liquid spawn, dissolve 50 grams of the prepared culture medium powder in one liter of water and mix well without heat using an air stirrer until a uniform mixture is obtained. Then pass through a 0.2 micron filter and transfer to an Erlenmeyer flask or fermenter for use. To form liquid spawn, inoculate the culture medium with the germinated mycelium of the fungus. The inoculated mycelium in the liquid culture medium is propagated for 14 days at a temperature of 27°C and on a shaker at a speed of 30 rpm. The mushroom fruiting medium is prepared using a mixture of compost, sphagnum moss and perlite. The culture medium is mixed with the liquid spawn of the medicinal mushroom psilocybe and the culture containers are kept in the dark for 2 weeks at a temperature of 25 degrees and 100% humidity. After the start of pinning, the culture medium is exposed to light with a wavelength of 460 nm at 8-hour intervals. After fruiting and before the radial grooves of the mushroom open, the mushroom is harvested. Explicit mention of the industrial application of the invention This invention provides a liquid culture medium with a formulation enriched with appropriate proportions of carbon and nitrogen sources for the production of liquid spawn and the cultivation of Psilocybe species of mushrooms with the aim of optimal production of the active ingredient psilocybin. The culture medium introduced in the present invention has the potential to be used with industrial application in a fermenter for the mass and cost-effective production of liquid mushroom spawn. Liquid culture medium with enriched formulation is in liquid form. For industrial and mass production of this culture medium, the necessary ingredients can be dissolved in appropriate proportions in autoclaved deionized water and then this liquid culture medium can be prepared, produced and packaged under sterile conditions, then stored in a refrigerator and delivered to the relevant pharmaceutical industries as needed. In the second method, the necessary ingredients can be prepared in the appropriate ratio in solid form (prepared culture medium powder) and provided to the pharmaceutical industry in the form of a ready-made powder. Then, the relevant industries can dissolve the powdered product in water for use and use it to produce liquid spawn. Also, for industrial and mass production of mushrooms, liquid spawn propagated in liquid culture medium is packaged under sterile conditions and made available to the pharmaceutical industry. By adding the prepared liquid spawn to the fruiting cultivation substrate (including compost, perlite, sphagnum moss in appropriate proportions) and placing the substrate in the conditions mentioned in the present invention, two kilograms of mushrooms can be harvested per square meter of cultivation per day. Using the culture medium mentioned in the present invention, bulk production of liquid spawn from various species of psilocybe mushrooms can be produced as an inoculant on an industrial scale and supplied to the pharmaceutical industry.

Claims

Claims What is claimed: Claim 1) What is claimed is a liquid culture medium with an enriched formulation for the propagation of mycelium and the production of liquid spawn of the medicinal mushroom species Psilocybe. This culture medium is a liquid solution that consists of three main parts. The first part consists of amino acids, which include (aspartate, serine, threonine, glycine, proline, alanine, histidine, lysine, glutamate, arginine, valine, tyrosine, leucine, isoleucine, phenylalanine, tryptophan). The second part consists of sugars, which include (glucose, fructose, maltose and sucrose). The third part consists of mineral salts, which include (magnesium sulfate, ferrous sulfate, calcium chloride and thiamine hydrochloride). These materials are in the form of solid powders that are dissolved in autoclaved deionized water without increasing the temperature, in the proportions mentioned in the table, and form a solution. Claim 2) According to claim 1, the proliferation of mycelium in a liquid culture medium with an enriched formulation leads to the formation of liquid spawn, which is used to transfer the reproductive organ to the fruiting culture medium.

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