SI20923A - Process of cultivation of fungus ganoderma lucidum on a solid cultivation substrate - Google Patents

Abstract

The invention refers to a new process of cultivation/growing of the fungus Ganoderma Lucidum on a solid cultivation substrate using the so-called "solid-state" cultivation in a horizontal stirred bioreactor. Beech sawdust is used as a solid cultivation substrate. The process enables a precise leading and monitoring of the fungus growth at sterile conditions. Great quantities of fungus biomass can be prepared according to the process to yield products applicable to pharmacy. The fungus biomass can be used also as a solid inoculum for the further cultivation of the fungus.

Description

Cultivation process of Ganoderma lucidum on solid medium

Technical field of the invention

The present invention relates to the field of biotechnology, in particular to a new method of cultivation / cultivation of Ganoderma lucidum on solid medium, with so-called "solid-state" cultivation, in a horizontal mixing bioreactor. Beech sawdust is used as a solid medium. The procedure allows accurate control and monitoring of fungal growth under sterile conditions. The process can produce large amounts of fungal biomass, which makes the products useful in pharmacy. Fungal biomass can also be used as a solid clamp to further grow the fungus.

The state of the art

Ganoderma lucidum (Curtis ex Fries) Karsten is a mushroom that has been used by various peoples for centuries, mainly for medicinal purposes. Because of its healing effects, many names have been given to it. The Japanese call it Reishi or Mannentake, which means a ten-year-old mushroom. The Chinese and Koreans named it Ling Chi, Ling Chih, or Ling Zhi because it is a sponge of immortality for them. It is traditionally associated with dignity, health and recovery, long life, sexual activity, wisdom and happiness.

In nature, Ganoderma lucidum grows as an annual in different trees. It grows primarily on deciduous trees (oak, maple, elm, willow, magnolia, acacia; in the East in plums). In the southeastern and southwestern parts of America, it is most common in oak forests and the northeastern parts of America on maple. It occurs less frequently on conifers (Stametes, P. 1993. Grovving gourment and medical mushrooms. A companion guide to the mushroom cultivator. Berkeley, Ten speed Press, pp. 355-369).

Due to the many substances contained, such as: polysaccharides, triterpenoids, proteins, adenosine and adenosine derivatives, RNAs that have a healing effect are mainly used in pharmacy. The market products of Ganoderma lucidum are used in the form of syrups, injections, tablets, capsules, tinctures, pills, teas and in combination with honey. They are used to treat various diseases: neurasthenia, indigestion, insomnia, chronic hepatitis, kidney inflammation, high serum cholesterol, hypertension, leukocytopenia, rhinitis, chronic bronchitis, angina, bronchial asthma, gastropathy, duodenal ulcer, duodenal ulcer, SP, Weis, AL 1997. Medicinal mushrooms - Ganoderma lucidum, Reishi mushroom Haifa, Pedeifus Publishing House, 29, Li, P., Xu R., Xia J., Zhang K., Production of tetracyclic triterpenoid acids by submerged fermentation of Ganoderma lucidum, Gongye VVeishengvvu (2000), 30 (1), 15-17).

G. lucidum and its active substances are also used in the treatment of the most dangerous diseases, cancer and AIDS today (Lee, S.-Y., Kang T.-S, Structural analysis of the antitumor active exo-polysaccharide produced by submerged cultivation of Ganoderma lucidum mycelium. Hariguk Kyunhakhoechi (1999), 27 (1), 76-81).

Many polysaccharides have antitumor and immunostimulatory activities (Lee, S.-Y., Kang T.-S, Structural analysis of the antitumor active exopolysaccharide produced by submerged cultivation of Ganoderma lucidum mycelium. Hariguk Kyunhakhoechi (1999), 27 (1). 76-81). They act cytotoxically on malignant cells and stimulate and strengthen the immune system (Mizuno, T. Wang.,

G., Zhang, J., Kavvagishi, H., Nishitoba, T., Li, J. 1995. Reishi, Ganoderma lucidum and Ganoderma tsuage: Bioactive substances and medicinal effects, Food Revievvs International, 11, (1), p. 151-166.).

In addition to these, isolated substances have other activities: cytotoxic, immunomodulatory (Liu, GT, Bao, TT, Niu, Χ.Υ., Li, SZ, 1979. Some pharmacological actions of the spores of Ganoderma lucidum and Ganoderma capense (Loyd) Teng cultivated by submerged fermentation, Chin Med. J., 92, pp. 496-500; Berovich, M., Boh B., Hodzar D., Pohleven F., Zore I., Submerged culture of the fungus Ganoderma lucidum and process for the Isolation of Active Agents, Ref.Consultations of the Consultation of the Letter of Chemistry Dnevi (1997), 695- 700, Editor (s): Glavic, Peter;

Faculty of Chemistry and Chemical Technology, University of Maribor, Maribor, Slovenia., Hypoglycemic (Hikino, H., Ishiyama, M., Suzuki, Y., Konno, C., 1989. Mechanism of Hypoglemic activity of Ganoderan B: Glycan of Ganoderma lucidum fruit bodies, Planta Medica, 55, pp. 423-428; Hodzar D., Boh, B., Berovich M., Habijanic J., Pohleven, F., Isolation and characterization of polysaccharides from the fungus Ganoderma lucidum cultured by submerged and solid - State fermentation, Ref Reflections on the Slov. Chem. Days (1998), 704-709. Editor (s); Glavič P., Brodnjak-Vončina D., Publisher: Faculty of Chemistry and Chemical Technology, University of Maribor, Maribor, Slovenia.), Radiation-Protective, Hypotensive (Kabir, Y., Kimura, S., Tamura, T. 1988. Dietary effect of Ganoderma lucidum Mushroom on Blood Pressure and Lipid Levels and Sponataneously Hypertensive Rats (SHR). Nutr. Sci. Vitaminol., 34, pp. 433-438; Morigiwa, A., Kibatake, K., Fujimoto, Y., Ikekavva, N. 1986. Angitensin convert ing enzyme-inhibiting triterpenes from Ganoderma lucidum. Chem. Pharm. Buli., 34, p. 3025-3028.), Hypocholesterolemic (Jong, SC, Birmingham, JM, 1992. Medicinal benefits of mushrooms Ganoderma, Adv. Appl. Microbiol., 37, pp. 101-134.) And others (VVasser, SP, Weis , AL 1997. Medicinal mushrooms - Ganoderma lucidum, Reishi mushroom. Haifa, Pedeifus Publishing House, p. 29; Li et al. 2000).

Except for classic medicines, the active ingredients of Ganoderma lucidum are also found in so-called over-the-counter or OCD medicines. The numerous successes of using active ingredients based on Ganoderma lucidum are the main cause of intensive research especially in Asia (Japan, China, Korea, Malaysia, Singapore, ...) where the production of active biomass Ganoderma lucidum and its active ingredients are products of strategic importance (Li , P., Xu R., Zhang K., Medium optimization for producing extrapolysaccharide by submerged fermentation of Ganoderma lucidum, Wuxi Qinggong Daxue Xuebao (1998), 17 (4), 26-30; Li, P., Xu R. , Xia J., Zhang K., Production of tetracyclic triterpenoid acids by submerged fermentation of Ganoderma lucidum, Gongye Weishengwu (2000), 30 (1), 1517; Li, Chun; Hong, Zhou; Zhu, Daohong; Li, Hui. Submerged fermentation of ediblefungi Shihezi DaxueXuebao, Ziran Kexueban (1998), 2 (2), 121- 124. Morigivva, A., Kibatake, K., Fujimoto, Y., Ikekawa, N. 1986. Angitensin converting enzyme-inhibiting triterpenes from Ganoderma lucidum. Chem. Pharm. Buli., 34, pp. 3025-3028).

US 5,721,134 describes the Ganoderma lucidum strain KCCM 10045, which produces proteoglycan (G009), which causes immunity to tumors. The cultivation of the fungus takes place only in a liquid, shaken medium.

US 5,744,138 describes the separation of organo-germanium compounds and inorganic germanium compounds from germanium-containing plants or products thereof. They also use the fungus Ganoderma lucidum. The fungus is grown on a solid medium, ie wood chippers, but only in polyethylene bags.

Cultivation of the fungus on solid medium but exclusively in polystyrene bags, chokas and logs is described in Wang, Qian; Ji, Hong; Wang, Hong; Chen, Venjie; Bi, Kewei; Li, Zongwu; Gu, Yajun, Preparation and analysis of the nutrient solution of Ganoderma lucidum by fermentation. Hebei Daxue Xuebao, Ziran Kexueban (1999), 19 (1), 50-53.

US 5,756,318 describes fungal polysaccharides and their preparation. The applications only mention the cultivation of the fungus in a liquid medium.

US 6,041,544 extensively describes the preparation of stubs for growing mushrooms on various media. However, it does not mention the conditions for cultivation of Ganoderma lucidum, nor does it address the quality of cultivated mushrooms as potential pharmaceutical raw materials.

US 6,197,573 deals with solid-state fermentation, but only from the standpoint of fermenter construction, and does not provide any information on the cultivation of Ganoderma lucidum for the production of pharmaceutical products.

Therefore, we can conclude that in the review of the professional and patent literature, we did not find any publications on the cultivation of the fungus Ganoderma lucidum or on the preparation of its inoculum in a bioreactor on a solid medium based on beech sawdust.

Description of solution to a technical problem with implementation examples

Now we have found a new procedure for growing Ganoderma lucidum on solid medium, that is, beech sawing. The process of the present invention makes it easy to prepare a large amount of fungal biomass in the form of the mycelium of the fungus Ganoderma lucidum, which can be used as a raw material for pharmaceutical products, or as a solid inoculum for the further cultivation of a fructified fungus-mushroom. The hook so prepared is suitable for inoculating sterile beech sawing.

The procedure allows accurate control and monitoring of fungal growth under sterile conditions. The process allows working in modern, controlled, sterile conditions in which we quickly produce the desired form of biomass or large amount of clamp / inoculum that can be used for further cultivation on beech sawing.

In the procedure, the fungus was used as a fungus strain Ganoderma lucidum Gal 4, deposited on April 15, 1996 at the depository institution BFWS (Microbiological Collection of the Department of Forestry at the Biotechnical Faculty, Ljubljana, Slovenia).

According to the subject process, we first prepare a vegetative inoculum of the fungus submerged on a shaker and multiply it in a submerged bioreactor, then transfer it to a horizontal mixing bioreactor where, on a solid medium based on complex natural ingredients, ie beech sawing (hydrocarbon source), glucose, olive oil and inorganic salts, fungal biomass growth and fungal biosynthesis take place.

The process of growing Ganoderma lucidum on solid medium in a horizontal mixing bioreactor is much simpler and more economical than the procedures described so far. Sterilization of the substrate takes place inside the bioreactor, so no additional apparatus for sterilization of the substrate is needed. Substrate inoculation is very easy. The conditions for the cultivation of the fungus (temperature, humidity, ρθ ₂ , PCO2) are controlled, so the production of biomass and products can be guided in the desired direction. Substrate mixing is possible, with more homogeneous substrate conditions between cultivation and homogeneous product. During cultivation, sampling and monitoring of fungal growth and product production are possible. Infections are very rare as both inoculation and cultivation are performed under sterile conditions. According to the presented process, large quantities of fungal biomass and products can be produced in a very short time, while utilizing secondary raw materials.

The process of the invention is carried out such that

a) prepare a solid substrate of 600 g to 1000 g, preferably 800 g of beech sawing, moistened with 1.2 L to 1.8 L, preferably 1.5 L of distilled water containing 60 g to 100 g, preferably 80 g of glucose , from 40 g to 80 g, preferably 50 mg (NH ^ SCU, from 150 mg to 250 mg, preferably 200 mg KH2PO4, from 25 mg to 75 mg, preferably 50 mg CaCI ₂ X 2H ₂ O, from 30 mg to 60 mg, preferably 50 mg MgSCU X 7H2O, from 120 to 180 mg, preferably 150 mg FeSC> 4 X 7H2O;

b) using the obtained wet sawing, fill the bioreactor and sterilize for 45 minutes to 90 minutes, preferably 60 minutes, at a temperature of 110 to 130 ° C, preferably 121 ° C (394 K), at a pressure of 1 to 1.4 Ί0 ⁵ Pa, preferably 1.2 10 ⁵ Pa;

c) cooled to 25 to 35 ° C, preferably 30 ° C, and the substrate is sterile inoculated with 1.5 L to 2.5 L, preferably 2 L, of the liquid clamp prepared according to the procedure described in Example 3 of our patent Sl 9700014 (Dawn , I., Berovich, M., Hodzhar, D., Boh, B., Pohleven, F. 1997, The submerged cultivation of Ganoderma lucidum in a laboratory bioreactor);

d) then grow the fungus 40 to 55 days, preferably 49 days, at a temperature of 25 to 35 ° C, preferably 30 °, with the first week of cultivation having an air flow of 0 to 1 liter / minute, preferably 0.5 liters / minute, for the next three weeks, 1 to 5 liters / minute, preferably 2 liters / minute, substrate mixing takes place at 50 to 150 rpm, preferably 80 rpm 1 to 4 minutes, preferably 2 minutes, with the first week of cultivation being mixed once a day to once every four days, preferably once every two days, and the next three weeks once a day to once every two days, preferably once a day.

At the end of the cultivation of the fungus, the amount of dry biomass produced is 0.5 mg glucosamine / g dry weight of the sample and polysaccharides is 5.77 mg / g.

The present invention is explained, but not limited to, by the following embodiments.

In all the following Examples, the cultivation conditions described above were analogous, the air flow in the first case being 5 L / min, in the second case the flow was 2 L / min, at air humidifier connected.

The first example

The solid substrate, which was inoculated with the mycelium, was prepared by soaking 800 g of beech sawdust with 1.5 liters of distilled water containing 80 g of glucose, 50 mg (NH ₄ ) ₂ SO ₄ , 200 mg KH2PO4, 50 mg CaCl2 X 2H ₂ O, 50 mg MgSO ₄ X 7H ₂ O, 150 mg FeSO ₄ X 7H ₂ O. With wet sawing, the bioreactor was filled and sterilized from 45 minutes to 90 minutes, preferably 60 minutes at 110 to 130 ° C. , preferably 121 ° C (394 K) and a pressure of 1 to 1.4 Ί O ⁵ Pa, preferably 1.2 -10 ⁵ Pa.

After cooling to 30 ° C, the substrate was sterile inoculated with 2 L of the liquid clamp prepared as described in Example 3 of our patent Sl 9700014 (Zore, I., Berovic, M., Hodzar, D., Boh, B., Greedy , F. 1997, The submerged cultivation of Ganoderma lucidum in a laboratory bioreactor). Cultivation is carried out at a temperature of 30 °. During the first week of cultivation, the flow rate was 0.5 liters / minute, the following weeks 5 liters / minute. Substrate stirring was performed at 80 rpm for 2 minutes. The first week of cultivation was mixed once every two days and the following weeks once a day.

Cultivation of the fungus by this procedure lasted 35 days. The substrate dried after 35 days. The amount of dry biomass produced at the end of cultivation was 0.21 mg glucosamine / g dry weight of the sample and polysaccharides 1.27 mg / g.

Another example

The solid substrate inoculated with mycelium was prepared by soaking 800 g of beech sawdust with 1.5 liters of distilled water containing 80 g of glucose, 50 mg (NH ₄ ) ₂ SO ₄ , 200 mg KH ₂ PO ₄ . 50 mg CaCI ₂ X 2H ₂ O, 50 mg MgSO ₄ X 7H ₂ O, 150 mg FeSO ₄ X 7H ₂ O. With humid sawing, the bioreactor was filled and sterilized from 45 minutes to 90 minutes, preferably 60 minutes at 110 to 130 ° C, preferably 121 ° C (394 K) and a pressure of 1 to 1.4 ΊO ⁵ Pa, preferably 1.2 -10 ⁵ Pa.

After cooling to 30 [deg.] C., the substrate was sterile inoculated with 2 L of the liquid clamp prepared as described in Example 3 of our patent Sl 9700014 (Zore, I., Berovic, M., Hodzar, D., Boh, B., Pohleven, F. 1997, The submerged cultivation of Ganoderma lucidum in a laboratory bioreactor). The cultivation was carried out at 30 ° C. During the first week of cultivation, the flow rate was 0.5 liters / minute, the following weeks 2 liters / minute. Substrate stirring was performed at 80 rpm for 2 minutes. The first week of cultivation was mixed once every two days and the following weeks once a day.

Cultivation of the fungus by this procedure lasted 42 days, the amount of dry biomass produced at the end of cultivation was 0.39 mg glucosamine / g dry weight of the sample and polysaccharides 2.24 mg / g.

The third example

The solid substrate inoculated with the mycelium was prepared by soaking 800 g of beech sawdust with 1.5 liters of distilled water containing 80 g of glucose, 50 mg (NH ₄ ) ₂ SO4, 200 mg KH ₂ PO ₄ , 50 mg CaCI ₂ X 2H ₂ O, 50 mg MgSO ₄ X 7H ₂ O, 150 mg FeSO ₄ X 7H ₂ O. With wet sawing, the bioreactor was filled and sterilized from 45 minutes to 90 minutes, preferably 60 minutes at 110 to 130 ° C, preferably 121 ° C (394 K) and a pressure of 1 to 1.4 Ί0 ⁵ Pa, preferably 1.2 -10 ⁵ Pa.

After cooling to 30 ° C, the substrate was sterile inoculated with 2 L of the liquid clamp prepared as described in Example 3 of our patent Sl 9700014 (Zore, I., Berovic, M., Hodzar, D., Boh, B., Greedy , F. 1997, The submerged cultivation of Ganoderma lucidum in a laboratory bioreactor). The cultivation was carried out at 30 ° C. During the first week of cultivation, the flow rate was 0.5 liters / minute, and the following weeks 2 liters / minute. Substrate stirring was performed at 80 rpm for 2 minutes. The first week of cultivation was mixed once every two days and the following weeks once a day. After 28 days of cultivation, substrate moistening began.

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Cultivation of the fungus by this procedure lasted 49 days and the amount of dry biomass produced at the end of cultivation was 0.65 mg glucosamine / g dry weight of the sample and polysaccharides 5.77 mg / g.

Claims (4)

PATENT APPLICATIONS 1. A process for the cultivation / cultivation of Ganoderma lucidum on a solid medium, characterized in that it is carried out by e) prepare a solid substrate of 600 g to 1000 g, preferably 800 g of beech sawing, moistened with 1.2 L to 1.8 L, preferably 1.5 L of distilled water containing 60 g to 100 g, preferably 80 g of glucose , from 40 g to 80 g, preferably 50 mg (NH 2 SO 4 from 150 mg to 250 mg, preferably 200 mg KH2PO4, from 25 mg to 75 mg, preferably 50 mg CaCI ₂ X 2H ₂ O, from 30 mg to 60 mg, preferably 50 mg MgSO4 X 7H ₂ O, from 120 to 180 mg, preferably 150 mg FeSO ₄ X 7H ₂ O; f) Using the obtained wet sawing, fill the bioreactor and sterilize for 45 minutes to 90 minutes, preferably 60 minutes, at a temperature of 110 to 130 ° C, preferably 121 ° C (394 K), at a pressure of 1 to 1.4 -10 ⁵ Pa. , preferably 1.2 • 10 ⁵ Pa; g) cooled to 25 to 35 ° C, preferably 30 ° C, and the substrate is sterile inoculated with 1.5 L to 2.5 L, preferably 2 L, of the liquid clamp of Ganoderma lucidum ^- , h) then grow the fungus 40 to 55 days, preferably 49 days, at a temperature of 25 to 35 ° C, preferably 30 °, with the first week of cultivation having an air flow of 0 to 1 liter / minute, preferably 0.5 liters / minute, for the next three weeks, 1 to 5 liters / minute, preferably 2 liters / minute, substrate mixing takes place at 50 to 150 rpm, preferably 80 rpm 1 to 4 minutes, preferably 2 minutes, with the first week of cultivation being mixed once a day to once every four days, preferably once every two days, and the next three weeks once a day to once every two days, preferably once a day. Method according to claim 1, characterized in that the Ganoderma lucidum strain is used as a strain of Ganoderma lucidum Gal 4 deposited on April 15, 1996 at the deposit facility BFVVS (Microbiology Collection of the Department of Forestry at the Biotechnical Faculty, Ljubljana, Slovenia. -1212 Method according to claims 1 and 2, characterized in that it can be carried out on a laboratory scale. Process according to claims 1 and 2, characterized in that it can be carried out on an industrial scale.