Method for producing mycelium sheets

Abstract

The present invention relates to a method for producing a mycelium sheet. The invention discloses a method for producing a mycelium sheet, which comprises the following steps of: standing and culturing fungi in a liquid culture medium containing cellulose, so that the mycelium sheet is formed on the surface of the liquid culture medium; and collecting the mycelium sheet from the surface of the liquid culture medium containing cellulose.

Description

Technical Field

[0001] This invention relates to a method for producing sheets of mycelium. Background Technology

[0002] Mycelium sheets (also known as mycelium mats) are sheet-like materials produced using fungi. Due to their good mechanical properties (typically a tensile strength of approximately 1-5 MPa), they have been used as an alternative to leather or fabric. Mycelium sheets are primarily composed of mycelium. Some studies have also explored adding cellulose to mycelium sheets to further improve their mechanical properties, creating mycelium-cellulose composites. Common production methods include solid-state culture of fungi and harvesting the resulting mycelium sheets from the surface of the culture medium, and liquid shaken culture of fungi followed by filtration (or further compression) of the liquid culture to produce mycelium sheets. However, the former is more time-consuming, while the latter is more complex in operation. Summary of the Invention

[0003] In this invention, the applicant accidentally discovered through experiments that static culture of fungi in a liquid culture medium containing cellulose can cause mycelial sheets to form on the surface of the liquid culture medium. This is not only quick and easy to operate, but also results in mycelial sheets with intact morphology and better tensile strength.

[0004] Therefore, the present invention provides a method for producing mycelial sheets, comprising:

[0005] Fungi are statically cultured in a liquid medium containing cellulose, causing mycelial sheets to form on the surface of the liquid medium; and

[0006] The mycelial sheet is collected from the surface of the liquid culture medium containing cellulose.

[0007] Preferably, the cellulose is selected from the group consisting of bacterial cellulose, plant cellulose, and combinations thereof.

[0008] Preferably, the plant cellulose is selected from the group consisting of potato cellulose, wheat cellulose, oat cellulose, and combinations thereof.

[0009] Preferably, based on the total weight of the liquid culture medium, the cellulose has a content ranging from 0.05 to 0.5 wt%.

[0010] Preferably, the mycelial sheet does not contain bacterial cellulose or plant cellulose.

[0011] Preferably, the fungus is selected from the group consisting of: species of the genus *Cordyceps*, species of the genus *Trametes*, *Ganoderma multipileum*, *Inonotustabacinus*, *Irpex brevis*, *Pleurotus cornucopiae*, *Schizophyllum commune*, *Terana coerulea*, and combinations thereof.

[0012] More preferably, the species of the genus Cordyceps are selected from the group consisting of: Cordyceps cateniobliqua, Cordyceps farinosa, Cordyceps militaris, Cordyceps tenuipes, and combinations thereof.

[0013] More preferably, the species of the genus Trametes are selected from the group consisting of Trametes hirsuta, Trametes gibbosa, Trametes versicolor, and combinations thereof. Attached Figure Description

[0014] The above and other objects, features and advantages of the present invention will become apparent from the following detailed description and preferred embodiments and the accompanying drawings, wherein:

[0015] Figure 1 The dry weight of each group of mycelial sheets in Example 1 is shown. Detailed Implementation

[0016] For the purposes of this instruction manual, it will be clearly understood that the word "comprising" means "including but not limited to", and the word "comprises" has the corresponding meaning.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art. Those skilled in the art will recognize many similar or equivalent methods and materials that can be used to practice the invention. Of course, the invention is by no means limited to the methods and materials described.

[0018] This invention provides a method for producing sheets of mycelium, comprising:

[0019] The fungus is statically cultivated in a liquid medium containing cellulose, resulting in the formation of mycelial sheets on the surface of the liquid medium; and

[0020] The mycelial sheet is collected from the surface of the liquid culture medium containing cellulose.

[0021] As used herein, the terms "mycelium sheet," "mycelium mat," "fungal sheet," and "fungal mat" may be used interchangeably and refer to a continuous planar sheet formed by interconnected fungal mycelia. Preferably, the mycelium sheet does not contain bacterial cellulose or plant cellulose. More preferably, the mycelium sheet does not contain bacterial cellulose or plant cellulose. In a preferred embodiment of the invention, the mycelium sheet consists solely of fungal mycelia.

[0022] As used herein, the term "fungi" is intended to encompass any fungus capable of producing mycelium [i.e., mycelium-producing fungi or filamentous fungi], including, but not limited to: species of the genus *Cordyceps*, species of the genus *Trametes*, *Ganoderma multipileum*, *Inonotus tabacinus*, *Irpexbrevis*, *Pleurotus cornucopiae*, *Schizophyllum commune*, and *Terana coerulea*.

[0023] Preferably, the fungus is selected from species of the genus Cordyceps comprising the group consisting of: *Cordyceps cateniobliqua*, *Cordyceps farinosa*, *Cordyceps militaris*, *Cordyceps tenuipes*, and combinations thereof. More preferably, the fungus is selected from species of the genus Cordyceps comprising the group consisting of: *Cordyceps farinosa* BCRC FU30829, *Cordyceps farinosa* BCRC FU31128, *Cordyceps tenuipes* BCRC FU31198, *Cordyceps cateniobliqua* BCRC FU31225, *Cordyceps militaris* BCRC FU31818, and combinations thereof.

[0024] Preferably, the fungus is selected from species of the genus *Trametes* comprising the group consisting of: *Trametes hirsuta*, *Trametes gibbosa*, *Trametes versicolor*, and combinations thereof. More preferably, the fungus is selected from species of the genus *Trametes* comprising the group consisting of: *Trametes gibbosa* BCRC MU30502, *Trametes hirsuta* BCRC MU30685, *Trametes versicolor* BCRC MU30810, and combinations thereof.

[0025] Preferably, the fungus is selected from the group consisting of: Ganoderma lucidum BCRC 37180, Phytotrichum citrinum BCRCMU30009, Pleurotus ostreatus BCRC 35360, Pleurotus ostreatus BCRC MU30825, Schizophyllum commune BCRC MU30359, Terbina bluea BCRC MU30808, and combinations thereof.

[0026] According to the present invention, the cellulose may be a commercially available product or obtained from a natural source using microbiological methods / isolation methods commonly used in the art. In this regard, references may be made, for example, to Wang J. et al. (2019), Carbohydr. Polym., 219:63-76 and Guo, Y. et al. (2021), Trans. Tianjin Univ., 27(5):385-393.

[0027] Alternatively, the cellulose can also be prepared by synthetic techniques known and commonly used by those skilled in the art.

[0028] Preferably, the cellulose is selected from the group consisting of bacterial cellulose, plant cellulose, and combinations thereof.

[0029] As used herein, the term "bacterial cellulose" means cellulose produced by bacteria. Bacteria suitable for use in this invention include, but are not limited to, species of the genera *Acetobacter*, *Alcaligenes*, *Pseudomonas*, *Rhizobium*, *Agrobacterium*, and *Sarcina*. In a preferred embodiment of the invention, the bacterium is *Glucoacetobacter xylinum*.

[0030] As used herein, the term "plant cellulose" means cellulose produced by plants. Plants suitable for use in this invention include, but are not limited to, species of the genera *Solanum*, *Triticum*, and *Avena*. In one preferred embodiment of the invention, the plant is potato (*Solanum tuberosum*). In another preferred embodiment, the plant is wheat (*Triticum aestivum* L.). In yet another preferred embodiment, the plant is oat (*Avena sativa*).

[0031] According to the present invention, the cellulose may have a particle size of 0.035 mm to 4 mm.

[0032] Preferably, the cellulose is bacterial cellulose with a particle size of 0.035 mm or more and less than 3 mm.

[0033] Preferably, the cellulose is plant cellulose with a particle size of 0.035 mm to 0.09 mm. In one preferred embodiment of the invention, the particle size is 0.035 mm. In another preferred embodiment of the invention, the particle size is 0.09 mm.

[0034] According to the present invention, the liquid culture medium containing cellulose can be prepared by adding cellulose to a basal culture medium suitable for the growth of fungal mycelia.

[0035] According to the present invention, based on the total weight of the liquid culture medium, the cellulose may have a content ranging from 0.05 to 0.5 wt%. In a preferred embodiment of the present invention, the cellulose content is 0.15 wt%. In another preferred embodiment of the present invention, the cellulose content is 0.5 wt%.

[0036] According to the present invention, the basic culture medium suitable for fungal growth is well known to those skilled in the art and can be self-prepared or a commercially available product, including, but not limited to, MEB liquid culture medium and PDB liquid culture medium.

[0037] According to the present invention, the basal culture medium suitable for use in the present invention may contain a carbon source selected from the group consisting of: glucose, fructose, lactose, sucrose, maltose, galactose, mannose, trehalose, starch, potato starch, malt extract, maltodextrin, dextrin, corn flour, and combinations thereof.

[0038] According to the present invention, the basal culture medium suitable for use in the present invention may contain a nitrogen source selected from the group consisting of: peptone, polypeptone, ammonium sulfate ((NH4)2SO4), ammonium nitrate (NH4NO3), ammonium chloride (NH4Cl), ammonium citrate, casamino acid, tryptone, meat extract, yeast extract, yeast powder, milk, skimmed milk powder, casein, soybean flour, whey, amino acids, and combinations thereof.

[0039] As used herein, the term "static cultivation" refers to cultivation carried out in a static state without stirring or agitation [e.g., in an incubator in stationary mode].

[0040] As used in this article, the terms “cultivation,” “culturing,” and “cultivation” may be used interchangeably.

[0041] The procedures and parameters involved in the cultivation process fall within the scope of expertise and routine technical knowledge of those skilled in the art. For reference, see, for example, El-Enshasy HA (2007), Bioprocessing for value-added products from renewable resources, 225-261.

[0042] According to the present invention, the culture can be carried out at a temperature of approximately 20 to 30°C for 7 to 28 days. In a preferred embodiment of the invention, the culture is carried out at a temperature of 25°C for 14 days.

[0043] The present invention will be further described with reference to the following embodiments, but it should be understood that these embodiments are for illustrative purposes only and should not be construed as limiting the implementation of the present invention.

[0044] Example

[0045] General experimental materials:

[0046] 1. Malt extract agar (MEA) plate:

[0047] The MEA culture plates used in the following examples have the formulations shown in Table 1 below.

[0048] Table 1. Formulation of MEA culture plates

[0049]

[0050] 2. Malt extract (MEB) liquid medium:

[0051] The MEB liquid culture medium used in the following examples has the formulation shown in Table 2 below.

[0052] Table 2. Formulation of MEB liquid culture medium

[0053]

[0054] 3. Fungal strains:

[0055] The fungal strains used in the following examples were all purchased from the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan (No. 331, Food Rd., Hsinchu City, Taiwan 300), including:

[0056] (1) Cordyceps species: Cordyceps farinosa

[0057] BCRC FU30829, Cordyceps powder BCRC FU31128, Cordyceps tenuipes BCRC FU31198, Cordyceps cateniobliqua BCRC FU31225, and Cordyceps militaris BCRC FU31818;

[0058] (2) Species of the genus Trametes: Trametes gibbosa BCRCMU30502, Trametes hirsuta BCRCMU30685, and Trametes versicolor BCRCMU30810;

[0059] (3) Terana coerulea BCRC MU30808;

[0060] (4) Inonotus tabacinus BCRC MU30009;

[0061] (5) Schizophyllum commune BCRC MU30359;

[0062] (6) White-yellow pleurotus cornucopiae BCRC MU30825;

[0063] (7) Irpex brevis BCRC 35360; and

[0064] (8) Ganoderma multipileum BCRC 37180.

[0065] 4. Preparation of inoculum:

[0066] The aforementioned fungal strains were inoculated into MEA culture plates (9 cm in diameter) and cultured at 25°C for 1 to 3 weeks. Next, the mycelial fragments formed in the culture plate were cut and added to 90 mL of sterile water, then homogenized using an Oster homogenizer (model BO-00001). The resulting homogenate was then inoculated into MEB liquid medium at a 10 vol% inoculum and cultured in a constant temperature shaking incubator (25°C, 150 rpm) for 5 to 7 days. The resulting culture was used as the inoculum for fungal strains.

[0067] 5. Cellulose:

[0068] In Example 1 below, the potato cellulose (particle size 0.09 mm) (product number Pot 90) used was purchased from Guanyue Technology Co., Ltd., the wheat cellulose (particle size 0.035 mm) (product number WF600-30) and the oat cellulose (particle size 0.035 mm) (product number HF600-30) were purchased from Zhenfang Co., Ltd., and the bacterial cellulose (particle size less than 3 mm) was obtained by producing a biofilm using Glucoacetobacter xylinum, washing it with water until neutral, and then homogenizing and freeze-drying it.

[0069] Additionally, the bacterial cellulose pulp (particle size less than 3 mm) (product number ND-A1018-HA-B7) used in Example 2 below was purchased from Jiaming Foods.

[0070] The industrial stock was homogenized before use, and the bacterial cellulose content was determined to be 1 wt%. Example 1. Static cultivation using cellulose from different sources.

[0071] Effects on the formation of mycelium sheets

[0072] Experimental methods and results:

[0073] First, the inoculum source of Cordyceps militaris BCRC FU31818 obtained in item 4 of "General Experimental Materials" above was divided into 5 groups, including 1 control group and 4 experimental groups (i.e., experimental groups 1 to 4). Next, the inoculum source of each group was inoculated at a 10 vol% inoculum into MEB liquid medium (8 cm in diameter at the bottom of the culture flask) containing different amounts of cellulose (all at a concentration of 0.5 wt%) as shown in Table 3 below, and then statically cultured in a constant temperature incubator (25°C) for 14 days. Afterwards, the mycelial sheets formed on the surface of the medium were observed and collected, washed with water, and then heated and dried overnight at 40°C. The dry weight of the mycelial sheets was then measured.

[0074] Table 3. Cellulose added to each group of liquid culture medium

[0075] Group Cellulose control group - Experimental group 1 Bacterial cellulose Experimental group 2 Potato cellulose Experimental group 3 Wheat cellulose Experimental group 4 oat cellulose

[0076] In addition, the inoculum sources for Cordyceps militaris BCRC FU31128, Cordyceps militaris FU30829, Cladosporium spp. BCRC FU31198, Paecilomyces obliqueiformis BCRC FU31225, Cordyceps militaris BCRC MU30502, Ganoderma lucidum BCRC 37180, Pleurotus ostreatus BCRC MU30825, and Cordyceps militaris BCRC MU30685 were also grouped, statically cultured, and had their mycelial dry weight measured, following the experimental procedures described above for Cordyceps militaris BCRC FU31818.

[0077] The experimental results showed that experimental groups 1 to 4 all formed complete mycelial sheets, while the control group did not form complete mycelial sheets (data not shown). The dry weight of the mycelial sheets in each group is shown in [data missing]. Figure 1 From. Figure 1 It is evident that, for different fungi, the dry weight of mycelial sheets in experimental groups 1 to 4 showed varying degrees of increase compared to the control group, with the most significant increase observed in experimental group 1 using *Cordyceps militaris* BCRC FU31818. Furthermore, the applicant also obtained similar results when using *Tetrandella blueifolia* BCRC MU30808 and *Bacillus brevis* BCRC 35360 as inoculants (data not shown).

[0078] In addition, in order to test the mechanical strength of the mycelial sheet, the applicant selected the mycelial sheet of Cordyceps militaris BCRC FU31818 as a representative, and referred to the method described in Appels FVWet al. (2020), Commun.Biol, 26; 3(1):334 and used a tensile testing machine (model Instron 5965) to test the tensile strength. The results showed that the measured tensile strength could reach as high as 6.11 MPa.

[0079] These experimental results show that static culture of fungi in liquid culture media containing different cellulose (especially bacterial cellulose) can produce complete mycelial sheets with good mechanical strength on the surface of the culture medium, and can achieve varying degrees of yield improvement.

[0080] Example 2. Effect of static culture using different concentrations of cellulose on mycelial sheet formation.

[0081] Experimental methods and results:

[0082] First, the inoculum source of *Cordyceps militaris* BCRC FU31818 obtained in item 4 of the "General Experimental Materials" section above was divided into 7 groups, including 1 control group and 6 experimental groups (i.e., experimental groups 1 to 6). Next, the inoculum source of each group was inoculated at a rate of 10 vol% into MEB liquid culture medium (8 cm in diameter at the bottom of the culture flask) with different bacterial cellulose concentrations as shown in Table 4 below, and then statically cultured in a constant temperature incubator (25°C) for 14 days. Afterwards, the mycelial sheets formed on the surface of the culture medium were observed and collected, washed with water, and then heated and dried overnight at 40°C. The dry weight of the mycelial sheets was then measured.

[0083] Table 4. Concentration of bacterial cellulose in each group of liquid culture medium

[0084]

[0085]

[0086] The experimental results showed that experimental groups 1 to 6 all produced complete mycelial sheets, while the control group did not (data not shown). The dry weight of the mycelial sheets in each group is shown in Table 5. As can be seen from Table 5, compared with the control group, the dry weight of the mycelial sheets in experimental groups 1 to 6 was significantly increased. This experimental result shows that static cultivation of fungi in liquid culture media containing different concentrations of cellulose can produce complete mycelial sheets on the surface of the culture medium, and a significant increase in yield can be obtained when the cellulose concentration is 0.15-0.30 wt%.

[0087] Table 5. Dry weight of mycelial sheets in each group

[0088]

[0089] Example 3. Production of large-area mycelial sheets

[0090] Experimental methods and results:

[0091] The following mycelial sheets were produced using the inoculum sources obtained from item 4 of the "General Experimental Materials" section above: Cordyceps militaris BCRC FU31818, Ganoderma lucidum BCRC 37180, Cordyceps powder BCRC FU30829, Cladosporium spp. BCRC FU31198, Paecilomyces obliqueiformis BCRC FU31225, Phytotrichum gloeosporioides BCRC MU30009, Schizophyllum commune BCRC MU30359, Pleurotus ostreatus BCRC MU30502, Trichophyton mentagrophytes BCRC MU30685, Trametes versicolor BCRC MU30810, and Pleurotus ostreatus BCRC MU30825. The mycelial sheets were produced in a manner largely consistent with the method described in Example 2 above, with the following differences: the concentration of bacterial cellulose in the liquid culture medium was 0.20 wt%, the bottom area of ​​the culture container was 20 cm × 20 cm, and the static culture time was 7 to 28 days.

[0092] The experimental results showed that all of these fungi formed morphologically complete mycelial sheets. Therefore, the applicant further selected a culture container with a bottom area of ​​54cm×41cm to repeat the above experiment, and found that morphologically complete mycelial sheets could also be formed (data not shown).

[0093] Based on the above experimental results, the applicant believes that: producing mycelial sheets by statically culturing fungi in a liquid culture medium containing cellulose is not only easy to operate, but also produces mycelial sheets with intact morphology and good mechanical strength, which can be used to replace existing mycelial-cellulose composite materials as alternative leather or fabrics.

[0094] All patents and documents cited in this specification are incorporated herein by reference in their entirety. In the event of any conflict, the detailed description of this application (including definitions) shall prevail.

[0095] Although the invention has been described with reference to the specific examples described above, it is apparent that many modifications and variations can be made without departing from the scope and spirit of the invention. Therefore, it is intended that the invention be limited only to those shown in the appended claims.

Claims

1. A method for producing mycelial sheets, characterized in that: The method includes: Fungi are statically cultured in a liquid medium containing cellulose, causing mycelial sheets to form on the surface of the liquid medium; and The mycelial sheet is collected from the surface of the liquid culture medium containing cellulose.

2. The method according to claim 1, characterized in that: The cellulose is selected from the group consisting of bacterial cellulose, plant cellulose, and combinations thereof.

3. The method according to claim 1, characterized in that: The plant cellulose is selected from the group consisting of: potato cellulose, wheat cellulose, oat cellulose, and combinations thereof.

4. The method according to claim 1, characterized in that: Based on the total weight of the liquid culture medium, the cellulose has a content ranging from 0.05 to 0.5 wt%.

5. The method according to claim 1, characterized in that: This mycelium sheet does not contain bacterial cellulose or plant cellulose.

6. The method according to claim 1, characterized in that: The fungi were selected from the group consisting of the following species: Cordyceps spp., Trametes spp., Ganoderma multipileum, Inonotus tabacinus, Irpex brevis, Pleurotus cornucopiae, Schizophyllum commune, Terana coerulea, and combinations thereof.

7. The method according to claim 6, characterized in that: The species of the genus Cordyceps are selected from the group consisting of: Cordyceps cateniobliqua, Cordyceps farinosa, Cordyceps militaris, Cordyceps tenuipes, and combinations thereof.

8. The method according to claim 6, characterized in that: The species of the genus Trametes are selected from the group consisting of the following: Trametes hirsuta, Trametes gibbosa, Trametes versicolor, and combinations thereof.

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