Method for the fermentative production of fungal mass for food

Abstract

The invention relates to a method for the fermentative production of fungal mycelium by providing an aqueous nutrient medium having a substrate content containing plant protein, inoculating the nutrient medium with at least one fungus, mixing and aerating the inoculated nutrient medium in order to cultivate fungal mass, wherein ammonium ions are added to the nutrient medium, such that the pH value is reduced by at least 2 pH units without controlled adjustment of the pH value by the fermentation, separating fungal mass from the fermentation broth in order to produce aqueous fungal mass and an aqueous residue, providing a further aqueous nutrient medium by mixing at least one portion of the aqueous residue, and repeating the aforementioned steps.

Description

[0001] T PAATENTRANWUALTTS ATNZLIESI

[0002] TARUTTIS - Aegidientorplatz 2b - D-30159 Hanover

[0003] Dr. rer. nat. Stefan Taruttis European Patent Office Graduate Engineer

[0004] Patent attorney

[0005] European Patent Attorney

[0006] 80298 MUNICH European Trademark Attorney

[0007] D-30159 Hannover, Aegidientorplatz 2b TORHAUS AM AEGI

[0008] Tel.: ++49511 12332670

[0009] Fax: +49 511 12332678 www.taruttis.com info@taruttis.com in cooperation with

[0010] Dr. rer. medic. Dirk Vollmer, Patent Attorney, European Patent Attorney, D-74523 Schwäbisch Hall, Hilde-Domin-Str. 8

[0011] Your Our

[0012] Symbol: Symbol: K1050PCT January 13, 2026

[0013] New international patent application for a process for the fermentative production of mushroom mass for food - Kynda Biotech GmbH

[0014] Method for the fermentative production of mushroom mass for food

[0015] The present invention relates to a process for the fermentative production of mushroom mass suitable for use as food. The fermentation carried out during production is preferably submerged fermentation in a bioreactor with aeration, preferably with stirring of the fermentation broth.

[0016] The process has the advantage that the substrate to be fermented is not necessarily sterile, and in particular, is not necessarily autoclaved before fermentation, but preferably only has a reduced microbial count, e.g., it is partially sterilized before fermentation, e.g., by pasteurization. This advantageously results in lower energy consumption for pasteurization than for sterilization by autoclaving. The substrate preferably contains or consists of plant protein and / or plant carbohydrates, optionally plant fibers, e.g., preferably plant by-products of food production. WO 2020 / 061502 Al describes an aerobic fermentation process using fungal mycelium for the production of meat analogues. In this process, a filamentous fungus is cultivated in a nutrient medium, harvested, optionally pasteurized, extruded, and dried. The nutrient medium can be a by-product of food production.

[0017] US Patent 2009 / 0148558 Al describes a process for producing meat analogues from fungal mycelium grown in a synthetic nutrient broth of potato dextrose broth, yeast extract, malt extract and Soytone (soy flour digested with papain) with cane sugar extract and sodium nitrate.

[0018] The WO 2022 / 229206 describes the fermentation of Rhizopus oligosporus in a medium that is sterilized before inoculation and whose pH is adjusted to 4.0 - 5.5 and controlled to pH 4.0 with ammonium ions for the production of a foodstuff.

[0019] SE 2151533 Al describes the fermentation of autoclaved food residues in medium containing ammonium sulfate, which is autoclaved after adjusting the pH to 5.5 and then inoculated with Rhizopus spores.

[0020] The invention aims to provide a method for producing mushroom mass that requires low energy consumption and, in particular, does not require sterilization of the substrate, for example by autoclaving. Preferably, the method should include a submerged fermentation step that is carried out without pH control.

[0021] The invention solves the problem with the features of the claims and in particular by means of a process for producing mushroom mass by fermentation, preferably submerged fermentation, which comprises the steps

[0022] 1) Providing an aqueous nutrient medium containing a substrate that includes vegetable protein and / or carbohydrate, the substrate being optionally pasteurized,

[0023] 2) Inoculating the nutrient medium with at least one fungus to produce a nutrient medium inoculated with the at least one fungus, 3) Mixing and aerating the inoculated nutrient medium to attract fungal mass of the at least one fungus for the aerobic fermentative production of fermenter broth containing attracted fungal mass, wherein ammonium ions are added to the nutrient medium, wherein the pH value is reduced by at least 2 pH units, preferably by at least 3 pH units or by at least 4 pH units, preferably by at least 4.5 or at least 5 pH units,

[0024] 4) preferably without controlled adjustment of the pH value, e.g. without adjusting the pH value by means of the controlled addition of acid,

[0025] 5) Separation of mushroom mass from the fermenter broth to produce aqueous mushroom mass and at least one aqueous residue,

[0026] 6) preferably providing a further aqueous nutrient medium by mixing at least a part of the aqueous residue, which optionally contains insoluble plant components or consists of soluble components, with plant protein and / or carbohydrate and repeating the preceding steps, in particular repeating steps 2) to 6).

[0027] exhibits or consists of.

[0028] The process for the fermentative production of fungal mycelium generally comprises providing an aqueous nutrient medium containing a substrate that includes plant protein, inoculating the nutrient medium with at least one fungus, mixing and aerating the inoculated nutrient medium to attract fungal mass by means of aerobic fermentation, whereby ammonium ions are added to the nutrient medium so that the pH is lowered by at least 2 pH units without controlled adjustment of the pH by the aerobic fermentation, separating fungal mass from the fermenter broth to produce aqueous fungal mass and an aqueous residue, providing another aqueous nutrient medium and mixing in at least a part of the aqueous residue, and preferably repeating the aforementioned steps, or consists thereof.

[0029] The process has the advantage that the fungus is suitable for food production, e.g., a fungus approved for food production by the relevant authorities, also known as GRAS. In particular, the fungus used in the process is not genetically modified or mutated. The substrate is optionally pasteurized, preferably not sterilized, but can be exclusively pasteurized. Preferably, before inoculation in step 2), the substrate has a microbial count that is the microbial count from the production, e.g., the microbial count of okara, which is preferably fresh, optionally frozen or unfrozen for up to 7 days, or up to 5 days, or up to 3 days at a maximum of 8°C, preferably at a maximum of 5°C or a maximum of 3°C. The substrate can contain vegetable protein and / or carbohydrate, e.g., in a mixture with cellulose, hemicellulose, and / or fat, or consist of vegetable protein and / or carbohydrate.

[0030] The vegetable protein and / or carbohydrate can be the water-insoluble residue from the aqueous extraction of pulses or cereals, also known as okara, e.g., residue from the aqueous extraction of beans, in particular soybeans, wheat, rice, or lentils, and in particular, the water-insoluble residue from the production of tofu from soybeans, also known as soy okara. Preferably, the aqueous nutrient medium contains vegetable protein at a concentration of at least 5 g / L, more preferably at least 6 g / L, and more preferably at least 10 g / L, e.g., up to 30 g / L or up to 25 g / L, and more preferably 10 to 20 g / L.

[0031] Alternatively or additionally, the substrate can consist of cereal and legume straw, sugar beet pulp, especially after sugar extraction, cereal bran, e.g., wheat bran, corn bran, and rice bran, potato peels, apple peels, grape skins, citrus peels, and carrot peels, cauliflower cuttings, leek greens, extracted tea leaves, press cake from edible oil production, especially rapeseed press cake, soybean press cake, and olive press cake, empty sunflower petals, sunflower skins, nutshells, optionally ground or unground and in chunks, corn cobs after kernel removal, red peanut shells, brewer's spent grain, wine pomace, optionally after distillation of alcohol, such as wine pomace left over after grappa production, sugar cane bagasse, sugar cane leaves, coffee grounds, etc.after aqueous extraction and / or after extraction of caffeine, leftover and residual bread, banana leaves or a combination of at least two of these, or consist of them.

[0032] In addition to or as an alternative to the aqueous nutrient medium containing pasteurized or microbial-reduced substrate, the aqueous phase of the aqueous nutrient medium can be pasteurized or partially sterilized separately before or after the introduction of the substrate. Preferably, at least a portion of the aqueous residue is added to the nutrient medium, optionally before or after the optional pasteurization or microbial reduction step. The aqueous residue is that which has been separated from a fermenter broth produced by the process, such that this aqueous residue acidifies the nutrient medium and the substrate contained therein.

[0033] It has been shown that the fermentative production of fermentation broth by mixing and aerating the nutrient medium inoculated with a fungus, upon addition of ammonium ions as an inorganic nitrogen source, preferably as the sole inorganic nitrogen source, e.g., as ammonium sulfate, leads to a reduction in the pH of the fermentation broth by at least 1 pH unit, preferably by at least 2 pH units, more preferably by at least 3 pH units, or by at least 4 pH units, more preferably by at least 4.5 to 5 pH units, without the need to control the pH by adding acid. The substrate preferably contains or consists of the water-insoluble residue from the aqueous extraction of pulses or cereals.According to the invention, the pH of the fermentation broth, to which ammonium ions have been added, is lowered during fermentation by at least 1 pH unit, preferably by at least 2 pH units, more preferably by at least 3 pH units or by at least 4 pH units, more preferably by at least 4.5 or at least 5 pH units, optionally additionally by adding acid. Preferably, the pH is lowered solely by the added ammonium ion content during fermentation, particularly within 48 hours, more preferably within 36 hours or within 24 hours after the start of fermentation, e.g., by inoculating the nutrient medium. Preferably, the pH of the fermentation broth is lowered by cultivating the fungus to a pH of 2.5 to 5, e.g., 3.5 to 4.5, particularly preferably within 48 hours, more preferably within 36 hours or within 24 hours after the start of fermentation. Ammonium ions, as a salt or in solution, are e.g.to achieve a concentration in the nutrient medium of 0.03 to 0.1 M, preferably 0.04 to 0.9 M, more preferably 0.05 to 0.085 M, e.g. 0.06 to 0.08 or up to 0.076 M (0.076 M corresponds to 10 g / L ammonium sulfate).

[0034] Therefore, in the fermentative production process, the pH of the fermentation broth is lowered by the fungus during fermentation by at least 1 pH unit, preferably by at least 2 pH units, more preferably by at least 3 pH units, or by at least 4 pH units, or by at least 5 pH units, by the addition of ammonium ions. Preferably, the inorganic nitrogen source added to the nutrient medium consists of ammonium ions, particularly in the case of a fungus selected from the group of Aspergillus oryzae (e.g., White Koji) or Aspergillus sojae (e.g., Yellow Koji). This is because it has been shown that, for example,During fermentation by Aspergillus oryzae (White Koji), when urea or sodium nitrate is added as the sole inorganic nitrogen source to the substrate, which essentially consists of 30 g / L of soy okara in water, or when no inorganic nitrogen source is added, the pH value from an initial 7 to 9 only decreases slightly, remains the same, or increases.

[0035] The substrate is pasteurized to such an extent that bacteria, yeasts, and molds contained in the substrate are reduced to a maximum of 10 6 CFU / mL, preferably a maximum of 10 5 CFU / mL or a maximum of 10 4 If the CFU / mL count is reduced, or preferably the substrate is inactivated, pasteurization or partial sterilization can be achieved by heat treatment, e.g., by steam injection, by treatment with pulsed electric fields, or a combination of these methods. The substrate can be suspended in water before or after pasteurization to provide the nutrient medium.

[0036] Preferably, fungal mass is separated from the fermentation broth during or after fermentation to obtain an aqueous residue that optionally contains insoluble components or consists entirely of soluble components. The separation of fungal mass from the fermentation broth can be achieved by separating a portion of the aqueous phase from the fermentation broth, e.g., an aqueous phase that contains essentially no insoluble components. The aqueous phase separated from the fermentation broth or remaining after the separation of fungal mass preferably has a pH of at most 5.5, at most 5.0, at most 4.5, more preferably at most 4.0, at most 3.5, or at most 3.0, and even more preferably at most 2.5.

[0037] In a preferred embodiment, this aqueous phase is mixed with vegetable protein and / or carbohydrate to provide a nutrient medium, preferably with the addition of ammonium ions, optionally without pasteurization or partial sterilization, also referred to as microbial reduction. Optionally, the vegetable protein and / or carbohydrate can be pasteurized after mixing with this aqueous phase. Preferably, the vegetable protein and / or carbohydrate is pasteurized before mixing with this aqueous phase, since it has been shown that mixing this aqueous phase, which originates from a fermentation, leads to a faster subsequent fermentative production of fermenter broth containing cultivated fungal mass.It is currently assumed that the faster fermentative production of fermenter broth containing grown fungal mass, when the nutrient medium contains an aqueous phase separated from a fermenter broth in mixture with vegetable protein and / or carbohydrate, is due to the lowering of the pH and the content of hydrolyzing enzymes in the aqueous phase.

[0038] Inoculation, also referred to here as seeding, of the substrate with a fungus can take place before or after suspending the pasteurized substrate in water, which preferably consists at least partially of an aqueous phase separated from a fermenter broth, e.g., by mixing in spores or mycelium of a fungus. Preferably, the inoculation with mycelium is carried out in a ratio of 1:5 to 1:25, e.g., 1:10 to 1:20 vol / vol to the substrate or to the suspended substrate. The mycelium used for inoculation is preferably produced by aerobic submerged fermentation, e.g., in an aqueous nutrient medium that is synthetic and / or an aqueous suspension of a substrate used according to the invention.

[0039] It has been shown that it is sufficient to pasteurize the substrate, optionally the nutrient medium including the substrate, before inoculation, e.g., by pasteurizing at 68 °C for 20 to 60 minutes, as the at least one fungus is not suppressed by bacteria or other fungi after inoculation, e.g., not overgrown, and preferably not contaminated by bacteria or other fungi. Preferably, the mycelium used for inoculation is comminuted before inoculation, e.g., by intensive shearing, such as with an Ultra-Turrax at 6400 to 13000 rpm for 3 to 30 seconds, particularly intensive shearing immediately before inoculation, e.g., within a maximum of 10 minutes or a maximum of 5 minutes before inoculation. The intensive shearing is carried out until the mycelium intended for inoculation is comminuted into individual hyphae or aggregates of a maximum of 10 hyphae, preferably a maximum of 5 or a maximum of 3 hyphae, as can be determined, e.g., by microscopic inspection.

[0040] Preferably, the substrate has a dry matter content of 3 to 40 wt%, more preferably 20 to 40 wt% or 20 to 30 wt% in the aqueous suspension. More preferably, the substrate has a protein content of at least 20 or at least 30 wt%, e.g., up to 60 wt%, e.g., up to 55 or up to 50 wt% of the dry matter. It has been shown that with such a substrate content in the aqueous suspension, which forms the nutrient medium, essentially mycelium is formed during or before inoculation by fermentation as a fermentation broth. This mycelium is still pumpable, but the fermentation broth contains hardly any or no free aqueous content. The mycelium produced has, for example, a dry matter content of approximately 10 to 30 g / L of the fermentation broth.

[0041] Submerged fermentation takes place, for example, in a closed vessel, also referred to here as a reactor, which is aerated with low-germ or sterile air, while the substrate suspended in the nutrient medium is mixed with a stirrer or solely by aeration. Aeration preferably continues until the dissolved oxygen concentration reaches at least 10%, preferably at least 20%. The stirrer can be a turbine, propeller, or anchor stirrer. Preferably, the vessel includes a temperature control device, in particular a cooling device. The vessel in which the fermentation takes place can, for example, be cooled to maintain a constant temperature of 15°C to 30°C for the fermentation broth during fermentation.

[0042] Preferably, mixing is effected solely by aeration, and the vessel has no mechanically or magnetically driven stirrer. It has been shown that the fungus, particularly when it has been fragmented by shearing prior to inoculation, is not overgrown or affected by other microorganisms even when aerated with non-sterile air; in fact, the fungus preferably suppresses other microorganisms. Fermentation can preferably take place in an open vessel. Optionally, the vessel is temperature-controlled, and in particular cooled, solely at its outer wall by ambient air.

[0043] The fermentation is preferably carried out until the aqueous fraction of the fermenter broth contains no insoluble residues of the substrate, wherein insoluble residues are, for example, those which are retained by a sieve with a mesh size of 1 mm, preferably a maximum of 0.5 mm or a maximum of 0.2 mm or a maximum of 0.05 mm.

[0044] In particular, fermentation is carried out until the fungus has degraded the substrate to such an extent that its residues have a maximum size of 1 mm, preferably a maximum of 0.5 mm or a maximum of 0.2 mm. Preferably, fermentation is continued until the substrate is completely degraded, or the fermenter broth is only drawn off when the substrate is completely degraded.

[0045] In general, the aqueous fraction of the fermenter broth remaining after mycelium removal can be freed from residues larger than 1 mm, preferably 0.5 mm or 0.2 mm, by sieving.

[0046] Generally, fermentation is preferred as a batch or fed-batch process. In the batch process, the entire raw material is placed in containers, inoculated, and fermented with aeration, without adding any further raw material before the mycelium is removed. In the fed-batch process, only a portion of the raw material is placed in containers, inoculated, and fermented with aeration, with additional raw material optionally added in batches or continuously until the mycelium is removed. Optionally, ammonium ions are added continuously or intermittently during fermentation, either as a powder or solution.

[0047] In general, and especially in the following examples, dry matter (DM) values ​​refer to mycelium, e.g. determined after washing mycelium with water to remove components of the nutrient medium from the mycelium.

[0048] Example 1: Submerged fermentative production of Aspergillus oryzae

[0049] The insoluble residue from soybean tofu production, also known as okara, was suspended in water at a concentration of 10 wt% to create a nutrient medium. The suspension was inoculated with the following microorganisms, which can naturally occur in such byproducts, to create an exemplary substrate contaminated by bacteria and molds: 10 5 Spores / mL of Bacillus cereus, approx. 10 each 5 Spores / mL of Rhizopus arrhizus and Mucor racemosus, approx. 10 5 CFU / mL of Lactiplantibacillus plantarum and approx. 10 5 CFU / mL of Salmonella enterica serovar. Typhimurium.

[0050] When the pH of the medium was lowered from an initial value of approximately 6 to 8 by adding acid, preferably aqueous residue separated from fermentation broth, to pH 3.0, followed by pasteurization at 80 °C for 120 min, the spores of Bacillus cereus were reduced by approximately 3 x 10 3The spore count decreased by 4 CFU / mL, and by a further order of magnitude during subsequent aerobic incubation with mixing. The spores of Rhizopus arrhizus and Mucor racemosus, as well as L. plantarum and S. enterica, were completely inactivated after fermentation.

[0051] For aerobic fermentation, aliquots of the nutrient medium were inoculated with A. oryzae in air-gassed containers, either without pH adjustment (with the addition of 10 g / L ammonium sulfate, pH not adjusted) or after acidification. The pH of the medium or fermenter broth was determined at the time of inoculation (pH 0 h) and after 48 h of aeration (pH 48 h). The dry matter content (DMT) of the separated fungal mycelium was determined after 48 h. No pH control, e.g., by adding acid, was used.

[0052] The results show that the nutrient medium with added ammonium sulfate acidified by approximately 4 pH units from the initial pH value solely through fungal growth (7.29 g / L dry matter). Adjusting the initial pH value to pH 4 (Al, A2, A3) or to pH 3 (Bl, B2, B3) without adding ammonium sulfate to the nutrient medium resulted in only a slight further acidification due to fungal growth, with similar or lower production of fungal dry matter.

[0053]

[0054] Example 2: Submerged fermentative production of A, oryzae (White Koji with various substrates)

[0055] Various substrates, including the insoluble residue from soybean tofu production (soyokara), the insoluble residue from oat tofu production (oatokara), the insoluble residue from rice tofu production (riceokara), oat flour, wheat flour, rye bran, spelt bran, or pea starch, were mixed with water containing either 10 g / L of ammonium sulfate or 30 g / L of ammonium sulfate without ammonium sulfate. This culture medium was not pasteurized or subjected to any antimicrobial treatment. The pH was measured at the beginning of inoculation and after 48 hours in two parallel containers. For inoculation, 10 to 20 mL of a pre-culture containing 10 3 up to 10 9Spores / mL were inoculated into LB medium and incubated for 24 h in a shake flask at 30 to 37 °C, and / or a preculture of A. oryzae mycelium, e.g., 30–120 mL with 0.2 to 0.8 g dry matter / L, preferably comminuted using Utra-Turrax, was added to inoculate 0.5 to 1.5 L of culture medium. Fermentation took place in air-gassed containers. pH control, e.g., by adding acid, was not used.

[0056] The results show that even with different plant substrates, the addition of ammonium sulfate alone has the effect of lowering the pH value of the fermenter broth by 3 to 5 pH units.

[0057]

[0058]

[0059] For comparison, two parallel containers were prepared with a nutrient medium consisting of 30 g / L soya koji, supplemented with 10 g / L ammonium sulfate, 10 g / L urea, 10 g / L sodium nitrate, or 20 g / L sodium nitrate as an additional nitrogen source. After inoculation with 30–120 mL of A. oryzae mycelium (white koji) with a dry matter content of 0.2–0.8 g / L, crushed using Ultra-Turrax, the mixtures were aerobically fermented for 48 h. The results show that of these added nitrogen sources, only ammonium ions, particularly ammonium sulfate, lowered the pH of the fermentation broth during aerobic fermentation, while the pH was only minimally affected by nitrate or urea.

[0060]

[0061] The effect of ammonium ions, which significantly lowers the pH value during fermentation, is also evident with longer fermentation durations. The following nutrient medium compositions were inoculated with Aspergillus oryzae in two containers each, without pasteurization and without pH adjustment. The pH value was measured at the beginning of inoculation (0 h), after 2 days (48 h), and after 5 days (120 h).

[0062] Container 1 + 2: Oat flour 20g / L, sucrose 10g / L, ammonium sulfate 10g / L

[0063] Containers 3 + 4: Oat flour 20g / L, sucrose 10g / L

[0064] Containers 5 + 6: Oat flour 20g / L, sucrose 10g / L, fresh yeast (Saccharomyces cerevisiae) 10g / L

[0065]

[0066] These results also show that when ammonium ions are added to the nutrient medium, the pH value is lowered by at least 2 pH units through aerobic fermentation alone.

Claims

Claims 1. Method for the fermentative production of mushroom mycelium with the following steps 1) Providing an aqueous nutrient medium containing a substrate that includes plant protein, 2) Inoculating the nutrient medium with at least one fungus to produce a nutrient medium inoculated with the at least one fungus, 3) Mixing and aerating the inoculated nutrient medium to attract fungal mass of at least one fungus for the fermentative production of fermenter broth containing attracted fungal mass, wherein ammonium ions are added to the nutrient medium at a concentration of 0.02 to 0.1 M, and the pH is lowered by at least 2 pH units through fermentation. 4) without controlled adjustment of the pH value, 5) Separation of mushroom mass from the fermenter broth to produce aqueous mushroom mass and at least one aqueous residue, 6) Providing a further aqueous nutrient medium by mixing at least part of the aqueous residue, which optionally contains insoluble plant components or consists of soluble components, with plant protein, and Repeat steps 2) to 6) exhibits.

2. Method according to claim 1, characterized in that in step 3) during the attraction of the mushroom mass in the nutrient medium with added ammonium ions, the pH value is lowered by at least 4 pH units.

3. Method according to one of the preceding claims, characterized in that the nutrient medium contains 5 to 30 g / L of vegetable protein.

4. A method according to any one of the preceding claims, characterized in that ammonium ions are added as ammonium sulfate.

5. A method according to any one of the preceding claims, characterized in that the nutrient medium in step 1) contains exclusively ammonium ions as an inorganic nitrogen source.

6. Method according to one of the preceding claims, characterized in that the substrate is the water-insoluble residue from the aqueous extraction of beans, in particular soybeans, oats, wheat, rice and / or lentils.

7. Method according to one of the preceding claims, characterized in that the pH value of the fermenter broth is lowered to a pH value of 2.5 to 4.5 by cultivating the fungus within 48 h.

8. Method according to one of the preceding claims, characterized in that only the substrate is pasteurized before inoculation and the pasteurized substrate is subsequently mixed with water to provide the aqueous nutrient medium.

9. Method according to one of the preceding claims, characterized in that the nutrient medium containing ammonium ions, substrate and aqueous residue is pasteurized before inoculation.

10. Method according to one of the preceding claims, characterized in that the substrate or the nutrient medium is heated to a maximum temperature of 70°C, preferably a maximum of 68°C, before inoculation.

11. Method according to one of the preceding claims, characterized in that the substrate and / or the nutrient medium has a bacterial content of a maximum of 10 6 exhibits KbE / mL.

12. Method according to one of the preceding claims, characterized in that, prior to step 2), the bacterial content of the substrate and / or the nutrient medium is reduced to a maximum of 10 by pasteurization or partial sterilization. 6 13. Method according to one of the preceding claims, characterized in that pasteurization is carried out by heating to 68 °C to 120 °C for 20 to 120 min, preferably up to 60 min.

14. Method according to one of the preceding claims, characterized in that inoculation is carried out by adding fragmented mycelium, which is fragmented by shearing into aggregates of a maximum of 10 hyphae.

15. Method according to one of the preceding claims, characterized in that the substrate is contained in the aqueous nutrient medium to a weight of 20 to 40% dry matter.

16. Method according to one of the preceding claims, characterized in that in step 3) the fermentative production of fermenter broth is carried out up to a content of the fermenter broth of cultivated mushroom mass of 10 to 30 g / L dry matter.

17. Method according to one of the preceding claims, characterized in that the fungus is Aspergillus oryzae.

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