Fusarium for producing mycelial protein and use thereof

Abstract

Provided in the present disclosure are Fusarium for producing a mycelial protein and the use thereof. Specifically disclosed in the present disclosure is the use of Fusarium or a microbial agent thereof in the production of a mycelial protein and / or a protein-rich mycelium. The Fusarium or the microbial agent thereof comprises any one or more of Fusarium avenaceum, Fusarium sp., and Fusarium equiseti.

Description

Fusarium species that produce mycelial protein and their applications Technical Field

[0001] This disclosure relates to the field of microbial technology, specifically to the application of one or more of Fusarium species, including Fusarium avenaceum, Fusarium sp., and Fusarium equiseti, in the production of mycelial protein and the resulting mycelial protein. Background Technology

[0002] Alternative proteins are proteins produced from plant or animal cells or through fermentation. These innovative foods are designed to taste the same as or better than traditional animal products and offer health benefits such as zero cholesterol, low fat, and low dietary fiber, while costing the same or less. Compared to conventionally produced animal products, alternative proteins require fewer inputs, such as land, fertilizer, and water, and produce far fewer negative externalities, such as greenhouse gas emissions and air pollution. They also reduce the contribution of food production to pandemic risk and antibiotic resistance.

[0003] Fusarium is a widely used filamentous fungus for fermenting and producing microbial protein. Its mycelial protein is more flavorful than single-celled proteins from yeast and bacteria, and has a meat-like texture. Furthermore, its rich dietary fiber aids digestion, making it a meat substitute that can meet the nutritional needs of modern people. Currently, in the field of filamentous fungal protein, *Fusarium venenatum* strains have been developed to produce meat products containing mycelial protein. However, naturally high-protein-producing *Fusarium venenatum* strains are extremely scarce. Therefore, finding more promising strains of the *Fusarium* genus that naturally produce high-protein filamentous fungi is a commercially valuable development opportunity. Summary of the Invention

[0004] In order to solve one of the aforementioned technical problems in the prior art, this disclosure provides a new application of Fusarium in the production of mycelial protein, as well as a method for producing mycelial protein.

[0005] According to one aspect of this disclosure, the use of Fusarium or its inoculum agents in the production of mycelial proteins and / or protein-rich mycelia is provided, wherein the Fusarium or its inoculum agents include any one or more of Fusarium avenaceum, Fusarium sp., and Fusarium equiseti.

[0006] According to another aspect of this disclosure, a Fusarium or its inoculum is provided, comprising one or more of Fusarium avenaceum, Fusarium sp., and Fusarium equiseti.

[0007] According to another aspect of this disclosure, a mycelial protein and / or protein-rich mycelium is provided, which is derived from the above-mentioned Fusarium or its inoculum agents of this disclosure.

[0008] In some embodiments, the Fusarium or its inoculum may include Fusarium avenaceum.

[0009] In some embodiments, the Fusarium or its inoculum may include Fusarium sp. MN113808.

[0010] In some embodiments, the Fusarium or its inoculum may include Fusarium equiseti.

[0011] In some embodiments, the Fusarium or its inoculum may include any two or three of Fusarium avenaceum, Fusarium sp. MN113808, and Fusarium equiseti.

[0012] In some embodiments, the translation elongation factor 1 (TEF1) of *Fusarium avenaceum* has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:9. In some embodiments, the translation elongation factor 1 (TEF1) of *Fusarium avenaceum* has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:9.

[0013] In some embodiments, the polymerase II subunit (RPB2) of *Fusarium avenaceum* has a nucleotide sequence that is at least 85% sequence identical to that of SEQ ID NO:10. In some embodiments, the RPB2 of *Fusarium avenaceum* has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to that of SEQ ID NO:10.

[0014] In some embodiments, the internal transcribed spacer (ITS) region of the *Fusarium avenaceum* has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:11. In some embodiments, the ITS of the *Fusarium avenaceum* has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:11.

[0015] In some embodiments, the Fusarium avenaceum may include strain MN133388, which was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41315.

[0016] In some embodiments, the TEF1 of the *Fusarium* sp. has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:14. In some embodiments, the TEF1 of the MN113808 (*Fusarium* sp.) has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:14.

[0017] In some embodiments, the internal transcribed spacer (ITS) region of the *Fusarium* sp. has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:15. In some embodiments, the ITS of the MN113808 (*Fusarium* sp.) has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:15.

[0018] In some embodiments, the RPB1 of the *Fusarium* sp. has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:16. In some embodiments, the RPB1 of the MN113808 (*Fusarium* sp.) has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:16.

[0019] In some embodiments, the RPB2 of the *Fusarium* sp. has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:17. In some embodiments, the RPB2 of the MN113808 (*Fusarium* sp.) has a nucleotide sequence that is at least 85%, at least 90%, 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:17.

[0020] In some embodiments, the Fusarium sp. may include strain MN113808, which was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41316.

[0021] In some embodiments, the TEF1 of *Fusarium equiseti* has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:18. In some embodiments, the TEF1 of *Fusarium equiseti* has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:18.

[0022] In some embodiments, the internal transcribed spacer (ITS) region of the *Fusarium equiseti* has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:19. In some embodiments, the ITS of the *Fusarium equiseti* has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:19.

[0023] In some embodiments, the RPB1 of *Fusarium equiseti* has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:20. In some embodiments, the RPB1 of *Fusarium equiseti* has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:20.

[0024] In some embodiments, the RPB2 of *Fusarium equiseti* has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:21. In some embodiments, the RPB2 of *Fusarium equiseti* has a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identical to SEQ ID NO:21.

[0025] In some embodiments, the *Fusarium equiseti* may include strain MN146565, which was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41317.

[0026] In some embodiments, the Fusarium can be in spore form and / or mycelial form.

[0027] In some embodiments, the mycelium comprises more than 45% protein based on its dry weight.

[0028] In some embodiments, the mycelium comprises about 45.5%, 50%, 50.5%, 51%, 51.5%, 52%, 52.5%, or 53% or more of protein, based on its dry weight.

[0029] In some embodiments, the mycelium comprises mycelial protein with a biomass concentration of about 0.4% or more, based on its dry weight. In some embodiments, the mycelium comprises protein with a biomass concentration of about 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, or 1.5% or more, based on its dry weight.

[0030] In some embodiments, the mycelium does not contain any harmful substances, such as aflatoxin (e.g., but not limited to aflatoxin B1, B2, G1, G2, etc.), vomitoxin (e.g., but not limited to deoxynivalenol), zearalenone, cucurbitacinol, fusarium ketone, ochratoxin, fumonisin (e.g., but not limited to fumonisin FA1, FA2, FB1, FB2, FB3, FB4, FC1, FC2, FC3, FC4, etc.), T-2 toxin, HT-2 toxin, fusarium citrate, etc., which are present or contained below the detection limit.

[0031] In some embodiments, the mycelium is filamentous; and / or, the filter cake is elastic or tough.

[0032] In some embodiments, the microbial agent includes the Fusarium spp. and excipients acceptable to the microbial agent. Those skilled in the art will understand that the excipients acceptable to the microbial agent and their amounts can be conventionally selected as needed, and there are no particular limitations in this disclosure.

[0033] According to another aspect of this disclosure, a method for producing mycelium is provided, the method comprising: 1) inoculating the Fusarium of this disclosure or its inoculum into a seed culture medium to obtain a seed liquid; 2) inoculating the seed liquid into a fermentation culture medium to obtain a fermentation broth; and 3) obtaining mycelium from the fermentation broth.

[0034] In some embodiments, the seed culture medium and / or fermentation culture medium may include a carbon source and a nitrogen source.

[0035] In some embodiments, the carbon source may include sugars (e.g., sucrose, maltose, glucose, fructose, rare Japanese sugars, etc.), sugar alcohols (e.g., glycerol, polyols, etc.), starch (e.g., corn starch, etc.), starch derivatives (e.g., maltodextrin, cyclodextrin, glucose syrup, hydrolysates, and modified starch), starch hydrolysates (e.g., corn starch saccharification liquid, rice starch saccharification liquid, sweet potato starch saccharification liquid, molasses, etc.), hydrogenated starch hydrolysates (HSH; e.g., hydrogenated glucose syrup, maltitol syrup, sorbitol syrup, etc.), lignocellulose pulp or raw materials (e.g., beet pulp, agricultural pulp, wood pulp, dried brewer's grains, brewery waste, etc.), potato starch extract, corn steep liquor, acid whey, sweet whey, whey, wheat steep liquor, carbohydrates, food waste, olive oil processing waste, hydrolysates of lignocellulose materials, wet-milled corn products (e.g., carbon-refined syrup, desalted syrup, enzyme-inverted syrup, etc.), and / or combinations thereof.

[0036] In some embodiments, the nitrogen source in the seed culture medium and / or fermentation culture medium can be an inorganic nitrogen source and / or an organic nitrogen source. In some embodiments, the organic nitrogen source may include, but is not limited to, peptone, yeast extract, yeast hydrolysate, yeast extract, soybean meal, etc. In some embodiments, the inorganic nitrogen source may include, but is not limited to, urea or ammonium compounds (e.g., ammonium sulfate, ammonium phosphate).

[0037] In some embodiments, in addition to nitrogen and carbon sources, the seed culture medium and / or fermentation culture medium may also include inorganic salts, such as sodium salts, potassium salts, calcium salts, magnesium salts, manganese salts, iron salts, zinc salts, etc. In some embodiments, the culture medium may include, but is not limited to, one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, ferrous sulfate, ferric sulfate, and zinc chloride.

[0038] In some embodiments, the seed culture medium may include 25-35 parts by weight of a carbon source, 20-35 parts by weight of a nitrogen source, and 0.1-2 parts by weight of an inorganic salt. In some embodiments, the seed culture medium may include 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 parts by weight of a carbon source. In some embodiments, the seed culture medium may include 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 parts by weight of a nitrogen source. In some embodiments, the seed culture medium may include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 parts by weight of an inorganic salt.

[0039] In a specific embodiment, the seed culture medium may include: 25-35 parts by weight of glucose, 20-35 parts by weight of yeast extract, 0.1-2 parts by weight of potassium dihydrogen phosphate, 0.1-2 parts by weight of magnesium sulfate heptahydrate, and 0.005-1 parts by weight of calcium chloride.

[0040] In a specific embodiment, the seed culture medium may include 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 parts by weight of glucose.

[0041] In a specific embodiment, the seed culture medium may include 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 parts by weight of yeast extract.

[0042] In a specific embodiment, the seed culture medium may include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 parts by weight of potassium dihydrogen phosphate.

[0043] In a specific embodiment, the seed culture medium may include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 parts by weight of magnesium sulfate heptahydrate.

[0044] In a specific embodiment, the seed culture medium may include 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 part by weight of calcium chloride.

[0045] In some embodiments, the fermentation medium may include 20 to 35 parts by weight of a carbon source, 0.01 to 10 parts by weight of a nitrogen source, and 0.01 to 10 parts by weight of an inorganic salt.

[0046] In some embodiments, the fermentation medium may include 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 parts by weight of a carbon source. In some embodiments, the fermentation medium may include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 parts by weight of a nitrogen source.

[0047] In some embodiments, the fermentation medium may include: 20-35 parts by weight of corn starch saccharification solution; 0.1-2 parts by weight of citric acid; 0.1-2 parts by weight of potassium dihydrogen phosphate; 0.01-10 parts by weight of ammonium sulfate; 0.01-2 parts by weight of magnesium sulfate heptahydrate; 0.001-1 parts by weight of calcium chloride; and, optionally, 0.001-0.1 parts by weight of trace elements.

[0048] In a specific embodiment, the fermentation culture medium may include 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 parts by weight of corn starch saccharification solution.

[0049] In a specific embodiment, the fermentation medium may include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 parts by weight of citric acid.

[0050] In a specific embodiment, the fermentation medium may include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 parts by weight of potassium dihydrogen phosphate.

[0051] In a specific embodiment, the fermentation culture medium may include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 parts by weight of ammonium sulfate.

[0052] In a specific embodiment, the fermentation medium may include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 parts by weight of magnesium sulfate heptahydrate.

[0053] In a specific embodiment, the fermentation culture medium may include 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 part by weight of calcium chloride.

[0054] In some embodiments, the fermentation medium may include 0.001 to 0.1 parts by weight of trace elements. In some embodiments, the trace elements include 0.5 to 2.5 parts by weight of manganese sulfate monohydrate, 1 to 10 parts by weight of ferrous sulfate heptahydrate, and 0.5 to 2.5 parts by weight of zinc chloride.

[0055] In a specific implementation, the trace element may include 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4 or 2.5 parts by weight of manganese sulfate monohydrate.

[0056] In a specific implementation, the trace element may include 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 parts by weight of ferrous sulfate heptahydrate.

[0057] In a specific embodiment, the fermentation culture medium may include 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4 or 2.5 parts by weight of zinc chloride.

[0058] In some embodiments, the culture temperature in step 2) can be approximately 20°C to 35°C. In some embodiments, the culture temperature in step 2) can be approximately 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0059] In some implementations, in step 2), the aeration rate during the culture process is approximately 5–15 L / h.

[0060] In some embodiments, in step 2), the pH of the fermentation medium is approximately 5.0 to 7.0. In some embodiments, in step 2), the pH of the culture is approximately 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0061] In some embodiments, the fermentation medium may further comprise a vitamin complex. In some embodiments, the fermentation medium may further comprise 0.0001 to 0.2 parts by weight of the vitamin complex. In some embodiments, the fermentation medium may further comprise 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, or 0.2 parts by weight of the vitamin complex.

[0062] In some embodiments, the vitamin complex may include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, or any combination thereof. In specific embodiments, the vitamin complex may include one or more of vitamin B1 (thiamine), vitamin B12 (cobalamin), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (D-calcium pantothenate), vitamin B6 (pyridoxine), vitamin B9 (folic acid and / or folate), vitamin C, and vitamin H (biotin). In specific embodiments, the components of the vitamin complex are mixed in equal parts by mass. In these embodiments, no spores are produced during the fermentation culture.

[0063] In some embodiments, in step 2), the aeration rate of the culture is approximately 1 L / min to 20 L / min. In some embodiments, in step 2), the aeration rate of the culture is approximately 1 L / min, 2 L / min, 3 L / min, 4 L / min, 5 L / min, 6 L / min, 7 L / min, 8 L / min, 9 L / min, 10 L / min, 11 L / min, 12 L / min, 13 L / min, 14 L / min, 15 L / min, 16 L / min, 17 L / min, 18 L / min, 19 L / min, or 20 L / min.

[0064] In some embodiments, the culture pressure in step 2) is approximately 0.005 MPa to 0.2 MPa. In some embodiments, the culture pressure in step 2) is approximately 0.005 MPa, 0.006 MPa, 0.007 MPa, 0.008 MPa, 0.009 MPa, 0.01 MPa, 0.02 MPa, 0.03 MPa, 0.04 MPa, 0.05 MPa, 0.06 MPa, 0.07 MPa, 0.08 MPa, 0.09 MPa, 0.1 MPa, 0.11 MPa, 0.12 MPa, 0.13 MPa, 0.14 MPa, 0.15 MPa, 0.16 MPa, 0.17 MPa, 0.18 MPa, 0.19 MPa, or 0.2 MPa.

[0065] In some embodiments, the culture temperature in step 2) can be approximately 20°C to 35°C. In some embodiments, the culture temperature in step 2) can be approximately 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0066] In some embodiments, in step 2), the pH of the fermentation medium can be 3.0 to 5.0. In some embodiments, in step 2), the pH of the fermentation medium can be 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0067] In some implementations, step 2) may include 2 to 10 rounds of fermentation, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 rounds of fermentation.

[0068] In some embodiments, the method may further include the following steps between steps 2) and 3): a) inoculating the first fermentation broth into a fermentation medium and culturing it at 0.01–0.1 mPa (e.g., 0.01 mPa, 0.02 mPa, 0.03 mPa, 0.04 mPa, 0.05 mPa, 0.06 mPa, 0.07 mPa, 0.08 mPa, 0.09 mPa, or 0.1 mPa) with an aeration rate of 800–1200 L / h for 5–20 h; and b) adding feed medium and culturing it at 0.1–0.5 mPa (e.g., 0.1 mPa, 0.15 mPa, 0.2 mPa, 0.25 mPa, 0.3 mPa, 0.35 mPa, 0.4 mPa, 0.45 mPa, or 0.5 mPa) with an aeration rate of 1300–2000 h / L for 24–48 h to obtain the second fermentation broth.

[0069] In some implementations, the ventilation rate in step a) can be 800 L / h, 900 L / h, 1000 L / h, 1100 L / h or 1200 L / h.

[0070] In some implementations, the ventilation rate in step b) can be 1300 L / h, 1400 L / h, 1500 L / h, 1600 L / h, 1700 L / h, 1800 L / h, 1900 L / h or 2000 L / h.

[0071] In some implementations, the incubation time for step a) can be, for example, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h or 20h.

[0072] In some embodiments, the supplemental culture medium may include the aforementioned carbon source and / or vitamin complex.

[0073] In some embodiments, the supplemental culture medium may include 10,000 to 50,000 parts by weight of corn starch saccharification solution and / or 0.001 to 0.2 parts by weight of vitamin complex.

[0074] In some embodiments, the feed medium may include 20,000 to 40,000 parts by weight of corn starch saccharification solution. In some embodiments, the feed medium may include 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 parts by weight of corn starch saccharification solution.

[0075] In some embodiments, the supplemental culture medium may include 0.001 to 0.2 parts by weight of a vitamin complex. In some embodiments, the supplemental culture medium may include 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, or 0.2 parts by weight of a vitamin complex.

[0076] According to another aspect of this disclosure, a composition is provided comprising the mycelial protein of this disclosure and / or protein-rich mycelium.

[0077] In some embodiments, the composition comprises 15 to 95 wt% (e.g., 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt% or 95 wt%) of mycelial protein and / or protein-rich mycelium, based on wet-based mycelial protein and / or protein-rich mycelium.

[0078] In some embodiments, the mycelial protein and / or protein-rich mycelium may also be provided in dry matter form.

[0079] In some embodiments, the composition may further comprise one or more of starch, konjac flour, vegetable or animal oil, alginate, arabinoxylan, carrageenan, carboxymethyl cellulose, cellulose, gelatin, gellan gum, dextran, galactomannan, guar gum, locust bean gum, tara gum, gum arabic, pectin, and xanthan gum.

[0080] This invention uses starch, edible gums and / or modified starch as thickeners, as specified in GB2760. The thickener absorbs water and swells, forming a network structure with the mycelial protein, which further enhances the organization and emulsification properties of the mycelial protein, thereby improving the texture and taste of the meat substitute. The resulting mycelial protein-based meat substitute can completely replace plant-based meat products, avoiding the defects of plant proteins, while also possessing excellent taste and texture. The taste is juicy, elastic and moderately firm, and the texture has good firmness, elasticity and chewiness, making it closer to real meat products. Compared with plant-based meat or pork and chicken, the mycelial protein-based meat substitute has a higher protein content and natural dietary fiber content, a lower fat content, and is cholesterol-free, making it more nutritious and healthy.

[0081] According to another aspect of this disclosure, the use of mycelial proteins and / or protein-rich mycelia of this disclosure, or the above-described compositions, in the preparation of meat substitutes is provided.

[0082] In some embodiments, the mycelial protein and / or protein-rich mycelium of this disclosure, or the above-described composition, can replace chicken, pork, beef, mutton, duck, etc. Attached Figure Description

[0083] Figure 1 shows the phylogenetic tree analysis results of strains MN133388, MN146565, and MN113808.

[0084] Figure 2 shows the mycelium prepared by fermentation of MN133388 strain.

[0085] Figure 3 shows the mycelium prepared by fermentation of MN146565 strain.

[0086] Figure 4 shows the mycelium prepared by fermentation of MN113808 strain.

[0087] Figure 5 shows the results of the first-generation fermentation culture of MN133388 strain. Figure 5A shows the microscopic examination results, and Figure 5B shows an image of the fermentation broth.

[0088] Figure 6 shows the results of the second-generation fermentation culture of MN133388 strain. Figure 6A shows the microscopic examination results, Figure 6B is an image of the fermentation broth, and Figure 6C is an image of the filter cake.

[0089] Figure 7 shows a picture of the filter cake obtained from the third-generation fermentation culture of MN133388 strain.

[0090] Figure 8 shows a picture of the filter cake obtained from the fifth generation fermentation culture of MN133388 strain.

[0091] Figure 9 shows the microscopic examination results of the third-generation fermentation culture of MN113808 strain.

[0092] Figure 10 shows a picture of the filter cake obtained from the third-generation fermentation culture of MN113808 strain.

[0093] Figure 11 shows the microscopic examination results of the third-generation fermentation culture of MN146565 strain.

[0094] Figure 12 shows a picture of the filter cake obtained from the third-generation fermentation culture of MN146565 strain.

[0095] Figure 13 shows the SGS detection results of mycelial protein from MN133388. Detailed Implementation

[0096] In the field of fungal mycelial proteins, naturally high-protein-producing *Fusarium* species are extremely rare, as are naturally high-protein-producing strains of the *Fusarium* genus. This publication discloses the development of a novel *Fusarium* species that naturally produces high-protein filamentous fungi.

[0097] Fusarium avenaceum, during scaled-up fermentation production, was tested and found to contain approximately 11.06% total fat, 48.37% crude protein, and 29.50% dietary fiber. Those skilled in the art know that a protein content of approximately 40% is suitable for product development. Currently, there are no reports of Fusarium avenaceum being used for the development and application of fungal proteins.

[0098] This disclosure identifies more than ten toxins in the mycelial proteins produced by *Fusarium avenaceum*, and the results show that the toxin content is below the limit, indicating that it is free of toxins. Therefore, the strain disclosed in this disclosure solves the safety issue for food consumption in food development.

[0099] In a specific implementation, this disclosure screened additives in the culture medium and found that adding compound vitamin solution to the culture medium can improve the quality of the mushroom cake.

[0100] It is known that spore production can affect the texture and shape of a product. This disclosure significantly controls spore production by improving the fermentation process. In a specific embodiment, no spores were observed under a microscope in the mycelial protein produced by the Fusarium spores of this disclosure. In a specific embodiment, the mycelial protein produced by the Fusarium spores of this disclosure has excellent filamentous structure, the filter cake is light yellow, elastic and resilient, and has no unpleasant odor.

[0101] In specific embodiments, the mycelial protein obtained from the *Fusarium* strain of this disclosure through fermentation culture exhibits well-defined filaments under microscopic examination, and the fermentation broth naturally displays a flesh-colored hue even without heating. The protein content of the mycelial protein produced by the *Fusarium* strain of this disclosure is all above 50%, even reaching over 55%.

[0102] Normally, strains can only utilize organic nitrogen sources and not inorganic nitrogen sources. In a specific embodiment, this disclosure enables the Fusarium strain to utilize inorganic nitrogen sources by adding trace elements. This also improves the shape, color, and texture of the mycelium.

[0103] This disclosure provides a method for producing mycelial protein, the method comprising: 1) inoculating the Fusarium spp. or its inoculum agent disclosed herein into a seed culture medium to obtain a seed liquid; 2) inoculating the seed liquid into a fermentation culture medium to obtain a fermentation broth; and 3) obtaining mycelial protein from the fermentation broth.

[0104] In a specific embodiment, the seed culture medium may include 25-35 g / L of glucose, 20-35 g / L of yeast extract, 0.1-2 g / L of potassium dihydrogen phosphate, 0.1-2 g / L of magnesium sulfate heptahydrate, and 0.005-1 g / L of calcium chloride.

[0105] In a specific embodiment, the seed culture medium may include 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, or 35 g / L of glucose.

[0106] In a specific embodiment, the seed culture medium may include yeast extract at concentrations of 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, or 35 g / L.

[0107] In a specific embodiment, the seed culture medium may include 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, or 2 g / L of potassium dihydrogen phosphate.

[0108] In a specific embodiment, the seed culture medium may include 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, or 2 g / L of magnesium sulfate heptahydrate.

[0109] In a specific embodiment, the seed culture medium may include 0.005 g / L, 0.006 g / L, 0.007 g / L, 0.008 g / L, 0.009 g / L, 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1 g / L of calcium chloride.

[0110] In some embodiments, the fermentation medium may include: 20-35 g / L corn starch saccharification solution, 0.1-2 g / L citric acid, 0.1-2 g / L potassium dihydrogen phosphate, 0.01-10 g / L ammonium sulfate, 0.01-2 g / L magnesium sulfate heptahydrate, 0.001-1 g / L calcium chloride, 0.5-2.5 mg / L manganese sulfate monohydrate, 1-10 mg / L ferrous sulfate heptahydrate, and 0.5-2.5 mg / L zinc chloride.

[0111] In a specific embodiment, the fermentation medium may include corn starch saccharification solution with a concentration of 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, or 35 g / L.

[0112] In a specific embodiment, the fermentation medium may include 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, or 2 g / L of citric acid.

[0113] In a specific embodiment, the fermentation medium may include 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, or 2 g / L of potassium dihydrogen phosphate.

[0114] In specific embodiments, the fermentation medium may include concentrations of 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, and 0. Ammonium sulfate at concentrations of 7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, or 10 g / L.

[0115] In a specific embodiment, the fermentation medium may include 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, or 2 g / L of magnesium sulfate heptahydrate.

[0116] In a specific embodiment, the fermentation medium may include 0.001 g / L, 0.002 g / L, 0.003 g / L, 0.004 g / L, 0.005 g / L, 0.006 g / L, 0.007 g / L, 0.008 g / L, 0.009 g / L, 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1 g / L of calcium chloride.

[0117] In a specific embodiment, the fermentation medium may include 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, or 2.5 mg / L of manganese sulfate monohydrate.

[0118] In a specific embodiment, the fermentation medium may include ferrous sulfate heptahydrate at concentrations of 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, 5 mg / L, 5.5 mg / L, 6 mg / L, 6.5 mg / L, 7 mg / L, 7.5 mg / L, 8 mg / L, 8.5 mg / L, 9 mg / L, 9.5 mg / L, or 10 mg / L.

[0119] In a specific embodiment, the fermentation medium may include zinc chloride at concentrations of 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, or 2.5 mg / L.

[0120] In some embodiments, the fermentation medium may further comprise a vitamin complex. In some embodiments, the fermentation medium may further comprise 0.01–2 mg / L of the vitamin complex. In some embodiments, the fermentation medium may further comprise a vitamin complex at concentrations of 0.01 mg / L, 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.05 mg / L, 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, or 2 mg / L.

[0121] In specific embodiments, the meat substitutes of this disclosure (e.g., meat chunks or minced meat) may include vegetable or animal oils, alginate, arabinoxylan, carrageenan, carboxymethyl cellulose, cellulose, gelatin, gellan gum, β-glucan, galactomannan, guar gum, locust bean gum, tara gum, gum arabic, pectin, konjac flour, starch, and xanthan gum.

[0122] In specific embodiments, the mycelial protein of this disclosure may comprise filaments with a length of less than 3000 μm, preferably less than 2500 μm, less than 2000 μm, less than 1500 μm, less than 1400 μm, less than 1300 μm, less than 1200 μm, less than 1100 μm, less than 1000 μm, less than 900 μm, less than 800 μm, or less than 700 μm. The mycelial protein of this disclosure may comprise filaments with a length greater than 100 μm, preferably greater than 200 μm.

[0123] In specific embodiments, the mycelial protein of this disclosure may comprise filaments with a diameter of less than 20 μm, preferably less than 10 μm, more preferably 5 μm or smaller. In specific embodiments, the mycelial protein of this disclosure may comprise filaments with a diameter greater than 1 μm, preferably greater than 2 μm.

[0124] In specific embodiments, the mycelial protein of this disclosure may comprise filaments with an aspect ratio (length / diameter) of less than 800, preferably less than 650, more preferably less than 400, and especially 250 or less. In specific embodiments, the mycelial protein of this disclosure may comprise filaments with an aspect ratio greater than 10, greater than 20, greater than 30, greater than 40, greater than 50, greater than 60, or greater than 70.

[0125] The Fusarium spores or their inoculants disclosed herein produce mycelial proteins with very few branches; the fermentation broth is flesh-colored, gradually deepening in color with increasing passage number; the fermentation broth has no obvious off-odor; the filter cake has good elasticity and no obvious off-odor after heating.

[0126] The mycelial protein produced by Fusarium or its inoculants disclosed herein can be used to prepare meat substitutes, etc.

[0127] In some embodiments, the vitamin complex may include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, or any combination thereof. In specific embodiments, the vitamin complex may include one or more of vitamin B1 (thiamine), vitamin B12 (cobalamin), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (D-calcium pantothenate), vitamin B6 (pyridoxine), vitamin B9 (folic acid and / or folate), vitamin C, and vitamin H (biotin). In specific embodiments, the components of the vitamin complex are mixed in equal parts by mass.

[0128] The Fusarium spores or their inoculants disclosed herein produce mycelial proteins with very few branches; the fermentation broth is flesh-colored, gradually deepening in color with increasing passage number; the fermentation broth has no obvious off-odor; the filter cake has good elasticity and no obvious off-odor after heating.

[0129] The mycelial protein produced by Fusarium or its inoculants disclosed herein can be used to prepare meat substitutes, etc.

[0130] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0131] definition

[0132] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0133] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0134] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0135] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0136] Example

[0137] Example 1. Isolation and screening of strains MN133388, MN113808, and MN146565

[0138] 1.1 Source of strains

[0139] The MN113808 strain was collected from humus soil samples from Tianchi Lake in Changbai Mountain.

[0140] The MN133388 strain was collected from quinoa seed samples from Tibet.

[0141] The MN146565 strain was collected from soil samples from the western Yunnan mountains.

[0142] 1.2 Separation Process

[0143] Mix the collected samples thoroughly, weigh 5g, and place them in an Erlenmeyer flask containing 45mL of sterile 0.7% sodium carboxymethyl cellulose solution and 15 glass beads. Shake at 30℃ and 150rpm for 30min. Take 1mL of the sample suspension and dilute with sterile water for 10 minutes. -2 -10 -3 Serial concentration gradient dilution, then take 10 -2 10 -3 Two dilutions were spread onto MGA2.5 (containing chloramphenicol and tetracycline hydrochloride) plates at a rate of 100 μL / plate. The plates were spread evenly and incubated upside down at 28°C for 2-7 days after the surface of the plates had dried.

[0144] The MGA2.5 flat panel consists of the following components:

[0145] Purification was performed using a stepwise transplantation method of hyphal tips. After colonies formed on the agar plate, hyphae from the edge of a single colony were picked and transferred to an MEA plate (purchased from Beijing Aoboxing Biotechnology Co., Ltd., catalog number: 02-183), and incubated at a constant temperature of 28°C until pure colonies were obtained. The obtained colonies were then stored at -80°C.

[0146] Example 2. Identification of strain MN133388

[0147] Genomic DNA was extracted from the mycelium of the strain and used as a template for gene amplification. Multiple genes were amplified and sequenced from the strain's DNA.

[0148] 2.1 Preparation of PCR system

[0149] Prepare a 25 μL PCR system in a PCR tube according to the table below:

[0150] The primers for each gene are shown in the table below:

[0151] The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0152] 2.2 PCR amplification

[0153] The prepared PCR system was placed in a PCR instrument (Dongsheng ETC811) for PCR amplification. The PCR reaction conditions are shown in the table below. After amplification, the PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0154] 2.3 The sequencing results for strain MN133388 are shown in the table below:

[0155] 2.4 MN133388 was identified by comparing it with the standard NT library in NCBI.

[0156] The four gene sequences were aligned using the NCBI online alignment system (BLAST), with the Nucleotide Collection (NR / NT) database as the alignment database. The most similar alignment results for the four genes are shown in the table below.

[0157] 2.6 Multigene identification of MN133388 using the MyCobank database

[0158] Identification was performed using the Polyphasic identification method provided by the online database mycobank (fusarium.mycobank.org) by simultaneously uploading four genes to the website. The results showed that, based on the TEF1, RPB1, and RPB2 genes, the closest species to the current strain is *Fusarium avenaceum* NRRL 54939, with a global similarity of 99.36%.

[0159] 2.7 Constructing a polygenic phylogenetic tree for MN133388 to determine its evolutionary position

[0160] Based on the two methods described above, since MN133388 was identified as *Fusarium avenaceum*, the species complex to which this species belongs is the *Fusarium tricinctum species complex* (FTSC). Sequences of the TEF1, RPB1, RPB2, and ITS genes of all FTSC strains were collected from the FUSARIOID-ID database (www.fusarium.org), and the evolutionary position of strain MN133388 was analyzed by constructing a polygene phylogenetic tree.

[0161] The steps for polygenic phylogenetic tree analysis are as follows: First, MAFFT (version v7.310) was used to perform multiple sequence alignment for each gene. Then, GBLOCKS (version 0.91b) was used to prune the multiple sequence alignment result file. Next, AliView was used for manual pruning. After pruning, the sequences of the four genes were tandemly spliced. IQ-TREE (version 2.2.5) was used to construct a polygenic phylogenetic tree from the spliced ​​sequences. The results are shown in Figure 1.

[0162] Based on the above three analytical methods, the species of MN133388 was identified as Fusarium avenaceum, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, with accession number CGMCC No.: 41315.

[0163] Example 3. Identification of MN113808 strain

[0164] 3.1 Preparation of DNA template

[0165] Prepare the DNA template according to the steps in 2.1.

[0166] 3.2 Preparation of PCR system

[0167] Configure the PCT system according to 2.2.

[0168] The amplified genes and primers are shown in the table below:

[0169] 3.3 The sequencing results of strain MN113808 are shown in the table below:

[0170] 3.4 MN113808 was identified by comparison with the standard NT library in NCBI;

[0171] The NCBI online alignment system (BLAST) was used to align the four gene sequences to the Nucleotide Collection (NR / NT) database. The most similar alignment results for the four genes are shown in the table below.

[0172] 3.5 MN113808 was identified using the MyCobank database;

[0173] Identification was performed using the Polyphasic identification method provided by the online database mycobank (fusarium.mycobank.org) by simultaneously uploading all four genes to the website. The results showed that, based on the TEF1, RPB2, and ITS genes, the closest species to the current strain is *Fusarium nelsonii* NRRL 28505, with a global similarity of 92.55%.

[0174] 3.6 Constructing a polygenic phylogenetic tree for MN113808 to determine its evolutionary position

[0175] Based on the two methods described above, among the identified species, the subspecies of *Fusarium chlamydosporum*, *Fusarium nelsonii*, and *Fusarium microconidium* are all classified as *Fusarium chlamydosporum* species complex (FCSC), while the subspecies of *Fusarium sibiricum* is classified as *Fusarium sambucinum* species complex (FSAMSC). Sequences of the TEF1, RPB1, RPB2, and ITS genes of all FCSC and FSAMSC strains were collected from the FUSARIOID-ID database (www.fusarium.org), and the evolutionary position of the current strains was analyzed by constructing a polygene phylogenetic tree.

[0176] The steps for polygenic phylogenetic tree analysis are as follows: First, MAFFT (version v7.310) was used to perform multiple sequence alignment for each gene. Then, GBLOCKS (version 0.91b) was used to prune the multiple sequence alignment result file. Next, AliView was used for manual pruning. After pruning, the sequences of the four genes were tandemly spliced. IQ-TREE (version 2.2.5) was used to construct a polygenic phylogenetic tree from the spliced ​​sequences. The results are shown in Figure 1.

[0177] As shown in the phylogenetic tree in Figure 1, MN113808 belongs to the genus *Fusarium*, but differs significantly from other FCSC subspecies strains. Combined with the analysis results from MyCoBank, this suggests that it may be a new species within the FCSC subspecies. MN113808 was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No.: 41316.

[0178] Example 4. Identification of MN146565 strain

[0179] 4.1 Preparation of DNA Template

[0180] Prepare the DNA template according to the steps in 2.1.

[0181] 4.2 Preparation of PCR system

[0182] Configure the PCT system according to 2.2.

[0183] The amplified genes and primers are shown in the table below:

[0184] 4.3 The sequencing results of strain MN146565 are shown in the table below:

[0185] 4.4 MN146565 was identified by comparing it with the standard NT library in NCBI.

[0186] The four gene sequences were aligned using the NCBI online alignment system (BLAST), with the Nucleotide Collection (NR / NT) database as the alignment database. The most similar alignment results for the four genes are shown in the table below.

[0187] 4.5 Multigene identification of MN146565 using the MyCobank database

[0188] The strain was identified using the Polyphasic identification method provided by the online database mycobank (fusarium.mycobank.org) by simultaneously uploading all four genes to the website. The results showed that, based on the TEF1, RPB2, and ITS genes, the closest species to the current strain is *Fusarium equiseti* NRRL 20697, with a global similarity of 99.77%.

[0189] 4.6 Constructing a polygenic phylogenetic tree for MN146565 to determine its evolutionary position

[0190] Based on the two methods described above, since all species identified except for *Fusarium subtropicale* belong to the *Fusarium incarnatum-equiseti* species complex (FIESC), the sequences of the TEF1, RPB1, RPB2, and ITS genes of all FIESC strains were collected from the FUSARIOID-ID database (www.fusarium.org), and the evolutionary position of the current strain was analyzed by constructing a polygene phylogenetic tree.

[0191] The steps for polygenic phylogenetic tree analysis are as follows: First, MAFFT (version v7.310) was used to perform multiple sequence alignment for each gene. Then, GBLOCKS (version 0.91b) was used to prune the multiple sequence alignment result file. Next, AliView was used for manual pruning. After pruning, the sequences of the four genes were tandemly spliced. IQ-TREE (version 2.2.5) was used to construct a polygenic phylogenetic tree from the spliced ​​sequences. The results are shown in Figure 1.

[0192] As can be seen from the phylogenetic tree shown in Figure 1, the species of MN146565 is Fusarium equiseti, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, with accession number CGMCC No.: 41317.

[0193] Example 5. Preparation of mycelial protein

[0194] 5.1 Initial Screening Culture

[0195] Activated strains of MN133388 (Fusarium avenaceum), MN113808 (Fusarium sp.), and MN146565 (Fusarium equiseti) were inoculated into 250 mL conical flasks containing 50 mL of primary screening medium and incubated at 200 rpm with shaking for 24–48 h. The primary screening medium consisted of: 30 g / L glucose, 30 g / L yeast extract, 1 g / L KH₂PO₄, 0.5 g / L MgSO₄·7H₂O, and 0.2 g / L CaCl₂, and was autoclaved at 121 °C for 20 min.

[0196] The filter cake was filtered using a vacuum filtration device and thoroughly washed. It was then dried in an electric heating drying oven at 60°C. The mass of the dried filter cake was accurately weighed and the biomass concentration was calculated [biomass concentration = mycelial dry weight (g) / culture medium mass (g) * 100%]. Then, referring to GB 5009.5-2016 National Food Safety Standard - Determination of Protein in Food, the crude protein in the mycelium was determined using the Kjeldahl method. The results are shown in the table below.

[0197] Results of observation and testing of mycelial morphology, filter cake color and texture, biomass and crude protein of the strain.

[0198] As can be seen from the table above, the mycelial protein prepared by fermentation using MN133388 can produce mycelial protein with filamentous morphology, light yellow color, elastic texture, and a biomass concentration of 0.66 wt%. Further analysis using the Kjeldahl method showed that the crude protein content of the mycelium reached 52.90 wt%.

[0199] Mycelial protein was prepared by fermentation using MN113808. The mycelial protein was obtained with filamentous morphology, light yellow color, elastic texture, and biomass concentration of 1.6%. The crude protein content of the mycelium was further determined by Kjeldahl method to be 53.50%.

[0200] Mycelial protein was prepared by fermentation using MN146565, and the mycelial protein was obtained with filamentous morphology, light yellow color, elastic texture, and biomass concentration of 0.46%. The crude protein content of the mycelium was further determined by Kjeldahl method to be 52.82%.

[0201] Figures 2 to 4 show the mycelia prepared by fermentation of strains MN133388, MN146565 and MN113808, respectively.

[0202] 5.2 Scale-up culture for the production of mycelial protein

[0203] 5.2.1 Fermenter double-screening culture method

[0204] The seed culture medium used has the following components:

[0205] Seed culture conditions: The primary seed culture was prepared using the seed culture medium shown in the table above. Activated MN133388, MN113808, and MN146565 strains were picked and inoculated into 250 mL conical flasks containing 50 mL of seed culture medium. The flasks were then incubated at a constant temperature of 200 rpm for 24 to 60 hours.

[0206] The secondary seed culture was prepared using the above-mentioned seed culture medium. The inoculum was transferred at a rate of 5% to a 3L concave conical flask containing 1L of seed culture medium, and cultured at 200rpm for 24h to 60h.

[0207] The composition of the secondary screening medium for producing mycelial protein is as follows:

[0208] Fermentation process conditions: Fermentation tank volume 15L, loaded with 10L of double-screened culture medium, sterilized at 121℃ for 30min, inoculum size 3%. Temperature 28℃, aeration 5-15L / h, tank pressure 0.08-0.12Mpa, rotation speed 200-600rpm, dissolved oxygen 20-50%, pH controlled at 6.0 with ammonia water. Ammonium sulfate in the table above can be replaced with ammonium phosphate. The corn starch saccharification solution is obtained by treating corn starch (Qinhuangdao Lihua Starch Co., Ltd.) with thermostable α-amylase and saccharifying enzyme from Angel Enzyme Preparations (Yichang) Co., Ltd.

[0209] Microscopic examination and determination by Kjeldahl method showed that the crude protein content of mycelium of MN133388 reached 50 wt%, the crude protein content of mycelium of MN113808 reached 53.4 wt%, and the crude protein content of mycelium of MN146565 reached 50.63 wt%.

[0210] Compared with the primary screening medium in Example 4, the secondary screening medium in this embodiment uses inorganic nitrogen (ammonium sulfate, ammonium phosphate) to replace the organic nitrogen source; however, the crude protein content can still maintain a high level. This also indicates that the Fusarium of the present invention can simultaneously utilize both organic and inorganic nitrogen sources as nitrogen sources for the fermentation culture of fungal proteins.

[0211] The results showed that replacing organic nitrogen with inorganic nitrogen reduced costs while maintaining the filamentous structure of the mycelium. The mycelium was also characterized by its slender length, few branches, and extremely low spore count. Furthermore, all mycelial species maintained a crude protein content of over 50 wt%. The mycelium exhibited a slender filamentous structure, and the resulting filter cake had an excellent texture, better mimicking the chewiness of meat.

[0212] Results of secondary screening of three bacterial strains:

[0213] 5.2.2 To determine the optimal number of subcultures, the subculture method is as follows:

[0214] Use the seed culture medium components from 5.2.1.

[0215] Seed culture conditions: Same as the seed culture conditions in 5.2.1 above.

[0216] The composition of the subculture medium is as follows:

[0217] Culture conditions: 15L tank, 10L liquid volume (completely submerging the paddles), 1L of seed culture transferred; rotation speed 100-200rpm; aeration rate 10L / min; tank pressure 0.05MPa; pH controlled at 4.0 with ammonia; culture at 28℃ for 24-60 hours; continuous culture for 5 generations, replacing 1 / 2 of the fermentation broth volume each time. The compound vitamins can be B-complex vitamins.

[0218] In this embodiment, the compound vitamin includes the following components in equal mass ratios: vitamin B1 / thiamine, vitamin B12 / cobalamin, vitamin B2 / riboflavin, vitamin B3 / niacin, vitamin B5 / D-calcium pantothenate, vitamin B6 / pyridoxine, vitamin B9 / folic acid, vitamin C, and vitamin H / D-biotin.

[0219] Comparison of mycelial protein results obtained from subculture of MN133388 with previous rescreening results:

[0220] 1. When passed to the second generation, no spores were observed under a microscope, the filter cake was light flesh-colored, had good toughness, and had no abnormal odor;

[0221] 2. As the number of generations increases, the flesh color gradually darkens;

[0222] 3. After several passages and sampling, the protein content is close to 50%.

[0223] First-generation culture results: No heat treatment was performed. Microscopic examination showed good filamentous structure. The fermentation broth was pale yellow (yeast powder base color) (Figures 5A and 5B). The filter cake showed good elasticity. The fermentation broth had no obvious off-odor.

[0224] Second-generation culture results: Microscopic examination revealed a small number of branches; the unheated fermentation broth was slightly flesh-colored; after heating, the filter cake was white with a yellowish tinge (Figures 6A-6C), with good elasticity; and the fermentation broth had no obvious off-odor.

[0225] Third-generation partial canning: heat treatment, the filter cake is slightly flesh-colored (Figure 7), the filter cake has good elasticity, and there is no obvious odor after heating.

[0226] Results of the fifth generation: After heat treatment, the color of the filter cake was darker than that of the third generation (Figure 8). The filter cake had good elasticity and no obvious odor after heating.

[0227] Conclusion: MN133388 showed the best fermentation state (color and filter cake quality) in the second and third generations of subculture, but the flesh color gradually darkened with the increase of subculture number.

[0228] MN113808 and MN146565 have similar results.

[0229] The images of mycelia and filter cake of MN113808 cultured to the third generation are shown in Figure 9 and Figure 10, respectively.

[0230] The images of mycelia and filter cake of MN146565 cultured to the third generation are shown in Figure 11 and Figure 12, respectively.

[0231] 5.2.3 Obtaining mycelial protein through fermentation culture

[0232] Fermentation process

[0233] Seed culture: Seed culture medium as described in 5.2.1 was used for inoculation, with an inoculation amount of 5% and a liquid volume of 1L / 3L; the shaking speed was 200rpm; and the culture was carried out for 24h.

[0234] Primary fermentation: Subculture was carried out using the subculture medium described in 5.2.2. A 15L tank was used, with a liquid volume of 10L. The culture was sterilized at 121℃ for 30 minutes, and the seed culture was completely transferred by flame inoculation. The rotation speed was 200 rpm, the aeration rate was 10L / min, and the tank pressure was 0.05 MPa. The pH was controlled at 4.0 with ammonia water. The culture was carried out at 28℃ for 24 hours. The culture was carried out for two to three generations, with 1 / 2 of the fermentation broth replaced each time.

[0235] Secondary fermentation: fermentation medium

[0236] Feeding culture medium

[0237] The fermentation medium formula is shown in the table above. The vitamin complex solution includes the following components in equal mass ratios: vitamin B1 / thiamine, vitamin B12 / cobalamin, vitamin B2 / riboflavin, vitamin B3 / niacin, vitamin B5 / D-calcium pantothenate, vitamin B6 / pyridoxine, vitamin B9 / folic acid, vitamin C, and vitamin H / D-biotin.

[0238] Use a 50L tank, fill with 30L of liquid, and sterilize at 121℃ for 30 minutes. After sterilization, add 0.1mg / L vitamin mixture (filtered for sterilization). Inoculate the fermentation broth from the second or third generation passaged in a 15L tank at a rate of 5-10% using a sterilized tubing pumped into the tank through the bottom valve. Aerate at 1000L / h for the first 15 hours, maintaining a tank pressure of 0.05MPa. After 15 hours, start feeding (adding culture medium) at 0.5-2g / L / h, and adjust the aeration to 1500L / h, maintaining a tank pressure of 0.1MPa. Control the pH to 4.0-6.0 with ammonia. Incubate at 28℃ with a rotation speed of 100-450rpm and dissolved oxygen at 20%-50%, for approximately 72 hours.

[0239] Fermentation was carried out using the fermentation process described in 5.2.3 to obtain mycelial protein.

[0240] 1) Heat treatment: After fermentation, heat the fermentation liquid to 65℃ and keep it warm for about 20 minutes, then heat it to 90℃ and keep it warm for about 10 minutes, and then cool it to room temperature.

[0241] 2) Filtration and refrigeration: The fermentation broth after heat treatment is filtered by a plate and frame filter press. After thorough washing and ventilation, the filter cake is taken out and stored at 4°C.

[0242] 3) Testing: For crude protein determination, the sample pretreatment involves drying, pulverizing, and passing through a 60-mesh sieve. The determination is then performed according to GB5009.5-2016 National Food Safety Standard for the Determination of Protein in Food. Common mycotoxins in food are tested by a third-party testing agency, SGS.

[0243] Physicochemical tests were performed on the mycelial protein sample (wet filter cake) obtained from the fermentation of MN133388. The SGS test results are shown in Figure 13.

[0244] Example 6. Preparation of meat chunks from Fusarium avenaceum MN133388 mycelial protein.

[0245] The mycelial protein prepared in Example 5 was used to prepare minced meat, shrimp paste, and fish balls, including the following raw materials: Fusarium avenaceum mycelial protein raw material, water, starch, gluten powder, carrageenan, konjac powder, sunflower seed oil or rapeseed oil; and seasonings: monosodium glutamate, yeast extract, white sugar, food flavoring, soybean oil, edible salt, spices, disodium 5'-ribonucleotide; and pigments: paprika oleoresin, red yeast rice red, sorghum red, allura red, tomato red, and carmine.

[0246] During the preparation of meat chunks, methylcellulose, sodium alginate, anhydrous calcium chloride, or calcium acetate can be added to the above-mentioned raw materials to adjust the viscosity of the meat chunks. The specific formula for preparing a meat substitute using Fusarium avenaceum is shown in the table below:

[0247] 6.1 The mycelial protein base in the form of minced meat is obtained through the above formulas 1-7. The steps for preparing sausage using the mycelial protein base are as follows:

[0248] 1. Mix the thickener with some ice water, chop and stir evenly, add mycelial protein base, umami agent, sweetener, food flavoring, vegetable oil, edible salt, spices, coloring and the remaining ice water, mix evenly to obtain vegetarian meat filling;

[0249] 2. Fill the vegetarian meat filling into cellulose casings using a filling machine, and place the resulting sausage hanging rods into a smoking oven. Dry at 55°C for 40 minutes and steam at 85°C for 30 minutes.

[0250] 3. Remove from the refrigerator and cool to below 30°C with cold air, then remove the casing and package.

[0251] 4. Quick-freeze and put into storage.

[0252] 6.2 The mycelial protein base in the form of meat paste is obtained through the above formulas 1-7. The steps for preparing shrimp paste using the mycelial protein base are as follows:

[0253] (1) Mix the guar gum and starch acetate evenly in advance, add ice water and chop evenly, add mycelial protein base, egg white powder, sunflower seed oil, food flavoring, edible salt, white sugar, yeast extract, monosodium glutamate, spices and TG enzyme (transglutaminase), and stir evenly.

[0254] (2) Pack the raw materials that have been mixed evenly in step (1) into bags and squeeze them out of the packaging bags onto the tray;

[0255] (3) Place the tray in the steamer and steam at 55°C for 30 minutes;

[0256] (4) Raise the temperature of the steamer to 85°C and steam for 30 minutes until cooked through. Then cool to room temperature.

[0257] 6.3 The following steps are taken to prepare fish sausage using the mycelial protein base obtained through the above formulas 1-7 in the form of minced meat:

[0258] 1. Mix carrageenan, guar gum, and tapioca starch evenly beforehand, add ice water and chop evenly, add mycelium protein base material and stir evenly;

[0259] 2. Fill the nylon casings with the filling material obtained in step 1 using a filling machine, and then fasten the casings with a clipping machine;

[0260] 3. Place in a boiling water bath and boil at 85℃ for 40 minutes;

[0261] 4. Cool down to below 30℃ using running water;

[0262] 5. Quick-freeze and put into storage.

[0263] 6.4 The mycelial protein base in the form of minced meat is obtained through the above formulas 1-7. The steps for preparing fish fillets using the mycelial protein base are as follows:

[0264] 1. Carrageenan, sodium alginate, and phosphate distarch are premixed evenly, then ice water is added and the mixture is chopped and stirred evenly. Mycelium protein base material is added and stirred evenly.

[0265] 2. Place the filling from step 1 into the fish fillet mold and press it into the shape of a fish fillet;

[0266] 3. Place the fish fillets in the refrigerator and freeze for 1 hour;

[0267] 4. Dip the fish fillets in egg wash and breadcrumbs, pre-fry in oil at 170℃ for 2 minutes, then cool to room temperature;

[0268] 5. Quick-freeze and put into storage.

[0269] 6.5 The sausages prepared from mycelial protein bases with meat-minced morphology obtained by (1) sensory evaluation and (2) texture analyzer were evaluated using formulations 1-7.

[0270] (1) The juiciness, elasticity, and firmness of the product were evaluated by ordinary consumers using sensory evaluation methods.

[0271] The evaluation results of the texture of mycelium protein-based meat chunks are shown in the table below:

[0272] The table above shows that sausages prepared using mycelial protein bases in formulas 1-5 exhibit excellent juiciness, elasticity, and firmness, resulting in a superior taste. In contrast, sausages made with mycelial protein bases in formula 6 have weak elasticity and firmness, leading to a poorer taste. Furthermore, sausages made with mycelial protein bases in formula 7 have weak juiciness, low elasticity, high firmness, and a poor taste. This demonstrates that, by selecting specific mycelial protein, thickener, and water, and using the selected amounts of each, this invention can produce sausages with mycelial protein as the sole protein source, exhibiting a superior taste and capable of completely replacing animal meat and plant protein sausages.

[0273] (2) Appearance inspection

[0274] Based on the formulas 1-7 above, further coloring is added to more closely resemble the color of meat. The specific formulas are as follows:

[0275] The appearance of sausages prepared using mycelial protein bases from formulations 8–14 and commercially available pork sausages was tested using a colorimeter (Minolta Konica Colorimeter CR400).

[0276] L*: The brightness of an object, 0-100 represents the range from black to white, indicating the lightness or darkness of the color;

[0277] a*: The red or green color of the object; a positive value represents red and a negative value represents green.

[0278] b*: The yellow or blue color of the object; a positive value represents yellow, and a negative value represents blue.

[0279] The colorimeter test results for mycelium protein-based sausages are shown in the table below:

[0280] The table above shows that the mycelium protein-based sausages prepared by formulas 8-12 are closer in appearance to commercially available pork sausages, while the mycelium protein-based sausages prepared by formulas 13-14 have a certain difference in appearance from commercially available pork sausages.

[0281] (3) Texture testing

[0282] Sausages prepared from mycelial protein bases according to formulas 1-7 and commercially available pork sausages were cut into cylinders with a diameter of φ2cm*2cm. The texture of the samples was repeatedly measured 6 times using a texture analyzer (Micro Stable TA.XTplus C, TPA mode, UK). The probe was P / 36R, the probe measurement speed was 1mm / s, the deformation (compression distance / sample height) was 40%, the trigger type was automatic, the trigger force was 5.0g, and the time interval between two compressions was 5s.

[0283] Elasticity: Elasticity refers to the ability of an object to return to its original state after it has been deformed by an external force and the force has been removed.

[0284] Cohesion: Cohesion reflects the property of food resisting damage and remaining tightly bound together during chewing, thus maintaining the integrity of the food.

[0285] Chewability: Used to describe solid test samples, it represents the energy required to chew a solid sample into a stable state for swallowing, and is numerically expressed as the product of hardness × cohesion × elasticity.

[0286] The texture analyzer test results for mycelial protein-based sausages are shown in the table below:

[0287] As can be seen from the table above, the sausages prepared with mycelium protein bases of formulas 1-5 of this invention are closer to commercially available pork sausages, and their hardness, elasticity, and chewiness are superior to those of commercially available pork sausages. However, the texture of the mycelium protein base sausages prepared with formulas 6-7 differs to some extent from that of commercially available pork sausages. The mycelium protein base sausage in formula 6 has too weak hardness, weak elasticity, and poor cohesion, resulting in poor chewiness and taste. Similarly, the mycelium protein base sausage in formula 7 has too high hardness, resulting in weak elasticity and poor cohesion, leading to excessively high chewiness and a significant difference in taste compared to commercially available pork sausages. This invention uses starch, edible gums and / or modified starch as thickeners, as specified in GB2760. The thickener absorbs water and swells, forming a network structure with the mycelial protein, which further enhances the organization and emulsification properties of the mycelial protein, thereby improving the texture and taste of the meat substitute. The resulting mycelial protein-based meat substitute can completely replace plant-based meat products, avoiding the defects of plant proteins, while also possessing excellent taste and texture. The taste is juicy, elastic and moderately firm, and the texture has good firmness, elasticity and chewiness, making it closer to real meat products. Compared with plant-based meat or pork and chicken, the mycelial protein-based meat substitute has a higher protein content and natural dietary fiber content, a lower fat content, and is cholesterol-free, making it more nutritious and healthy.

[0288] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. The use of Fusarium or its inoculum in the production of mycelial protein and / or protein-rich mycelium, wherein the Fusarium or its inoculum includes any one or more of Fusarium avenaceum, Fusarium sp., and Fusarium equiseti.

2. A Fusarium species or its inoculum, comprising one or more of Fusarium avenaceum, Fusarium sp., and Fusarium equiseti.

3. A mycelial protein and / or protein-rich mycelium derived from Fusarium or its inoculum as described in claim 2.

4. The application according to claim 1, the Fusarium or its inoculum agent according to claim 2, or the mycelial protein and / or protein-rich mycelium according to claim 3, characterized in that, The TEF1 of *Fusarium avenaceum* has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:9, and / or The RPB2 of *Fusarium avenaceum* has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:10, and / or The ITS of the *Fusarium avenaceum* species has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:

11. Preferably, the Fusarium avenaceum includes strain MN133388, which was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41315.

5. The application according to claim 1, the Fusarium or its inoculum agent according to claim 2, or the mycelial protein and / or protein-rich mycelium according to claim 3, characterized in that, The TEF1 of the Fusarium sp. has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:14, and / or The ITS of the Fusarium sp. has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:15, and / or The RPB1 of the Fusarium sp. has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:16, and / or The RPB2 of the Fusarium sp. has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:

17. Preferably, the Fusarium sp. may include strain MN113808, which was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.41316.

6. The application according to claim 1, the Fusarium or its inoculum agent according to claim 2, or the mycelial protein and / or protein-rich mycelium according to claim 3, characterized in that, The TEF1 of *Fusarium equiseti* has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:18, and / or The ITS of *Fusarium equiseti* has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:19, and / or The RPB1 of *Fusarium equiseti* has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:20, and / or The RPB2 of *Fusarium equiseti* has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:

21. Preferably, the *Fusarium equiseti* strain includes MN146565, which was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41317.

7. The application according to claim 1, the Fusarium or its inoculum agent according to claim 2, or the mycelial protein and / or protein-rich mycelium according to claim 3, characterized in that, The Fusarium species are in spore and / or mycelial form, and / or Based on the dry weight of the mycelium, the mycelium comprises approximately 45% or more protein, and / or Based on the dry weight of the mycelium, the mycelium comprises mycelial protein with a biomass concentration of approximately 0.4% or more, and / or In some embodiments, the mycelium is filamentous; and / or, the filter cake is elastic or tough.

8. A method for producing mycelium, the method comprising: 1) Inoculate the Fusarium spores or its inoculum as described in claim 2 into a seed culture medium and culture to obtain a seed solution; 2) Inoculate the seed culture into the fermentation medium and culture to obtain the fermentation broth; and 3) Mycelium is obtained from the fermentation broth.

9. The method according to claim 8, characterized in that, The seed culture medium and / or fermentation culture medium include a carbon source and a nitrogen source. Preferably, the carbon source includes sugars, sugar alcohols, starch, starch derivatives, starch hydrolysates, hydrogenated starch hydrolysates, lignocellulosic pulp or raw materials, potato starch, corn steep liquor, acid whey, sweet whey, whey, wheat steep liquor, carbohydrates, food waste, olive oil processing waste, hydrolysates of lignocellulosic materials, wet-milled corn products, and / or combinations thereof. Preferably, the nitrogen source is an inorganic nitrogen source and / or an organic nitrogen source. Preferably, the organic nitrogen source includes peptone, yeast extract, yeast hydrolysate, yeast extract, and soybean meal powder. Preferably, the inorganic nitrogen source includes urea or ammonium compounds.

10. The method according to claim 8, characterized in that, The seed culture medium comprises 25-35 parts by weight of a carbon source, 20-35 parts by weight of a nitrogen source, and 0.1-2 parts by weight of inorganic salts. Preferably, the seed culture medium comprises: 25-35 parts by weight of glucose, 20-35 parts by weight of yeast extract, 0.1-2 parts by weight of potassium dihydrogen phosphate, 0.1-2 parts by weight of magnesium sulfate heptahydrate, and 0.005-1 parts by weight of calcium chloride. Preferably, the fermentation medium comprises 20-35 parts by weight of a carbon source, 0.01-10 parts by weight of a nitrogen source, and 0.01-10 parts by weight of inorganic salts. Preferably, the fermentation medium comprises: 20-35 parts by weight of corn starch saccharification solution; 0.1-2 parts by weight of citric acid; 0.1-2 parts by weight of potassium dihydrogen phosphate; 0.01-10 parts by weight of ammonium sulfate; 0.01-2 parts by weight of magnesium sulfate heptahydrate; 0.001-1 parts by weight of calcium chloride; and, optionally, 0.001-0.1 parts by weight of trace elements. Preferably, the trace elements include 0.5–2.5 parts by weight of manganese sulfate monohydrate, 1–10 parts by weight of ferrous sulfate heptahydrate, and 0.5–2.5 parts by weight of zinc chloride. Optionally, the fermentation medium further includes 0.0001 to 0.2 parts by weight of a vitamin complex, which preferably includes vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, or any combination thereof.

11. The method according to claim 8, characterized in that, In step 2), the culture temperature is approximately 20°C to 35°C, and / or In step 2), the aeration rate during the culture process is approximately 5–15 L / h, and / or In step 2), the culture pressure is maintained at approximately 0.005–0.2 MPa, and / or In step 2), the pH of the fermentation medium is approximately 5.0–7.0, and / or In step 2), the aeration rate for the culture is approximately 1 L / min to 20 L / min, and / or In step 2), the culture temperature is approximately 20°C to 35°C, and / or In step 2), the pH of the fermentation medium is 3.0 to 5.

0.

12. The method according to claim 8, characterized in that, The method further includes the following steps between step 2) and step 3): a) Inoculate the first fermentation broth into the fermentation medium and incubate at 0.01–0.1 mPa and an aeration rate of 800–1200 L / h for 5–20 h; and b) Add feed medium and culture at 0.1–0.5 mPa and aeration rate of 1300–2000 h / L for 24–48 h to obtain the second fermentation broth.

13. The method according to claim 12, characterized in that, The supplemental culture medium comprises 10,000 to 50,000 parts by weight of corn starch saccharification solution and / or 0.001 to 0.2 parts by weight of vitamin complex.

14. A composition or food comprising the mycelial protein of claim 3 and / or protein-rich mycelium.

15. The use of the mycelial protein and / or protein-rich mycelium of claim 3, or the composition or food of claim 14, in the preparation of meat substitutes.

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