Fusarium compactum and use thereof

Abstract

The present disclosure relates to Fusarium compactum and the use thereof. Specifically, provided in the present disclosure are Fusarium or a microbial agent thereof, a mycelial protein and / or a protein-rich mycelium derived from the Fusarium or the microbial agent thereof, a composition containing the mycelial protein and / or protein-rich mycelium, and the use thereof.

Description

Fusarium tumefaciens and its applications Technical Field

[0001] This disclosure relates to the field of microbial technology, specifically to Fusarium compactum, a fungus that produces mycelial protein, mycelial protein produced by Fusarium compactum, and its applications. Background Technology

[0002] Fusarium is a widely used filamentous fungus that produces microbial protein. Its mycelial protein is delicious and has a meat-like texture, making it a meat substitute that can meet the nutritional needs of modern people. Currently, the strain *Fusarium venenatum* has been developed to produce meat products using mycelial protein. However, naturally high-protein *Fusarium venenatum* is extremely rare.

[0003] Therefore, finding more promising strains of naturally high-yielding filamentous fungi and developing them into food ingredients such as vegetarian meat, meat substitutes, baked goods, dairy products, and frozen desserts is a highly commercially valuable endeavor. 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 in the production of mycelial proteins and / or protein-rich mycelia is provided, wherein the Fusarium or its inoculum includes Fusarium compactum.

[0006] According to another aspect of this disclosure, a Fusarium or its inoculum agent is provided, comprising Fusarium compactum.

[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 translation elongation factor 1 (TEF1) of *Fusarium compactum* 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.

[0009] In some embodiments, the internal transcribed spacer (ITS) region of the *Fusarium compactum* has a nucleotide sequence that is at least 55% sequence identical to SEQ ID NO:10. 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:10.

[0010] In some embodiments, the polymerase I subunit (RPB1) of *Fusarium compactum* has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:11. 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 SEQ ID NO:11.

[0011] In some embodiments, the polymerase II subunit (RPB2) of *Fusarium compactum* has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:12. 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 SEQ ID NO:12.

[0012] In some embodiments, the Fusarium compactum may include one or more strains of MM-135, MN143240, MN142289, MM-581, MM-635, and MM-766.

[0013] In some embodiments, the MM-135 strain was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41312.

[0014] In some embodiments, the MN143240 strain was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41313.

[0015] In some embodiments, the MN142289 strain was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41314.

[0016] In some embodiments, the MM-581 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 26, 2024, with accession number CGMCC No. 41677;

[0017] In some embodiments, the MM-635 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 26, 2024, with accession number CGMCC No. 41679;

[0018] In some embodiments, the MM-766 strain was deposited on November 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41678.

[0019] In some embodiments, the *Fusarium compactum* can be in spore and / or mycelial form.

[0020] In some embodiments, the mycelium comprises more than 45 wt% protein, based on its dry weight.

[0021] In some embodiments, the mycelium comprises about 45.5 wt%, 50 wt%, 50.5 wt%, 51 wt%, 51.5 wt%, 52 wt%, 52.5 wt%, 53 wt%, 53.5 wt%, 54 wt%, 54.5 wt%, 55 wt%, 55.5 wt%, 56 wt%, 56.5 wt%, 57 wt%, 57.5 wt%, 58 wt%, 58.5 wt%, or 59 wt% or more of protein, based on its dry weight.

[0022] In some embodiments, the mycelium comprises mycelial protein with a biomass concentration of about 0.4 wt% or more, based on its dry weight. In some embodiments, the mycelium comprises protein with a biomass concentration of about 0.45 wt%, 0.5 wt%, 0.55 wt%, 0.6 wt%, 0.65 wt%, 0.7 wt%, 0.75 wt%, 0.8 wt%, 0.85 wt%, 0.9 wt%, 0.95 wt%, 1.0 wt%, or 1.5 wt% or more, based on its dry weight.

[0023] In some embodiments, the mycelium is filamentous; and / or the filter cake is white or orange-red in color; and / or the filter cake is elastic and / or has good toughness.

[0024] 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.

[0025] 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 first fermentation broth; and 3) obtaining mycelium from the fermentation broth.

[0026] In some embodiments, the seed culture medium and / or fermentation culture medium may include a carbon source and a nitrogen source.

[0027] 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.

[0028] 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 salts (e.g., ammonium sulfate, ammonium phosphate).

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] In some embodiments, the fermentation medium may include 20 to 35 parts by weight of a carbon source, 1 to 10 parts by weight of a nitrogen source, and 0.01 to 10 parts by weight of an inorganic salt.

[0038] 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 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts by weight of a nitrogen source.

[0039] 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 inorganic salt.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] In some implementations, in step 2), the rotation speed of the culture can be approximately 100 to 300 rpm.

[0053] 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.

[0054] In some embodiments, the fermentation medium may further comprise a vitamin complex. In some embodiments, the fermentation medium may further comprise 0.001 to 0.2 parts by weight of the vitamin complex. In some embodiments, the fermentation medium may further comprise 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.

[0055] 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.

[0056] In some embodiments, the fermentation medium may further comprise 0.01 to 10 parts by weight of yeast powder. In some embodiments, the fermentation medium may further comprise 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 yeast powder.

[0057] 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.

[0058] In some embodiments, the culture pressure in step 2) is approximately 0.005 MPa to 0.1 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, or 0.1 MPa.

[0059] 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.

[0060] In some embodiments, in step 2), the pH of the fermentation medium can be 5.0 to 7.0. In some embodiments, in step 2), the pH of the fermentation medium can be 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 implementations, in step 2), the culture time can be 10 to 30 hours, preferably 16 to 20 hours.

[0062] 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 and 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 and an aeration rate of 1300–2000 h / L for 24–48 h to obtain the second fermentation broth.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] In some embodiments, the supplemental culture medium may include the aforementioned carbon source and / or vitamin complex.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In some embodiments, the mycelium can be used to prepare meat substitutes.

[0071] According to another aspect of this disclosure, mycelium obtained by the above-described method of this disclosure is provided.

[0072] According to another aspect of this disclosure, a composition is provided comprising the mycelial protein of this disclosure and / or protein-rich mycelium.

[0073] 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.

[0074] In some embodiments, the composition comprises, based on wet-based mycelial protein and / or protein-rich mycelium, 30-60 parts by weight of mycelial protein and / or protein-rich mycelium; and 5-20 parts by weight of coating powder. In some embodiments, the composition comprises 30, 35, 40, 45, 50, 55, or 60 parts by weight of mycelial protein and / or protein-rich mycelium. In some embodiments, the composition comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 parts by weight of coating powder.

[0075] In some embodiments, the mycelial protein and / or protein-rich mycelium may also be provided in dry matter form.

[0076] In some embodiments, the coating powder may include coating powders conventionally used in the art, such as egg white powder, starch, etc. In some embodiments, the coating powder may include egg white powder and starch in an appropriate ratio, for example, the mass ratio of egg white powder to starch may be 1:(0.1-2). In some embodiments, the mass ratio of egg white powder to starch in the coating powder may be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.0.

[0077] In some embodiments, the composition may further comprise one or more of the following: alginate, arabinoxylan, carrageenan, carboxymethyl cellulose, cellulose, gelatin, gellan gum, dextran, galactomannan, guar gum, locust bean gum, tara gum, gum arabic, pectin, konjac gum, starch, and xanthan gum.

[0078] According to another aspect of this disclosure, mycelial proteins and / or protein-rich mycelia of this disclosure, or the use of the above compositions in the preparation or as food substitutes, are provided.

[0079] In some embodiments, the mycelial proteins and / or protein-rich mycelia of this disclosure, or the above-described compositions, are provided for use in the preparation of meat substitutes. In some embodiments, the mycelial proteins and / or protein-rich mycelia of this disclosure, or the above-described compositions, can replace chicken meat.

[0080] In some embodiments, the food is selected from at least one of plant-based meat, meat substitutes, baked goods, dairy products, and frozen beverages.

[0081] According to another aspect of this disclosure, an edible preparation, food ingredient or food component is provided, comprising (1) at least 10% w / w of cultured Fusarium tumefaciens biomass; (2) at least one of calcium salt, alginate, methylcellulose or carboxymethylcellulose; and (3) water and / or a thickener.

[0082] In some embodiments, the *Fusarium spp.* is selected from the *Fusarium spp.* described above in this disclosure.

[0083] In some embodiments, the Fusarium compactum biomass is the mycelial protein and / or protein-rich mycelium described above in this disclosure, or the mycelium described above in this disclosure.

[0084] In some embodiments, the alginate includes at least one of sodium alginate, calcium alginate, potassium alginate, propylene glycol alginate (PGA), and ammonium alginate.

[0085] In some embodiments, the thickener includes at least one selected from gelatin, sodium caseinate, gum arabic, tamarind gum, guar gum, agar, sodium alginate, carrageenan, konjac flour, pectin, locust bean gum, guar bean gum, starch acetate, pectin, xanthan gum, β-cyclodextrin, sodium carboxymethyl cellulose, sodium starch phosphate, sodium carboxymethyl starch, hydroxypropyl starch, and propylene glycol alginate.

[0086] According to another aspect of this disclosure, the use of the above-described edible preparations, food ingredients or additives of this disclosure in the preparation of alternative foods is provided.

[0087] According to another aspect of this disclosure, a method for preparing a food is provided, the method comprising contacting the mycelial proteins and / or protein-rich mycelia of the present disclosure, the mycelia of the present disclosure, a composition, an edible preparation, a food ingredient or additive, with other components of the food. In some embodiments, the food is selected from at least one of plant-based meat, meat substitutes, baked goods, dairy products, and frozen desserts.

[0088] This invention has found that existing fungal protein-based meat substitutes cannot completely replace plant-based meat substitutes because common fungal proteins, such as yeast proteins, have poor texture and emulsification properties. Meat substitutes prepared solely as the main protein ingredient have poor texture and mouthfeel. Therefore, plant proteins with stronger texture, such as legume proteins, are needed as the main or auxiliary protein ingredients to improve the texture and mouthfeel of the fungal protein-containing meat substitutes. Thus, common fungal protein-based meat substitutes cannot completely replace plant proteins. This invention provides a mycelium protein-based meat substitute food, comprising the following ingredients by weight: mycelium protein, thickener, and water; wherein the mycelium protein contains filaments with a length less than 1100 μm and filaments with a length greater than 100 μm; therefore, this invention has developed a mycelium protein-based meat substitute food. On one hand, this invention selects mycelium proteins containing filaments with a length less than 1100 μm and filaments with a length greater than 100 μm, such as the fungus *Fusarium oxysporum*. Mycelial protein obtained from the fermentation mycelium of *Avenaceum* is used as a protein raw material. Due to its excellent texturizing and emulsifying properties, this mycelial protein can be used alone to prepare meat substitutes without combining with plant proteins, avoiding the problems associated with using plant protein raw materials, such as off-flavors, complex processes, and low nutrient content. Furthermore, to further improve the texture and taste of meat substitutes, this invention enhances the texture of meat substitutes by using a selected amount of thickener and water. Specifically, the thickener absorbs water and swells; the swollen thickener and mycelial protein form a network. The mycelial protein's structure further enhances its organization and emulsification properties, thereby improving the texture and taste of the meat substitute. In summary, the mycelial protein-based meat substitute of the present invention can completely replace plant-based meat sausages, avoiding the defects brought by 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.

[0089] This invention provides a mycelium protein-based meat substitute food, wherein the thickener is starch, edible gums permitted under GB2760, and / or modified starch. Research in this invention has found that using starch and edible gums in combination as thickeners enhances the texturing and emulsifying properties of the mycelium protein. Starch can gelatinize, and edible gums can absorb water and swell. When combined with the mycelium protein, the resulting network structure of the mycelium protein exhibits superior texturing and emulsifying properties, resulting in a mycelium protein-based meat substitute food with excellent texture and mouthfeel characteristics. Attached Figure Description

[0090] Figure 1 shows the phylogenetic tree analysis results of strains MM-135, MN143240, MN142289, MM-581, MM-635, and MM-766.

[0091] Figure 2 shows images of the mycelium (Figure 2A) and hyphae (Figure 2B) of strain MM-135.

[0092] Figure 3 shows images of the mycelium (Fig. 3A) and hyphae (Fig. 3B) of strain MN143240.

[0093] Figure 4 shows images of the mycelium (Figure 4A) and hyphae (Figure 4B) of strain MN142289.

[0094] Figure 5 shows images of the mycelium (Figure 5A) and hyphae (Figure 5B) of strain MM-635.

[0095] Figure 6 shows images of the mycelium (Figure 6A) and hyphae (Figure 6B) of strain MM-581.

[0096] Figure 7 shows images of the mycelium (Figure 7A) and hyphae (Figure 7B) of strain MM-766.

[0097] Figure 8 shows the growth curves of strain MN143240 (Figure 8A) and strain MM-135 (Figure 8B). Detailed Implementation

[0098] In the field of fungal mycelial protein, naturally high-protein-producing Fusarium species are extremely rare, and naturally high-protein-producing strains of the Fusarium genus are also very scarce. This publication discloses the development of a novel naturally high-protein-producing filamentous fungal species, *Fusarium compactum*.

[0099] In some embodiments, this disclosure provides Fusarium compactum strains MM-135, MN143240, MN142289, MM-581, MM-635 and / or MM-766.

[0100] In some embodiments, this disclosure provides a microbial agent comprising one or more of the following Fusarium compactum strains: MM-135, MN143240, MN142289, MM-581, MM-635 and / or MM-766.

[0101] In some embodiments, the TEF1 of strain MM-135 has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:9. In some embodiments, the TEF1 of strain MM-135 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.

[0102] In some embodiments, the internal transcribed spacer (ITS) of strain MM-135 has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:10. In some embodiments, the ITS of 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:10.

[0103] In some embodiments, RPB1 of strain MM-135 has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:11. In some embodiments, RPB2 of strain MM-135 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.

[0104] In some embodiments, the RPB2 of strain MM-135 has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO:12. In some embodiments, the RPB2 of strain MM-135 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:12.

[0105] 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.

[0106] 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 *Fusarium compactum* of this disclosure. In a specific embodiment, the mycelial protein produced by *Fusarium compactum* of this disclosure has excellent filamentous structure, and the filter cake is light yellow and lacks elasticity.

[0107] In specific embodiments, the mycelial protein obtained from the fermentation culture of *Fusarium compactum* disclosed herein exhibits good filamentous structure 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 compactum* disclosed herein is all higher than 50%, even reaching over 55%.

[0108] Normally, bacterial strains can only utilize organic nitrogen sources and not inorganic nitrogen sources. In a specific embodiment, this disclosure enables the Fusarium compactum strain to utilize inorganic nitrogen sources by adding trace elements. This also improves the shape, color, and texture of the mycelium.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, and 0.45 g / L. Magnesium sulfate heptahydrate in concentrations of 0.5 g / L, 0.55 g / L, 0.6 g / L, 0.65 g / L, 0.7 g / L, 0.75 g / L, 0.8 g / L, 0.85 g / L, 0.9 g / L, 0.95 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[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] In some embodiments, the fermentation medium may further comprise 0.01 g / L to 10 g / L of yeast extract. In some embodiments, the fermentation medium may further comprise 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... 7g / L, 0.8g / L, 0.9g / L, 1g / L, 1.5g / L, 2g / L, 2.5g / L, 3g / L, 3.5g / L, 4g / L, 4.5g / L, 5 g / L, 5.5g / L, 6g / L, 6.5g / L, 7g / L, 7.5g / L, 8g / L, 8.5g / L, 9g / L, 9.5g / L or 10g / L yeast powder.

[0129] In a specific embodiment, the supplemental culture medium may include 100 g / L to 500 g / L of corn starch saccharification solution and / or 0.01 to 2 mg / L of vitamin complex by weight.

[0130] In a specific embodiment, the supplemental culture medium may include corn starch saccharification solution of 100 g / L, 150 g / L, 200 g / L, 250 g / L, 300 g / L, 350 g / L, 400 g / L, 450 g / L or 500 g / L.

[0131] In a specific embodiment, the supplemental culture medium may include 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.

[0132] In specific embodiments, the meat substitutes of this disclosure (e.g., meat chunks or minced meat) may contain one or more of the following: alginate, arabinoxylan, carrageenan, carboxymethyl cellulose, cellulose, gelatin, gellan gum, dextran, galactomannan, guar gum, locust bean gum, tara gum, gum arabic, pectin, konjac gum, starch, and xanthan gum.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] The mycelial protein produced by Fusarium or its inoculants disclosed herein can be used to prepare meat substitutes, etc.

[0138] 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.

[0139] definition

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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 invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0144] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0145] Example

[0146] Example 1: Screening of Fusarium compactum strains

[0147] 1.1 Source of strains

[0148] The MM-135 strain was isolated from humus soil samples from Shennongjia, Hubei Province.

[0149] Strain MN143240 was isolated from humus soil samples from Tianchi Lake in Changbai Mountain.

[0150] The MN142289 strain was isolated from soil samples from the western Yunnan mountains.

[0151] MM-581 strain was isolated from the soil around the roots of kiwifruit in Guantang Village, Meixian County, Baoji City.

[0152] MM-635 strain was isolated from tomato planting soil in Huanian Town, Eshan County, Yuxi City.

[0153] MM-766 strain was isolated from the soil around the roots of kiwifruit in Guantang Village, Meixian County, Baoji City.

[0154] 1.2 Separation Process

[0155] 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.

[0156] The specific components of the MGA2.5 flat panel are shown in the table below;

[0157] Purification was performed using a stepwise transplantation method at the hyphal tips. After colonies formed on the agar plate, hyphae from the edge of a single colony were picked and placed on 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.

[0158] Example 2. Identification of Fusarium compactum strains

[0159] 2.1 Preparation of DNA template

[0160] 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.

[0161] 2.2 PCR system preparation

[0162] Prepare a 25 μL PCR system in a PCR tube according to the table below:

[0163] The amplified gene and primers are as follows:

[0164] The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0165] 2.3 PCR amplification

[0166] 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.

[0167] The sequencing results of strain MM-135 are shown in the table below.

[0168] The sequencing results of strain MN143240 are shown in the table below.

[0169] The sequencing results of strain MN142289 are shown in the table below.

[0170] The sequencing results of strain MM-581 are shown in the table below.

[0171] The sequencing results of strain MM-635 are shown in the table below.

[0172] The sequencing results of strain MM-766 are shown in the table below.

[0173] 2.4 Species identification analysis based on polyphasic identification

[0174] The species of strains MM-135, MN143240, MN142289, MM-581, MM-635, and MM-766 were identified using the Polyphasic identification method provided by the online database mycobank (fusarium.mycobank.org). The results showed that based on the TEF1, RPB2, and ITS genes, the closest species to strain MM-135 was Fusarium compactum NRRL 36318, with a global similarity of 99.65%.

[0175] Using the Polyphasic identification method provided by the online database mycobank (fusarium.mycobank.org), by simultaneously uploading multiple genes obtained from the above sequencing to the webpage, the results showed that based on the TEF1, RPB2, and ITS genes, the closest species to strain MN143240 is Fusarium compactum NRRL 36318, with a global similarity of 99.65%.

[0176] The Polyphasic identification method provided by the online database mycobank (fusarium.mycobank.org) was used to identify the strain. By uploading multiple genes obtained from the above sequencing to the webpage at the same time, the results showed that based on the TEF1 and RPB2 genes, the closest species to strain MN142289 was Fusarium compactum NRRL 36318, with a global similarity of 99.65%.

[0177] Based on the TEF1, RPB2, and ITS genes, the closest species to strain MM-581 is Fusarium compactum NRRL 36318, with a global similarity of 99.65%.

[0178] Based on the TEF1 and ITS genes, the closest species to strain MM-635 is Fusarium compactum NRRL 36318, with a global similarity of 100%.

[0179] Based on the TEF1, RPB2, and ITS genes, the closest species to strain MM-766 is Fusarium compactum NRRL 36318, with a global similarity of 99.65%.

[0180] Therefore, strains MM-135, MN143240, MN142289, MM-581, MM-635, and MM-766 were all identified as Fusarium compactum.

[0181] 2.5 Species identification analysis based on polygenic phylogenetic tree

[0182] The species identification analysis method based on polyphasic identification is used because the species identified belong to the Fusarium incarnatum-equiseti species complex (FIESC). 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.

[0183] 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.

[0184] As shown in Figure 1, strains MM-135, MN143240, MN142289, MM-581, MM-635, and MM-766 belong to the species Fusarium compactum.

[0185] Strain MM-135 was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41312.

[0186] Strain MN143240 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, with accession number CGMCC No. 41313.

[0187] Strain MN142289 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, with accession number CGMCC No. 41314.

[0188] Strain MM-581 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 26, 2024, with accession number CGMCC No. 41677.

[0189] Strain MM-635 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 26, 2024, with accession number CGMCC No. 41679.

[0190] Strain MM-766 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 26, 2024, with accession number CGMCC No. 41678.

[0191] Example 3. Fermentation preparation of mycelial protein

[0192] The activated Fusarium compactum strains MM-135, MN143240, MN142289, MM-581, MM-635, and MM-766 were inoculated into 250 mL conical flasks containing 50 mL of primary screening medium and cultured at a constant temperature of 200 rpm for 24 h.

[0193] The initial screening culture medium formula was: glucose 30 g / L, yeast extract 30 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.2 g / L, autoclaved at 121℃ for 20 min.

[0194] 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.

[0195] Results of observation and testing of mycelial morphology, filter cake color and texture, biomass and crude protein of the strain.

[0196] The mycelial protein content of MM-135 reached 1.62 wt% in biomass, and the crude protein content of the mycelium was further determined by the Kjeldahl method to be 59.17 wt%.

[0197] The mycelial protein content of MN143240 reached 1.88 wt% in biomass, and the crude protein content of the mycelium was further determined by the Kjeldahl method to be 62.18 wt%.

[0198] The mycelial protein content of MN142289 reached 1.56 wt%, and the crude protein content of the mycelium was further determined by the Kjeldahl method to be 53.11 wt%.

[0199] The mycelial protein content of MM-581 reached 2.08 wt% in biomass, and further analysis using the Kjeldahl method showed that the crude protein content of the mycelium reached 52.10 wt%.

[0200] The biomass concentration of MM-635 reached 1.18 wt% mycelial protein, and the crude protein content of the mycelium was further determined by the Kjeldahl method to be 55.90 wt%.

[0201] The biomass concentration of MM-766 reached 1.98 wt% mycelial protein, and the crude protein content of the mycelium was further determined by the Kjeldahl method to be 52.67 wt%.

[0202] Example 4. Scale-up culture for the production of mycelial protein

[0203] 4.1 Fermenter double-screening culture method

[0204] The components of the seed culture medium used are shown in the table below:

[0205] Seed culture conditions: For the preparation of the primary seed culture, the above-mentioned seed culture medium was used. Activated Fusarium compactum strains MM-135, MN143240, MN142289, MM-581, MM-635, and MM-766 were inoculated into 250 mL conical flasks containing 50 mL of seed culture medium and cultured at a constant temperature of 200 rpm for 24 h with shaking. For the preparation of the secondary seed culture, the above-mentioned seed culture medium was used, with 5% inoculum transferred to 3 L conical flasks containing 1 L of seed culture medium and cultured at 200 rpm for 24-30 h. Observations showed that the culture time of the seed culture medium affected the color of the filter cake formed by mycelial protein; therefore, the color and texture of the fermented filter cake could be controlled by controlling the culture time of the seed culture medium.

[0206] Then, inoculate the secondary screening medium at a rate of 3%. The components of the secondary screening medium for producing mycelial protein are as follows:

[0207] Fermentation process conditions: Fermentation tank volume 15L, liquid volume 10L, sterilization at 121℃ for 30min. Temperature 28℃, aeration 5-15L / h, tank pressure 0.05-0.10Mpa, rotation speed 100-400rpm, dissolved oxygen 20-50%, pH controlled at 6.0 with ammonia. The shape of the mycelium has a significant impact on the texture of the prepared product (e.g., meat substitute); cottonseed-like mycelium has a poor texture. In this embodiment, inorganic nitrogen is used to replace organic nitrogen, achieving cost reduction while optimizing the filamentous structure and reducing balling.

[0208] Ammonium sulfate in the table above can be replaced with ammonium phosphate.

[0209] Using the same method as in Example 3, the color, texture, odor, mycelial morphology, mycelial length, mycelial diameter, mycelial branching, spore number, and crude protein content of the filter cake obtained from the re-screening of *Fusarium compactum* strain were tested. The results showed that the granular and cottonseed-like structures in the initial screening were effectively improved, the mycelia exhibited a slender filamentous structure, and the prepared filter cake had an excellent texture, better mimicking the chewiness of meat (Figures 2 to 4). The crude protein content of the mycelium in MM-135 was determined to be 55.29 wt% by the Kjeldahl method, while that in MN143240 and MN142289 was 54.88 wt%.

[0210] The table below shows the rescreening results of strains MM-135, MN143240, MN142289, MM-581, MM-635, and MM-766.

[0211] Compared with the primary screening medium in Example 3, 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. Furthermore, this also indicates that the strain *Fusarium compactum* of this invention can utilize both organic and inorganic nitrogen sources as nitrogen sources for the fermentation culture of fungal proteins.

[0212] 4.2 Fermentation process corresponding to fermentation medium:

[0213] 4.2.1 Seed culture

[0214] Use a 3L conical flask, PDB medium (potato extract powder 300g / L, glucose g / L, purchased from Guangdong Huankai Microbial Technology Co., Ltd.); inoculum size 5%, liquid volume 1L; shaker speed 200rpm; incubate for 24h.

[0215] 4.2.2 Primary Fermentation

[0216] The composition of the culture medium for primary fermentation is shown in the table below.

[0217] Subculture: Use a 15L tank with a 10L culture volume, sterilize at 121℃ for 30 min, and transfer the entire seed culture by flame inoculation; rotation speed 100-300 rpm; aeration rate 10L / min; tank pressure 0.05 MPa; pH controlled at 6.0 with ammonia; incubate at 28℃ for 16-20 h. Ammonium sulfate in the primary culture medium can be replaced with ammonium phosphate.

[0218] 4.3.3 Secondary fermentation

[0219] The composition of the secondary fermentation medium is shown in the table below.

[0220] Feeding culture medium

[0221] The secondary fermentation medium formula is shown in the table above (ammonium sulfate can be replaced with ammonium phosphate). Use a 50L tank, fill with 30L of liquid, and sterilize at 121℃ for 30min. After sterilization, add 0.1mg / L vitamin mixture (filtered for sterilization). Inoculate at a rate of 5-10% by pumping the seed culture from a 15L tank through a sterilized tubing through the bottom valve. Aerate at 1000L / h for the first 15 hours, with a tank pressure of 0.05MPa. After 15 hours, start feeding at 0.5-2g / L / h and adjust the aeration to 1500L / h, with a tank pressure of 0.1MPa. Control the pH with ammonia water to 4.0-6.0. Incubate at 28℃, with a rotation speed of 100-300rpm and dissolved oxygen at 20%-50%, for 24-48 hours. The multivitamin includes B-complex vitamins, which consist of 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. It increases dissolved oxygen levels and efficiency, promoting mycelial growth without damaging the mycelium.

[0222] During the fermentation and subculture process, the strain grew well, and the strain growth curve is shown in Figure 8.

[0223] Example 5. Optimization of Fusarium compactum culture medium during production scale-up process

[0224] This embodiment investigated the effects of the components and their content in the fermentation medium on fermentation mycelium and protein yield during the scale-up process. During the scale-up process, a large inoculum size resulted in low conversion efficiency.

[0225] Using the fermentation medium formulation shown in the table below, strains MM-135, MN143240, MN142289, MM-581, MM-635 and / or MM-766 were cultured according to the method in Example 4.

[0226] Using the same method as in Example 3, the color, texture, odor, hyphal morphology, hyphal length, hyphal diameter, hyphal branching, spore number, and crude protein content of filter cakes obtained from three strains of *Fusarium compactum* were tested. The results showed that:

[0227] When the culture medium does not contain ammonium sulfate, the clumping phenomenon is alleviated, but the crude protein content of the resulting mycelium decreases significantly, by about 5% to 15%.

[0228] Ca in fermentation medium 2+ The content of calcium ions affects the mycelial balling phenomenon. Reducing the calcium ion content can effectively control the mycelial balling phenomenon, but too low a calcium ion content will affect the crude protein content and cause foaming.

[0229] When the calcium ion content in the fermentation medium is high (e.g., 0.01 g / L), adding yeast powder (or yeast hydrolysate, yeast extract, or yeast extract) will significantly reduce the mycelial clumping phenomenon.

[0230] The presence of an appropriate amount of phosphate in the culture medium will increase the biomass of the product. For example, when the phosphate concentration is increased from 0.2 g / L to 1 g / L, the biomass of the product increases by 20% to 40%.

[0231] Adding Zn to the culture medium 2+ Mn 2+ Fe 2+ Increasing the concentration of trace element solutions can improve protein content.

[0232] Example 6. Preparation of meat chunks from Fusarium compactum mycelial protein

[0233] Meat chunks were prepared using the mycelial protein obtained in Example 4, including the following raw materials: mycelial protein of Fusarium compactum strain, water, egg white powder, starch, and other seasonings (e.g., monosodium glutamate, yeast extract, white sugar, food flavoring, soybean oil, edible salt, spices, disodium 5'-inosinate, gluten, etc.).

[0234] During the preparation of meat chunks, methylcellulose or sodium alginate, anhydrous calcium chloride, and calcium acetate can be added to the above ingredients to adjust the viscosity of the meat chunks. The specific formula is shown in the table below:

[0235] The steps for preparing meat chunks are as follows:

[0236] 1. Mix egg white powder and starch to obtain coating powder, and divide it into a first part and a second part by weight ratio of 75:25; mix the first part of coating powder with ice water, stir well to make a batter, and set aside.

[0237] 2. Mix the second part of the coating powder with the mycelium protein and stir well. Then add the seasonings and ice water, stir well, and you will get the vegetarian meat filling.

[0238] 3. Press the vegetarian meat filling into rectangular meat blocks, each weighing 20g. Steam at 85℃ for 30 minutes to set the shape, then cool to room temperature.

[0239] 4. Coat the meat pieces with the batter from step 1, deep-fry at 160℃ for 30 seconds, and cool to room temperature;

[0240] 5. Packaging, quick-freezing, and warehousing.

[0241] Evaluation was conducted using (1) sensory evaluation and (2) texture analyzer.

[0242] (1) Use sensory evaluation methods to assess its juiciness, elasticity, and firmness.

[0243] The evaluation results are shown in the table below:

[0244] The texture of mycelium protein-based meat chunks

[0245] The table above shows that the mycelium protein-based meat chunks of the present invention have excellent juiciness, elasticity, and firmness, resulting in a superior taste. In contrast, the mycelium protein-based meat chunks in formulation 14 have weak elasticity and firmness, leading to a poorer taste. The mycelium protein-based meat chunks in formulations 15-16 have weak juiciness, high firmness, and a poor taste. This demonstrates that the mycelium protein, thickener, and water, along with the selected amounts thereof, prepared according to the present disclosure, produce a superior taste and can completely replace meat chunks made from animal and plant proteins.

[0246] (2) Measuring texture structure with a texture analyzer

[0247] Meat chunks prepared with mycelial protein from formulations 10-16 and commercially available chicken chunks were cut into 1cm × 1cm × 1cm cylindrical pieces. 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.

[0248] 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.

[0249] Cohesion: Cohesion reflects the property of food resisting damage and remaining tightly bound together during chewing, thus maintaining the integrity of the food.

[0250] 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.

[0251] The following table shows the texture analyzer test results of mycelial protein-based meat blocks:

[0252] The table above shows that the mycelium protein-based meat chunks prepared by formulas 10-13 are closer to commercially available chicken chunks, while the texture of the mycelium protein-based meat chunks prepared by formulas 14-16 differs from that of commercially available chicken chunks. The mycelium protein-based meat chunks prepared by formula 14 are too weak in hardness, elasticity, and cohesion, resulting in poor chewiness and taste. The mycelium protein-based meat chunks prepared by formulas 15-16 are too hard, making their taste significantly different from that of commercially available chicken chunks.

[0253] Calcium acetate and calcium chloride are highly water-soluble, and calcium acetate itself has a sweet taste. When calcium acetate is combined with anhydrous calcium chloride, it produces a mixture with an acceptable slightly salty taste. Using a mixture of anhydrous calcium chloride, calcium acetate, and microbial protein allows for rheological effects on the mycelial protein, as well as an acceptable slightly salty taste.

[0254] The formula contains colloids or coagulants; methylcellulose can also be used as a colloid to replace sodium alginate, calcium chloride, and calcium acetate. In formula 16, an excess of methylcellulose was used as a colloid to replace sodium alginate, calcium chloride, and calcium acetate. The results showed that the combination of egg white powder and methylcellulose affected the textural structure. Egg white powder combined with methylcellulose, and a high proportion of methylcellulose, resulted in a harder texture and poorer chewiness.

[0255] 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 associated with plant proteins, while also possessing excellent taste and texture. The taste is juicy, elastic, and moderately firm, and the texture exhibits good firmness, elasticity, and chewiness, more closely resembling real meat. Compared to plant-based meat or pork / chicken, the mycelial protein-based meat substitute has higher protein and natural dietary fiber content, lower fat content, and is cholesterol-free, making it more nutritious and healthy.

[0256] Example 7. Preparation of cold beverages from Fusarium compactum mycelial protein

[0257] Fungal protein ice cream was prepared using the mycelial protein obtained in Example 4. After fermentation, the mycelial protein was heat-treated at 60℃~90℃ to reduce RNA, and then filtered or centrifuged to reduce moisture, resulting in a fungal mycelial enrichment. The enrichment was then washed with water to remove off-odors. The fungal protein ice cream contained the following ingredients: mycelial protein from the Fusarium compactum strain, water, emulsifier, thickener, sweetener, and vegetable oil.

[0258] The emulsifier is a blend of mono- and diglyceride fatty acid esters and polyglyceride fatty acid esters in a 2:1 mass ratio. The thickener is a blend of sodium alginate, xanthan gum, and carrageenan in a 3:2:1 mass ratio. 15g of sweetener is a blend of xylitol and sucrose in a 4:1 mass ratio. 8g of vegetable oil is a blend of refined coconut oil and low-temperature palm oil in a 1:1 mass ratio.

[0259] The specific formula is shown in the table below.

[0260] The method for preparing the fungal protein ice cream includes the following steps:

[0261] A. Mix the mycelial protein with water and stir at 40°C using a high-speed stirrer (1200-1500 rpm; the higher the mycelial protein content, the higher the speed should be to ensure thorough stirring) for 15 minutes to fully dissolve it and form a uniform protein solution.

[0262] B. Add emulsifier, some sweetener, and vegetable oil to the protein solution in step A in sequence, and continue stirring for 12-15 minutes at a stirring speed of 1000-1500 rpm to ensure that all components are mixed evenly to obtain the liquid.

[0263] C. Heat the liquid from step B to 60-70℃, dry mix the remaining sweetener and thickener evenly, and slowly add them to the liquid. Use a high-shear device (speed 2500-3000rpm) to shear for 15-20 minutes to form a stable colloidal system.

[0264] D. After filtering the liquid from step C through an 80-mesh filter, homogenize it at a pressure of 15-20 MPa to further refine the fat globules and make them more evenly distributed. Then, sterilize it at 90-100℃ for 10-20 minutes to kill any microorganisms that may be present and ensure product quality and safety.

[0265] E. Quickly cool the sterilized liquid to 1-6℃, age it for 4-20 hours, and then pour it into ice cream molds to allow the liquid to initially solidify and take shape using the low temperature.

[0266] F. Place the mold containing the liquid into a quick-freezing machine and freeze it rapidly until the center temperature is ≤-18℃ for 20-40 minutes to completely freeze the ice cream, forming a stable ice crystal structure and a good taste.

[0267] G. Finally, remove the ice cream from the mold and seal it with food-grade plastic film or cardboard to prevent contamination and moisture loss during storage and sales.

[0268] 1. Product Evaluation:

[0269] The fungal protein ice cream products prepared according to formulas 17-19 were evaluated separately, and the specific test steps were as follows:

[0270] Twenty randomly selected evaluators (half male and half female, aged 20-50) were blind-tested based on acceptability and likability. The evaluation was based on appearance, taste, flavor, texture, and likability. Higher scores were better (each item was out of 10), and the average score was taken.

[0271] Table 1 shows the evaluation criteria and scoring rules as follows:

[0272] The evaluation results are shown in the table below:

[0273] Table 2. Evaluation Results

[0274] In the preparation process of fungal protein ice cream, the mycelial protein raw material is a dough-like solid with a moisture content of about 70%. Dissolving and dispersing the mycelial protein first can greatly increase its contact area in water, making the subsequent ingredients blend better.

[0275] In ice cream preparation, emulsifiers and thickeners are typically added simultaneously. In this application, because the surface of fungal proteins has a polysaccharide structure, resulting in a higher viscosity of the liquid, the emulsifier, some sweetener, and vegetable oil are added first, mixed thoroughly, and then the remaining sweetener and thickener are added. Emulsification before adding the thickener improves the mixing effect of the ingredients, allowing for better mixing of the components and enhancing the texture and smoothness of the ice cream.

[0276] Compared to traditional dairy-based ice cream, Fusarium fungal protein ice cream scores similarly in appearance, richness of texture, uniqueness of flavor, and fineness of structure. Overall, it significantly outperforms plant-based ice cream. Furthermore, while offering a delicious taste, fungal protein ice cream also boasts health benefits, making it stand out among plant-based ice creams and a new, delicious, and healthy ice cream option.

[0277] 2. Optimization of emulsifiers and thickeners

[0278] In the ice cream making process, emulsifiers promote uniform mixing and enhance the texture. Thickeners improve the texture and mouthfeel of the ice cream. The combined use of emulsifiers and thickeners can further improve product performance: emulsifiers stabilize the fat-water system, while thickeners optimize the overall structure. Together, they give the ice cream resistance to melting, a smooth texture, and stable shape.

[0279] In traditional ice cream making, whey protein exists as small molecules in the ingredient liquid. However, fungal proteins have different characteristics from whey protein, including differences in molecular structure, isoelectric point, and hydrophilic / hydrophobic properties. Fungal proteins have larger particles in the ingredient liquid and are a mixture mainly composed of protein and fiber. Their surface polysaccharide structure makes them more hydrophilic, requiring optimization in the selection of emulsifiers and thickeners.

[0280] This experimental example investigated the formulation of different emulsifiers and thickeners to improve the melt resistance of fungal protein ice cream.

[0281] Compared to formula 17, formulas 20, 21, and 22 have different emulsifier and thickener formulations. The anti-melting properties of the fungal protein ice cream prepared using these formulas were tested. The specific test steps are as follows: The fungal protein ice cream was placed in a constant temperature and humidity chamber at 30℃±1℃ and 60%RH±1%RH. The time when the surface began to melt was recorded, with visible droplets as the standard. Each group was repeated three times, and the average value was taken.

[0282] The test results are as follows:

[0283] Table 3. Results of the heat resistance test

[0284] Based on the above results, it is clear that formulation 22, which omits the emulsifier and thickener, begins to melt within 1.2 minutes, exhibiting poor melt resistance. Among formulations 17, 20, and 21, formulation 17 demonstrates the best melt resistance (6.8 minutes), indicating that its emulsifier and thickener combination is the most ideal. Therefore, the optimal emulsifier and thickener combination is: mono- and diglyceride fatty acid esters + polyglycerol esters, and sodium alginate + xanthan gum + carrageenan.

[0285] Mono- and diglycerides of fatty acids are water-in-oil emulsifiers, while polyglycerol fatty acid esters are oil-in-water emulsifiers. The polysaccharide structure on the surface of fungal proteins makes them more hydrophilic; therefore, using a combination of both water-in-oil and oil-in-water emulsifiers achieves better emulsification. The use of a combination of mono- and diglycerides of fatty acids and polyglycerol fatty acid esters effectively promotes uniform mixing of the components and prevents oil-water separation.

[0286] The thickener is a blend of sodium alginate, xanthan gum, and carrageenan. Sodium alginate can control the formation of ice crystals, improve the texture and taste of the ice cream, making it more delicate and uniform. Carrageenan can form a thermally reversible gel, giving the ice cream a good shape and resistance to melting. Xanthan gum has thickening and stabilizing effects. The three ingredients work synergistically to enhance the stability of the ice cream system.

[0287] 3. Optimization of aging process

[0288] This experiment investigated different aging processes to improve the quality of fungal protein ice cream, reduce ice crystal formation, and enhance the texture of the ice cream.

[0289] The ice crystal texture of fungal protein ice cream prepared by different aging processes was evaluated. The specific steps were as follows: 20 evaluators (half male and half female, aged 20-50) were randomly selected to conduct blind tests. The scoring criteria were: no ice crystals (10 points), slight (7-9 points), obvious (4-6 points), and severe (1-3 points). The higher the score, the better. The average score was taken.

[0290] The test results are as follows:

[0291] Table 4. Ice-like sensation rating

[0292] Based on the above results, it is evident that the fungal protein ice cream prepared without the aging process exhibits a significant icy texture, which negatively impacts the overall quality of the ice cream. In contrast, the icy texture score of the ice cream prepared with the aging process is significantly higher than that without the aging process. Based on the above experiments, the optimal range for the aging process is 1°C to 4°C, with a duration of 4 to 16 hours.

[0293] Example 8. Preparation of baked goods from Fusarium compactum mycelial protein

[0294] Cookies were prepared using the mycelial protein obtained in Example 4. After fermentation, the mycelial protein was heat-treated at 60℃~90℃ to reduce RNA, and then filtered or centrifuged to reduce moisture, resulting in a fungal mycelium enrichment. The mycelium was then washed to remove off-flavors. Fungal protein ice cream comprised the following ingredients: mycelial protein from the Fusarium compactum strain, flour, butter, milk, granulated sugar, and nut powder. It was prepared using the following weight ratios: 5 parts Fusarium mycelial protein (dry powder), 20 parts low-gluten flour, 31 parts unsalted butter, 13 parts whole milk, 18 parts icing sugar, 0.3 parts edible salt, 2.5 parts cocoa powder, and 8 parts almond powder.

[0295] The production method includes the following steps:

[0296] 1. Preparation: Take the butter out of the refrigerator and let it soften at room temperature. Add the powdered sugar to the milk and stir until completely dissolved.

[0297] 2. Creaming the butter: Place the softened butter in a bowl, add salt, and beat with an electric mixer on low speed until smooth. Then add the milk and sugar syrup to the butter in three batches, beating until the butter fully incorporates each time before adding the next batch, until the butter is whipped to a light, fluffy consistency.

[0298] 3. Mixing dry ingredients: Sift the Fusarium mycelium protein (dry powder), low-gluten flour and almond flour into the whipped butter. Use a silicone spatula to mix them evenly by folding, avoiding overmixing to prevent the flour from developing gluten.

[0299] 4. Piping: Put the mixed batter into a piping bag and pipe out the desired shapes onto a baking sheet lined with parchment paper. Make sure to leave some space between the cookies to prevent them from sticking together during baking.

[0300] 5. Bake the cookies: Bake at 150 degrees Celsius (300 degrees Fahrenheit) for 20 minutes using both top and bottom heat.

[0301] 6. Cooling: After baking, remove the cookies from the oven and place them on a cooling rack to cool completely. They will become crispy after cooling.

[0302] 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

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 Fusarium compactum. A Fusarium species or its inoculum, including Fusarium compactum. A mycelial protein and / or protein-rich mycelium derived from Fusarium oxysporum or its inoculum according to claim 2, preferably wherein the mycelial protein or mycelium has a length of less than 2000 μm and / or an aspect ratio of less than 800. The application according to claim 1, the Fusarium or its inoculum according to claim 2, or the mycelial protein and / or protein-rich mycelium according to claim 3, is characterized in that, The TEF1 of *Fusarium compactum* has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:9; and / or The ITS of the Fusarium compactum has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:10; The RPB1 of *Fusarium compactum* has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:11; and / or The RPB2 of the Fusarium compactum species has a nucleotide sequence that is at least 85% identical to that of SEQ ID NO:

12. The application according to claim 1, the Fusarium or its inoculum according to claim 2, or the mycelium according to claim 3, is characterized in that, The Fusarium compactum is selected from at least one of the following (1) to (6): (1) Strain MM-135 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, with accession number CGMCC No. 41312. (2) Strain MN143240, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, with accession number CGMCC No. 41313. (3) The MN142289 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, with accession number CGMCC No. 41314; (4) The MM-581 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 26, 2024, with accession number CGMCC No. 41677; (5) The MM-635 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 26, 2024, with the accession number CGMCC No. 41679; (6) The MM-766 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 26, 2024, with accession number CGMCC No.41678. The application according to claim 1, the Fusarium or its inoculum according to claim 2, or the mycelium according to claim 3, is characterized in that, The *Fusarium compactum* is in spore, mycelial, and / or powder form, and / or Based on the dry weight of the mycelium, the mycelium comprises more than 45 wt% protein, and / or Based on the dry weight of the mycelium, the mycelium comprises mycelial protein with a biomass concentration of approximately 0.4 wt% or more, and / or The mycelium is filamentous, and / or the filter cake is white or orange-red in color, and / or the filter cake is elastic and / or has good toughness. A method for producing mycelium, characterized in that, The method includes: 1) Inoculate the Fusarium or its inoculum into a seed culture medium and culture to obtain a seed solution; 2) The seed culture was inoculated into the fermentation medium and cultured to obtain the first fermentation broth; and 3) Mycelium is obtained from the fermentation broth. The method according to claim 7, 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 carbon source includes sucrose, maltose, glucose, fructose, glycerol, polyol, corn starch, maltodextrin, cyclodextrin, glucose syrup, modified starch, corn starch saccharification liquid, rice starch saccharification liquid, sweet potato starch saccharification liquid, molasses, hydrogenated glucose syrup, maltitol syrup, sorbitol syrup, beet pulp, agricultural pulp, wood pulp, dried brewer's grains, brewery waste, carbon-refined syrup, desalted syrup, enzyme-inverted syrup, and / or combinations thereof; Preferably, the nitrogen source in the seed culture medium and / or fermentation culture medium is an inorganic nitrogen source and / or an organic nitrogen source. Preferably, the organic nitrogen source includes peptone, yeast extract, soybean meal, and / or combinations thereof. Preferably, the inorganic nitrogen source includes urea and / or ammonium compounds, more preferably ammonium sulfate. The method according to claim 8, characterized in that, The seed culture medium and / or fermentation medium further include inorganic salts, preferably potassium salts, calcium salts, magnesium salts, manganese salts, iron salts, zinc salts and / or combinations thereof. Preferably, the seed culture medium and / or fermentation culture medium comprises one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, ferrous sulfate, ferric sulfate, and zinc chloride. Preferably, 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, 1-10 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; and 0.1-2 parts by weight of citric acid; 0.1 to 2 parts by weight of potassium dihydrogen phosphate; 0.01 to 10 parts by weight of ammonium sulfate; 0.01 to 2 parts by weight of magnesium sulfate heptahydrate; 0.001 to 1 part by weight of calcium chloride; and, optionally, 0.001 to 0.1 parts by weight of trace elements. Preferably, the fermentation medium further includes a vitamin complex, more preferably 0.001 to 0.2 parts by weight of the vitamin complex. Preferably, the fermentation medium further includes 0.01 to 10 parts by weight of yeast powder. The method according to claim 8, characterized in that, In step 2), The culture temperature is 20℃~35℃, and / or The culture rotation speed is 100–300 rpm, and / or The pH of the fermentation medium is 5.0 to 7.

0. The method according to claim 7, characterized in that, The method further includes step a) between steps 2) and 3): inoculating the first fermentation broth into a fermentation medium for cultivation to obtain the second fermentation broth. Optionally, in step a), a supplementary culture medium is added, preferably comprising 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. A mycelium obtained by the method according to any one of claims 7 to 11. A composition or food comprising the mycelial protein of claim 3 and / or protein-rich mycelium. The use of Fusarium or its inoculum agent as described in claim 2, mycelial protein and / or protein-rich mycelium as described in claim 3, or the composition as described in claim 13 in the preparation or as a food substitute; preferably, the food is selected from at least one of plant-based meat, meat substitutes, baked goods, dairy products, and frozen beverages. An edible preparation, food ingredient or food component comprising (1) at least 10% w / w of cultured Fusarium tumefaciens biomass; (2) at least one of calcium salt, alginate, methylcellulose or carboxymethylcellulose; and (3) water and / or a thickener. Preferably, the *Fusarium oxysporum* is selected from at least one of the following: 1) the *Fusarium oxysporum* is the *Fusarium oxysporum* according to any one of claims 4-6; 2) The Fusarium compactum biomass is the mycelial protein and / or protein-rich mycelium as described in claim 3, or the mycelium as described in claim 12; Preferably, the alginate includes at least one of sodium alginate, calcium alginate, potassium alginate, propylene glycol alginate (PGA), and ammonium alginate; Preferably, the thickener comprises at least one selected from gelatin, sodium caseinate, gum arabic, tamarind gum, guar gum, agar, sodium alginate, carrageenan, konjac flour, pectin, locust bean gum, guar bean gum, starch acetate, pectin, xanthan gum, β-cyclodextrin, sodium carboxymethyl cellulose, sodium starch phosphate, sodium carboxymethyl starch, hydroxypropyl starch, and propylene glycol alginate. The use of the edible preparation, food ingredient or additive of claim 15 in the preparation of alternative foods. A method for preparing food, the method comprising contacting the mycelial protein and / or protein-rich mycelium of claim 3, the mycelium of claim 12, the composition of claim 13, the edible preparation of claim 15, food ingredients or additives with other components of the food; The food is selected from at least one of plant-based meat, meat substitutes, baked goods, dairy products, and frozen beverages.

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