Fusarium sp. For producing hypha protein and application thereof

Abstract

The invention provides fusarium sp. For producing hypha protein and application of the fusarium sp. The invention specifically discloses application of fusarium sp. Or a bacterial agent of the fusarium sp. To production of mycelium protein and / or mycelium rich in protein. The fusarium sp. Or the bacterial agent of the fusarium sp. Comprises any one or more of fusarium avenaceum, fusarium sp. And fusarium equiseti. The fusarium avenaceum or the bacterial agent of the fusarium avenaceum or the bacterial agent of the fusarium avenaceum or the bacterial agent of the fusarium avenaceum or the bacterial agent of the fusarium avenaceum or the bacterial agent of the fusarium avenaceum or the bacterial agent of the fusarium avenaceum or the bacterial agent of the fusarium avenaceum comprises any one or more of the fusarium avenaceum and the fusarium equiseti.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular to the use of any one or more Fusarium species selected from Fusarium avenaceum, Fusarium sp. and Fusarium equiseti in the production of mycelial protein, and the mycelial protein produced. Background Art

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

[0003] Fusarium is a widely used filamentous fungus for fermentation and production of microbial protein. The mycelial protein produced by it is more delicious than single-cell proteins such as yeast and bacteria, and has a meat-like tissue structure. At the same time, the rich edible crude fiber it contains helps human gastrointestinal digestion, and is a meat substitute that can meet the nutritional needs of modern people. At present, in the field of filamentous fungal protein, Fusarium venenatum strains have been developed and mycelial protein-forming meat products have been formed. However, it is currently found that Fusarium venenatum with natural high protein yield is extremely scarce, so finding more potential strains of natural high-yield filamentous fungal protein from the genus Fusarium is a development with great commercial value. Summary of the invention

[0004] In order to solve one of the above-mentioned technical problems existing in the prior art, the present disclosure provides a new application of Fusarium in producing mycelial protein, and a method for producing mycelial protein.

[0005] According to one aspect of the present disclosure, there is provided the use of Fusarium or its bacterial agent in producing mycelial protein and / or protein-rich mycelium, wherein the Fusarium or its bacterial agent includes any one or more of Fusarium avenaceum, Fusarium sp. MN113808 and Fusarium equiseti.

[0006] According to another aspect of the present disclosure, a Fusarium or a bacterial agent thereof is provided, which includes any one or more of Fusarium avenaceum, Fusarium sp. MN113808 and Fusarium equiseti.

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

[0008] In some embodiments, the Fusarium or its agent may include Fusarium spp.

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

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

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

[0012] In some embodiments, the translation elongation factor 1 (TEF1) of Fusarium avenaceum has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 9. In some embodiments, the translation elongation factor 1 (TEF1) of Fusarium avenaceum has a nucleotide sequence with 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 identity to SEQ ID NO: 9.

[0013] In some embodiments, the polymerase II subunit (RPB2) of Fusarium avenaceum has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, the RPB2 of Fusarium avenaceum has a nucleotide sequence with 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 identity to SEQ ID NO: 10.

[0014] In some embodiments, the internal transcribed spacer (ITS) of Fusarium avenaceum has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 11. In some embodiments, the ITS of Fusarium avenaceum has a nucleotide sequence with 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 identity to SEQ ID NO: 11.

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

[0016] In some embodiments, the TEF1 of Fusarium sp. MN113808 has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, the TEF1 of MN113808 (Fusarium sp.) has a nucleotide sequence with 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 identity to SEQ ID NO: 14.

[0017] In some embodiments, the internal transcribed spacer (ITS) of MN113808 (Fusarium sp.) has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 15. In some embodiments, the ITS of MN113808 (Fusarium sp.) has a nucleotide sequence with 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 identity to SEQ ID NO: 15.

[0018] In some embodiments, the RPB1 of MN113808 (Fusarium sp.) has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 16. In some embodiments, the RPB1 of MN113808 (Fusarium sp.) has a nucleotide sequence with 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 identity to SEQ ID NO: 16.

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

[0020] In some embodiments, the MN113808 (Fusarium sp.) may include the MN113808 strain, which was deposited in the China General Microbiology Center (CGMCC) on May 21, 2024, with a deposit number of CGMCC No.41316.

[0021] In some embodiments, the TEF1 of Fusarium equiseti has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 18. In some embodiments, the TEF1 of Fusarium equiseti has a nucleotide sequence with 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 identity to SEQ ID NO: 18.

[0022] In some embodiments, the internal transcribed spacer (ITS) of Fusarium equiseti has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 19. In some embodiments, the ITS of Fusarium equiseti has a nucleotide sequence with 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 identity to SEQ ID NO: 19.

[0023] In some embodiments, the RPB1 of Fusarium equiseti has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 20. In some embodiments, the RPB1 of Fusarium equiseti has a nucleotide sequence with 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 identity to SEQ ID NO: 20.

[0024] In some embodiments, the RPB2 of Fusarium equiseti has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 21. In some embodiments, the RPB2 of Fusarium equiseti has a nucleotide sequence with 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 identity to SEQ ID NO: 21.

[0025] In some embodiments, the Fusarium equiseti may include the MN146565 strain, which was deposited in the China General Microbiology Center (CGMCC) on May 21, 2024, with a deposit number of CGMCC No.41317.

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

[0027] In some embodiments, the mycelium comprises greater than about 45% protein based on dry weight of the mycelium.

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

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

[0030] In some embodiments, the mycelium does not contain any harmful substances, for example, it does not contain or contains below the detection limit aflatoxins (for example, but not limited to aflatoxins B1, B2, G1, G2, etc.), vomitoxins (for example, but not limited to deoxynivalenol), zearalenone, fusaritoxin, fusaritoxin, ochratoxin, fumonisins (for example, but not limited to fumonisins FA1, FA2, FB1, FB2, FB3, FB4, FC1, FC2, FC3, FC4, etc.), T-2 toxin, HT-2 toxin, fusaritoxin C, etc.

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

[0032] In some embodiments, the microbial agent includes the Fusarium and an adjuvant acceptable to the microbial agent. Those skilled in the art should understand that the adjuvant acceptable to the microbial agent and its dosage can be conventionally selected as needed and are not particularly limited in the present disclosure.

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

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

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

[0036] In some embodiments, the nitrogen source in the seed culture medium and / or fermentation medium may 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 hydrolyzate, yeast extract, bean cake powder, etc. In some embodiments, the inorganic nitrogen source may include, but is not limited to, urea or ammonium compounds (e.g., ammonium sulfate, ammonium phosphate).

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

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

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

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

[0041] In specific 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 yeast extract.

[0042] In specific 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 potassium dihydrogen phosphate.

[0043] In specific 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 magnesium sulfate heptahydrate.

[0044] In specific embodiments, the seed culture medium can 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 weight portion of calcium chloride.

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

[0046] In some embodiments, the fermentation medium can 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 can include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 parts by weight of a nitrogen source.

[0047] In some embodiments, the fermentation medium may include: 20 to 35 parts by weight of corn starch saccharification liquid; 0.1 to 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.

[0048] In a specific embodiment, the fermentation medium can 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 liquid.

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

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

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

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

[0053] In specific embodiments, the fermentation medium can 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 parts by weight of calcium chloride.

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

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

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

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

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

[0059] In some embodiments, in step 2), the ventilation rate during the culture is about 5 to 15 L / h.

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

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

[0062] In some embodiments, the vitamin complex may include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, or any combination thereof. In a specific embodiment, 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 a specific embodiment, the components in the vitamin complex are mixed in equal parts by mass. In these embodiments, no spores are produced during the fermentation culture process.

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

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

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

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

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

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

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

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

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

[0072] In some embodiments, the feed medium may include the above-mentioned carbon source and / or vitamin complex.

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

[0074] In some embodiments, the feed medium may include 20,000 to 40,000 parts by weight of corn starch saccharification liquid. 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 liquid.

[0075] In some embodiments, the feed medium may include 0.001 to 0.2 parts by weight of a vitamin complex. In some embodiments, the feed 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 parts by weight of a vitamin complex.

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

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

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

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

[0080] The present invention uses, for example, starch, edible gum specified in GB2760 and / or modified starch as thickeners; the thickener absorbs water and swells, and the swollen thickener and mycelial protein form a network structure, so that the organization and emulsification of the mycelial protein are further enhanced, thereby improving the texture and taste of the meat substitute food; that is, the obtained mycelial protein-based meat substitute food can completely replace plant protein vegetarian meat products, avoiding the defects brought by plant protein, and at the same time has excellent taste and texture, the taste is juicy, elastic and moderate hardness, the texture has good hardness, elasticity and chewiness, and is closer to real meat food. Compared with plant protein vegetarian meat or pork and chicken, the mycelial protein-based meat substitute food has high protein content and natural dietary fiber content, low fat content, and does not contain cholesterol, and is more nutritious and healthy.

[0081] According to another aspect of the present disclosure, provided is the use of the mycelial protein and / or protein-rich mycelium of the present disclosure, or the above composition in the preparation of meat substitutes.

[0082] In some embodiments, the mycelial protein and / or protein-rich mycelium of the present disclosure, or the above-mentioned composition can replace chicken, pork, beef, mutton, duck, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The phylogenetic tree analysis results of the bacterial species MN133388, MN146565 and MN113808 are shown.

[0084] Figure 2 The mycelium prepared by fermentation of MN133388 strain is shown.

[0085] Figure 3 The mycelium prepared by fermentation of strain MN146565 is shown.

[0086] Figure 4 The mycelium prepared by fermentation of strain MN113808 is shown.

[0087] FIG5 shows the results of the first generation fermentation culture of strain MN133388. Figure 5A The results of microscopy are Figure 5B This is a picture of the fermentation broth.

[0088] FIG6 shows the results of the second generation fermentation culture of MN133388 strain. Fig. 6A The results of microscopy are Figure 6B This is a picture of the fermentation broth. Figure 6C This is a picture of the filter cake.

[0089] Figure 7 A picture of the filter cake obtained from the third generation fermentation culture of the MN133388 strain is shown.

[0090] Figure 8 A picture of the filter cake obtained from the fifth generation fermentation culture of the MN133388 strain is shown.

[0091] Fig. 9 The microscopic examination results of the third generation fermentation culture of the MN113808 strain are shown.

[0092] Fig.10 A picture of the filter cake obtained from the third generation fermentation culture of the MN113808 strain is shown.

[0093] Fig.11 The microscopic examination results of the third generation fermentation culture of the MN146565 strain are shown.

[0094] Fig.12 A picture of the filter cake obtained from the third generation fermentation culture of the MN146565 strain is shown.

[0095] Fig.13 The results of SGS testing of MN133388 mycelial protein are shown. DETAILED DESCRIPTION

[0096] In the field of fungal mycelial protein, natural high-protein-producing Fusarium venenatum is extremely scarce, and natural high-protein-producing Fusarium strains are also very scarce. The present invention develops a new natural high-yield filamentous fungal protein Fusarium.

[0097] Fusarium avenaceum, after testing in amplified fermentation production, has a total fat content of about 11.06%, a crude protein content of about 48.37%, and a dietary fiber content of about 29.50%. Those skilled in the art know that a protein content of about 40% can be developed into a product. At present, there is no report that Fusarium avenaceum can be used for the development and application of fungal protein.

[0098] The present disclosure has identified more than ten toxins in the mycelial protein produced by Fusarium avenaceum, and the results show that the toxins contained in it are all below the limit, and it can be considered to be toxin-free. Therefore, the strains disclosed in the present disclosure solve the problem of safe consumption in food development.

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

[0100] It is known that the production of spores will affect the texture and shape of the product. The present disclosure has achieved the control of spore production to a great extent by improving the fermentation process. In a specific embodiment, no spores were observed in the mycelial protein produced by the Fusarium of the present disclosure under microscopic examination. In a specific embodiment, the mycelial protein produced by the Fusarium of the present disclosure has excellent filaments, the filter cake is light yellow, elastic and tough, and has no bad smell.

[0101] In a specific embodiment, the mycelial protein obtained by fermentation culture of the Fusarium of the present disclosure has good filaments under microscopic examination, and the fermentation liquid can naturally present flesh color without heating. The protein content of the mycelial protein produced by the Fusarium of the present disclosure is higher than 50%, even reaching more than 55%.

[0102] Usually, strains can only utilize organic nitrogen sources, but not inorganic nitrogen sources. In a specific embodiment, the present invention can enable the Fusarium of the present invention to utilize inorganic nitrogen sources by adding trace elements, while improving the shape and color texture of the mycelium.

[0103] The present disclosure provides a method for producing mycelial protein, the method comprising: 1) inoculating the Fusarium or its bacterial agent of the present disclosure into a seed culture medium for culturing to obtain a seed liquid; 2) inoculating the seed liquid into a fermentation culture medium for culturing to obtain a fermentation liquid; and, 3) obtaining mycelial protein from the fermentation liquid.

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

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

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

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

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

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

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

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

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

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

[0114] In a specific embodiment, the fermentation medium may include 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0. 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, 5g / 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 of ammonium sulfate.

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

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

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

[0118] In a specific embodiment, the fermentation medium may include 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 of ferrous sulfate heptahydrate.

[0119] 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 zinc chloride.

[0120] In some embodiments, the fermentation medium may further include a vitamin complex. In some embodiments, the fermentation medium may further include 0.01 to 2 mg / L of a vitamin complex. In some embodiments, the fermentation medium can also include 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 of vitamin complex.

[0121] In a specific embodiment, the meat substitute (e.g., meat chunks or meat emulsion) of the present disclosure may include vegetable or animal oils, alginate, arabinoxylan, carrageenan, carboxymethylcellulose, cellulose, curdlan, gelatin, gellan gum, -glucan, galactomannan, guar gum, locust bean gum, tara gum, gum arabic, pectin, konjac flour, starch and xanthan gum.

[0122] In a specific embodiment, the hyphal protein of the present disclosure may comprise filaments having 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 hyphal protein of the present disclosure may comprise filaments having a length of greater than 100 μm, preferably greater than 200 μm.

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

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

[0125] The mycelial protein produced by the Fusarium or its bacterial agent disclosed in the present invention has very few bifurcations; the fermentation liquid is flesh-colored, and the flesh color gradually deepens with the increase in the number of subcultures; the fermentation liquid has no obvious odor; the filter cake has good elasticity and has no obvious odor after heating.

[0126] The mycelial protein produced by the Fusarium or its fungal agent disclosed in the present invention can be used to prepare meat substitutes.

[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 a specific embodiment, 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 a specific embodiment, the components in the vitamin complex are mixed in equal parts by mass.

[0128] The mycelial protein produced by the Fusarium or its bacterial agent disclosed in the present invention has very few bifurcations; the fermentation liquid is flesh-colored, and the flesh color gradually deepens with the increase in the number of subcultures; the fermentation liquid has no obvious odor; the filter cake has good elasticity and has no obvious odor after heating.

[0129] The mycelial protein produced by the Fusarium or its fungal agent disclosed in the present invention can be used to prepare meat substitutes.

[0130] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0131] definition

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

[0133] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

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

[0135] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0136] Example

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

[0138] 1.1 Strain source

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

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

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

[0142] 1.2 Separation process

[0143] Mix the collected samples, weigh 5 g, and place them in a conical flask containing 45 mL of sterile 0.7% sodium carboxymethyl cellulose solution and 15 glass beads, and shake at 30°C and 150 rpm for 30 min. Take 1 mL of the sample suspension and sieve it with sterile water for 10 min. -2 -10 -3 Serial concentration gradient dilution, then take 10 -2 , 10 -3 The two dilutions were spread on MGA2.5 (containing chloramphenicol and tetracycline hydrochloride) plates at a drop size 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 dried.

[0144] Among them, the specific composition of the MGA2.5 flat panel is:

[0145] Peptone 15g <![CDATA[KH2PO4]]> 1.0g <![CDATA[MgSO4·7H2O]]> 0.5g Malachite Green Oxalate 0.0025g Agar 20g Distilled water 1L

[0146] Purification was performed by the step-by-step transplantation method of hyphae ends. After the colonies were formed on the plate, hyphae at the edge of a single colony were picked and placed on a MEA plate (purchased from: Beijing Aoboxing Biotechnology Co., Ltd., item number: 02-183), and continued to be cultured at 28°C until pure colonies were obtained, and the obtained colonies were stored at -80°C.

[0147] Example 2. Identification of MN133388 strain

[0148] The genomic DNA was extracted from the mycelium of the strain and used as a template for gene amplification. The strain DNA was subjected to multi-gene amplification and sequencing.

[0149] 2.1 PCR system preparation

[0150] According to the table below, prepare 25 μL of PCR system in a PCR tube:

[0151] 2×Taq PCR Master Mix (Takara) 12μL DMSO 1μL Forward primer (10 μmol / L) 0.6μL Reverse primer (10 μmol / L) 0.6μL Template DNA 2μL Ultrapure sterile water 8.8μL

[0152] The primers for each gene are shown in the following table:

[0153]

[0154] The above primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0155] 2.2 PCR amplification

[0156] The prepared PCR system was placed in a PCR instrument (Dongsheng ETC811) for PCR amplification. The PCR reaction conditions are shown in the following table. After amplification, the PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0157]

[0158] 2.3 The sequencing results for strain MN133388 are shown in the following table:

[0159]

[0160]

[0161]

[0162] 2.4 Identification of MN133388 by comparing with the standard NT library in NCBI

[0163] The NCBI online alignment system (blast) was used to align the four gene sequences respectively. The alignment database was the Nucleotide collection (nr / nt). The most similar alignment results of the four genes are shown in the following table.

[0164] Gene Accession Scientific Name Per.Ident Query Cover E value TEF1 MK572769.1 Fusarium avenaceum 100％ 100％ 0 ITS OR808063.1 Fusarium acuminatum 100％ 100％ 0 RPB1 LC724045.1 Fusarium avenaceum 99.29％ 98％ 0 RPB2 CP109670.1 Fusarium avenaceum 98.39％ 100％ 0

[0165] 2.6MN133388 multi-gene identification using the mycobank database

[0166] The polyphasic identification method provided by the mycobank (fusarium.mycobank.org) online database was used to upload the four genes to the webpage at the same time. The results showed that based on the TEF1, RPB1, and RPB2 genes, the closest species to the current strain was Fusarium avenaceum NRRL 54939, with a global similarity of 99.36%.

[0167] 2.7MN133388 Constructing a polygenic species evolutionary tree to determine its evolutionary position

[0168] Based on the above two methods, since MN133388 was identified as Fusarium avenaceum, the species complex to which this species belongs is Fusarium tricinctum species complex (FTSC). The sequences of TEF1 gene, RPB1 gene, RPB2 gene and ITS gene of all FTSC strains were collected from FUSARIOID-ID database (www.fusarium.org), and the evolutionary position of MN133388 strain was analyzed by constructing a multi-gene evolutionary tree.

[0169] The steps of multi-gene evolutionary tree analysis are as follows: first, use MAFFT (version v7.310) to perform multiple sequence alignment for each gene, then use GBLOCKS (version 0.91b) to trim the multiple sequence alignment result file, and then use AliView to manually trim it. After trimming, the sequences of the four genes are spliced ​​in series, and the spliced ​​sequences are used to construct a multi-gene joint phylogenetic tree using IQ-TREE (version 2.2.5). The results are shown in Figure 1 shown.

[0170] Based on the above three analysis methods, the species of MN133388 was identified as Fusarium solani (Fusariumavenaceum), which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on May 21, 2024, with the deposit number CGMCC No.: 41315.

[0171] Example 3. Identification of MN113808 strain

[0172] 3.1 Preparation of DNA template

[0173] Prepare DNA template as in step 2.1

[0174] 3.2 PCR system preparation

[0175] Configure the PCT system according to 2.2.

[0176] The amplified genes and primers are shown in the following table:

[0177]

[0178] 3.3 The sequencing results of MN113808 strain are shown in the following table:

[0179]

[0180]

[0181]

[0182] 3.4 Identification of MN113808 by comparison with the standard NT library in NCBI;

[0183] The NCBI online alignment system (blast) was used to align the four gene sequences respectively. The alignment database was the Nucleotide collection (nr / nt). The most similar alignment results of the four genes are shown in the following table:

[0184] Gene Accession Scientific Name Per.Ident Query Cover E value TEF1 HM134858.1 Fusarium chlamydosporum 99.37％ 92％ 0 ITS ON408997.1 Fusarium nelsonii 99.27％ 97％ 0 RPB1 MN120721.1 Fusarium microconidium 93.81％ 97％ 0 RPB2 HQ154463.1 Fusarium sibiricum 91.06％ 94％ 0

[0185] 3.5MN113808 was used for multi-gene identification using the mycobank database;

[0186] The polyphasic identification method provided by the mycobank (fusarium.mycobank.org) online database was used to upload four genes to the webpage at the same time. The results showed that based on the TEF1, RPB2, and ITS genes, the closest species to the current strain was Fusarium nelsonii NRRL 28505, with a global similarity of 92.55%.

[0187] 3.6MN113808 Constructing a polygenic species evolutionary tree to determine its evolutionary position

[0188] Based on the above two methods, the subspecies of Fusarium chlamydosporum, Fusarium nelsonii and Fusarium microconidium were all Fusarium chlamydosporum species complex (FCSC), while the subspecies of Fusarium sibiricum was Fusarium sambucinum species complex (FSAMSC). The sequences of TEF1 gene, RPB1 gene, RPB2 gene and ITS gene of all FCSC and FSAMSC strains were collected from FUSARIOID-ID database (www.fusarium.org), and the evolutionary position of the current strain was analyzed by constructing a multi-gene evolutionary tree.

[0189] The steps of multi-gene evolutionary tree analysis are as follows: first, use MAFFT (version v7.310) to perform multiple sequence alignment for each gene, then use GBLOCKS (version 0.91b) to trim the multiple sequence alignment result file, and then use AliView to manually trim it. After trimming, the sequences of the four genes are spliced ​​in series, and the spliced ​​sequences are used to construct a multi-gene joint phylogenetic tree using IQ-TREE (version 2.2.5). The results are shown in Figure 2. Figure 1 shown.

[0190] from Figure 1 As can be seen from the phylogenetic tree shown, MN113808 belongs to the genus Fusarium sp., but is quite different from other FCSC subspecies strains. Combined with the analysis results of mycobank, it suggests that it may be a new species under the FCSC subspecies. MN113808 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC) on May 21, 2024, with the deposit number CGMCC No.: 41316.

[0191] Example 4. Identification of MN146565 strain

[0192] 4.1 Preparation of DNA template

[0193] Prepare DNA template as in step 2.1

[0194] 4.2 PCR system preparation

[0195] Configure the PCT system according to 2.2.

[0196] The amplified genes and primers are shown in the following table:

[0197]

[0198] 4.3 The sequencing results of strain MN146565 are shown in the following table:

[0199]

[0200]

[0201]

[0202] 4.4 Identification of MN146565 by comparison with the standard NT library in NCBI

[0203] The NCBI online alignment system (blast) was used to align the four gene sequences respectively. The alignment database was the Nucleotide collection (nr / nt). The most similar alignment results of the four genes are shown in the following table.

[0204] Gene Accession Scientific Name Per.Ident Query Cover E value TEF1 GQ505599.1 Fusarium equiseti 100％ 99％ 0 ITS NR_121457.1 Fusarium equiseti 100％ 99％ 0 RPB1 MN120717.1 Fusarium coffeatum 96.63％ 98％ 0 RPB2 MH706973.1 Fusarium subtropicale 89.84％ 94％ 0

[0205] 4.5MN146565 Multi-gene identification using the mycobank database

[0206] The polyphasic identification method provided by the mycobank (fusarium.mycobank.org) online database was used to upload four genes to the webpage at the same time. The results showed that based on the TEF1, RPB2, and ITS genes, the closest species to the current strain was Fusarium equiseti NRRL 20697, with a global similarity of 99.77%.

[0207] 4.6MN146565 Constructing a polygenic species evolutionary tree to determine its evolutionary position

[0208] Based on the above two methods, except for Fusarium subtropicale, all other species belong to the Fusarium incarnatum-equiseti species complex (FIESC). The sequences of TEF1 gene, RPB1 gene, RPB2 gene and ITS gene of all FIESC strains were collected from FUSARIOID-ID database (www.fusarium.org), and the evolutionary position of the current strain was analyzed by constructing a multi-gene evolutionary tree.

[0209] The steps of multi-gene evolutionary tree analysis are as follows: first, use MAFFT (version v7.310) to perform multiple sequence alignment for each gene, then use GBLOCKS (version 0.91b) to trim the multiple sequence alignment result file, and then use AliView to manually trim it. After trimming, the sequences of the four genes are spliced ​​in series, and the spliced ​​sequences are used to construct a multi-gene joint phylogenetic tree using IQ-TREE (version 2.2.5). The results are shown in Figure 2. Figure 1 shown.

[0210] from Figure 1 It can be seen from the phylogenetic tree shown that the species of MN146565 is Fusarium equiseti, which was deposited in the China General Microbiology Center (CGMCC) on May 21, 2024, with the deposit number CGMCC No.: 41317.

[0211] Example 5. Preparation of mycelial protein

[0212] 5.1 Primary screening culture

[0213] The activated MN133388 (Fusarium avenaceum), MN113808 (Fusarium sp.), and MN146565 (Fusarium equiseti) strains were inoculated into 250 mL three-concave conical flasks containing 50 mL of the primary screening medium, and cultured at 200 rpm for 24 to 48 hours. The primary screening medium formula is: glucose 30 g / L, yeast extract 30 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.2 g / L, and sterilized by high pressure steam at 121°C for 20 minutes.

[0214] Filter with a vacuum filtration device and wash the filter cake thoroughly, place it in an electric blast drying oven at 60°C to dry it, accurately weigh the mass of the dry filter cake and calculate the biomass concentration [biomass concentration = mycelium dry weight (g) / culture medium mass (g) * 100%], and then refer to "GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food" and use the Kjeldahl nitrogen method to determine the mycelium crude protein. The results are shown in the following table.

[0215] Observation and test results of mycelial morphology, filter cake color and texture, biomass and crude protein of the strain.

[0216] Bacteria Mycelium morphology Filter cake color Filter cake texture Biomass wt% Crude protein wt% MN133388 Filamentous Light yellow Flexible 0.66 52.90 MN113808 Filamentous Light yellow Flexible 1.60 53.50 MN146565 Filamentous Light yellow Flexible 0.46 52.82

[0217] As can be seen from the above table, the mycelial protein prepared by fermentation with MN133388 can obtain mycelial protein with filamentous morphology, light yellow color, elastic texture, and biomass concentration of 0.66wt%. The crude protein content of the mycelium is further determined by the Kjeldahl method to be 52.90wt%.

[0218] By using MN113808 to ferment and prepare mycelial protein, a mycelial protein with a filamentous morphology, light yellow color, elastic texture, and a biomass concentration of 1.6% can be obtained. The crude protein content of the mycelium is further determined by the Kjeldahl method to be 53.50%.

[0219] Using MN146565 to ferment and prepare mycelial protein, a mycelial protein with a filamentous morphology, light yellow color, elastic texture, and a biomass concentration of 0.46% can be obtained. The crude protein content of the mycelium is further determined by the Kjeldahl method to be 52.82%.

[0220] Figures 2 to 4 The mycelia prepared by fermentation of strains MN133388, MN146565 and MN113808 are shown respectively.

[0221] 5.2 Scaling up the culture to produce mycelial protein

[0222] 5.2.1 Fermentation tank screening culture method

[0223] The composition of the seed culture medium used was as follows:

[0224] Element Seed culture medium g / L glucose 30 Yeast extract powder 25 Potassium dihydrogen phosphate 1 Magnesium sulfate heptahydrate 0.2 Calcium chloride 0.01

[0225] Seed solution culture conditions: The first-level seed solution was prepared using the seed culture medium shown in the table above. Activated MN133388, MN113808, and MN146565 strains were picked and inoculated into 250 mL tri-conical flasks containing 50 mL of seed culture medium, and cultured at 200 rpm for 24 to 60 h.

[0226] Preparation of secondary seed solution: Use the above seed culture medium, transfer to a 3L three-concave conical flask containing 1L seed culture medium at a 5% inoculation rate, rotate at 200rpm, and culture for 24h to 60h;

[0227] The composition of the rescreening medium for producing mycelial protein is as follows:

[0228] Element Fermentation medium g / L Corn starch saccharification liquid 25 Citric acid 1 Potassium dihydrogen phosphate 1 Ammonium sulfate 5 Magnesium sulfate heptahydrate 0.2g / L Calcium chloride 0.01 Manganese sulfate monohydrate 0.0017 Ferrous Sulfate Heptahydrate 0.005 Zinc chloride 0.0017

[0229] Fermentation process conditions: fermentation tank volume 15L, loaded with 10L of rescreened culture medium, sterilized at 121℃ for 30min, inoculation volume 3%. Temperature 28℃, ventilation 5-15L / h, tank pressure 0.08-0.12Mpa, rotation speed 200-600rpm, dissolved oxygen 20-50%, pH 6.0 controlled by ammonia water. Ammonium sulfate in the above table can be replaced with ammonium phosphate. Corn starch saccharification liquid is obtained by treating corn starch (Qinhuangdao Lihua Starch Co., Ltd.) with high temperature resistant α-amylase and saccharifying enzyme of Angel Enzyme Preparation (Yichang) Co., Ltd.

[0230] Microscopic examination and Kjeldahl nitrogen determination showed that the crude protein content of mycelium of MN133388, MN113808 and MN146565 reached 50.63wt%, respectively.

[0231] Compared with the primary screening medium in Example 4, the secondary screening medium in this example uses inorganic nitrogen (ammonium sulfate, ammonium phosphate) to replace the organic nitrogen source; however, the crude protein content can still maintain a high protein content level. In addition, it also shows that the Fusarium of the present invention can simultaneously use organic nitrogen sources and inorganic nitrogen sources as nitrogen sources for the fermentation culture of fungal protein.

[0232] The results show that the use of inorganic nitrogen to replace organic nitrogen can reduce costs while maintaining the filamentous structure of the mycelium. The mycelium is slender, has few branches, and has a very low number of spores. At the same time, different species of bacteria can maintain a crude protein content of more than 50wt%. The mycelium presents a slender filamentous structure, and the prepared filter cake has an excellent texture and can better simulate the chewing feeling of meat.

[0233] Results of rescreening of three strains:

[0234] strain MN133388 MN113808 MN146565 Fermentation time 66h 65h 60h Filter cake color Flesh color Orange White Filter cake texture Flexible Flexible Flexible Filter cake odor slight slight slight Mycelium morphology Filamentous Filamentous Filamentous Mycelium length long long long Mycelium diameter thin thin thin Mycelium branching few few few Spore count few many none Crude Protein 50％ 53.40％ 50.63％

[0235] 5.2.2 To determine the optimal number of subcultures, the subculture method is as follows:

[0236] Use the seed medium composition from 5.2.1.

[0237] Seed solution culture conditions: the same as the seed solution culture conditions in 5.2.1 above.

[0238] The composition of the subculture medium is as follows:

[0239] Element Subculture medium g / L Corn starch saccharification liquid 25 Citric acid 1 Potassium dihydrogen phosphate 1 Ammonium sulfate 5 Magnesium sulfate heptahydrate 0.2 Calcium chloride 0.01 Manganese sulfate monohydrate 0.0017 Ferrous Sulfate Heptahydrate 0.005 Zinc chloride 0.0017 Vitamin complex liquid 0.0001

[0240] Culture conditions: 15L tank, 10L liquid volume (completely covering the paddles), 1L seed liquid is fully transferred; rotation speed 100-200rpm; ventilation volume 10L / min, tank pressure 0.05mpa; ammonia water to control pH 4.0; culture at 28℃ for 24h-60h; continuous culture for 5 generations, replacing 1 / 2 of the fermentation liquid volume each time. The complex vitamins can be B complex vitamins.

[0241] The complex vitamin in this embodiment includes the following components in equal mass ratios: vitamin B1 / thiamine, vitamin B12 / cobalamin, vitamin B2 / riboflavin, vitamin B3 / niacin, vitamin B5 / D-calcium pantothenate, vitamin B6 / pyridoxine, vitamin B9 / folic acid, vitamin C and vitamin H / D-biotin.

[0242] Comparison of the mycelial protein results obtained by subculture of MN133388 with the previous re-screening results:

[0243] 1. When it is passed to the second generation, there are no spores under microscopic examination, the filter cake is light flesh color, has good toughness, and has no abnormal odor;

[0244] 2. As the number of passages increases, the flesh color gradually deepens;

[0245] 3. After several passages, the protein content was close to 50%.

[0246] First generation culture results: No heat treatment, good filaments under microscopic examination, fermentation liquid light yellow (yeast powder base color) ( Figure 5A and 5B ), the filter cake has good elasticity and the fermentation liquid has no obvious odor.

[0247] Second generation culture results: Microscopic examination showed a small amount of bifurcation, the color of the unheated fermentation liquid was slightly flesh-colored, and the color of the filter cake after heating was white with a yellowish tint ( Figures 6A to 6C ), good elasticity, and the fermentation liquid has no obvious odor.

[0248] The third generation of partial canning: heating treatment, the filter cake is slightly flesh-colored ( Figure 7 ), the filter cake has good elasticity and no obvious odor after heating.

[0249] The fifth generation culture results: After heating, the filter cake color became darker than that of the third generation ( Figure 8 ), the filter cake has good elasticity and no obvious odor after heating.

[0250] Conclusion: The fermentation state (color, cake quality) of MN133388 was the best when it was subcultured to the second and third generations, but with the increase of subculture times, the meat color gradually deepened.

[0251] MN113808 and MN146565 had similar results.

[0252] The pictures of mycelium and filter cake of MN113808 cultured to the third generation are as follows Fig. 9 and Fig.10 shown.

[0253] The pictures of mycelium and filter cake of MN146565 cultured to the third generation are as follows Fig.11 and Fig.12 shown.

[0254] 5.2.3 Fermentation to obtain mycelial protein

[0255] Fermentation process

[0256] Seed liquid culture: Use the seed culture medium of 5.2.1 for inoculation, inoculation volume 5%, liquid volume 1L / 3L; shaker speed 200rpm; culture for 24h.

[0257] Primary fermentation: Use the subculture medium in 5.2.2 for subculture, use a 15L tank with a liquid volume of 10L, sterilize at 121℃ for 30min, and transfer all the seed liquid by flame inoculation; rotation speed 200rpm; ventilation volume 10L / min, tank pressure 0.05mpa; ammonia water to control pH 4.0; culture at 28℃ for 24h; culture continuously for two to three generations, replacing 1 / 2 of the fermentation liquid each time.

[0258] Secondary fermentation: Fermentation medium

[0259] Element Fermentation medium (g / L) Corn starch saccharification liquid 25 Citric acid 1 Potassium dihydrogen phosphate 1 Magnesium sulfate heptahydrate 5 Calcium chloride 0.2 Manganese sulfate monohydrate 0.01 Ferrous Sulfate Heptahydrate 0.0017 Zinc chloride 0.005 Vitamin complex liquid 0.0001

[0260] Feed medium

[0261] Element Feed medium Corn starch saccharification liquid / glucose 300g / L Vitamin complex liquid 0.1mg / L

[0262] The formula of the fermentation medium is shown in the table above, wherein the vitamin complex solution includes the following components in equal mass ratios: vitamin B1 / thiamine, vitamin B12 / cobalamin, vitamin B2 / riboflavin, vitamin B3 / niacin, vitamin B5 / D-calcium pantothenate, vitamin B6 / pyridoxine, vitamin B9 / folic acid, vitamin C, and vitamin H / D-biotin.

[0263] Use a 50L tank with a liquid volume of 30L and sterilize at 121°C for 30 minutes; add 0.1mg / L vitamin mixture after sterilization (filter sterilization); pump the fermentation liquid that has been subcultured to the second or third generation in the 15L tank through a sterilized hose from the bottom valve at an inoculation rate of 5-10% to complete the inoculation; ventilate at 1000L / h for the first 15 hours and the tank pressure at 0.05mpa; start feeding (i.e., adding feed culture medium) at 0.5-2g / L / h after 15 hours, and adjust the ventilation to 1500L / h and the tank pressure to 0.1mpa; use ammonia water to control the pH at 4.0-6.0; culture at 28°C; rotate at 100-450rpm coupled with dissolved oxygen 20%-50% and culture for about 72 hours.

[0264] The fermentation process in 5.2.3 was used for fermentation to obtain mycelial protein.

[0265] 1) Heat treatment: After fermentation, heat the fermentation liquid to 65°C and keep it warm for about 20 minutes, then heat it to 90°C and keep it warm for about 10 minutes, and then cool it to room temperature.

[0266] 2) Filtration and refrigeration: The fermentation liquid after heat treatment is filtered using a plate and frame filter press. After sufficient washing and ventilation, the filter cake is taken out and stored at 4°C.

[0267] 3) Detection: Samples for crude protein determination are pre-treated, dried and crushed, and then sieved through a 60-mesh sieve. Then the determination is carried out in accordance with the "GB5009.5-2016 National Food Safety Standard Determination of Protein in Food". Common fungal toxins in food are sent to a third party SGS for testing.

[0268] The mycelial protein sample (wet base filter cake) obtained by fermentation of MN133388 was subjected to physical and chemical testing. The SGS test results are as follows: Fig.13 shown.

[0269] Example 6. Preparation of meat chunks using Fusarium avenaceum MN133388 mycelial protein

[0270] Minced meat, shrimp paste and fish balls are prepared using the mycelium protein prepared in Example 5, including the following raw materials: Fusariumavenaceum mycelium protein raw material, water, starch, gluten, carrageenan, konjac flour, sunflower oil or rapeseed oil; and seasonings: monosodium glutamate, yeast extract, white sugar, food flavors, soybean oil, edible salt, spices, 5'-flavor ribonucleotide disodium; pigments: capsicum red, red yeast rice, sorghum red, allura red, tomato red, and cochineal red.

[0271] During the preparation of meat, methylcellulose or sodium alginate, anhydrous calcium chloride, and calcium acetate can be further added to the above raw materials to adjust the viscosity of the meat. The specific formula for preparing alternative minced meat with Fusarium avenaceum is shown in the following table:

[0272]

[0273] 6.1 The steps of obtaining the mycelial protein base in the form of minced meat by the above formulas 1 to 7 and preparing sausages by using the mycelial protein base are as follows:

[0274] 1. Mix the thickener with part of the ice water, chop and stir evenly, add the mycelium protein base, umami agent, sweetener, food flavor, vegetable oil, edible salt, spices, pigment and the remaining ice water, mix evenly to obtain the vegetarian meat filling;

[0275] 2. Pour the vegetarian meat filling into the cellulose casing through a filling machine, and put the obtained sausage hanging rod into a smoking oven, dry at 55°C for 40 minutes, and steam at 85°C for 30 minutes;

[0276] 3. Take out and blow with cold air to below 30℃, remove casing and pack;

[0277] 4. Quick freezing and storage.

[0278] 6.2 The mycelial protein base in the form of minced meat is obtained by the above formulas 1 to 7. The steps of preparing shrimp paste using the mycelial protein base are as follows:

[0279] (1) Premix the curdlan and acetate starch, add ice water and chop evenly, add mycelium protein base, egg white powder, sunflower oil, food flavor, edible salt, white sugar, yeast extract, monosodium glutamate, spices and TG enzyme (glutamine transaminase), and stir evenly;

[0280] (2) bagging the raw materials mixed evenly in step (1) and squeezing them out of the packaging bags onto a tray;

[0281] (3) Place the tray in a steamer and steam at 55°C for 30 minutes;

[0282] (4) Raise the steamer temperature to 85°C and steam for 30 minutes until cooked through, then cool to room temperature.

[0283] 6.3 The steps of obtaining the mycelial protein base in the form of minced meat by the above formulas 1 to 7 and preparing fish sausage using the mycelial protein base are as follows:

[0284] 1. Mix carrageenan, curdlan and tapioca starch in advance, add ice water and chop evenly, add mycelium protein-based raw materials and stir evenly;

[0285] 2. Pour the stuffing obtained in step 1 into the nylon casing through a filling machine and fasten the casing with a clip punching machine;

[0286] 3. Put it in a boiling tank and cook at 85℃ for 40 minutes;

[0287] 4. Use running water to cool down to below 30℃;

[0288] 5. Quick freezing and storage.

[0289] 6.4 The steps of obtaining the mycelial protein base in the form of minced meat by using the above formulas 1 to 7 and preparing fish fillets using the mycelial protein base are as follows:

[0290] 1. Mix carrageenan, sodium alginate and distarch phosphate evenly in advance, add ice water and chop evenly, add mycelial protein-based raw materials and stir evenly;

[0291] 2. Put the fillings in step 1 into the fish fillet mold and squeeze it into the shape of fish fillet;

[0292] 3. Put the fish fillet into the refrigerator and freeze for 1 hour;

[0293] 4. Dip the fish fillet in egg liquid and bread crumbs, pre-fry at 170℃ for 2 minutes, and cool to room temperature;

[0294] 5. Quick freezing and storage.

[0295] 6.5 The mycelial protein-based sausages prepared in the form of minced meat were evaluated by (1) sensory evaluation and (2) texture analysis.

[0296] (1) Through tasting by ordinary consumers, the juiciness, elasticity and hardness indicators are evaluated using sensory evaluation methods.

[0297] The evaluation results of the taste of the mycelial protein-based meat chunks are shown in the following table:

[0298]

[0299]

[0300] It can be concluded from the above table that the sausages prepared from the mycelial protein base of formulas 1-5 have excellent juiciness, elasticity, hardness, and good taste, while the mycelial protein-based sausages in formula 6 have weak elasticity and hardness and poor taste, and the mycelial protein-based sausages in formula 7 have weak juiciness, weak elasticity, high hardness, and poor taste. The above can illustrate that the present invention can prepare sausages whose protein raw material is only mycelial protein under the selected mycelial protein, thickener and water and the selected respective amounts, and have a better taste, and can completely replace sausages made of animal meat and plant protein.

[0301] (2) Appearance inspection

[0302] Based on the above formulas 1 to 7, pigments are further added to make the color closer to meat. The specific formula is as follows:

[0303]

[0304] The appearance of sausages prepared from mycelial protein base of recipes 8 to 14 and commercially available pork sausages were tested using a colorimeter (Minolta Konica colorimeter CR400).

[0305] L*: The brightness of an object, 0-100 represents from black to white, referring to the depth of the color;

[0306] a*: the red and green color of the object, positive values ​​represent red, and negative values ​​represent green;

[0307] b*: The yellow-blue color of the object, positive values ​​represent yellow and negative values ​​represent blue.

[0308] The colorimeter test results of mycelial protein-based sausages are shown in the following table:

[0309]

[0310]

[0311] It can be concluded from the above table that the appearance of the mycelial protein-based sausages prepared by formulas 8 to 12 is closer to the commercially available pork sausages, while the appearance of the mycelial protein-based sausages prepared by formulas 13 to 14 is somewhat different from that of the commercially available pork sausages.

[0312] (3) Texture testing

[0313] The sausages prepared from mycelial protein base of recipes 1 to 7 and commercially available pork sausages were cut into φ2cm*2cm cylinders, and the texture of the samples was repeatedly tested 6 times using a texture analyzer (UK Micro Stable TA.XT plus C, TPA mode). 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.

[0314] Elasticity: Elasticity refers to the ability of an object to deform under the action of external force and return to its original state after the external force is removed.

[0315] Cohesion: Cohesion reflects the property of food that resists damage and connects tightly when chewing, keeping the food intact.

[0316] Chewability: It is used to describe solid test samples. It indicates the energy required to chew the solid sample into a stable state when swallowed. The value is expressed as the product of hardness × cohesion × elasticity.

[0317] The texture analyzer test results of mycelial protein-based sausages are shown in the following table:

[0318] sample hardness elasticity Cohesion Chewability Recipe 1 4877.17±36.83 0.88±0.002 0.52±0.04 2349.10±151.65 Recipe 2 4899.32±30.86 0.87±0.01 0.55±0.01 2336.36±140.12 Recipe 3 4626.17±34.53 0.89±0.01 0.52±0.03 2124.01±143.81 Recipe 4 3956.05±29.84 0.89±0.01 0.50±0.02 1764.42±146.34 Recipe 5 5291.06±32.09 0.83±0.02 0.62±0.01 2907.38±142.56 Recipe 6 1586.75±43.36 0.66±0.03 0.58±0.01 612.00±64.39 Recipe 7 7658.09±191.01 0.82±0.01 0.49±0.01 3058.89±235.91 Commercial pork sausage 3001.16±75.92 0.84±0.01 0.62±0.02 1712.22±228.23

[0319] It can be concluded from the above table that the mycelial protein-based sausages prepared according to formulas 1 to 5 of the present invention are closer to commercially available pork sausages, and their hardness, elasticity and chewiness are better than those of commercially available pork sausages, while the texture of the mycelial protein-based sausages prepared according to formulas 6 to 7 is somewhat different from that of commercially available pork sausages. The mycelial protein-based sausages of formula 6 have too weak hardness, weak elasticity, and poor cohesion, resulting in poor chewiness and poor taste, while the mycelial protein-based sausages in formula 7 have too high hardness, which also results in weak elasticity and poor cohesion, resulting in too high chewiness, which makes the taste too different from that of commercially available pork sausages. The present invention uses, for example, starch, edible gum specified in GB2760 and / or modified starch as thickeners; the thickener absorbs water and swells, and the swollen thickener and mycelial protein form a network structure, so that the organization and emulsification of the mycelial protein are further enhanced, thereby improving the texture and taste of the meat substitute food; that is, the obtained mycelial protein-based meat substitute food can completely replace plant protein vegetarian meat products, avoiding the defects brought by plant protein, and at the same time has excellent taste and texture, the taste is juicy, elastic and moderate hardness, the texture has good hardness, elasticity and chewiness, and is closer to real meat food. Compared with plant protein vegetarian meat or pork and chicken, the mycelial protein-based meat substitute food has high protein content and natural dietary fiber content, low fat content, and does not contain cholesterol, and is more nutritious and healthy.

[0320]

[0321] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of Fusarium or its bacterial agent in producing mycelial protein and / or protein-rich mycelium, wherein the Fusarium or its bacterial agent comprises any one or more of Fusarium avenaceum, Fusarium sp. and Fusarium equiseti.

2. A Fusarium or a bacterial agent thereof, comprising any one or more of Fusarium avenaceum, Fusarium sp. and Fusarium equiseti.

3. A mycelial protein and / or protein-rich mycelium derived from the Fusarium or its bacterial agent according to claim 2.

4. The use according to claim 1, the Fusarium or its bacterial agent according to claim 2, or the mycelial protein and / or protein-rich mycelium according to claim 3, characterized in that: The TEF1 of Fusarium avenaceum has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 9, and / or The RPB2 of Fusarium avenaceum has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 10, and / or The ITS of Fusarium avenaceum has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 11, Preferably, the Fusarium avenaceum includes the MN133388 strain, which was deposited in the China General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on May 21, 2024, with a deposit number of CGMCC No.41315.

5. The use according to claim 1, the Fusarium or its bacterial agent according to claim 2, or the mycelial protein and / or protein-rich mycelium according to claim 3, characterized in that: The TEF1 of Fusarium sp. has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 14, and / or The ITS of Fusarium sp. has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 15, and / or The RPB1 of Fusarium sp. has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 16, and / or The RPB2 of Fusarium sp. has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 17, Preferably, the Fusarium sp. may include the MN113808 strain, which was deposited in the China General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on May 21, 2024, with a deposit number of CGMCC No.41316.

6. The use according to claim 1, the Fusarium or its bacterial agent according to claim 2, or the mycelial protein and / or protein-rich mycelium according to claim 3, characterized in that: The TEF1 of Fusarium equiseti has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 18, and / or The ITS of Fusarium equiseti has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 19, and / or The RPB1 of Fusarium equiseti has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 20, and / or The RPB2 of Fusarium equiseti has a nucleotide sequence with at least 85% sequence identity to SEQ ID NO: 21, Preferably, the Fusarium equiseti includes the MN146565 strain, which was deposited in the China General Microbiology Center (CGMCC) on May 21, 2024, with a deposit number of CGMCC No.41317.

7. The use according to claim 1, the Fusarium or its bacterial agent according to claim 2, or the mycelial protein and / or protein-rich mycelium according to claim 3, characterized in that: The Fusarium is in spore form and / or mycelium form, and / or The mycelium comprises greater than about 45% protein based on the dry weight of the mycelium, and / or The mycelium comprises mycelial protein at a biomass concentration of about 0.4% or more based on the dry weight of the mycelium, and / or In some embodiments, the mycelium is filamentous; and / or the filter cake is elastic or tough.

8. A method for producing mycelium, the method comprising: 1) inoculating the Fusarium or the bacterial agent thereof according to claim 2 into a seed culture medium for cultivation to obtain a seed solution; 2) inoculating the seed liquid into a fermentation medium to obtain a fermentation liquid; and 3) obtaining mycelium from the fermentation broth.

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

10. The method according to claim 8, characterized in that The seed culture medium comprises 25 to 35 parts by weight of a carbon source, 20 to 35 parts by weight of a nitrogen source and 0.1 to 2 parts by weight of an inorganic salt. Preferably, the seed culture medium comprises: 25 to 35 parts by weight of glucose, 20 to 35 parts by weight of yeast extract powder, 0.1 to 2 parts by weight of potassium dihydrogen phosphate, 0.1 to 2 parts by weight of magnesium sulfate heptahydrate, and 0.005 to 1 parts by weight of calcium chloride. Preferably, the fermentation medium comprises 20 to 35 parts by weight of a carbon source, 0.01 to 10 parts by weight of a nitrogen source and 0.01 to 10 parts by weight of an inorganic salt. Preferably, the fermentation medium comprises: 20 to 35 parts by weight of corn starch saccharification liquid; 0.1 to 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 parts by weight of calcium chloride; and, optionally, 0.001 to 0.1 parts by weight of trace elements, Preferably, 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. Optionally, the fermentation medium further comprises 0.0001 to 0.2 parts by weight of a vitamin complex, wherein the vitamin complex preferably comprises vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H or any combination thereof.

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

0.

12. The method according to claim 8, characterized in that The method further comprises the following steps between step 2) and step 3): a) inoculating the first fermentation liquid into a fermentation medium, and culturing for 5 to 20 hours at 0.01 to 0.1 mPa and a ventilation volume of 800 to 1200 L / h; and b) adding feed medium, culturing at 0.1-0.5 mPa and aeration rate of 1300-2000 h / L for 24-48 hours to obtain a second fermentation liquid.

13. The method according to claim 12, characterized in that The feed culture medium comprises 10000-50000 parts by weight of corn starch saccharification liquid and / or 0.001-0.2 parts by weight of vitamin complex.

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

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

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