Fusarium compactum and application thereof

Abstract

The invention relates to fusarium compactum and application thereof. Specifically, the invention provides fusarium sp. Or a bacterial agent thereof, mycelium protein and / or protein-rich mycelium derived from the fusarium sp. Or the bacterial agent thereof, a composition containing the mycelium protein and / or the protein-rich mycelium and application of the composition.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular to Fusarium compactum for producing mycelial protein, mycelial protein produced by Fusarium compactum and applications thereof. Background Art

[0002] Fusarium is a widely used filamentous fungus for producing microbial protein. The mycelial protein produced by it is delicious and has a meat-like tissue structure. At the same time, the rich edible crude fiber it contains helps human gastrointestinal digestion, and it is a meat substitute that can meet the nutritional needs of modern people. At present, the strain of Fusarium venenatum has been developed to produce meat products with mycelial protein. However, the natural high-yield protein Fusarium venenatum is extremely scarce.

[0003] Therefore, finding more potential strains that naturally produce high-yield filamentous fungal proteins 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 use of Fusarium compactum MM-135 or a bacterial agent thereof in producing mycelial protein and / or protein-rich mycelium.

[0006] According to another aspect of the present disclosure, a Fusarium or a bacterial agent thereof is provided, which includes Fusarium compactum MM-135.

[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 compactum MM-135 or its bacterial agent of the present disclosure.

[0008] In some embodiments, the translation elongation factor 1 (TEF1) of Fusarium compactum MM-135 has a nucleotide sequence as shown in SEQ ID NO:9.

[0009] In some embodiments, the internal transcribed spacer (ITS) of Fusarium compactum MM-135 has a nucleotide sequence as shown in SEQ ID NO:10.

[0010] In some embodiments, the polymerase I subunit (RPB1) of Fusarium compactum MM-135 has a nucleotide sequence as shown in SEQ ID NO:11.

[0011] In some embodiments, the polymerase II subunit (RPB2) of Fusarium compactum MM-135 has a nucleotide sequence as shown in SEQ ID NO:12.

[0012] In some embodiments, the Fusarium compactum can include MM-135.

[0013] In some embodiments, the MM-135 strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on May 21, 2024, with the deposit number of CGMCC No.41312.

[0014] In some embodiments, the Fusarium compactum MM-135 may be in a spore form and / or a mycelium form.

[0015] In some embodiments, the mycelium comprises greater than about 45 wt % protein based on the dry weight of the mycelium.

[0016] 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 more of protein based on the dry weight of the mycelium.

[0017] In some embodiments, the mycelium comprises mycelial protein at a biomass concentration of about 0.4 wt% 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 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 the dry weight of the mycelium.

[0018] In some embodiments, the microbial agent includes the Fusarium MM-135 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.

[0019] 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 first fermentation liquid; and, 3) obtaining mycelium from the fermentation liquid.

[0020] In some embodiments, the seed medium and / or fermentation medium may include a carbon source and a nitrogen source.

[0021] 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.

[0022] 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 salts (e.g., ammonium sulfate, ammonium phosphate).

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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 an inorganic salt.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In some embodiments, in step 2), the culture rotation speed may be about 100 to 300 rpm.

[0047] 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.

[0048] In some embodiments, the fermentation medium may further include a vitamin complex. In some embodiments, the fermentation medium may further include 0.001 to 0.2 parts by weight of a vitamin complex. In some embodiments, the fermentation medium may further 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.

[0049] 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.

[0050] In some embodiments, the fermentation medium may further include 0.01 to 10 parts by weight of yeast powder. In some embodiments, the fermentation medium may further 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 yeast powder.

[0051] 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.

[0052] In some embodiments, in step 2), the culture pressure is about 0.005 MPa to 0.1 MPa. In some embodiments, in step 2), the culture pressure 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 or 0.1 MPa.

[0053] 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.

[0054] In some embodiments, in step 2), the pH of the fermentation medium may be 5.0 to 7.0. In some embodiments, in step 2), the pH of the fermentation medium may 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.

[0055] In some embodiments, in step 2), the culturing time may be 10 to 30 hours, preferably 16 to 20 hours.

[0056] 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 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 and an aeration rate of 1300 to 2000 h / L to obtain a second fermentation broth.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] In some embodiments, the feed medium may include the above-mentioned carbon source and / or vitamin complex.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In some embodiments, the MM-135 mycelium can be used to prepare a meat substitute.

[0065] According to yet another aspect of the present disclosure, provided is the MM-135 mycelium obtained by the above method of the present disclosure.

[0066] According to yet another aspect of the present disclosure, a composition is provided, comprising the MM-135 mycelial protein and / or protein-enriched MM-135 mycelium of the present disclosure.

[0067] 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-enriched mycelium based on a wet basis of MM-135 mycelial protein and / or protein-enriched MM-135 mycelium.

[0068] In some embodiments, the composition comprises, based on the wet basis of MM-135 mycelial protein and / or protein-rich MM-135 mycelium, 30 to 60 parts by weight of MM-135 mycelial protein and / or protein-rich MM-135 mycelium; and, 5 to 20 parts by weight of powder coating. In some embodiments, the composition comprises 30, 35, 40, 45, 50, 55, 60 parts by weight of MM-135 mycelial protein and / or protein-rich MM-135 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 powder coating.

[0069] In some embodiments, the mycelial protein and / or protein-enriched mycelium may also be provided in the form of dry matter.

[0070] 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 proportion, 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.

[0071] In some embodiments, the composition may further include one or more commonly used food additives or food supplements.

[0072] According to another aspect of the present disclosure, the MM-135 mycelial protein and / or the protein-rich MM-135 mycelium of the present disclosure, or the use of the above composition in preparing a meat substitute is provided. In some embodiments, the MM-135 mycelial protein and / or the protein-rich MM-135 mycelium of the present disclosure, or the above composition can replace chicken.

[0073] The present invention's research found that the reason why existing fungal protein vegetarian meat is difficult to completely replace plant protein vegetarian meat is that general fungal proteins such as yeast protein have poor organization and emulsification properties, and the vegetarian meat prepared as the main protein ingredient alone has poor texture and taste. Strongly organized plant proteins such as bean protein are required as the main protein ingredient or auxiliary protein raw material to improve the texture and taste of vegetarian meat containing fungal protein. Therefore, general fungal protein vegetarian meat is difficult to completely replace plant protein.

[0074] The mycelium protein-based meat substitute food provided by the present invention uses mycelium protein prepared by fermentation mycelium of Fusarium compactum MM-135 as a protein raw material. Since the mycelium protein has good organization and emulsification properties, it can be used alone as a protein raw material to prepare meat substitute food, without the need to be combined with plant protein, avoiding the problems of peculiar smell, complex process and low nutrient content caused by the use of plant protein raw materials. In summary, the mycelium protein-based meat substitute food of the present invention can completely replace the plant protein vegetarian sausage, avoiding the defects caused by plant protein, and at the same time having excellent taste and texture, the taste is juicy, elastic and moderate hardness, the texture is good hardness, elasticity and chewiness, closer to real meat food, and compared with plant protein vegetarian meat or pork and chicken, the mycelium protein-based meat substitute food has high protein content and natural dietary fiber content, low fat content, no cholesterol, and is more nutritious and healthy. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 The phylogenetic tree analysis results of the MM-135 strain are shown.

[0076] FIG. 2 shows the bacterial cake of MM-135 strain ( Figure 2A ) and hyphae ( Figure 2B ) images.

[0077] Figure 3 Shown is a growth curve graph of the MM-135 strain. DETAILED DESCRIPTION

[0078] In the field of fungal mycelial protein, natural high-protein-producing Fusarium 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 compactum.

[0079] In some embodiments, the present disclosure provides Fusarium compactum strain MM-135.

[0080] In some embodiments, the present disclosure provides a bacterial agent comprising Fusarium compactum strain MM-135.

[0081] In some embodiments, the TEF1 of the strain MM-135 has a nucleotide sequence as shown in SEQ ID NO:9.

[0082] In some embodiments, the internal transcribed spacer (ITS) of the strain MM-135 has a nucleotide sequence as shown in SEQ ID NO:10.

[0083] In some embodiments, the RPB1 of the strain MM-135 has a nucleotide sequence as shown in SEQ ID NO:11.

[0084] In some embodiments, the RPB2 of strain MM-135 has a nucleotide sequence as shown in SEQ ID NO:12.

[0085] 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.

[0086] It is known that the production of spores will affect the texture and shape of the product. The present disclosure achieves control of spore production to a great extent by improving the fermentation process. In a specific embodiment, no spores are observed in the mycelial protein produced by Fusarium compactum of the present disclosure under microscopic examination. In a specific embodiment, the mycelial protein produced by Fusarium compactum MM-135 of the present disclosure has excellent filaments, the filter cake is light yellow, and has good elasticity.

[0087] In a specific embodiment, the mycelial protein obtained by fermentation culture of Fusarium compactum MM-135 disclosed in the present invention has good filaments under microscopic examination, and the fermentation liquid can naturally present a flesh-colored color without heating. The protein content of the mycelial protein produced by Fusarium compactum MM-135 disclosed in the present invention is higher than 40%, and even reaches more than 55%.

[0088] Usually, strains can only utilize organic nitrogen sources, but not inorganic nitrogen sources. In a specific embodiment, the present disclosure can enable the Fusarium compactum MM-135 of the present disclosure to utilize inorganic nitrogen sources by adding trace elements, while improving the shape and color texture of the mycelium.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, 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 of magnesium sulfate heptahydrate.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] In some embodiments, the fermentation medium may further include 0.01 g / L to 10 g / L of yeast powder. In some embodiments, the fermentation medium may further 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, 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.

[0109] In a specific embodiment, the feed medium may include 100 g / L to 500 g / L of corn starch saccharification liquid and / or 0.01 to 2 mg / L of vitamin complex by weight.

[0110] In a specific embodiment, the feed medium may include 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 of corn starch saccharification liquid.

[0111] In a specific embodiment, the feed medium may 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.

[0112] In a specific embodiment, the meat substitute (e.g., meat chunks or meat emulsion) of the present disclosure may include one or more of alginate, arabinoxylan, carrageenan, carboxymethyl cellulose, cellulose, curdlan, gelatin, gellan gum, dextran, galactomannan, guar gum, locust bean gum, tara gum, gum arabic, pectin, konjac gum, starch and xanthan gum.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] The MM-135 mycelial protein produced by the Fusarium MM-135 or its bacterial agent disclosed in the present invention can be used to prepare meat substitutes.

[0117] 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.

[0118] definition

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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 scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention. The reagents and / or kits used in the following examples are all commercially available or can be synthesized by known methods.

[0123] It should be noted that, if the specific conditions are not specified in the examples, the experimental conditions are carried out according to conventional conditions, manufacturer recommendations or publicly reported experimental conditions. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially. If the manufacturer of the reagents used is specified, similar products from other manufacturers are substitutable.

[0124] Example

[0125] Example 1: Screening of Fusarium compactum MM-135 strain

[0126] 1.1 Strain source

[0127] The MM-135 strain was isolated from humus soil samples in Shennongjia, Hubei.

[0128] 1.2 Separation process

[0129] 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 to 7 days after the surface of the plates dried.

[0130] Among them, the specific composition of the MGA2.5 flat panel is shown in the following table;

[0131] Peptone 15g <![CDATA[KH2PO4]]> 1.0g <![CDATA[MgSO4·7H2O]]> 0.5g Malachite Green Oxalate 0.0025g Agar 20g Distilled water 1L

[0132] Purification was performed by the hyphae end step-by-step transplantation method. After the colonies were formed on the plate, the hyphae at the edge of the 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.

[0133] Example 2. Identification of Fusarium compactum MM-135 strain

[0134] 2.1 Preparation of DNA template

[0135] 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.

[0136] 2.2 PCR system preparation

[0137] According to the table below, prepare 25 μL of PCR system in a PCR tube:

[0138]

[0139]

[0140] The genes and primers amplified are as follows:

[0141]

[0142] The above primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0143] 2.3 PCR amplification

[0144] 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.

[0145]

[0146] The sequencing results of strain MM-135 are shown in the following table.

[0147]

[0148]

[0149]

[0150] 2.4 Species identification analysis based on Polyphasic identification

[0151] The strain MM-135 was identified by the Polyphasic identification method provided by the mycobank (fusarium.mycobank.org) online database. The results showed that based on TEF1, RPB2, and ITS genes, the closest species to the MM-135 strain was Fusarium compactum NRRL 36318, with a global similarity of 99.65%.

[0152] Therefore, strain MM-135 was identified as Fusarium compactum.

[0153] 2.5 Species identification analysis based on multi-gene evolutionary trees

[0154] The species identification analysis method based on polyphasic identification was used. Since the species complex to which the identified species belonged was 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 the FUSARIOID-ID database (www.fusarium.org), and the evolutionary position of the current strain was analyzed by constructing a multi-gene evolutionary tree.

[0155] 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.

[0156] like Figure 1 As shown, the MM-135 strain belongs to the species Fusarium compactum.

[0157] Strain MM-135 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on May 21, 2024, with the deposit number CGMCC No.41312.

[0158] Example 3. Preparation of mycelial protein by fermentation

[0159] The activated Fusarium compactum strain MM-135 was inoculated into a 250 mL three-concave conical flask containing 50 mL of the primary screening medium and cultured at 200 rpm for 24 h.

[0160] The formula of the primary screening culture medium is: glucose 30g / L, yeast extract powder 30g / L, KH2PO4 1g / L, MgSO4·7H2O0.5g / L, CaCl2 0.2g / L, sterilized by high-pressure steam at 121℃ for 20min.

[0161] 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.

[0162] Observation and test results of mycelial morphology, filter cake color and texture, biomass and crude protein of the strain.

[0163] strains Mycelium morphology Filter cake color Filter cake texture Biomass wt% Crude protein wt% MM-135 Cottonseed Light yellow No elasticity 1.62 59.17

[0164] The biomass concentration of MM-135 reached 1.62 wt% mycelial protein, and the crude protein content of mycelium was further determined by Kjeldahl method to be 59.17 wt%.

[0165] Example 4. Scale-up of culture to produce mycelial protein

[0166] 4.1 Fermentation tank rescreening culture method

[0167] The composition of the seed culture medium used is shown in the following table:

[0168] Formula Materials g / L glucose 30 Yeast extract powder 25 Potassium dihydrogen phosphate 1 Magnesium sulfate heptahydrate 0.2 Calcium chloride 0.01

[0169] Seed liquid culture conditions: The first-level seed liquid was prepared using the above seed culture medium, and the activated Fusarium compactum strain MM-135 was picked and inoculated into a 250mL three-concave conical flask containing 50mL seed culture medium, and cultured at 200rpm for 24h. The second-level seed liquid was prepared using the above seed culture medium, and transferred to a 3L three-concave conical flask containing 1L seed culture medium at a 5% inoculation amount, with a rotation speed of 200rpm and cultured for 24h to 30h. It was observed that the culture time of the seed culture medium affects the color of the filter cake formed by the mycelial protein; the culture time of the seed culture medium can be controlled to control the color and texture of the filter cake obtained by fermentation.

[0170] Then, the inoculation amount is 3% and inoculated into the rescreening medium. The components of the rescreening medium for producing mycelial protein are as follows:

[0171] Element Fermentation medium Corn starch saccharification liquid 25g / L Citric acid 1g / L Potassium dihydrogen phosphate 1g / L Ammonium sulfate 5g / L Magnesium sulfate heptahydrate 0.2g / L Calcium chloride 0.01g / L Manganese sulfate monohydrate 1.7mg / L Ferrous Sulfate Heptahydrate 5mg / L Zinc chloride 1.7mg / L

[0172] Fermentation process conditions: fermenter volume 15L, liquid volume 10L, sterilization at 121℃ for 30min. Temperature 28℃, ventilation 5-15L / h, tank pressure 0.05-0.10Mpa, rotation speed 100-400rpm, dissolved oxygen 20-50%, pH 6.0 controlled by ammonia water. The shape of mycelium has a great influence on the taste of the prepared product (such as meat substitute), and cottonseed-shaped mycelium has a poor taste. This embodiment uses inorganic nitrogen to replace organic nitrogen, thereby reducing costs while optimizing the filamentous structure and reducing the balling phenomenon.

[0173] The same method as in Example 3 was used to test the color, texture, smell, mycelium morphology, mycelium length, mycelium diameter, mycelium branching, spore number and crude protein content of the filter cake obtained by the rescreening of Fusarium compactum strain MM-135. The results showed that the granular and cottonseed shapes in the initial screening were effectively improved, the mycelium showed a slender filamentous structure, and the prepared filter cake had an excellent texture and could better simulate the chewing feeling of meat (Figure 2). The crude protein content of MM-135 mycelium was determined by the Kjeldahl method to reach 55.29wt%.

[0174] The results of the rescreening of strain MM-135 are shown in the table below.

[0175] Results of strain rescreening MM-135 Fermentation time 30h Filter cake color White Filter cake texture Excellent toughness Filter cake odor none Mycelium morphology Filamentous Mycelium length long Mycelium diameter thin Mycelium branching few Spore count none Crude Protein 55.29wt%

[0176] Compared with the primary screening medium in Example 3, 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 strain Fusarium compactum of the present invention can simultaneously use organic nitrogen sources and inorganic nitrogen sources as nitrogen sources for the fermentation culture of fungal protein.

[0177] FIG. 2 shows the bacterial cake of MM-135 strain ( Figure 2A ) and hyphae ( Figure 2B ) images.

[0178] 4.2 Fermentation process corresponding to fermentation medium:

[0179] 4.2.1 Seed liquid culture

[0180] A 3L three-concave conical flask was used, PDB medium (potato extract powder 300g / L, glucose 5g / L, purchased from Guangdong Huankai Microbiological Technology Co., Ltd.); inoculation volume 5%, liquid volume 1L; shaker speed 200rpm; culture for 24h.

[0181] 4.2.2 Primary fermentation

[0182] The composition of the culture medium for primary fermentation is shown in the following table.

[0183] Element Primary fermentation medium Corn starch saccharification liquid 25g / L Citric acid 1g / L Potassium dihydrogen phosphate 1g / L Ammonium sulfate 5g / L Magnesium sulfate heptahydrate 0.2g / L Calcium chloride 0.01g / L Manganese sulfate monohydrate 1.7mg / L Ferrous Sulfate Heptahydrate 5mg / L Zinc chloride 1.7mg / L Vitamin complex liquid 0.1mg / L

[0184] Subculture: Use a 15L tank with 10L liquid, sterilize at 121℃ for 30min, transfer all the seed liquid by flame inoculation; rotation speed 100-300rpm; ventilation 10L / min, tank pressure 0.05mpa; pH 6.0 controlled by ammonia water; culture at 28℃ for 16-20h. Ammonium sulfate in the primary culture medium can be replaced by ammonium phosphate.

[0185] 4.3.3 Secondary fermentation

[0186] The composition of the secondary fermentation medium is shown in the following table.

[0187] Element Secondary fermentation medium Corn starch saccharification liquid 25g / L Citric acid 1g / L Potassium dihydrogen phosphate 1g / L Ammonium sulfate 5g / L Magnesium sulfate heptahydrate 0.2g / L Calcium chloride 0.01g / L Manganese sulfate monohydrate 1.7mg / L Ferrous Sulfate Heptahydrate 5mg / L Zinc chloride 1.7mg / L Vitamin complex liquid 0.1mg / L

[0188] Feed medium

[0189]

[0190]

[0191] The formula of the secondary fermentation medium is shown in the table above (ammonium sulfate can be replaced by ammonium phosphate). Use a 50L tank with a liquid volume of 30L and sterilize at 121°C for 30min; add 0.1mg / L vitamin mixture after sterilization (filter sterilization); at an inoculation rate of 5-10%, pump the seed liquid cultured in the 15L tank through the sterilized hose from the bottom valve to complete the inoculation; ventilate 1000L / h for the first 15h, and the tank pressure is 0.05mpa; after 15h, start feeding at 0.5-2g / L / h, and adjust the ventilation to 1500L / h and the tank pressure to 0.1mpa; control the pH to 4.0-6.0 with ammonia water; culture at 28°C; rotate at 100-300rpm coupled with dissolved oxygen 20%-50%, and culture for 24-48h. The complex vitamins include B complex vitamins, and the complex vitamins include 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 can improve the dissolved oxygen content and dissolved oxygen efficiency, and promote the growth of mycelium without destroying the mycelium.

[0192] During the fermentation and subculture process, the strain grew well, and the growth curve of the strain is as shown in Figure 3 shown.

[0193] Example 5. Optimization of Fusarium compactum MM-135 culture medium during production scale-up

[0194] This example studies the effects of the components and content of the fermentation medium in the production scale-up process on the fermentation mycelium and protein yield. During the scale-up process, the strain inoculation amount is large and the conversion efficiency is low.

[0195] The same method as in Example 3 was used to test the color, texture, odor, hyphae morphology, hyphae length, hyphae diameter, hyphae branching, spore count and crude protein content of the filter cake obtained from Fusarium compactum strain MM-135. The results showed that:

[0196] When ammonium sulfate is not present in the culture medium, the balling phenomenon is alleviated;

[0197] The presence of an appropriate amount of phosphate in the culture medium will increase the biomass in the product. For example, when the phosphate concentration increases from 0.2 g / L to 1 g / L, the biomass in the product increases by 20% to 40%;

[0198] Adding Zn to the culture medium 2+ The concentration of trace element solution can increase the protein content.

[0199] Example 6. Preparation of meat chunks using Fusarium compactum MM-135 mycelial protein

[0200] Meat chunks were prepared using mycelial protein prepared in Example 4, including the following raw materials: mycelial protein of Fusarium compactum strain MM-135, water, egg white powder, starch, and other seasonings (such as monosodium glutamate, yeast extract, white sugar, food flavors, soybean oil, edible salt, spices, 5'-flavor ribonucleotide disodium, gluten, etc.).

[0201] During the preparation of the meat, methylcellulose or sodium alginate, anhydrous calcium chloride, and calcium acetate may be further added to the above raw materials to adjust the viscosity of the meat. The steps for preparing the meat are as follows:

[0202] 1. Mix egg white powder and starch to obtain a coating powder, and divide it into a first portion of coating powder and a second portion of coating powder at a mass ratio of 75:25; mix the first portion of coating powder with ice water, stir evenly to obtain a slurry, and set aside;

[0203] 2. Mix the second portion of the coating powder with the mycelium protein and stir evenly, then add seasoning and ice water, stir evenly to obtain vegetarian meat filling;

[0204] 3. Press the vegetarian meat filling into rectangular pieces, 20g per piece, steam at 85℃ for 30min to set, and cool to room temperature;

[0205] 4. Coat the meat with the slurry from step 1, fry at 160°C for 30 seconds, and cool to room temperature;

[0206] 5. Packaging, quick freezing and storage.

[0207] The evaluation was conducted using (1) sensory evaluation and (2) texture analyzer.

[0208] It can be concluded that the mycelium protein-based meat chunks of the present invention have excellent juiciness, elasticity, hardness, and good taste, and can completely replace meat chunks made of animal meat and plant protein.

[0209] (2) Texture analyzer to measure texture structure

[0210] The meat blocks prepared from mycelial protein and commercially available chicken blocks were cut into 1 cm × 1 cm × 1 cm columns, 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 1 mm / s, the deformation (compression distance / sample height) was 40%, the trigger type was automatic, the trigger force was 5.0 g, and the time interval between two compressions was 5 s.

[0211] 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.

[0212] Cohesion: Cohesion reflects the property of food that resists damage and connects tightly when chewing, keeping the food intact.

[0213] 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.

[0214] The table below shows the texture analyzer test results of mycelial protein based meat nuggets:

[0215]

[0216] It can be concluded from the above table that mycelial protein based meat nuggets are close to commercially available chicken nuggets.

[0217] The organization and emulsification of mycelial protein can be further enhanced, thereby improving the texture and taste of meat substitute foods; that is, the obtained mycelial protein-based meat substitute foods can completely replace plant protein vegetarian meat products, avoiding the defects brought by plant protein, while having excellent taste and texture, with a juicy, elastic and moderate hardness taste, and a texture with good hardness, elasticity and chewiness, which is closer to real meat food. Compared with plant protein vegetarian meat or pork and chicken, the mycelial protein-based meat substitute foods have high protein content and natural dietary fiber content, low fat content, and do not contain cholesterol, and are more nutritious and healthy.

[0218] 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 compactum MM-135 or its bacterial agent in producing mycelial protein and / or protein-rich mycelium.

2. The use according to claim 1, characterized in that The TEF1 of Fusarium compactum MM-135 has the nucleotide sequence shown in SEQ ID NO: 9; and / or The ITS of Fusarium compactum MM-135 has the nucleotide sequence shown in SEQ ID NO: 10; The RPB1 of Fusarium compactum MM-135 has the nucleotide sequence shown in SEQ ID NO: 11; and / or The RPB2 of Fusarium compactum MM-135 has a nucleotide sequence as shown in SEQ ID NO:

12.

3. The use according to claim 1 or 2, characterized in that The length of the hyphae protein or mycelium of Fusarium compactum MM-135 is less than 2000 μm, and / or the aspect ratio is less than 800; Preferably, the MM-135 strain was deposited in the China General Microbiology Center (CGMCC) on May 21, 2024, with the deposit number CGMCC No.41312.

4. The use according to claim 1 or 2, characterized in that The Fusarium compactum MM-135 is in spore form and / or mycelium form, and / or The mycelium comprises more than about 45 wt % protein based on the dry weight of the mycelium, and / or The mycelium includes mycelial protein at a biomass concentration of about 0.4 wt % or more based on the dry weight of the mycelium.

5. A method for producing mycelium of Fusarium compactum MM-135, characterized in that: The method comprises: 1) inoculating Fusarium compactum MM-135 or its bacterial agent into a seed culture medium for cultivation to obtain a seed solution; 2) inoculating the seed liquid into a fermentation medium for culturing to obtain a first fermentation liquid; and 3) obtaining mycelium from the fermentation broth.

6. The method according to claim 5, 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 carbon source comprises 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 distiller's grains, brewery waste, carbon refined syrup, desalted syrup, enzyme-converted syrup and / or a combination thereof; Preferably, the nitrogen source in the seed culture medium and / or fermentation medium is an inorganic nitrogen source and / or an organic nitrogen source. Preferably, the organic nitrogen source comprises peptone, yeast extract powder, soybean cake powder and / or a combination thereof. Preferably, the inorganic nitrogen source includes urea and / or ammonium compounds, more preferably ammonium sulfate.

7. The method according to claim 6, characterized in that In step 2), The culture temperature is 20°C to 35°C, and / or The culture speed is 100-300 rpm, and / or The pH of the fermentation medium is 5.0-7.

0.

8. A mycelium of Fusarium compactum MM-135 obtained by the method according to any one of claims 5 to 7.

9. A composition or food comprising mycelial protein and / or protein-enriched mycelium of Fusarium compactum MM-135.

10. Use of mycelial protein of Fusarium compactum MM-135 and / or protein-rich mycelium of Fusarium compactum MM-135 or the composition according to claim 9 in the preparation or as a meat substitute.

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