Fusarium compactum and uses thereof

Abstract

The present disclosure relates to Fusarium compactum and its applications. Specifically, the present disclosure provides Fusarium compactum or its inoculum, mycelial protein derived from the Fusarium compactum or its inoculum and / or protein-enriched mycelium, compositions containing the mycelial protein and / or protein-enriched mycelium, and applications thereof.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of microbiology, in particular to a Fusarium compactum for producing mycelial protein, mycelial protein produced by the Fusarium compactum, and application thereof. BACKGROUND

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

[0003] Therefore, it is of great commercial value to find more potential and naturally high-yield filamentous fungal protein-producing strains. SUMMARY

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

[0005] According to one aspect of the present disclosure, there is provided an application of Fusarium compactum MM-135 or a microbial agent thereof in producing mycelial protein and / or protein-rich mycelium.

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

[0007] According to still another aspect of the present disclosure, there is provided a mycelial protein and / or protein-rich mycelium derived from the above-mentioned Fusarium compactum MM-135 or a microbial agent thereof of the present disclosure.

[0008] In some embodiments, the translation elongation factor 1 (TEF1) of the 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 the 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 the Fusarium compactum MM-135 has a nucleotide sequence as set forth in SEQ ID NO: 11.

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

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

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

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

[0015] In some embodiments, the mycelium comprises about 45 wt% or more of 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 59 wt% or more of protein, based on the dry weight of the mycelium.

[0017] In some embodiments, the mycelium comprises a biomass concentration of about 0.4 wt% or more of mycelium protein, based on the dry weight of the mycelium. In some embodiments, the mycelium comprises 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 of protein, based on the dry weight of the mycelium.

[0018] In some embodiments, the bacterial agent includes the Fusarium MM-135 and a bacterial agent acceptable adjuvant. Those skilled in the art will understand that the bacterial agent acceptable adjuvant and the amount thereof can be routinely selected as desired and are not particularly limited in the present disclosure.

[0019] According to another aspect of the present disclosure, there is provided a method for producing mycelium, the method comprising: 1) inoculating the Fusarium of the present disclosure or a bacterial agent thereof into a seed culture medium to culture, to obtain a seed liquid; 2) inoculating the seed liquid into a fermentation culture medium to culture, to obtain a first fermentation liquid; and, 3) obtaining mycelium from the fermentation liquid.

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

[0021] In some embodiments, the carbon source can include sugars (e.g., sucrose, maltose, glucose, fructose, rare sugar of Japan, etc.), sugar alcohols (e.g., glycerol, polyol, etc.), starches (e.g., corn starch, etc.), starch derivatives (e.g., maltodextrin, cyclodextrin, glucose syrup, hydrolyzate, and modified starch), starch hydrolyzates (e.g., corn starch saccharification liquid, rice starch saccharification liquid, sweet potato starch saccharification liquid, molasses, etc.), hydrogenated starch hydrolyzates (HSH; e.g., hydrogenated glucose syrup, maltitol syrup, sorbitol syrup, etc.), lignocellulosic pulp or raw material (e.g., sugar beet pulp, agricultural pulp, wood pulp, distiller’s dried grain, brewery waste, etc.), potato infusion powder, corn pulp, acid whey, sweet whey, whey, wheat infusion liquid, carbohydrates, food waste, olive oil processing waste, hydrolyzate of lignocellulosic material, corn wet milling product (e.g., carbon refined sugar syrup, desalted sugar syrup, enzyme conversion sugar syrup, etc.), and / or combinations thereof.

[0022] In some embodiments, the nitrogen source in the seed culture medium and / or the fermentation culture medium can be an inorganic nitrogen source and / or an organic nitrogen source. In some embodiments, the organic nitrogen source can include, but is not limited to, peptone, yeast infusion powder, yeast hydrolysate, yeast extract, soybean meal, etc. In some embodiments, the inorganic nitrogen source can include, but is not limited to, urea or ammonium salt (e.g., ammonium sulfate, ammonium phosphate).

[0023] In some embodiments, in addition to the nitrogen source and the carbon source, the seed culture medium and / or the fermentation culture medium can further include inorganic salts, such as, for example, sodium salts, potassium salts, calcium salts, magnesium salts, manganese salts, iron salts, zinc salts, etc. In some embodiments, the culture medium can 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 medium can 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 medium can 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 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 nitrogen source. In some embodiments, the seed 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 an inorganic salt.

[0025] In a specific embodiment, the seed medium can include 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.

[0026] In a specific embodiment, the seed medium can include 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 parts by weight of glucose.

[0027] In a specific embodiment, the seed medium can include 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 parts by weight of yeast extract powder.

[0028] In a specific embodiment, the seed 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.

[0029] In a specific embodiment, the seed 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 magnesium sulfate heptahydrate.

[0030] In specific embodiments, the seed 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 parts by weight of calcium chloride.

[0031] In some embodiments, the fermentation medium can 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 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 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 can include 20 to 35 parts by weight of a corn starch mashing 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.

[0035] In specific 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 corn starch mashing 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 particular 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 particular 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 ammonium sulfate.

[0039] In particular 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, 0.15, 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 particular 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 can include 0.001-0.1 parts by weight of trace elements. In some embodiments, the trace elements include 0.5-2.5 parts by weight of manganese sulfate monohydrate, 1-10 parts by weight of ferrous sulfate heptahydrate, and 0.5-2.5 parts by weight of zinc chloride.

[0042] In particular embodiments, the trace elements 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 manganese sulfate monohydrate.

[0043] In specific embodiments, the microelement can 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, the temperature of the culture in step 2) can be about 20-35°C. In some embodiments, the temperature of the culture in step 2) can 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, the rotation speed of the culture in step 2) can be about 100-300 rpm.

[0047] In some embodiments, the pH of the fermentation medium in step 2) can be about 5.0-7.0. In some embodiments, the pH of the culture in step 2) can be 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 can further include a vitamin compound. In some embodiments, the fermentation medium can further include 0.001-0.2 parts by weight of the vitamin compound. In some embodiments, the fermentation medium can 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 the vitamin compound.

[0049] In some embodiments, the vitamin complex can include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, or any combination thereof. In specific embodiments, the vitamin complex can include one or more of vitamin Bl (thiamine), vitamin B 12 (cobalamin), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (D-calcium pantothenate), vitamin B6 (pyridoxine), vitamin B9 (folic acid and / or folate), vitamin C, and vitamin H (biotin). In specific embodiments, the components of the vitamin complex are mixed in equal mass parts. In these embodiments, no spores are produced during the fermentation process.

[0050] In some embodiments, the fermentation medium can further include 0.01 to 10 parts by weight of yeast powder. In some embodiments, the fermentation medium can 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, the culture in step 2) is aerated at a rate of about 1 L / min to about 20 L / min. In some embodiments, the culture in step 2) is aerated at a rate of 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, the culture in step 2) is aerated at a pressure of about 0.005 mpa to about 0.1 mpa. In some embodiments, the culture in step 2) is aerated at a pressure of 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, the temperature of the culture in step 2) can be about 20-35°C. In some embodiments, the temperature of the culture in step 2) can 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, the pH of the fermentation medium in step 2) can be 5.0-7.0. In some embodiments, the pH of the fermentation medium in step 2) can be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0.

[0055] In some embodiments, the time of the culture in step 2) can be 10-30h, preferably 16-20h.

[0056] In some embodiments, the method can further comprise the following steps between step 2) and step 3): a) inoculating the first fermentation broth into a fermentation medium, culturing at 0.01-0.1 mPa, aeration amount of 800-1200 L / h for 5-20h; and, b) adding a feed medium, culturing at 0.1-0.5 mPa, aeration amount of 1300-2000 h / L for 24-48h to obtain a second fermentation broth.

[0057] In some embodiments, the aeration amount 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 aeration amount 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, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h or 20h.

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

[0061] In some embodiments, the feed medium can comprise 10000-50000 parts by weight of corn starch saccharification liquid and / or 0.001-0.2 parts by weight of vitamin complex.

[0062] In some embodiments, the feed medium can include 20000-40000 parts by weight of corn starch liquefied solution. In some embodiments, the feed medium can include 20000, 25000, 30000, 35000, 40000, 45000 or 50000 parts by weight of corn starch liquefied solution.

[0063] In some embodiments, the feed medium can include 0.001-0.2 parts by weight of vitamin complex. In some embodiments, the feed 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 parts by weight of 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, there is provided an MM-135 mycelium obtained from the above method of the present disclosure.

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

[0067] In some embodiments, the composition comprises 15-95 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 the wet basis MM-135 mycelial protein and / or protein-enriched MM-135 mycelium.

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

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

[0070] In some embodiments, the breading powder can include breading powder conventionally used in the art, such as egg white powder, starch, etc. In some embodiments, the breading powder can include egg white powder and starch in appropriate proportions, for example, the mass ratio of egg white powder to starch can be 1:(0.1-2). In some embodiments, the mass ratio of egg white powder to starch in the breading powder can 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 can further include one or more of commonly used food additives or food adjuvants.

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

[0073] The present disclosure found that the reason why existing mycoprotein meat is difficult to completely replace plant protein meat is that general mycoprotein such as yeast protein has poor texturization and emulsification, and the texture and taste of the meat substitute prepared by using mycoprotein as the main protein material are poor, and thus plant protein such as legume protein with strong texturization is needed as the main protein material or auxiliary protein material to improve the texture and taste of the meat substitute containing mycoprotein, and thus it is difficult for general mycoprotein meat to completely replace plant protein.

[0074] The mycelial protein-based meat substitute food provided by the present application adopts mycelial protein prepared from the fermented mycelium of Fusarium compactum MM-135 as a protein raw material. Due to the good texturization and emulsification properties of the mycelial protein, it can be used as a protein raw material alone to prepare meat substitute food, without the need to be combined with plant protein, thereby avoiding the problems of odor, complex process and low content of nutritional components caused by the use of plant protein raw materials. In summary, the mycelial protein-based meat substitute food of the present application can completely replace plant protein sausages, avoiding the defects caused by plant protein, while having excellent taste and texture, with a juicy, elastic and moderate hardness taste, a good hardness, elasticity and chewiness texture, which is closer to real meat food. Compared with plant protein meat or pork and chicken, the mycelial protein-based meat substitute food has high protein and natural dietary fiber contents, low fat content and no cholesterol, and is more nutritious and healthy. BRIEF DESCRIPTION OF DRAWINGS

[0075] Figure 1 A phylogenetic tree analysis result of the MM-135 strain is shown.

[0076] Figure 2 shows images of MM-135 strain's fungus cake ( Figure 2A ) and mycelium ( Figure 2B ).

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

[0078] In the field of fungal mycelial protein, naturally high-yield protein-producing fusarium is extremely rare, and naturally high-yield protein-producing fusarium strains are also very rare. The present disclosure develops a new naturally high-yield filamentous fungal protein-producing Fusarium compactum.

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

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

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

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

[0085] In specific embodiments, the present disclosure screens additives in the culture medium and finds that adding a complex vitamin solution in the culture medium can improve the quality of the fungus cake.

[0086] It is known that the production of spores will affect the texture and shape of the product, and the present disclosure greatly achieves the control of spore production by improving the fermentation process. In specific embodiments, the mycelial protein produced by the Fusarium compactum of the present disclosure has no spores observed under a microscope. In specific embodiments, the mycelial protein produced by the Fusarium compactum MM-135 of the present disclosure has excellent filamentous shape, light yellow filter cake, and good elasticity.

[0087] In specific embodiments, the mycelial protein produced by the Fusarium compactum MM-135 of the present disclosure obtained by fermentation culture has good filamentous shape under a microscope, and the fermentation broth can naturally present a meat color without heating. The protein content of the mycelial protein produced by the Fusarium compactum MM-135 of the present disclosure is higher than 40%, and even reaches more than 55%.

[0088] Generally, strains can only utilize organic nitrogen sources, and cannot utilize inorganic nitrogen sources. In specific embodiments, the present disclosure can make the Fusarium compactum MM-135 of the present disclosure utilize inorganic nitrogen sources by adding trace elements. Meanwhile, the shape and color texture of the mycelium are improved.

[0089] The present disclosure provides a method for producing mycelial protein, which comprises: 1) inoculating the Fusarium of the present disclosure or its inoculant into a seed culture medium for cultivation to obtain a seed liquid; 2) inoculating the seed liquid into a fermentation culture medium for cultivation to obtain a fermentation broth; and 3) obtaining mycelial protein from the fermentation broth.

[0090] In specific embodiments, the seed culture medium can comprise 25-35 g / L of glucose, 20-35 g / L of yeast extract powder, 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 particular embodiments, the seed medium can 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 particular embodiments, the seed medium can include 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, or 35 g / L of yeast extract.

[0093] In particular embodiments, the seed medium can 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 particular embodiments, the seed medium can 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 particular embodiments, the seed medium can include 0.005 g / L, 0.006 g / L, 0.007 g / L, 0.008 g / L, 0.009 g / L, 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1 g / L of calcium chloride.

[0096] In some embodiments, the fermentation medium can include 20-35 g / L of corn starch mashing, 0.1-2 g / L of citric acid, 0.1-2 g / L of potassium dihydrogen phosphate, 0.01-10 g / L of ammonium sulfate, 0.01-2 g / L of magnesium sulfate heptahydrate, 0.001-1 g / L of calcium chloride, 0.5-2.5 mg / L of manganese sulfate monohydrate, 1-10 mg / L of ferrous sulfate heptahydrate, and 0.5-2.5 mg / L of zinc chloride.

[0097] In specific embodiments, the fermentation medium can include 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, or 35 g / L of corn starch mashing.

[0098] In specific embodiments, the fermentation medium can 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 specific embodiments, the fermentation medium can 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 specific embodiments, the fermentation medium can 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, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, or 10 g / L of ammonium sulfate.

[0101] In particular embodiments, the fermentation medium can 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 particular embodiments, the fermentation medium can 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 particular embodiments, the fermentation medium can 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 particular embodiments, the fermentation medium can 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 specific embodiments, the fermentation medium can 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 can further include a vitamin complex. In some embodiments, the fermentation medium can further include 0.01-2 mg / L of the vitamin complex. In some embodiments, the fermentation medium can further 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 the vitamin complex.

[0107] In some embodiments, the vitamin complex can include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, or any combination thereof. In specific embodiments, the vitamin complex can include one or more of vitamin Bl (thiamine), vitamin B12 (cobalamin), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (D-calcium pantothenate), vitamin B6 (pyridoxine), vitamin B9 (folic acid and / or folate), vitamin C, and vitamin H (biotin). In specific embodiments, the components in the vitamin complex are mixed in equal mass parts.

[0108] In some embodiments, the fermentation medium can further include 0.01 g / L to 10 g / L of yeast powder. In some embodiments, the fermentation medium can 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, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, or 10 g / L of yeast powder.

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

[0110] In specific embodiments, the feed medium can 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 liquefact.

[0111] In specific embodiments, the feed medium can 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 a vitamin complex.

[0112] In specific embodiments, the meat substitute (e.g., a meat chunk or a meat emulsion) of the present disclosure can comprise one or more of alginates, arabinoxylan, carrageenan, carboxymethylcellulose, cellulose, curdlan, gelatin, gellan gum, dextran, galactomannan, guar gum, locust bean gum, tara gum, acacia gum, pectin, konjac gum, starch, and xanthan gum.

[0113] In particular embodiments, the mycelin protein of the present disclosure can comprise filaments having a length of less than 3000 pm, preferably less than 2500 pm, less than 2000 pm, less than 1500 pm, less than 1400 pm, less than 1300 pm, less than 1200 pm, less than 1100 pm, less than 1000 pm, less than 900 pm, less than 800 pm, or less than 700 pm. The mycelin protein of the present disclosure can comprise filaments having a length of more than 100 pm, preferably more than 200 pm.

[0114] In particular embodiments, the mycelin protein of the present disclosure can comprise filaments having a diameter of less than 20 pm, preferably less than 10 pm, more preferably 5 pm or less. In particular embodiments, the mycelin protein of the present disclosure can comprise filaments having a diameter of more than 1 pm, preferably more than 2 pm.

[0115] In particular embodiments, the mycelin protein of the present disclosure can comprise filaments having an aspect ratio (length / diameter) of less than 800, preferably less than 650, more preferably less than 400, especially 250 or less. In particular embodiments, the mycelin protein of the present disclosure can comprise filaments having an aspect ratio of more than 10, more than 20, more than 30, more than 40, more than 50, more than 60, or more than 70.

[0116] The MM-135 mycelin protein produced by Fusarium MM-135 or a microbial agent thereof of the present disclosure can be used for the preparation of meat alternatives and the like.

[0117] In order to make the objects, technical solutions, and advantages of the present application clearer, the following further describes the present application in conjunction with embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of well-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] Definitions

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions of the present application and throughout this document, and apply equally whether the singular or plural form of the terms is used.

[0120] The expressions "a" and "an" used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes more than one such cell, and equivalents thereof known to those skilled in the art, and so forth.

[0121] As used herein, the term "about" means ±20% of the value that follows. In some embodiments, the term "about" means ±10% of the value that follows. In some embodiments, the term "about" means ±5% of the value that follows.

[0122] The following examples and drawings are provided to aid the understanding of the present application, but are not intended to construe as limiting the present application in any way. The present application is set forth in the claims. It is understood that any modifications and changes can be made to any of the examples without departing from the spirit of the present application. The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0123] It should be noted that, in the examples, the specific conditions are not specified, and the conventional conditions, the manufacturer's recommendations or the publicly reported experimental conditions are used. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be obtained by commercial purchase. The reagents used are specified by the manufacturer, and similar products from other manufacturers have substitutability.

[0124] Examples

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

[0126] 1.1 Strain source

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

[0128] 1.2 Isolation process

[0129] The collected sample was mixed and 5 g was weighed into a triangular flask containing 45 mL of sterile 0.7% sodium carboxymethyl cellulose solution and 15 glass beads, and was shaken at 30°C and 150 rpm for 30 min. 1 mL of the sample suspension was taken and 9 mL of sterile water was added to dilute it to 10 -2 ~10 -3 Serial dilution gradient was performed, and then 10 -2 , 10 -3 Two dilutions were spread on MGA2.5 (containing chloramphenicol and tetracycline hydrochloride) plates, with a dropwise amount of 100 μL / plate, and were evenly spread. After the surface of the plates was dried, they were incubated at 28°C for 2-7 days.

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

[0131] Peptone 15g KH2PO4 1.0g MgSO4.7H2O 0.5g Malachite green oxalate 0.0025g Agar 20g Distilled water 1L

[0132] Purification was performed by stepwise transfer of mycelium tips. After colonies were formed on plates, the mycelium at the edge of single colony was picked and transferred to MEA plates (purchased from Beijing Aobosxing Biotechnology Co., Ltd.; Catalog No. 02-183) and incubated at 28°C until pure colonies were obtained. 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] Genomic DNA was extracted from mycelium of the strain and used as a template for gene amplification. The DNA of the strain was subjected to multi-gene amplification and sequencing.

[0136] 2.2 Preparation of PCR system

[0137] A 25 μL PCR system was prepared in a PCR tube according to the following table:

[0138]

[0139]

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

[0141]

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

[0143] 2.3 PCR amplification

[0144] The prepared PCR system was subjected to PCR amplification in a PCR instrument (Dongsheng ETC811). The PCR reaction conditions are shown in the following table. After amplification, the PCR products were sent to Beijing Qikexin 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 Polyphasic identification method provided by the online database mycobank (fusarium.mycobank.org) was used to identify the strain MM-135, and the results showed that based on the TEF1, RPB2, and ITS genes, the MM-135 strain was closest to the species Fusarium compactum NRRL 36318, with a global similarity of 99.65%.

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

[0153] 2.5 Species identification analysis based on multi-gene phylogenetic tree

[0154] The species identification analysis method based on Polyphasic identification showed that the identified species belonged to the Fusarium incarnatum-equiseti species complex (FIESC). The sequences of the 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 phylogenetic tree.

[0155] The steps of multi-gene phylogenetic 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, after trimming, concatenate the sequences of the four genes, and use IQ-TREE (version 2.2.5) to construct a multi-gene joint phylogenetic tree. The results are shown in Figure 1 .

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

[0157] The strain MM-135 was preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, with the preservation number CGMCC No. 41312.

[0158] Example 3. Fermentation preparation of mycelial protein

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

[0160] The primary screening medium was prepared with the following ingredients: glucose 30 g / L, yeast extract powder 30 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, CaCl20.2 g / L, and sterilized at 121 ℃ for 20 min.

[0161] The filter cake was filtered and washed thoroughly using a vacuum filtration device, dried in an electric heating air drying oven at 60 ℃, and the mass of the dried filter cake was accurately measured to calculate the biomass concentration [biomass concentration = mycelium dry weight (g) / medium mass (g) * 100%]. Then, the crude protein of the mycelium was determined by the Kjeldahl method according to GB 5009.5-2016 National Food Safety Standard-Determination of Protein in Foods. The results are shown in the following table.

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

[0163] Strain Mycelium morphology Filter cake color Filter cake texture Biomass wt% Crude protein wt% MM-135 Cotton seed-like Light yellow Inelastic 1.62 59.17

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

[0165] Example 4. Production of mycelial protein by scale-up culture

[0166] 4.1 Fermentor re-screening culture method

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

[0168] Formulation materials g / L Glucose 30 Yeast extract powder 25 Monopotassium phosphate 1 Magnesium sulfate heptahydrate 0.2 Calcium chloride 0.01

[0169] Seed liquid culture conditions: The first-stage seed liquid was prepared using the above seed medium, and the activated Fusarium compactum strain MM-135 was inoculated into a 250 mL three-cone flask containing 50 mL of the seed medium at a 200 rpm constant temperature and vibration for 24 h. The second-stage seed liquid was prepared using the above seed medium, and inoculated into a 3L three-cone flask containing 1L of seed medium at a 200 rpm rotation speed for 24-30 h. It was observed that the length of the seed medium culture time affected the color of the filter cake formed by mycelial protein; the culture time of the seed medium could be controlled to further control the color and texture of the filter cake obtained by fermentation.

[0170] Then inoculate into the re-screening medium at 3% inoculation amount. The re-screening medium for producing mycelial protein has the following components:

[0171] Ingredients Fermentation medium Corn starch hydrolysate 25 g / L Citric acid 1 g / L Monopotassium phosphate 1 g / L Ammonium sulfate 5 g / L Magnesium sulfate heptahydrate 0.2 g / L Calcium chloride 0.01 g / L Manganese sulfate monohydrate 1.7 mg / L Ferrous sulfate heptahydrate 5 mg / L Zinc chloride 1.7 mg / L

[0172] Fermentation process conditions: the volume of the fermentation tank is 15 L, the liquid loading is 10 L, and sterilization is performed at 121 ℃ for 30 min. The temperature is 28 ℃, the aeration is 5-15 L / h, the tank pressure is 0.05-0.10 Mpa, the rotation speed is 100-400 rpm, the dissolved oxygen is 20-50%, and the pH is controlled to 6.0 by using ammonia water. The shape of the mycelium has a great influence on the taste of the product (for example, meat substitute), and the mycelium in the shape of cotton seeds tastes poor. In this embodiment, inorganic nitrogen is used to replace organic nitrogen, so as to reduce the cost, optimize the filamentous structure, and reduce the balling phenomenon.

[0173] Using the same method as in Example 3, the color, texture, odor, mycelial morphology, mycelial length, mycelial diameter, mycelial branching, spore number, and crude protein content of the filter cake obtained by re-screening of the Fusarium compactum strain MM-135 are detected. It is found that the granular and cotton seed-like shape in the primary screening is effectively improved, the mycelium presents a slender filamentous structure, and the prepared filter cake has an excellent texture and can better simulate the chewiness of meat (Figure 2). The crude protein content of the MM-135 mycelium is determined by the Kjeldahl method to be 55.29 wt%.

[0174] The re-screening results of the strain MM-135 are shown in the following table.

[0175] Strain re-screening results MM-135 Fermentation time 30h Filter cake color White Filter cake texture Very tough Filter cake odor None Mycelium morphology Filamentous Mycelium length Long Mycelium diameter Thin Mycelium branching Few Spore number None Crude protein 55.29 wt%

[0176] Compared with the primary screening medium in Example 3, the re-screening medium in this embodiment uses inorganic nitrogen (ammonium sulfate, ammonium phosphate) to replace organic nitrogen source; but 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 application can use both organic nitrogen source and inorganic nitrogen source as the nitrogen source for fermentation culture of fungal protein.

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

[0178] 4.2 Fermentation process corresponding to the fermentation medium:

[0179] 4.2.1 Seed liquid culture

[0180] A 3L three-cone flask is used, and PDB medium (potato infusion powder 300 g / L, glucose g / L, purchased from Guangdong Huan Kai Microbial Technology Co., Ltd.) is used; the inoculation amount is 5%, the liquid loading is 1L, and the rotation speed of the shaker is 200 rpm; and the culture is performed for 24 h.

[0181] 4.2.2 Primary fermentation

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

[0183] Ingredients Primary fermentation medium Corn starch hydrolysate 25 g / L Citric acid 1 g / L Monopotassium phosphate 1 g / L Ammonium sulfate 5 g / L Magnesium sulfate heptahydrate 0.2 g / L Calcium chloride 0.01 g / L Manganese sulfate monohydrate 1.7 mg / L Ferrous sulfate heptahydrate 5 mg / L Zinc chloride 1.7 mg / L Vitamin complex solution 0.1 mg / L

[0184] Subculture: use 15L tank, liquid volume 10L, 121℃ sterilization 30min, through the way of flame inoculation seed liquid all transfer; speed 100-300rpm; ventilation 10L / min, tank pressure 0.05mpa; ammonia control pH 6.0; 28℃ culture 16-20h. Ammonium sulfate in the primary culture medium can be replaced by ammonium phosphate.

[0185] 4.3.3 Secondary fermentation

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

[0187] Ingredients Secondary fermentation medium Corn starch hydrolysate 25 g / L Citric acid 1 g / L Monopotassium phosphate 1 g / L Ammonium sulfate 5 g / L Magnesium sulfate heptahydrate 0.2 g / L Calcium chloride 0.01 g / L Manganese sulfate monohydrate 1.7 mg / L Ferrous sulfate heptahydrate 5 mg / L Zinc chloride 1.7 mg / L Vitamin complex solution 0.1 mg / L

[0188] Feed medium

[0189]

[0190]

[0191] The secondary fermentation medium formula is shown in the above table (ammonium sulfate can be replaced by ammonium phosphate), use 50L tank, liquid volume 30L, 121℃ sterilization 30min; after sterilization, add 0.1mg / L vitamin mixture (filter sterilization); according to 5-10% inoculation amount, seed liquid cultured in 15L tank is pumped into through the bottom valve after sterilization to complete inoculation; before 15h, ventilation 1000L / h, tank pressure 0.05mpa; after 15h, start feeding according to 0.5-2g / L / h, and adjust ventilation to 1500L / h, tank pressure to 0.1mpa; ammonia control pH 4.0-6.0; 28℃ culture; speed 100-300rpm coupled with dissolved oxygen 20%-50%, culture 24-48h. The composite vitamin includes B group composite vitamin, and the composite vitamin includes the following components in equal mass ratio: vitamin B1 / thiamine, vitamin B12 / cobalamin, vitamin B2 / riboflavin, vitamin B3 / niacin, vitamin B5 / D-pantothenic acid calcium, vitamin B6 / pyridoxine, vitamin B9 / folic acid, vitamin C and vitamin H / D-biotin. Increasing the dissolved oxygen amount and the dissolved oxygen efficiency can promote the growth of mycelium without damaging the mycelium.

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

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

[0194] This example investigates the effect of the components and their concentrations in the fermentation medium on the mycelium and protein yield in the production scale-up process. In the scale-up process, the inoculum size is large and the transformation efficiency is low.

[0195] Using the same method as in Example 3, the color, texture, odor, mycelium morphology, mycelium length, mycelium diameter, mycelium branching, spore number, and crude protein content of the filter cake obtained from the Fusarium compactum strain MM-135 were determined. The results showed that:

[0196] The balling phenomenon was alleviated when the medium did not contain ammonium sulfate;

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

[0198] Increasing the concentration of Zn 2+ and other trace element solutions in the medium increased the protein content.

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

[0200] Meat chunks were prepared from the mycelium protein prepared in Example 4, using the following raw materials: Fusarium compactum strain MM-135 mycelium protein, water, egg white powder, starch, and other seasonings (e.g., monosodium glutamate, yeast extract, white granulated sugar, food essence, soybean oil, edible salt, spices, 5'-disodium inosine nucleotide, and gluten).

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

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

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

[0204] 3. Press the vegetarian meat filling into meat chunks with a length of 20 g each, and cook at 85°C for 30 min to set the shape, and then cool to room temperature.

[0205] 4. The meat pieces are coated with the slurry of step 1, and deep fried at 160°C for 30 seconds, and cooled to room temperature.

[0206] 5. Packaged, quick-frozen, and stored.

[0207] (1) Sensory evaluation and (2) Texture analyzer are used for evaluation.

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

[0209] (2) Texture analyzer measurement of texture structure

[0210] The mycoprotein-based meat pieces and commercially available chicken meat pieces are cut into 1 cm x 1 cm x 1 cm columnar bodies, and the texture of the samples is repeatedly detected 6 times using a texture analyzer (British Micro Stable TA.XT plus C, TPA mode). The probe is P / 36R, the probe measurement speed is 1 mm / s, the deformation amount (compression distance / sample height) is 40%, the trigger type is automatic, the trigger force is 5.0 g, and the two compression time intervals are 5 s.

[0211] Elasticity: Elasticity refers to the ability of an object to deform under the action of an external force and return to its original state after the external force is removed.

[0212] Cohesiveness: Cohesiveness reflects the property of food resisting damage and close connection when chewing food, so that the food remains intact.

[0213] Chewiness: Used to describe solid test samples, it indicates the energy required to chew a solid sample into a stable state for swallowing, and is represented by the product of hardness x cohesiveness x elasticity in numerical value.

[0214] The following table shows the texture analyzer test results of the mycoprotein-based meat pieces:

[0215]

[0216] From the above table, it can be concluded that the mycoprotein-based meat pieces are close to commercially available chicken meat pieces.

[0217] The texturization and emulsification of the mycelial protein can be further enhanced, so as to improve the texture and taste of the meat substitute food; that is, the obtained mycelial protein-based meat substitute food can completely replace plant protein-based meat products, avoiding the defects brought by plant protein, while having excellent taste and texture, the taste is juicy, elastic and moderate in hardness, the texture is good in hardness, elasticity and chewiness, which is closer to real meat food, and compared with plant protein meat or pork, chicken, the mycelial protein-based meat substitute food has high protein content, high natural dietary fiber content and low fat content, does not contain cholesterol, and is more nutritious and healthy.

[0218] The technical scheme of the present application is not limited to the above specific embodiments, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.

Claims

1. Use of Fusarium reticulatum (F. venenatum) ATCC 38534, MM-135 or an inoculum thereof for the production of mycelian protein for food and / or protein-rich mycelium for food. Fusarium compactum ) The MM-135 strain was preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, and the preservation number is CGMCC No. 41312.

2. Use according to claim 1, characterized in that, The Fusarium reticulatum (F. reticulatum) Fusarium compactum TEF1 of MM-135 has a nucleotide sequence as set forth in SEQ ID NO: 9; and / or The Fusarium compactum (Fusarium compactum) Fusarium compactum The ITS of MM-135 has a nucleotide sequence as set forth in SEQ ID NO: 10; The Fusarium tsicily ( Fusarium compactum MM-135's RPB1 has the nucleotide sequence shown in SEQ ID NO: 11; and / or The Fusarium compactum (Fusarium compactum) Fusarium compactum RPB2 of MM-135 has a nucleotide sequence as set forth in SEQ ID NO:

12.

3. Use according to claim 1 or 2, characterized in that, The Fusarium reticulatum (F. reticulatum) Fusarium compactum The hyphal protein or mycelium of MM-135 has a length of less than 2000 μm and / or an aspect ratio of less than 800.

4. Use according to claim 1 or 2, characterized in that, The Fusarium reticulatum (F. reticulatum) Fusarium compactum ) MM-135 is in spore form and / or mycelium form, and / or The mycelium comprises 45 wt% or more of protein, and / or The mycelium comprises 0.4 wt% or more of mycelium protein in terms of dry weight of the mycelium.

5. A method of producing mycelium of Fusarium venenatum (ATCC 31280) MM-135 for use in food production, characterized by, Fusarium compactum The method comprises: ​ 1) inoculating Fusarium compactum (Fusarium reticulatum) Fusarium compactum ) MM-135 or its bacterial agent into the seed culture medium to obtain a seed liquid; 2) inoculating the seed liquid into a fermentation medium to culture, to obtain a first fermentation liquid; and 3) obtaining mycelium from the fermentation liquid; The fermentation medium does not contain ammonium sulfate. The MM-135 strain was preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, and the preservation number is CGMCC No. 41312.

6. The method of claim 5, wherein, The seed medium and / or the fermentation medium comprises a carbon source and a nitrogen source.

7. The method of claim 6, wherein, The carbon source comprises a sugar, a sugar alcohol, a starch, a starch derivative, a lignocellulosic pulp or raw material, whey, wheat soaking liquid, a hydrolysis product of lignocellulosic material, a corn wet milling product, and / or a combination thereof.

8. The method of claim 7, wherein, The starch comprises potato powder, corn pulp, and / or a combination thereof.

9. The method of claim 7, wherein, The starch derivative comprises a starch hydrolysate, a hydrogenated starch hydrolysate, and / or a combination thereof.

10. The method of claim 7, wherein, The whey comprises acid whey, sweet whey, and / or a combination thereof.

11. The method of claim 6, wherein, The carbon source comprises sucrose, maltose, glucose, fructose, glycerol, a 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, sugar beet pulp, agricultural pulp, wood pulp, dried distiller's grains, carbon refined sugar syrup, desalted sugar syrup, enzyme conversion sugar syrup, and / or a combination thereof.

12. The method of claim 6, wherein, The nitrogen source in the seed medium and / or the fermentation medium is an inorganic nitrogen source and / or an organic nitrogen source.

13. The method of claim 12, wherein, The organic nitrogen source comprises peptone, yeast extract, soybean meal, and / or a combination thereof.

14. The method of claim 12, wherein, The inorganic nitrogen source comprises urea and / or an ammonium compound.

15. The method according to any one of claims 6 to 14, characterized in that, In step 2), The temperature of the culture is 20-35°C, and / or The rotation speed of the culture is 100-300 rpm, and / or The pH of the fermentation medium is 5.0-7.

0.

16. A Fusarium torulosum (F. torulosum) obtained by the method of any one of claims 5 to 15. Fusarium compactum ) MM-135 mycelium.

17. A composition or food comprising mycelial proteins and / or protein-rich mycelium of Fusarium reticulatum (MM-135) for use in food. Fusarium compactum ) The MM-135 strain was preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, and the preservation number is CGMCC No. 41312.

18. Fusarium torulosum (ATCC® MM-135) mycelial protein and / or Fusarium torulosum (ATCC® MM-135) protein-rich mycelium for food use. Fusarium compactum ) MM-135) mycelium for food use or the composition of claim 17 for use in the preparation of or as a meat substitute. Fusarium compactum ) MM-135) mycelium for food use or the composition of claim 17 for use in the preparation of or as a meat substitute. The MM-135 strain was preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, and the preservation number was CGMCC No. 41312.

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