Use of fusarium brachygibbosum in production of mycelium protein

Abstract

The present invention belongs to the technical field of microbial fermentation. Provided is the use of Fusarium brachygibbosum in the production of mycelium protein. The present invention proposes for the first time that Fusarium brachygibbosum has the property of of high production of mycelium protein, and the protein yield is significantly higher than that of other types of Fusaria currently reported. The mycelium protein produced by Fusarium brachygibbosum is filamentous, meat-colored, elastic and non-toxic, and is an ideal alternative protein. Therefore, the present invention provides a new means for industrial production of alternative proteins, and provides a basis for developing safe food. Moreover, production on the basis of a fermentation method can greatly improve protein production efficiency, reduce carbon emissions, and protect the environment.

Description

Application of Fusarium brevicaulis in the production of mycelial protein

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on January 11, 2024, with application number 202410044012.8 and invention name “Application of Fusarium brevicaulis in the production of mycelial protein”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention belongs to the technical field of functional microorganisms, and particularly relates to the application of Fusarium brevicaulis in the production of mycelial protein. Background Art

[0003] Alternative proteins are proteins produced from plant or animal cells or through microbial fermentation. These innovative foods are designed to taste the same or better than traditional animal products and offer health benefits such as zero cholesterol, low fat, and a small amount of dietary fiber, all at the same or lower cost. Compared to traditionally produced animal products, alternative proteins require fewer inputs, such as land, fertilizer, and water, and produce fewer negative externalities, such as greenhouse gas emissions and air pollution. They also reduce the contribution of food production to pandemic risk and antimicrobial resistance.

[0004] As one of the forms of alternative protein production, microbial fermentation protein uses microbial or algae fermentation to produce protein. It contains cellulose, rich enzymes and bioactive substances, and does not contain cholesterol, which is more friendly to patients with cardiovascular and cerebrovascular diseases. In industry, the main industrial strains used for fermentation production of microbial protein meat include algae, yeast and filamentous fungi. Algae have problems such as slow growth rate and low density, and so far mainly carry out small-scale protein production. Yeast grows fast, has a high cell density, and requires simple production equipment. However, the low cell wall digestibility and high nucleic acid content have restricted the further development of yeast fermentation protein to a certain extent. The filamentous fungi that produce protein are mainly concentrated in Fusarium. The mycelium produced by Fusarium is more delicious than single-cell proteins such as yeast and bacteria, and has a meat-like tissue structure. At the same time, the rich edible crude fiber it contains helps human gastrointestinal digestion. It is a meat substitute that can meet the nutritional needs of modern people. Currently, a strain of Fusarium venenatum has been developed and its mycelium, which is high in protein, has been used to produce meat products (CN 115851458 A). However, the yield of mycelial protein produced by this strain of Fusarium venenatum needs to be improved. Furthermore, patent CN116640753 discloses that mycelial protein production in Fusarium venenatum can be effectively increased by deleting the pyruvate decarboxylase gene FvPDC6 from the thallus. Therefore, naturally high-yielding filamentous fungal strains are extremely scarce. Finding more promising strains that naturally produce high yields of filamentous fungal protein is a highly commercially valuable development.

[0005] Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a new type of Fusarium for use in the production of mycelial protein.

[0007] The present invention provides application of Fusarium brachygibbosum or a microbial agent thereof in producing mycelium protein.

[0008] Preferably, the Fusarium brevicaulis is a Fusarium brevicaulis strain having a TEF1 gene sequence at least 95% homologous to SEQ ID NO: 5 and / or an ITS1 gene sequence at least 97% homologous to SEQ ID NO: 6, or a composition formed by the Fusarium brevicaulis strains.

[0009] Preferably, the Fusarium brevicaulis includes the Fusarium brevicaulis strain FXFB001;

[0010] The deposit number of the Fusarium brevicaulis strain FXFB001 is CGMCC No: 41066.

[0011] Preferably, the Fusarium brevicaulis includes spore form and / or mycelial form.

[0012] The present invention provides an edible mycelium, wherein, based on the dry mass of the edible mycelium, the protein content of the edible mycelium is greater than 40%; and / or the dietary fiber content is greater than 20%; and / or the fat content is less than 8%; the edible mycelium is derived from Fusarium brachygibbosum;

[0013] The Fusarium brevicaulis is the Fusarium brevicaulis used in the application.

[0014] Preferably, the edible mycelium is elastic and / or tough.

[0015] The invention provides a Fusarium brachygibbosum strain FXFB001, with a preservation number of CGMCC No: 41066.

[0016] The present invention provides a microbial agent for producing protein or protein-rich mycelium, comprising the Fusarium brevicaulis strain FXFB001 and an auxiliary material acceptable to the agent.

[0017] Preferably, the mass ratio of the Fusarium brevicaulis strain FXFB001 to the auxiliary material is 1-10:1-100.

[0018] Preferably, the auxiliary materials include one or more of culture medium, additives and carriers.

[0019] The present invention provides the use of the Fusarium brevicaulis strain FXFB001 or the microbial agent in producing protein or mycelium.

[0020] The present invention provides a method for producing edible mycelium using Fusarium brachygibbosum, comprising the following steps:

[0021] inoculating a seed solution prepared from Fusarium brevicaulis or a microbial agent thereof into a fermentation medium for fermentation and culturing to obtain a fermentation solution;

[0022] After sterilizing the fermentation liquid, separating the fermentation product to obtain edible mycelium;

[0023] The Fusarium brevicaulis is the Fusarium brevicaulis used in the application.

[0024] Preferably, the fermentation medium includes fermentation medium No. 1 and / or fermentation medium No. 2;

[0025] The fermentation medium No. 1 comprises the following components: 23-27 g / L of starch, 0.8-1.2 g / L of citric acid, 0.8-1.2 g / L of potassium dihydrogen phosphate, 4.5-5.5 g / L of ammonium sulfate, 17-21 mg / L of magnesium salts (calculated as Mg), 2.8-43 mg / L of calcium salts (calculated as Ca), 0.48-0.62 mg / L of manganese salts (calculated as Mn), 0.96-1.05 mg / L of ferric salts or ferrous salts (calculated as Fe), 0.72-0.91 mg / L of zinc salts (calculated as Zn), 0.44-0.54 mg / L of cobalt salts (calculated as Co), and 0.08-0.12 mg / L of multivitamins;

[0026] The fermentation medium No. 2 includes the following components: glucose 15-60 kg / m 3 、K2SO40.5~1.5kg / m 3 、H3PO40.4~0.8kg / m 3 、Calculate the concentration of magnesium salt in terms of Mg: 9.75~58.5g / m 3 , zinc salt with concentration calculated as Zn 2.2~22.5g / m 3 , manganese salt concentration calculated as Mn 2.46 ~ 9.85g / m 3 Calcium acetate 0.1~0.3kg / m 3 and defoaming agent 0.08~0.12kg / m 3 .

[0027] Preferably, when fermentation medium No. 1 is used for fermentation, the fermentation conditions include:

[0028] During the first 15 hours of fermentation, the ventilation rate was 900-1100 L / h and the pressure was controlled at 0.04-0.06 MPa.

[0029] After 15 hours of fermentation, feed was started at a rate of 0.5-2.0 mL / L / h, the aeration rate was 1400-1600 L / h, and the pressure was 0.08-0.12 MPa; the pH value of the fermentation broth was 4.0-6.0;

[0030] The fermentation temperature is 27°C to 29°C, the rotation speed is 100 to 450 rpm, the coupled dissolved oxygen is 20% to 50%, and the fermentation time is 68 to 76 hours.

[0031] The feeding ingredients during the feeding process include 280-320 g / L of glucose and 0.08-0.12 mg / L of multivitamins;

[0032] The vitamin complex comprises the following components in parts by weight: 0.08 to 0.12 parts of vitamin B1, 120.08~0.12 parts, vitamin B2 0.08~0.12 parts, vitamin B3 0.08~0.12 parts, vitamin B5 0.08~0.12 parts, vitamin B6 0.08~0.12 parts, vitamin B9 0.08~0.12 parts, vitamin C 0.08~0.12 parts and vitamin H 0.08~0.12 parts.

[0033] Preferably, when the fermentation culture is carried out using fermentation medium No. 2, the fermentation culture conditions are as follows: aeration rate of 900 to 1600 L / h, pressure control of 0.04 to 0.12 MPa; pH value of the fermentation liquid is 4.0 to 6.0;

[0034] The fermentation temperature is 27°C to 29°C, the rotation speed is 100 to 450 rpm, the coupled dissolved oxygen is 20% to 50%, and the fermentation time is 72 to 480 hours.

[0035] When the reducing sugar content in the fermentation system is lower than 2-5 g / L, 20%-90% of the volume fraction of the fermentation liquid is discharged, and fermentation medium No. 2 is added to the original volume.

[0036] Preferably, the method for preparing seed liquid from Fusarium brevicaulis comprises the following method:

[0037] inoculating the Fusarium brevicaulis into a first seed culture medium and sequentially performing primary seed culture and secondary seed culture to obtain a secondary seed solution;

[0038] The secondary seed liquid is inoculated into the second seed culture medium for tertiary seed culture to obtain the tertiary seed liquid.

[0039] Preferably, the first seed culture medium contains the following components: glucose 28-32 g / L, yeast extract 23-27 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, magnesium salt calculated as Mg 17-21 mg / L and calcium salt calculated as Ca 3-4 mg / L; the second seed culture medium contains the following components: starch 23-27 g / L, citric acid 0.8-1.2 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L , ammonium sulfate 4.8~5.2g / L, magnesium salts calculated as Mg 17~21mg / L, calcium salts calculated as Ca 0.003~0.004g / L, manganese salts calculated as Mn 0.48~0.62mg / L, ferric or ferrous salts calculated as Fe 0.96~1.05mg / L, zinc salts calculated as Zn 0.72~0.91mg / L and cobalt salts calculated as Co 0.44~0.54mg / L.

[0040] Preferably, the culture temperature of the primary seed culture, the secondary seed culture or the tertiary seed culture is 25° C. to 30° C.;

[0041] The cultivation time of the first-level seed culture, the second-level seed culture or the third-level seed culture is 22 to 26 hours;

[0042] The rotation speed of the first-stage seed culture, the second-stage seed culture or the third-stage seed culture is 100-200 rpm;

[0043] The ventilation rate of the three-stage seed culture is 8 to 12 L / min;

[0044] The pressure of the three-stage seed culture is 0.04-0.06 MPa;

[0045] The pH value of the tertiary seed culture system is 4.0-6.0.

[0046] Preferably, the method for sterilizing the fermentation liquid is heating sterilization;

[0047] The heating sterilization procedure is as follows: keeping the temperature at 60° C. to 70° C. for 18 to 22 minutes, raising the temperature to 88° C. to 92° C. and keeping the temperature for 8 to 15 minutes.

[0048] Preferably, the method for separating the fermentation product comprises separating the fermentation liquid into solid and liquid, washing the solid phase and then drying it to obtain edible mycelium.

[0049] The present invention provides use of the edible mycelium or the edible mycelium prepared by the method in preparing a meat substitute.

[0050] Preferably, the preparation method does not include structural processing.

[0051] The present invention provides the use of Fusarium brachygibbosum in the production of mycelial protein. The present invention develops Fusarium brachygibbosum for the fermentation production of mycelial protein for the first time. Experiments have shown that the Fusarium brachygibbosum is superior to other Fusarium species (such as Fusarium venezuelae and Fusarium venezuelae) in terms of the content of mycelial protein produced by fermentation. The mycelial protein content detected by the Kjeldahl nitrogen determination method is above 55.39%. It also has the characteristics of low fat content and high dietary fiber content, with a total fat content of 7.97% and a dietary fiber content of 26.01%. The mycelial protein produced does not contain toxins and is an ideal alternative protein. It can be seen that the mycelium produced by Fusarium brachygibbosum in the present invention has a high mycelial protein content, good morphology, and no toxic side effects. Therefore, the present invention provides a new means for the industrial production of alternative proteins and provides a basis for the development of safe foods. At the same time, based on the production method of fermentation, it can greatly improve protein production efficiency, reduce carbon emissions, and protect the environment.

[0052] The present invention also provides a Fusarium brachygibbosum strain FXFB001, with a deposit number of CGMCC No: 41066. The present invention isolated strain FXFB001 from a forest humus soil sample in the Tibet Autonomous Region. After morphological and molecular identification, the results showed that strain FXFB001 is Fusarium brachygibbosum. Through preliminary screening, the strain produced light yellow, filamentous and elastic mycelium, and the protein content was above 55.39% as determined by the Kjeldahl nitrogen determination method. Through fermentation and cultivation, the protein-rich mycelium produced by strain FXFB001 was well-filamentous, and the fermentation broth was able to naturally present a flesh-colored color without any unpleasant odor even in an unheated state. It can be seen that the Fusarium brachygibbosum strain FXFB001 provided by the present invention is a naturally high-protein-yielding microbial strain, with a protein yield significantly higher than that of other types of Fusarium strains currently reported, and the protein produced does not contain toxins, is safe and has no side effects. Therefore, the present invention provides a new means for the industrial production of alternative proteins and also provides a basis for the development of safe foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG1 shows the colony morphology of the strain FXFB001 isolated and purified in Example 1 of the present invention;

[0054] FIG2 is a phylogenetic tree of strain FXFB001 constructed based on multiple genes in Example 1 of the present invention;

[0055] FIG3 is a bacterial cake obtained by preliminary screening and culture of strain FXFB001 in Example 2 of the present invention;

[0056] FIG4 is a fermentation product obtained by fermentation culture of strain FXFB001 in Example 3 of the present invention;

[0057] FIG5 shows the results of microscopic observation of protein-rich mycelia obtained by fermentation of strain FXFB001 in Example 3 of the present invention.

[0058] Biomaterial deposit information

[0059] The Fusarium brachygibbosum strain FXFB001 provided by the present invention was deposited in the China General Microbiological Culture Collection Center on December 13, 2023. The depository is abbreviated as CGMCC. The depository address is No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No: 41066. DETAILED DESCRIPTION

[0060] The present invention provides application of Fusarium brachygibbosum or a microbial agent thereof in producing mycelium protein.

[0061] In the present invention, the Fusarium brevicaulis comprises a single Fusarium brevicaulis strain or a combination of multiple Fusarium brevicaulis strains. The present invention does not impose any special restrictions on Fusarium brevicaulis strains. All strains taxonomically identified as Fusarium brevicaulis fall within the scope of protection of the present invention. For example, the Fusarium brevicaulis strains having a TEF1 gene sequence that is 95%, 96%, 97%, 98%, 99% and 100% homologous to SEQ ID NO: 5 and an ITS1 gene sequence that is 97%, 98%, 99% and 100% homologous to SEQ ID NO: 6 can be used, such as the Fusarium brevicaulis strain FXFB001. The Fusarium brevicaulis strain FXFB001 has a deposit number of CGMCC No: 41066 and has excellent performance in producing protein-rich mycelium.

[0062] In the present invention, the production is preferably fermentation production. The fermentation production is a process of preparing the microbial cell itself or direct metabolites or secondary metabolites by means of the life activities of microorganisms under aerobic or anaerobic conditions. In one embodiment of the present invention, the fermentation production is a process of growing Fusarium brevicaulis in large quantities under the conditions of nutrients provided by the fermentation medium to obtain protein-rich mycelium. The present invention has no special restrictions on the morphology of the Fusarium brevicaulis, and any live bacterial form of Fusarium brevicaulis known in the art can be used, for example, including spore form and / or hyphae form.

[0063] The invention provides a Fusarium brachygibbosum strain FXFB001, with a preservation number of CGMCC No: 41066.

[0064] In the present invention, the Fusarium brevicaulis strain FXFB001 was isolated and purified from a forest humus soil sample in Shannan City, Tibet Autonomous Region. Morphological identification showed that the aerial hyphae grown on PSA culture medium were felt-like, initially white and gradually turning pink, with the back of the plate being orange-red to dark red. Conidia morphology: large conidia were sickle-shaped, slightly pointed at both ends, with 3-5 septa, and measuring 10.8-25.1 μm x 2.0-3.6 μm. Small conidia were fusiform, slightly curved, slightly flattened at both ends, and with 0-2 septa. Chlamydospores were spherical, solitary within the hyphae, and measured 5.2-7.5 μm. Molecular identification based on TEF1 and ITS sequences revealed that the TEF1 sequence of strain FXFB001 shared 99.84% identity with Fusarium brachygibbosum (NCBI: MK752485.1), and the ITS1 sequence shared 100% identity with Fusarium brachygibbosum (NCBI: GQ505450.1). A multi-gene phylogenetic tree constructed also revealed that strain FXFB001 and Fusarium brachygibbosum were clustered on the same branch. Based on the morphological and molecular identification results, strain FXFB001 was classified as Fusarium brachygibbosum.

[0065] In one embodiment of the present invention, the protein production characteristics of the strain FXFB001 are initially screened. The initial screening method preferably comprises inoculating the strain FXFB001 into a first seed culture medium for constant temperature shaking culture, isolating the solid-phase culture product, drying it, and measuring the crude protein content. The first seed culture medium preferably contains the following components: 28-32 g / L glucose, 23-27 g / L yeast extract, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.18-0.22 g / L magnesium sulfate heptahydrate, and 0.009-0.011 g / L calcium chloride; more preferably, 30 g / L glucose, 25 g / L yeast extract, 1.0 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, and 0.01 g / L calcium chloride. The constant temperature shaking culture temperature is preferably 25-30°C, more preferably 28°C. The constant temperature shaking culture duration is preferably 45-50 hours, more preferably 48 hours. The rotation speed of the constant temperature shaking culture is preferably 180 to 220 rpm, more preferably 200 rpm. The method for separating the solid phase culture product is preferably to filter by vacuum filtration and thoroughly wash the filter cake. The drying is preferably oven drying. The oven drying temperature is preferably 55°C to 65°C, more preferably 60°C. The protein-rich mycelium prepared by fermentation using strain FXFB001 has a filamentous mycelium morphology and a light yellow filter cake color. The filter cake is elastic and has a dry weight biomass percentage of 0.63%. The protein content determined by Kjeldahl nitrogen determination is above 55.39%.

[0066] The invention provides a microbial agent for producing mycelium protein, comprising the Fusarium brevicaulis strain FXFB001 and auxiliary materials.

[0067] In the present invention, the mass ratio of the Fusarium brevicaulis strain FXFB001 to the excipient is preferably 1-10:1-100, more preferably 1-10:5-80, and even more preferably 1:10-50. The type of the excipient varies depending on the dosage form of the microbial agent.

[0068] In the present invention, the microbial agent preferably comprises a freeze-dried powder or an aqueous solution. The method for preparing the aqueous solution of the microbial agent preferably comprises preparing a seed solution of the Fusarium brevicaulis strain FXFB001 and preparing the aqueous solution. The method for preparing the freeze-dried powder of the microbial agent preferably comprises preparing a seed solution of the Fusarium brevicaulis strain FXFB001 and freeze-drying it.

[0069] In one embodiment of the present invention, the method for preparing the seed liquid of the microbial inoculant of the Fusarium brevicaulis strain FXFB001 preferably includes the following steps: inoculating the activated strain FXFB001 into a first culture medium for primary seed culture to obtain a first seed liquid; inoculating the first seed liquid into the first culture medium again for secondary seed culture to obtain a second seed liquid; inoculating the second seed liquid into a second culture medium for tertiary seed culture to obtain a tertiary seed liquid.

[0070] In the present invention, the culture method involves the use of artificially formulated culture media and artificially created culture conditions (such as culture temperature) to enable the rapid growth and reproduction of certain microorganisms. The first seed culture medium contains the following components: 28-32 g / L glucose, 23-27 g / L yeast extract, 0.8-1.2 g / L potassium dihydrogen phosphate, 17-21 mg / L magnesium salt (calculated as Mg), and 3-4 mg / L calcium salt (calculated as Ca); more preferably, 30 g / L glucose, 25 g / L yeast extract, 1.0 g / L potassium dihydrogen phosphate, 19 mg / L magnesium salt (calculated as Mg), and 3.5 mg / L calcium salt (calculated as Ca). The second seed culture medium contains the following components: 23-27 g / L starch, 0.8-1.2 g / L citric acid, 0.8-1.2 g / L potassium dihydrogen phosphate, 4.8-5.2 g / L ammonium sulfate, 17-21 mg / L magnesium salt calculated as Mg, 3-4 mg / L calcium salt calculated as Ca, 0.48-0.62 mg / L manganese salt calculated as Mn, 0.96-1.05 mg / L ferric salt or ferrous salt calculated as Fe, 0.72-0. 91 mg / L and 0.44-0.54 mg / L of cobalt salt calculated as Co, more preferably 25 g / L of starch, 1 g / L of citric acid, 1 g / L of potassium dihydrogen phosphate, 5 g / L of ammonium sulfate, 19 mg / L of magnesium salt calculated as Mg, 3.5 mg / L of calcium salt calculated as Ca, 0.56 mg / L of manganese salt calculated as Mn, 1 mg / L of ferric salt or ferrous salt calculated as Fe, 0.81 mg / L of zinc salt calculated as Zn, and 0.49 mg / L of cobalt salt calculated as Co. The present invention has no particular limitation on the type of starch, and any starch known in the art may be used, such as sweet potato starch, corn starch, cassava starch, potato starch, and the like. The present invention imposes no particular restrictions on the form of calcium, cobalt, and zinc salts; any form of each metal salt known in the art may be employed. For example, calcium salts may be present in one or more forms of calcium chloride, calcium sulfate, or calcium bicarbonate; cobalt salts may preferably be present in the form of cobalt chloride; and zinc salts may be present in one or more forms of zinc chloride or zinc sulfate. The magnesium salts may preferably include one or more of magnesium sulfate, magnesium chloride, and magnesium nitrate. The manganese salts may preferably include one or more of magnesium sulfate, manganese chloride, potassium permanganate, and potassium manganate. The present invention imposes no particular restrictions on the sources of the fermentation medium components; commercially available sources of components known in the art may be employed. The present invention imposes no particular restrictions on the preparation methods of the first seed culture medium and the second seed culture medium; methods known in the art for microbial culture may be employed. After preparation, the culture medium is preferably sterilized. The sterilization method is preferably high-pressure steam sterilization.In an embodiment of the present invention, the first seed culture medium preferably contains the following components: 28-32 g / L of glucose, 23-27 g / L of yeast extract powder, 0.8-1.2 g / L of potassium dihydrogen phosphate, 0.18-0.22 g / L of magnesium sulfate heptahydrate, and 0.003-0.004 g / L of calcium chloride; more preferably, 30 g / L of glucose, 25 g / L of yeast extract powder, 1.0 g / L of potassium dihydrogen phosphate, 0.2 g / L of magnesium sulfate heptahydrate, and 0.005 g / L of calcium chloride. The second seed culture medium contains the following components: starch 23-27 g / L, citric acid 0.8-1.2 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, ammonium sulfate 4.8-5.2 g / L, magnesium sulfate heptahydrate 0.18-0.22 g / L, calcium salt 0.003-0.004 g / L, manganese sulfate monohydrate 1.5-1.9 mg / L, ferrous sulfate heptahydrate 4.8-5.2 mg / L, zinc chloride 0.72-0.9 1mg / L and 0.44-0.54mg / L of cobalt chloride; more preferably, 25g / L of starch, 1.0g / L of citric acid, 1.0g / L of potassium dihydrogen phosphate, 5.0g / L of ammonium sulfate, 0.017-0.021g / L of magnesium sulfate heptahydrate, 0.01g / L of calcium chloride, 1.5mg / L of manganese sulfate monohydrate, 5.0mg / L of ferrous sulfate heptahydrate, 1.9-2.0mg / L of zinc sulfate, and 1.8-2.2mg / L of cobalt chloride. In the embodiments of the present invention, the error in weighing each reagent in each culture medium preparation shall not exceed 10%. The culture temperature of the primary seed culture, secondary seed culture, or tertiary seed culture is preferably 25°C-30°C, more preferably 28°C. The culture time of the primary and secondary seed culture or tertiary seed culture is preferably 22-26h, more preferably 24h. The primary and secondary seed culture or tertiary seed culture is preferably carried out in a culture bottle. The rotation speed of the primary and secondary seed cultures is preferably 180-220 rpm, more preferably 200 rpm. For the secondary seed culture, the inoculum size is preferably 4%-6%, more preferably 5%. The rotation speed of the tertiary seed culture is preferably 100-220 rpm, more preferably 150 rpm. During the culture period, the culture flask is preferably placed in a shaker for shaking culture. For the tertiary seed culture, the inoculum size is preferably 5%-10%, more preferably 8%. The present invention does not specifically limit the method of seed solution inoculation; any inoculation method known in the art may be employed, such as, under sterile conditions, using a sterile pipette to transfer a small amount of seed solution to the corresponding culture medium. The aeration rate of the tertiary seed culture is preferably 8-12 L / min, more preferably 10 L / min. The pressure of the tertiary seed culture is preferably 0.04-0.06 MPa, more preferably 0.05 MPa. The pH value of the tertiary seed culture system is preferably 4.0-6.0, more preferably 5.0. The tertiary seed culture is preferably carried out in a culture tank equipped with a pressure valve.The present invention has no special restrictions on the specifications and brands of the culture tanks, and any culture tank known in the art can be used.

[0071] In the present invention, when preparing the microbial agent, spores are separated from the third-level seed liquid, and the separated spores are mixed with an aqueous solution solvent or a freeze-dried protective agent to prepare an aqueous solution or a freeze-dried powder. The molecular spore method is preferably centrifugation. The present invention does not impose any special restrictions on the types of auxiliary materials of the aqueous solution, and the types of auxiliary materials of aqueous solutions well known in the art can be used, such as culture medium. In the microbial agent aqueous solution, the number of effective spores of the strain FXFB001 is (1 to 100) × 10 7 / mL, more preferably 5×10 7 / mL. In the microbial agent solution, the effective spore count of the strain FXFB001 is (1-100)×10 7 / g, more preferably (1 to 100)×10 7 / g.

[0072] The present invention provides a method for producing edible mycelium using Fusarium brevicaulis, comprising the following steps:

[0073] inoculating a seed solution prepared from Fusarium brevicaulis or a microbial agent thereof into a fermentation medium for fermentation and culturing to obtain a fermentation solution;

[0074] After sterilizing the fermentation liquid, separating the fermentation product to obtain edible mycelium;

[0075] The Fusarium brevicaulis is the Fusarium brevicaulis used in the application described in the above technical solution.

[0076] In the present invention, the preparation method of the seed liquid prepared from Fusarium brevicaulis is the same as described above and will not be described in detail here.

[0077] In the present invention, fermentation is the process of producing microbial cells themselves, or direct metabolites or secondary metabolites, through the life activities of microorganisms under aerobic or anaerobic conditions. Fermentation, as it is commonly referred to, generally refers to the decomposition process of organic matter by an organism. Fusarium brevicaulis fermentation is the process of utilizing the nutrients of the fermentation medium to continuously grow and form a large amount of mycelium. The fermentation medium comprises a nitrogen source, a carbon source, an inorganic salt, and water. The fermentation medium includes Fermentation Medium No. 1 and / or Fermentation Medium No. 2. The fermentation medium No. 1 preferably includes the following components: 23-27 g / L starch, 0.8-1.2 g / L citric acid, 0.8-1.2 g / L potassium dihydrogen phosphate, 4.5-5.5 g / L ammonium sulfate, 17-21 mg / L magnesium salt calculated as Mg, 2.8-43 mg / L calcium salt calculated as Ca, 0.48-0.62 mg / L manganese salt calculated as Mn, 0.96-1.05 mg / L ferric salt or ferrous salt calculated as Fe, 0.72-0.91 mg / L zinc salt calculated as Zn, 0.72-0.91 mg / L cobalt salt calculated as Co, 0.8-1.2 g / L ferrous salt calculated as F ... The fermentation medium No. 2 preferably comprises the following components: 15-60 kg / m 2 of glucose; 15-60 kg / m 2 of glucose; 19-100 kg / m 3 of magnesium salt; 25-20 mg / m 3 of calcium salt; 0.54-100 mg / m 4 of manganese salt; 10-15 mg / m 4 of ferrous salt; 10-15 mg / m 5 of zinc salt; 0.81-10 mg / m 2 of zinc salt; 0.49-10 mg / m 3 of cobalt salt; 0.1-10 mg / m 4 of cobalt salt; 0.1-10 mg / m 4 of glucose; 0.1-10 mg / m 4 of glucose; 0.1-10 mg / m 4 of cobalt salt; 0.4-10 mg / m 4 of cobalt salt ... 3 、K2SO40.5~1.5kg / m 3 、H3PO40.4~0.8kg / m 3 、Calculate the concentration of magnesium salt in terms of Mg: 9.75~58.5g / m 3 , zinc salt with concentration calculated as Zn 2.2~22.5g / m 3 , manganese salt concentration calculated as Mn 2.46 ~ 9.85g / m 3 Calcium acetate 0.1~0.3kg / m 3 and defoaming agent 0.08~0.12kg / m 3 , more preferably glucose 25-55 kg / m 3 、K2SO40.8~1.3kg / m 3 、H3PO40.5~0.7kg / m 3 、Calculate the concentration of magnesium salt in Mg form: 12.5~50.5g / m 3 , zinc salt with a concentration of 4.5 to 20.5 g / m3 , manganese salt concentration calculated as Mn 5.2 ~ 7.5g / m 3 Calcium acetate 0.15~0.25kg / m 3 and defoaming agent 0.1kg / m 3 .

[0078] The present invention does not impose any particular restrictions on the type of starch; any starch known in the art may be used, such as sweet potato starch, corn starch, tapioca starch, and potato starch. The present invention does not impose any particular restrictions on the form of calcium, cobalt, and zinc salts; any metal salt known in the art may be used. For example, calcium salts may be present in one or more of the forms of calcium chloride, calcium sulfate, and calcium bicarbonate; cobalt salts may be present in the form of cobalt chloride; and zinc salts may be present in one or more of the forms of zinc chloride and zinc sulfate. The magnesium salts may preferably include one or more of magnesium sulfate, magnesium chloride, and magnesium nitrate. The iron or ferrous salts (calculated as Fe) may preferably include one or more of ferrous sulfate heptahydrate, ferric chloride, or ferrous chloride. The manganese salts may preferably include one or more of magnesium sulfate, manganese chloride, potassium permanganate, and potassium manganate. The present invention does not impose any particular restrictions on the sources of the fermentation medium components; any components known in the art may be purchased from these sources.

[0079] In an embodiment of the present invention, the fermentation medium No. 1 preferably includes the following components: 23-27 g / L starch, 0.8-1.2 g / L citric acid, 0.8-1.2 g / L potassium dihydrogen phosphate, 4.5-5.5 g / L ammonium sulfate, 0.18-0.22 g / L magnesium sulfate heptahydrate, 0.008-0.012 g / L calcium chloride, 1.5-1.9 mg / L manganese sulfate monohydrate, 4.5-5.5 mg / L ferrous sulfate heptahydrate, 1.8-2.2 mg / L zinc chloride, and 1.8-2.2 mg / L cobalt chloride. and multivitamins 0.08-0.12 mg / L; more preferably, starch 25 g / L, citric acid 1.0 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 5.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, calcium salt 0.0034 g / L, manganese sulfate monohydrate 1.7 mg / L, ferrous sulfate heptahydrate 5.0 mg / L, zinc salt 0.8 mg / L, cobalt chloride 0.5 mg / L and multivitamins 0.1 mg / L. In an embodiment of the present invention, the fermentation medium No. 2 preferably includes the following components: glucose 35 kg / m 3 、K2SO41.0kg / m 3 、H3PO40.6kg / m 3 , magnesium sulfate heptahydrate 0.25kg / m 3 , zinc sulfate heptahydrate 0.05g / m 3 , Manganese sulfate tetrahydrate 0.03g / m 3 Calcium acetate 0.1~0.3kg / m3 and defoaming agent 0.1kg / m 3 .

[0080] The present invention has no special restrictions on the preparation method of the fermentation medium, and the preparation method well known in the art can be used. After the preparation is completed, the fermentation medium is preferably sterilized. The present invention has no special restrictions on the sterilization method of the fermentation medium, and the sterilization method well known in the art can be used, such as high-pressure steam sterilization. After the sterilization is completed, the fermentation medium is placed at room temperature and then inoculated under sterile conditions. The present invention has no special restrictions on the inoculation method, and the inoculation method well known in the art can be used. For example, in a sterile environment, a certain amount of seed liquid is accurately measured with a sterile container and transferred to the fermentation medium, and mixed. The fermentation is preferably completed in a fermenter with a pressure valve. The present invention has no special restrictions on the specifications and brands of the fermenter, and a fermenter well known in the art can be used.

[0081] In the present invention, in addition to the fermentation medium affecting the fermentation process and results, the fermentation conditions are also important factors affecting the fermentation results. For example, temperature, fermentation time, system pH, dissolved oxygen, rotation speed, pressure, and feeding timing and feeding amount will affect the fermentation results. When fermentation is carried out using fermentation medium No. 1, the fermentation culture conditions are preferably: within the first 15 hours of fermentation culture, the ventilation volume is 900-1100 L / h, and the pressure is controlled at 0.04-0.06 MPa; after 15 hours of fermentation culture, the feeding is started at a rate of 0.5-2.0 mL / L / h, the ventilation volume is 1400-1600 L / h, and the pressure is 0.08-0.12 MPa; the pH value of the fermentation liquid is 4.0-6.0; the fermentation culture temperature is 27°C-29°C, and the rotation speed is 100-4 50rpm, coupled dissolved oxygen is 20% to 50%, and the fermentation culture time is 68 to 76h; more preferably, within the first 15h of fermentation culture, the ventilation volume is 1000L / h and the pressure is controlled at 0.05MPa; after 15h of fermentation culture, feeding is started at a rate of 1.2mL / L / h, the ventilation volume is 1500L / h, and the pressure is 0.1MPa; the pH value of the fermentation broth is 5.0; the fermentation culture temperature is 28°C, the rotation speed is 280rpm, the coupled dissolved oxygen is 35%, and the fermentation culture time is 72h. The feeding rate refers to the volume of feed medium added per liter of culture medium per hour. When the fermentation culture is carried out using fermentation medium No. 1, the feed ingredients during the feeding include 280-320g / L of glucose and 0.08-0.12mg / L of multivitamins; more preferably, 300g / L of glucose and 0.1mg / L of multivitamins. The vitamin complex comprises the following components in parts by weight: 0.08 to 0.12 parts of vitamin B1, 120.08-0.12 parts of vitamin B, 0.08-0.12 parts of vitamin B2, 0.08-0.12 parts of vitamin B3, 0.08-0.12 parts of vitamin B5, 0.08-0.12 parts of vitamin B6, 0.08-0.12 parts of vitamin B9, 0.08-0.12 parts of vitamin C and 0.08-0.12 parts of vitamin H. The addition of the above-mentioned complex vitamins during the fermentation process is beneficial to improving the elasticity and toughness of the mycelium. The present invention has no particular limitation on the source of the vitamins in the fermentation medium, and the vitamins can be purchased from sources well known in the art.

[0082] In the present invention, when the fermentation culture is carried out using fermentation medium No. 2, the fermentation culture conditions are as follows: an aeration rate of 900 to 1600 L / h, a pressure control of 0.04 to 0.12 MPa, a fermentation broth pH of 4.0 to 6.0, a fermentation culture temperature of 27° C. to 29° C., a rotation speed of 100 to 450 rpm, a coupled dissolved oxygen of 20% to 50%, and a fermentation culture time of 72 to 480 h; more preferably, an aeration rate of 1000 to 1400 L / h, a pressure control of 0.05 to 0.1 MPa, a fermentation broth pH of 5.0, a fermentation culture temperature of 28° C., a rotation speed of 280 rpm, and a coupled dissolved oxygen of 35%. During the fermentation culture, when the reducing sugar in the fermentation system is lower than 2 to 5 g / L or 3 to 4 g / L, 20% to 90% of the fermentation broth by volume is discharged and fermentation medium No. 2 is added to the original volume. The volume of the discharged fermentation broth preferably accounts for 30% to 80% of the original fermentation broth volume, more preferably 40% to 70%, and most preferably 50%. The addition of fermentation medium No. 2 to the original volume is preferably completed within 10 to 30 minutes, preferably within 20 minutes. During the fermentation culture, feeding is initiated once reducing sugar levels fall below 2 to 5 g / L. This feeding operation can be repeated multiple times.

[0083] In the present invention, after the fermentation is completed, the fermentation broth is preferably sterilized. The fermentation broth sterilization is beneficial for inactivating the live bacteria in the fermentation broth, and is also beneficial for further degradation of the nucleic acid molecules produced during the fermentation process, reducing the nucleic acid content in the fermented mycelium, and facilitating subsequent processing and application. The method for sterilizing the fermentation broth is preferably heat sterilization. The heat sterilization procedure is preferably to keep the temperature at 60°C to 70°C for 18 to 22 minutes, and to keep the temperature at 88°C to 92°C for 8 to 15 minutes; more preferably, to keep the temperature at 65°C for 20 minutes, and to keep the temperature at 90°C for 12 minutes.

[0084] In the present invention, after sterilization, the fermentation product is separated. The method for separating the fermentation product preferably includes separating the solid and liquid of the fermentation liquid, and drying the solid phase after washing. The solid-liquid separation method includes filtration, centrifugation, etc. In one embodiment of the present invention, the method for separating the fermentation product is preferably to filter with a vacuum filtration device and thoroughly wash the filter cake. After solid-liquid separation, the solid phase is collected and washed with pure water. The number of washings is preferably 1 to 3 times, more preferably 2 times. The drying is preferably oven drying. The drying temperature is preferably 55°C to 65°C, more preferably 60°C. Observing the above-mentioned filter cake, the mycelium morphology is good, filamentous, light yellow filter cake color, the filter cake is elastic, and the dry weight biomass percentage is 0.63% or more. The protein content determined by Kjeldahl nitrogen determination is 55.39% or more. After toxin detection, the filter cake does not contain toxins. At the same time, the mycelium produced by the Fusarium brevicaulis strain FXFB001 has excellent characteristics of being toxin-free and protein-rich mycelium. Therefore, the present invention provides the use of the Fusarium brevicaulis strain FXFB001 or the microbial agent in producing mycelium protein.

[0085] The present invention provides an edible mycelium. Calculated on the dry mass of the edible mycelium, the protein content in the edible mycelium is greater than 40%; and / or the dietary fiber content is greater than 20%; and / or the fat content is less than 8%. The edible mycelium is derived from Fusarium brachygibbosum, for example, the edible mycelium is obtained by fermenting Fusarium brachygibbosum.

[0086] In the present invention, the protein content of the edible mycelium is preferably 45%, 50%, 55%, 60% or 65% or more. The dietary fiber content of the edible mycelium is preferably 25%, 30%, 35%, 40% or 45% or more. The fat content of the edible mycelium is preferably 7%, 6%, 5%, 4% or 3% or less.

[0087] In the present invention, the edible mycelium preferably comprises a strain derived from Fusarium brachygibbosum with a deposit number of CGMCC No: 41066. The elasticity and toughness of the edible mycelium are improved by adding the complex vitamins during the fermentation and cultivation process.

[0088] In the present invention, the fermentation method is the same as the method for producing edible mycelium using Fusarium brevicaulis described in the aforementioned technical solution. The edible mycelium produced by fermentation is preferably filamentous in appearance, flesh-colored, and elastic. Physical and chemical testing has shown that the protein-rich mycelium is toxin-free and safe for consumption.

[0089] Since the edible mycelium is rich in protein, it can be used as a raw material for protein replacement in industrial production. The present invention provides the use of the edible mycelium or the edible mycelium prepared by the above method in the preparation of meat substitutes.

[0090] In the present invention, the meat substitute as a processed product of edible mycelium is a meat substitute prepared from the edible mycelium without undergoing a structural process, such as a nutritional food, a health product, etc. The structural process preferably includes shearing.

[0091] In the present invention, a method for preparing a meat substitute is to process or further process protein-rich edible mycelium as a raw material to obtain various different products. The method for preparing the protein-rich mycelium into a nutritious food preferably includes preparing the nutritious food directly or with other auxiliary materials by extracting protein with higher purity from the protein-rich mycelium. Because the mycelium is rich in elasticity and toughness, and the rich protein contains a variety of essential amino acids, the nutritional value of the prepared food product can be greatly improved. The food product is preferably presented in the form of a nutritional supplement. Similarly, food products can be formulated to enhance the taste or to be made into food products that are more attractive to consumers by combining with ordinary foods. In certain embodiments, the composition of the present invention is formulated as a milk-based product or a meat-based product. In one embodiment of the present invention, edible mycelium obtained by fermentation is used to prepare ham or sausage instead of meat raw materials (pork, beef, chicken, fish, mutton, rabbit meat and donkey meat, etc.). The term "milk-based product" means any liquid or semi-solid milk or whey-based product with a varying fat content. Milk-based products may be, for example, cow's milk, goat's milk, sheep's milk, skimmed milk, whole milk, milk reconstituted from milk powder and whey without any processing, or processed products such as yogurt, coagulated milk, curd, yogurt, acidified whole milk, buttermilk and other sour milk products. Another important group includes milk beverages such as whey beverages, fermented milk, condensed milk, baby or toddler milk; flavored milk, ice cream; milk-containing foods such as candy.

[0092] In the present invention, the method for preparing the protein-rich mycelium into a health product preferably includes preparing the health product directly or by extracting a higher purity protein from the protein-rich mycelium or by combining it with other active ingredients and excipients. The health product includes an acceptable excipient or carrier. Acceptable carriers or diluents for health care purposes are well known in the field of health care products. For example, the carrier preferably includes any one or more of the following: lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol and sorbitol, etc. or their analogs. The diluent preferably includes ethanol, glycerol and water or other solvents. The selection of pharmaceutical carriers, excipients or diluents can be selected taking into account the intended route of administration and standard pharmaceutical / health care product practices. In addition to the carrier, excipient or diluent, the health product may contain any suitable adhesive, lubricant, suspending agent, coating agent, solubilizer. Binders include starch, gelatin, and natural sugars such as glucose, anhydrous lactose, free-flowing lactose, and beta-lactose; corn sweeteners; natural and synthetic gums such as gum arabic, gum tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Preservatives, stabilizers, dyes, and even flavorings may be provided in the health supplement. Preservatives include sodium benzoate, sorbic acid, cysteine, and esters of parahydroxybenzoic acid. Antioxidants and suspending agents may also be used. Another example of a suitable carrier is sucrose. Another example of a preservative is cysteine.

[0093] When used in conjunction with the term "comprising" in the claims and / or the specification, the word "a" or "an" can mean "one", but can also mean "one or more", "at least one" and "one or more than one".

[0094] As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0095] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or as mutually exclusive between alternatives.

[0096] When used in the claims or description, a selected / optional / preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

[0097] "Edible mycelium" refers to a fungal fermentation culture or concentrate, or an edible mycelium concentrate obtained by dehydrating and removing nucleic acids from the fungal fermentation culture. Depending on the moisture content of the dehydrated concentrate, the edible mycelium can be in a solid state or a mixture of solid and liquid.

[0098] "Mycelium" refers to the vegetative growth portion of a fungus, composed of hyphae, and is an important source of fungal protein. The mycelium in the present disclosure is derived from a fungus of the genus Fusarium, and the edible mycelium is obtained after the fungus is fermented. In some embodiments, the edible mycelium is derived from a fermentation culture of a single species of fungus. In some embodiments, the edible mycelium is derived from a combined fermentation culture of multiple species of fungi.

[0099] The application of the Fusarium brevicaulis provided by the present invention in the production of mycelial protein is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0100] Example 1

[0101] Isolation and Screening Method of a Fusarium brevicaulis Strain FXFB001

[0102] 1. Strain Source

[0103] 1.1 Sample Collection: Strain FXFB001 was collected from a forest humus soil sample in Shannan City, Tibet Autonomous Region in June 2018;

[0104] 1.2 Separation process:

[0105] Mix the forest humus soil sample, weigh 5 g, and place it in a triangular flask containing 45 mL of sterile 0.7% sodium carboxymethyl cellulose solution and 15 glass beads. Oscillate at 30°C and 150 rpm for 30 min. Take 1 mL of the soil suspension and shake it with sterile water for 10 min. -1 , 10 -2 , 10 -3 Serial concentration gradient dilution, then take 10 -2 , 10 -3 Spread both dilutions onto MGA2.5 plates (containing chloramphenicol and tetracycline hydrochloride) at a rate of 100 μL per plate. Spread evenly, allow the plates to dry, and then incubate inverted at 28°C for 2–7 days. MGA2.5 medium contains the following components: peptone 15 g / L, KH2PO4 1.0 g, MgSO4·7H2O 0.5 g, malachite green oxalate 0.0025 g, agar 20 g, and distilled water 1 L.

[0106] 1.3 Purification process:

[0107] Purification was performed using the hyphal tip transplantation method. After colonies formed on the plate, hyphae at the edge of a single colony were picked and plated on malt extract agar / wort agar (MEA, purchased from Beijing Aoboxing Biotechnology Co., Ltd.). Culture was continued at 28°C until pure colonies were obtained. The resulting colonies were stored at -80°C and designated strain FXFB001.

[0108] 2. Strain Identification

[0109] 2.1 Morphological characteristics of strain FXFB001

[0110] Strain FXFB001 was inoculated into MEA medium and cultured at 28°C for 4 days. The strain was identified based on growth rate, hyphal shape, colony color, the number and shape of microconidia and macroconidia, conidial cells, the shape and presence of chlamydospores, and fruiting body type.

[0111] Culture characteristics: The diameter of the colony after 4 days is 3.8 cm. The aerial hyphae growing on PSA medium are felt-like, white at first and then gradually turn pink. The back of the plate is orange-red to dark red (see Figure 1).

[0112] Morphological characteristics:

[0113] Conidia: Large conidia are sickle-shaped, slightly pointed at both ends, with 3–5 septa, and measure 10.8–25.1 μm x 2.0–3.6 μm. Small conidia are fusiform, slightly curved, slightly flattened at both ends, and have 0–2 septa. Chlamydospores are spherical, solitary within the hyphae, and measure 5.2–7.5 μm.

[0114] 2.2 Molecular biological identification of strain FXFB001

[0115] 2.2.1 TEF1 and ITS sequence analysis

[0116] 1. DNA Template Preparation

[0117] Transfer an appropriate amount of mycelium from the strain to a PCR tube containing 50 μL of lysis buffer. Place the tube in a PCR instrument (Dongsheng ETC811), heat at 95°C for 30 minutes, and freeze at -20°C for 15 minutes. Centrifuge the tube (SIGMA 3K15) at 3000 rpm for 2 minutes to allow cell debris and lysis buffer to settle to the bottom of the tube. The supernatant should be nearly clear, and the fungal genetic material should be dissolved in the supernatant.

[0118] 2. PCR System Preparation

[0119] Prepare 25 μL of PCR system: 2 μL of supernatant, 8.8 μL of ultrapure sterile water, 12 μL of 2×PCR Mix, 1 μL of DMSO, 0.6 μL of 10 μmol / L upstream primer, and 0.6 μL of 10 μmol / L downstream primer. The primer sequences are shown in Table 1:

[0120] Table 1 Amplification primer sequences of two genes

[0121] 3. PCR Amplification

[0122] Add 2 μL of supernatant and the prepared PCR system to a PCR tube and perform PCR amplification using a PCR instrument (Dongsheng ETC811). The PCR reaction conditions are shown in the table below. After amplification, the PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing using a sequencer (abi3730XL).

[0123] PCR reaction conditions are as follows:

[0124] TEF1: pre-denaturation at 94°C for 3 min; 34 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and amplification at 72°C for 1 min; and then maintaining at 72°C for 5 min.

[0125] ITS1: pre-denaturation at 94°C for 2.5 min; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, amplification at 72°C for 1 min, 30 cycles; and maintaining at 72°C for another 5 min.

[0126] 4. The PCR product was sequenced. The resulting PCR product sequence is as follows:

[0127] TEF1 sequence:

[0128] ITS1 sequence:

[0129] The two gene sequences were aligned using the NBCI online alignment system (Blast), and the alignment database was the Nucleotide collection (nr / nt). The most similar alignment results of the two genes are shown in Table 2 ;

[0130] Table 2 Comparison results of the two gene sequences of the strains

[0131] The sequences of TEF1 and ITS1 genes of all FSAMSC 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.

[0132] The steps of multi-gene phylogenetic tree analysis are as follows: first, MAFFT (version v7.310) was used to perform multiple sequence alignment for each gene, and then GBLOCKS (version 0.91b) was used to trim the multiple sequence alignment result file. Then, AliView was used for manual trimming. After trimming, the sequences of the two genes were concatenated and spliced. The spliced ​​sequences were used to construct a multi-gene joint phylogenetic tree using IQ-TREE (version 2.2.5) as shown in Figure 2.

[0133] Based on morphological characteristics, ITS1 and TEF1 sequence analysis, strain FXFB001 was identified as Fusarium brachygibbosum. The strain was deposited with the China General Microbiological Culture Collection on December 13, 2023, with the deposit number CGMCC No: 41066.

[0134] Example 2

[0135] Preliminary screening experiment of strains producing mycelial protein

[0136] 1. Preliminary screening method for preparing mycelial protein

[0137] Strain FXFB001 isolated and identified in Example 1 and other microbial strains isolated from the same batch were inoculated into 50 mL of screening medium in 250 mL concave Erlenmeyer flasks and cultured with shaking at 200 rpm for 48 hours. The screening medium formulation consisted of 30 g / L glucose, 30 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 0.2 g / L CaCl2. The medium was sterilized by high-pressure steam at 121°C for 20 minutes.

[0138] Filter with a vacuum filtration device and wash the filter cake thoroughly, place it in an electric blast drying oven at 60°C, accurately weigh the mass of the dried filter cake and calculate the biomass concentration, and then refer to "GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food" and use the Kjeldahl method to determine the crude protein of mycelium.

[0139] 2 The results of the initial screening and preparation of mycelium are shown in Table 3.

[0140] Table 3 Summary of mycelia produced by primary screening strains Note: Biomass (%) = mycelial dry weight / culture medium volume × 100%; Protein content (%) = (crude protein mass / total cell mass) × 100%.

[0141] Strain No. 41 is strain FXFB001. The mycelium produced by fermentation with strain FXFB001 exhibits filamentous hyphae, a light yellow filter cake color, and a springy texture (Figure 3). The dry weight biomass reaches 0.63%, and the mycelial protein content, as determined by Kjeldahl nitrogen determination, is 55.39%. Overall, the protein-rich mycelium produced by strain FXFB001 exhibits no spores under microscopic examination, excellent filamentous texture, and a light yellow, springy filter cake with no unpleasant odor.

[0142] Example 3

[0143] Amplification and fermentation of mycelium from strain FXFB001 and several microbial strains obtained from its initial screening

[0144] 1. Culture method

[0145] 1.1 The shake flask seed culture medium used is shown in Table 4 (culture medium for primary seed culture and secondary seed culture) and Table 5 (culture medium for tertiary seed culture).

[0146] Table 4 Culture medium for primary seed culture and secondary seed culture

[0147] Table 5 Three-level seed culture medium

[0148] 1.2 Seed liquid culture conditions: The preparation of the first-level seed liquid uses the seed culture medium of Table 4 above, and the strain of the present invention of picking activation (select the mycelial morphology in the seed liquid and fermentation liquid, the strain with good various indicators, see Table 7) is inoculated into a 250mL three-concave conical flask containing 50mL primary screening culture medium, and cultured at 200rpm constant temperature shaking for 24h. The preparation of the second-level seed liquid uses the seed culture medium of Table 4 above, and is transferred to a 3L three-concave conical flask containing 1L seed culture medium at a 5% inoculum size, and the shaking table speed is 200rpm for 24h; The second-level seed liquid is inoculated into a fermenter with a liquid volume of 10L and the seed culture medium shown in Table 5 according to an inoculum size of 8%, with a ventilation rate of 10L / min, a tank pressure of 0.05Mpa, a stirring speed of 150rpm, a temperature of 28°C, an online ammonia solution for adjusting the pH to 5.0, and a fermentation time of about 24h.

[0149] 1.3 Fermentation and cultivation to produce protein-rich mycelium:

[0150] 1.3.1 Fermentation medium No. 1 for producing protein-rich mycelium is shown in Table 6.

[0151] Table 6 Fermentation medium No. 1

[0152] Among them, the multivitamin used in this embodiment includes the following components in equal mass ratios: vitamin B1 / thiamine, vitamin B12 / cobalamin, vitamin B2 / riboflavin, vitamin B3 / niacin, vitamin B5 / D-calcium pantothenate, vitamin B6 / pyridoxine, vitamin B9 / folic acid, vitamin C, and vitamin H / D-biotin.

[0153] 1.4 Fermentation culture method is as follows:

[0154] The third-grade seed liquid cultured for 24 hours was pumped into a 50-L fermentation tank with a liquid capacity of 30 L through a sterilized hose from the bottom valve at a 5% inoculation rate; the ventilation rate for the first 15 hours was 1000 L / h and the tank pressure was 0.05 MPa; after 15 hours, feeding was started at a rate of 0.5 mL / L / h, and the feed ingredients included 300 g / L of glucose and 0.1 mg / L of multivitamins, and the ventilation was adjusted to 1500 L / h and the tank pressure was 0.1 MPa; ammonia water was used to control the pH to 6.0; the culture was carried out at 28° C.; the rotation speed was 100 rpm and the dissolved oxygen was 50% for 72 hours.

[0155] 1.5 Fermentation post-processing process

[0156] 1) Heat treatment: After fermentation, heat the fermentation liquid to 65°C and keep it warm for about 20 minutes, then heat it to 90°C and keep it warm for about 10 minutes, and then cool it to room temperature.

[0157] 2) Filtration and Refrigeration: The fermentation broth after heat treatment is filtered using a plate and frame filter press. After thorough washing and ventilation, the filter cake is removed and stored at 4°C.

[0158] 3) Testing: For crude protein determination, samples are pre-treated, dried, crushed, and passed through a 60-mesh sieve. Determination is then performed according to GB5009.5-2016 National Food Safety Standard - Determination of Protein in Foods. Common mycotoxins in food are tested by a third-party testing company, SGS.

[0159] The fermentation results of each strain are shown in Table 7.

[0160] Table 7 Fusarium 50L fermenter rescreening results

[0161] The mycelia obtained by fermenting Fusarium brevis strain FXFB001 in fermentation medium No. 1 supplemented with multivitamins were compared with the results of the preliminary screening and preparation of protein-rich mycelia in Example 2:

[0162] 1. Preliminary screening and preparation of mycelium: no spores are found under microscopic examination, the filter cake is grayish white, has good elasticity and toughness, and no unpleasant odor;

[0163] 2. The mycelial protein obtained by continuous fermentation culture was well-filamented under microscopic examination (Figures 4 and 5), allowing the fermentation liquid to naturally appear flesh-colored without being heated; the protein content was approximately 55%.

[0164] From the toxin test results, it can be seen that under the above fermentation culture conditions, the fungal toxins contained in the strain FXFB001 were not detected (below the limit, see Table 8, and the detection method refers to the SN / T3136-2012 standard), the total fat content was 7.97%, the crude protein content was 60.51%, and the dietary fiber content was 26.01%.

[0165] Table 8 Summary of physical and chemical test results

[0166] Example 4

[0167] A method for producing protein-rich mycelium by fermenting Fusarium brevicaulis

[0168] 1. Culture method

[0169] 1.1 The components of the shake flask seed culture medium used are shown in Table 8 (culture medium for primary seed culture and secondary seed culture) and Table 9 (culture medium for tertiary seed culture).

[0170] Table 8 Culture medium for primary seed culture and secondary seed culture

[0171] Table 9 Three-level seed culture medium

[0172] 1.2 Seed liquid culture conditions: Preparation of the first-level seed liquid Use the seed culture medium shown in Table 8, pick the activated strain FXFB001 and inoculate it into a 250mL three-concave conical flask containing 50mL of the culture medium, and culture it at a constant temperature of 180rpm for 26h. Preparation of the second-level seed liquid Use the seed culture medium shown in Table 8, transfer it to a 3L three-concave conical flask containing 1L of seed culture medium at a 6% inoculum, shake at 220rpm, and culture for 22h; According to the inoculum of 10%, the second-level seed liquid is inoculated into a fermenter with a liquid volume of 10L filled with the culture medium described in Table 9, with an aeration rate of 11L / min, a tank pressure of 0.06Mpa, a stirring speed of 220rpm, a temperature of 26°C, an online ammonia solution to adjust the pH to 6.0, and a fermentation time of 36h.

[0173] 1.3 Fermentation and cultivation to produce protein-rich mycelium:

[0174] 1.3.1 The fermentation medium for producing protein-rich mycelium is shown in Table 10.

[0175] Table 10 Fermentation medium No. 2

[0176] 1.4 Fermentation culture method is as follows:

[0177] The tertiary seed liquid cultured for 22 hours was pumped from the bottom valve into a 50-L fermentation tank containing fermentation medium No. 2 shown in Table 10 with a 30-L liquid capacity at a 7% inoculum rate through a sterilized hose. The fermentation conditions were an aeration rate of 1100 L / h and a tank pressure of 0.05 MPa. When the reducing sugar in the fermentation medium was less than 2 g / L, 80% of the medium was discharged and then fed to the original volume within 20 minutes. The feed medium was fermentation medium No. 2, and the aeration rate was adjusted to 1400 L / h and the tank pressure was 0.1 MPa. The pH value of the system was controlled to 5.2 with ammonia water. The culture was carried out at 28° C. The rotation speed was 350 rpm and the dissolved oxygen was 40%, and the culture was carried out for 480 hours.

[0178] 1.5 Fermentation post-processing process

[0179] 1) Heat treatment: After fermentation, heat the fermentation liquid to 65°C and keep it warm for about 20 minutes, then heat it to 90°C and keep it warm for about 10 minutes, and then cool it to room temperature.

[0180] 2) Filtration and refrigeration: The fermentation broth after heat treatment is filtered using a plate and frame filter press. After thorough washing and ventilation, the filter cake is removed and stored at 4°C.

[0181] 3) Testing: For crude protein determination, samples are pre-treated, dried, crushed, and passed through a 60-mesh sieve. Determination is then performed according to GB5009.5-2016 National Food Safety Standard - Determination of Protein in Foods. Common mycotoxins in food are tested by a third-party testing company, SGS.

[0182] Under the above fermentation culture conditions, no toxins were detected in the mycelium produced by the strain FXFB001. On a dry basis, the mycelium contained 5.8% total fat, 54.2% crude protein and 20.4% dietary fiber.

[0183] Example 5

[0184] 1. Culture method

[0185] 1.1 The components of the shake flask seed culture medium used are shown in Table 11 (culture medium for primary and secondary seed culture) and Table 12 (culture medium for tertiary seed culture).

[0186] Table 11 Culture medium for primary seed culture and secondary seed culture

[0187] Table 12 Tertiary seed culture medium

[0188] 1.2 Seed liquid culture conditions: The preparation of the first-level seed liquid uses the culture medium shown in Table 11, picks the activated strain FXFB001 and inoculates it into a 250mL three-concave conical flask containing 50mL of the culture medium, and cultures it at a constant temperature of 200rpm for 24h. The preparation of the second-level seed liquid uses the seed culture medium shown in Table 11, transfers it to a 3L three-concave conical flask containing 1L seed culture medium at a 5% inoculum, shakes it at 200rpm, and cultures it for 24h; the second-level seed liquid is inoculated into a fermenter with a liquid volume of 10L and the culture medium shown in Table 12 at an 8% inoculum, with an aeration rate of 10L / min, a tank pressure of 0.05Mpa, a stirring speed of 150rpm, a temperature of 27°C, and an online ammonia solution to adjust the pH to 5.5. The fermentation time is 24h.

[0189] 1.3 Fermentation and cultivation to produce protein-rich mycelium:

[0190] 1.3.1 The composition of the fermentation medium for producing protein-rich mycelium is shown in Table 13.

[0191] Table 13 Fermentation medium No. 1

[0192] The vitamin complex comprises vitamins in the following mass ratios: vitamin B1 / thiamine 0.1 mg, vitamin B12 / cobalamin 0.1 mg, vitamin B2 / riboflavin 0.1 mg, vitamin B3 / niacin 0.1 mg, vitamin B5 / D-calcium pantothenate 0.1 mg, vitamin B6 / pyridoxine 0.1 mg, vitamin B9 / folic acid 0.1 mg, vitamin C 0.1 mg, and vitamin H / D-biotin 0.1 mg.

[0193] 1.4 Fermentation culture method is as follows:

[0194] The tertiary seed liquid cultured for 24 hours was pumped from the bottom valve into a 50-L fermenter containing 25 L of fermentation medium No. 1 shown in Table 13 at a 10% inoculum rate through a sterilized hose; the ventilation rate for the first 15 hours was 900 L / h, and the tank pressure was 0.04 MPa; after 15 hours, feeding was started at 2 ml / L / h with a feed medium consisting of 300 g / L glucose and 0.1 mg / L multivitamins, and the ventilation was adjusted to 1600 L / h and the tank pressure to 0.1 MPa; the pH was controlled at 4.5 with ammonia water; the culture was carried out at 28°C; the rotation speed was 100 rpm, coupled with 30% dissolved oxygen, and the culture was carried out for 70 hours.

[0195] 1.5 Fermentation post-processing process

[0196] 1) Heat treatment: After fermentation, heat the fermentation liquid to 65°C and keep it warm for about 20 minutes, then heat it to 90°C and keep it warm for about 10 minutes, and then cool it to room temperature.

[0197] 2) Filtration and refrigeration: The fermentation broth after heat treatment is filtered using a plate and frame filter press. After thorough washing and ventilation, the filter cake is removed and stored at 4°C.

[0198] 3) Testing: For crude protein determination, samples are pre-treated, dried, crushed, and passed through a 60-mesh sieve. Determination is then performed according to GB5009.5-2016 National Food Safety Standard - Determination of Protein in Foods. Common mycotoxins in food are tested by a third-party testing company, SGS.

[0199] Under the above fermentation culture conditions, no toxins were detected in the mycelium produced by the strain FXFB001. On a dry basis, the mycelium contained 8% total fat, 60.5% crude protein and 26.0% dietary fiber.

[0200] Example 6

[0201] A method for producing protein-rich mycelium by fermenting Fusarium brevicaulis

[0202] 1. Culture method

[0203] 1.1 The components of the shake flask seed culture medium used are shown in Table 14 (culture medium for primary and secondary seed culture) and Table 15 (culture medium for tertiary seed culture).

[0204] Table 14 Culture medium for primary seed culture and secondary seed culture

[0205] Table 15 Three-level seed culture medium

[0206] 1.2 Seed liquid culture conditions: Preparation of the first-level seed liquid Using the seed culture medium shown in Table 14, the activated strain FXFB001 of the present invention was picked and inoculated into a 250mL three-concave conical flask containing 50mL of the culture medium, and cultured at a constant temperature of 200rpm for 22h. Preparation of the second-level seed liquid Using the seed culture medium shown in Table 14, it was transferred to a 3L three-concave conical flask containing 1L of seed culture medium at a 6% inoculum, shaken at 220rpm, and cultured for 22h; According to the inoculum of 10%, the second-level seed liquid was inoculated into a fermenter with a liquid volume of 10L and the culture medium shown in Table 15, with an aeration rate of 11L / min, a tank pressure of 0.06Mpa, a stirring speed of 220rpm, a temperature of 28°C, an online ammonia adjustment system pH of 5.0, and a fermentation time of 42h.

[0207] 1.3 Fermentation and cultivation to produce protein-rich mycelium:

[0208] 1.3.1 The fermentation medium for producing protein-rich mycelium is shown in Table 16.

[0209] Table 16 Fermentation medium No. 2

[0210] 1.4 Fermentation culture method is as follows:

[0211] The tertiary seed liquid cultured for 22 hours was pumped from the bottom valve into a 50-L fermentation tank containing fermentation medium No. 2 shown in Table 16 with a liquid capacity of 30 L at a 7% inoculum rate through a sterilized hose; the fermentation conditions were an aeration rate of 1100 L / h and a tank pressure of 0.05 MPa; when the reducing sugar in the fermentation system was lower than 5 g / L, 50% of the culture medium was released and then fed to the original volume within 20 minutes using fermentation medium No. 2, and the aeration was adjusted to 1400 L / h and the tank pressure was 0.1 MPa; the pH value of the system was controlled to 5.0 with ammonia water; the culture was cultured at 28°C; the rotation speed was 350 rpm and the dissolved oxygen was 40% for 120 hours.

[0212] 1.5 Fermentation post-processing process

[0213] 1) Heat treatment: After fermentation, heat the fermentation liquid to 65°C and keep it warm for about 20 minutes, then heat it to 90°C and keep it warm for about 10 minutes, and then cool it to room temperature.

[0214] 2) Filtration and Refrigeration: The fermentation broth after heat treatment is filtered using a plate and frame filter press. After thorough washing and ventilation, the filter cake is removed and stored at 4°C.

[0215] 3) Testing: For crude protein determination, samples are pre-treated, dried, crushed, and passed through a 60-mesh sieve. Determination is then performed according to GB5009.5-2016 National Food Safety Standard - Determination of Protein in Foods. Common mycotoxins in food are tested by a third-party testing company, SGS.

[0216] Under the above fermentation culture conditions, no toxins were detected in the mycelium produced by the strain FXFB001. On a dry basis, the mycelium contained 8.0% total fat, 60.5% crude protein and 26.0% dietary fiber.

[0217] Example 7

[0218] A microbial agent comprising strain FXFB001

[0219] The Fusarium brevicaulis strain FXFB001 was expanded and cultured according to the method described in Example 3 to obtain a third-grade seed solution, which was then added with 50% glycerol and stored at -80°C.

[0220] Example 8

[0221] A method for preparing a meat substitute (edible fungus protein-based sausage) using the mycelium of Example 5

[0222] Accurately weigh the following raw materials: 40 kg of edible mycelium, 58 kg of ice water, 0.1 kg of MSG, 0.7 kg of yeast extract, 1 kg of edible sugar, 0.5 kg of meat flavoring, 3 kg of sunflower oil, 0.6 kg of edible salt, 0.6 kg of spices, 2 kg of starch, 2.5 kg of carrageenan, and 0.106 kg of paprika.

[0223] The preparation method comprises the following steps:

[0224] 1. Mix carrageenan, starch and part of the ice water, chop and stir evenly, add fungal protein, MSG, sugar, food flavoring, vegetable oil, edible salt, spices, pigment and the remaining ice water, and mix evenly;

[0225] 2. The meat filling is filled into nylon casings through a filling machine, the sausages are hung on a rod, put into a smoking oven, dried at 60℃ for 20 minutes, and steamed at 95℃ for 20 minutes;

[0226] 3. Take out and blow with cold air to below 25℃ and then pack;

[0227] 4. Quick freezing and storage.

[0228] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of Fusarium brachygibbosum or its microbial inoculum in the production of mycelial protein.

2. The application according to claim 1, characterized in that The Fusarium brachygibbosum is a Fusarium brachygibbosum strain having a TEF1 gene sequence with at least 95% homology to SEQ ID NO: 5 and / or an ITS1 gene sequence with at least 97% homology to SEQ ID NO: 6, or a composition formed by the Fusarium brachygibbosum strain.

3. The application according to claim 1, wherein The Fusarium brachygibbosum includes the Fusarium brachygibbosum strain FXFB001; The preservation number of the Fusarium brachygibbosum strain FXFB001 is CGMCC No: 41066.

4. The application according to any one of claims 1 to 3, characterized in that The Fusarium brachygibbosum includes spore morphology and / or mycelial morphology.

5. An edible mycelium, characterized in that, Based on the dry mass of the edible mycelium, the mass percentage of protein in the edible mycelium is more than 40%; and / or the mass percentage of dietary fiber is more than 20%; and / or the mass percentage of fat is less than 8%; The edible mycelium is derived from Fusarium brachygibbosum; The Fusarium brachygibbosum is the Fusarium brachygibbosum in any one of the applications described in claims 2 to 4.

6. The edible mycelium according to claim 5, characterized in that, The edible mycelium is elastic and / or tough.

7. A strain of Fusarium brachygibbosum, namely FXFB001, characterized in that, The preservation number is CGMCC No: 41066.

8. A microbial inoculum for producing proteins or protein-rich mycelia, characterized in that, It includes the Fusarium brachygibbosum strain FXFB001 described in claim 7 and excipients acceptable for the inoculum.

9. The microbial inoculum for producing a protein or a protein-rich mycelium according to claim 8, characterized in that, The mass ratio of the Fusarium brachygibbosum strain FXFB001 to the excipients is 1 - 10:1 - 100.

10. The microbial inoculum for producing protein or protein-rich mycelia according to claim 8 or 9, characterized in that, The excipients include one or several of culture medium, additives and carriers.

11. Use of the Fusarium brachygibbosum strain FXFB001 described in claim 7 or the microbial inoculum described in any one of claims 8 to 10 in the production of protein or protein-rich mycelium.

12. A method for producing edible mycelia using Fusarium brachygibbosum, characterized in that, It includes the following steps: Inoculate the seed liquid prepared from Fusarium brachygibbosum or its microbial inoculum into the fermentation medium for fermentation culture to obtain a fermentation broth; After sterilizing the fermentation broth, separate the fermentation product to obtain edible mycelium; The Fusarium brachygibbosum is the Fusarium brachygibbosum in any one of the applications described in claims 2 to 4.

13. The method according to claim 12, wherein The fermentation medium includes Fermentation Medium No. 1 and / or Fermentation Medium No. 2; Fermentation Medium No. 1 includes the following components in the following contents: starch 23 - 27 g / L, citric acid 0.8 - 1.2 g / L, potassium dihydrogen phosphate 0.8 - 1.2 g / L, ammonium sulfate 4.5 - 5.5 g / L, magnesium salt calculated by Mg concentration 17 - 21 mg / L, calcium salt calculated by Ca concentration 2.8 - 43 mg / L, manganese salt calculated by Mn concentration 0.48 - 0.62 mg / L, iron salt or ferrous salt calculated by Fe concentration 0.96 - 1.05 mg / L, zinc salt calculated by Zn concentration 0.72 - 0.91 mg / L, cobalt salt calculated by Co concentration 0.44 - 0.54 mg / L, and complex vitamins 0.08 - 0.12 mg / L; The fermentation medium No. 2 comprises the following components in the following amounts: glucose 15 - 60 kg / m 3 , K2SO4 0.5 - 1.5 kg / m 3 , H3PO4 0.4 - 0.8 kg / m 3 , magnesium salt calculated by the concentration of Mg 9.75 - 58.5 g / m 3 , zinc salt calculated by the concentration of Zn 2.2 - 22.5 g / m 3 , manganese salt calculated by the concentration of Mn 2.46 - 9.85 g / m 3 , calcium acetate 0.1 - 0.3 kg / m 3 and antifoaming agent 0.08 - 0.12 kg / m 3 .

14. The method according to claim 13, wherein When using Fermentation Medium No. 1 for fermentation, the conditions for the fermentation culture include: Within the first 15 h of fermentation culture, the ventilation rate is 900 - 1100 L / h, and the pressure is controlled at 0.04 - 0.06 MPa; After 15 h of fermentation culture, feeding starts at a rate of 0.5 - 2.0 mL / L / h. The ventilation rate is 1400 - 1600 L / h, and the pressure is 0.08 - 0.12 MPa; the pH value of the fermentation broth is 4.0 - 6.0; The temperature of fermentation culture is 27°C - 29°C, the rotation speed is 100 - 450 rpm, the coupled dissolved oxygen is 20% - 50%, and the fermentation culture time is 68 - 76 h; When feeding, the feeding components include 280 - 320 g / L of glucose and 0.08 - 0.12 mg / L of compound vitamins; The compound vitamins include the following components by mass: 0.08 - 0.12 parts of vitamin B1, vitamin Vitamin B 12 0.08 - 0.12 parts, riboflavin 0.08 - 0.12 parts, niacin 0.08 - 0.12 parts, pantothenic acid 0.08 - 0.12 parts, pyridoxine 0.08 - 0.12 parts, folic acid 0.08 - 0.12 parts, ascorbic acid 0.08 - 0.12 parts and biotin 0.08 - 0.12 parts.

15. The method according to claim 13, wherein When the fermentation culture is carried out using fermentation medium No. 2, the fermentation culture conditions are that the ventilation rate is 900 - 1600 L / h, and the pressure is controlled at 0.04 - 0.12 MPa; the pH value of the fermentation broth is 4.0 - 6.0; The temperature of fermentation culture is 27°C - 29°C, the rotation speed is 100 - 450 rpm, the coupled dissolved oxygen is 20% - 50%, and the fermentation culture time is 72 - 480 h; When the reducing sugar content in the fermentation system is lower than 2 - 5 g / L, 20% - 90% of the fermentation broth is discharged, and fermentation medium No. 2 is added to make up to the original volume.

16. The method according to claim 12, wherein The method for preparing the seed liquid of Fusarium oxysporum f. sp. cubense includes the following steps: Inoculate the Fusarium oxysporum f. sp. cubense into the first seed medium and carry out primary seed culture and secondary seed culture in sequence to obtain the secondary seed liquid; Inoculate the secondary seed liquid into the second seed medium for tertiary seed culture to obtain the tertiary seed liquid.

17. The method according to claim 16, wherein The first seed medium contains the following components: 28 - 32 g / L of glucose, 23 - 27 g / L of yeast extract powder, 0.8 - 1.2 g / L of potassium dihydrogen phosphate, 17 - 21 mg / L of magnesium salt calculated by Mg, and 3 - 4 mg / L of calcium salt calculated by Ca; The second seed medium contains the following components: 23 - 27 g / L of starch, 0.8 - 1.2 g / L of citric acid, 0.8 - 1.2 g / L of potassium dihydrogen phosphate, 4.8 - 5.2 g / L of ammonium sulfate, 17 - 21 mg / L of magnesium salt calculated by Mg, 0.003 - 0.004 g / L of calcium salt calculated by Ca, 0.48 - 0.62 mg / L of manganese salt calculated by Mn, 0.96 - 1.05 mg / L of iron salt or ferrous salt calculated by Fe, 0.72 - 0.91 mg / L of zinc salt calculated by Zn, and 0.44 - 0.54 mg / L of cobalt salt calculated by Co.

18. The method according to claim 16 or 17, wherein The culture temperature for the primary seed culture, secondary seed culture or tertiary seed culture is 25°C - 30°C; The culture time for the primary seed culture, secondary seed culture or tertiary seed culture is 22 - 26 h; The rotation speed for the primary seed culture, secondary seed culture or tertiary seed culture is 100 - 200 rpm; The aeration rate of the tertiary seed culture is 8 - 12 L / min; The pressure of the tertiary seed culture is 0.04 - 0.06 MPa; The pH value of the system of the tertiary seed culture is 4.0 - 6.

0.

19. The method according to claim 12, wherein The method for sterilizing the fermentation broth is heat sterilization; The procedure of the heat sterilization is to keep the temperature at 60°C - 70°C for 18 - 22 min, and then raise the temperature to 88°C - 92°C and keep it for 8 - 15 min.

20. The method according to claim 12, wherein The method for separating the fermentation product includes solid-liquid separation of the fermentation broth, and the solid phase is washed and then dried to obtain edible mycelia.

21. Use of the edible mycelia as claimed in claim 5 or 6 or the edible mycelia prepared by the method as claimed in any one of claims 12 - 20 in the preparation of meat substitutes.

22. A meat substitute, characterized in that, Prepared by using the edible mycelia as claimed in claim 5 or 6 or the edible mycelia prepared by the method as claimed in any one of claims 12 - 20 as a meat raw material.

23. The meat substitute according to claim 22, wherein Including ham sausage or sausage.

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