Aspergillus niger with high mycelial protein production, bacterial agent, application and method for high mycelial protein production

Abstract

The present invention belongs to the technical field of Aspergillus niger genetic engineering, and specifically relates to an Aspergillus niger with high mycelial protein production, a bacterial agent, an application and a method for high mycelial protein production. An ΔABSU The mycelial protein content of the strain increased by about 107.3% compared with the wild type, from 27.49% to 47.00%. An ΔABSU The hyphae of the strain are completely dispersed and no longer aggregate into balls, which is conducive to the recovery and film-forming of hyphae protein. AnΔ ABSU The growth rate of the strain was significantly accelerated, and the biomass reached 16.5g / L, which was 113.1% higher than that of the wild type. This strain has broad application prospects in microbial protein production.

Description

Technical Field

[0001] The invention belongs to the technical field of Aspergillus niger genetic engineering, and specifically relates to an Aspergillus niger with high mycelial protein production, a bacterial agent, an application and a method for high mycelial protein production. Background Art

[0002] As the global population continues to grow, protein demand is also increasing. It is estimated that by 2050, the global protein demand gap will reach more than 50% of current consumption. However, the existing protein sources mainly rely on animal husbandry, which not only causes serious environmental pollution, but also occupies a large amount of land and water resources. Therefore, finding new ways to produce protein has become an urgent need.

[0003] As a recognized safe fungus, Aspergillus niger has been certified as a safe strain by the US FDA. It has the advantages of good safety, short fermentation cycle, and continuous production. It is an ideal cell factory for producing mycelial protein. However, the mycelial protein content of wild-type Aspergillus niger is low (about 25%, on a dry basis), and the mycelium will cross-link to form a spherical structure, causing the mycelium in the ball to age excessively, resulting in too low mycelial protein content and high production costs. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] The present invention aims to provide a genetically modified Aspergillus niger strain. AnΔABSU The strain can significantly increase the yield of mycelial protein. The present invention uses CRISPR / Cas9 technology to modify the α-1,3-glucan synthase ( agS , aiZd ) and the key gene for galactosamine galactan synthase ( sph3 , uge3 ) were specifically knocked out in order to change the cell wall structure, thereby changing the mycelial morphology and increasing the mycelial protein content.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] Screening and identification of Aspergillus niger: Wild-type Aspergillus niger strains were screened out from soy sauce residue and confirmed by ITS sequencing.

[0008] Obtaining the target gene: Download the Aspergillus niger gene from NCBI agS , aiZd , sph3 , uge3 The gene sequence was obtained, and primers were designed using SnapGene, which was then amplified and verified using the Aspergillus niger genome in the laboratory as a template.

[0009] Construction of gene editing vector: Using CRISPR / Cas9 technology to construct agS , aiZd , sph3 , uge3 Gene knockout plasmid.

[0010] Obtaining Aspergillus niger mutants: The constructed knockout plasmid was introduced into Aspergillus niger through PEG-mediated protoplast transformation to obtain candidate gene-edited transformants, and positive gene-edited mutants were identified through sequencing comparison AnΔA , AnΔAB , AnΔABS and AnΔABSU .

[0011] Fermentation evaluation: Fermentation evaluation was performed on the wild type and mutants to measure indicators such as biomass, glucose consumption, crude protein and phenotype.

[0012] The invention provides a kind of Aspergillus niger with high mycelial protein production, wherein the Aspergillus niger is Aspergillus niger AnΔABSU , which is classified as Aspergillus niger ( Aspergillus niger ), Aspergillus niger ( Aspergillus niger ) AnΔABSU , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short), with the deposit number CGMCC NO.41621, the deposit time is November 8, 2024, and the address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] Provided is a bacterial agent containing the Aspergillus niger.

[0014] Provided is the use of the Aspergillus niger with high mycelial protein production or the bacterial agent in producing mycelial protein.

[0015] Provided is a method for high-yield mycelial protein, which comprises fermenting and culturing the Aspergillus niger with high mycelial protein yield or the bacterial agent in a fermentation medium, wherein the nitrogen source of the fermentation medium is an inorganic nitrogen source.

[0016] Preferably, the fermentation culture temperature is 26-30° C., the culture method is shaking culture, and the culture time is 3 days.

[0017] Preferably, the inorganic nitrogen source is ammonium sulfate 1-12 g / L.

[0018] Preferably, the inorganic nitrogen source is 10 g / L ammonium sulfate.

[0019] Preferably, the fermentation medium comprises: 1-80 g / L glucose, 1-20 g / L ammonium sulfate, 0.1-2 g / L potassium chloride, 0.1-3 g / L anhydrous magnesium sulfate, 0.1-1 g / L anhydrous sodium sulfate, 0.1-4 g / L potassium dihydrogen phosphate, 0.1-0.5 g / L calcium carbonate, and 0.1-1 g / L corn steep liquor powder.

[0020] Preferably, the bacterial cells after fermentation and culture are collected and dried.

[0021] The present invention has at least the following beneficial effects:

[0022] First, significantly increase crude protein content: through genetic modification, AnΔABSU The mycelial protein content of the strain increased by 107.3% compared with the wild type, from 27.49% to 47.00%.

[0023] Second, improve the mycelium morphology: AnΔABSU The mycelium of the strain is completely dispersed and no longer aggregates into balls, which is conducive to the extraction and utilization of mycelial protein.

[0024] Third, speed up the growth rate: AnΔABSU The growth rate of the strain was significantly accelerated, and the biomass reached 16.5g / L, an increase of 113.1% compared with the wild type.

[0025] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the morphology of Aspergillus niger hyphae;

[0027] Figure 2 This is the evolutionary tree of Aspergillus niger strains;

[0028] Figure 3 Schematic diagram for constructing mutant strains of Aspergillus niger (including schematic diagram of CRISPR / Cas9 gene knockout mechanism and plasmid construction process);

[0029] Figure 4 For liquid culture WT , AnΔA , AnΔAB , AnΔABS and AnΔABSU Phenotype diagram;

[0030] Figure 5 For transmission electron microscopy WT , AnΔABSU Diagram of the cell wall structure;

[0031] Figure 6In a stressful environment WT , AnΔABSU Colony growth status diagram;

[0032] Figure 7 This is the 250mL shake flask fermentation evaluation chart (including glucose consumption and biomass);

[0033] Figure 8 This is the evaluation chart of 250mL shake flask fermentation (including extracellular protein concentration and crude protein content);

[0034] Fig. 9 The volcano plot is for differentially expressed genes;

[0035] Fig.10 This is a GO enrichment analysis diagram;

[0036] Fig.11 This is the KEGG enrichment analysis diagram;

[0037] Fig.12 Optimize shake flask fermentation evaluation chart for carbon source (including biomass, crude protein content);

[0038] Fig.13 Optimization of shake flask fermentation evaluation chart for inorganic nitrogen source (including biomass, crude protein content);

[0039] Fig.14 Optimization of shake flask fermentation evaluation graphs (including biomass and crude protein content) for ammonium sulfate concentration;

[0040] Fig.15 Optimization of shake flask fermentation evaluation chart for organic nitrogen source (including biomass, crude protein content);

[0041] Fig.16 Schematic diagram for constructing mutant strains of Aspergillus niger ( agS , aiZd , sph3 , uge3 Mutation site alignment diagram).

[0042] Aspergillus niger Aspergillus niger ) AnΔABSU , its deposit number is CGMCC NO. 41621, and it was deposited in the General Microbiology Center of China Culture Collection Administration on November 8, 2024. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0044] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified; in the description of the present invention, the orientation or positional relationship indicated by the terms is based on the orientation or positional relationship shown in the accompanying drawings, which is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0045] The experimental scheme of the present invention includes:

[0046] Example 1

[0047] Bacteria screening and identification:

[0048] Take 1 g of soy sauce residue, dilute it into 100 mL of PDA saline, and culture it in a shaker at 28°C for 12 h. Take 10 μL of the dilution and spread it on PDA medium for 2 days. Pick out the filamentous fungi in a new medium, and screen out the wild-type Aspergillus niger species through ITS sequencing and comparison.

[0049] After screening, 12 strains of filamentous fungi were obtained. After ITS sequencing, the wild type Aspergillus niger was obtained. Its hyphae morphology is as follows Figure 1 As shown, the ITS comparison diagram is as follows Figure 2 shown.

[0050] Example 2

[0051] Construction of gene editing vector for Aspergillus niger mutants: Construction of gene editing vector for Aspergillus niger mutants agS , aiZd , sph3 , uge3 Gene knockout plasmid, gene knockout mechanism and plasmid construction process Figure 4 a and Figure 4 As shown in b.

[0052] Specific methods:

[0053] Obtain the target gene and download the Aspergillus niger gene from NCBI agS , agsB, sph3, uge3 The gene sequence was obtained by SnapGene, and primers were designed to verify the wild-type Aspergillus niger obtained in Example 1 as a template. agS , agsB、sph3、 uge3 Whether the four genes can be amplified in this genome.

[0054] The endogenous 5SrRNA promoter sequence was amplified from the DNA genome of wild-type Aspergillus niger using primer pair 5SrRNA-F / R, and the sgRNA was amplified using primer pair 5SrRNA-F / R. agsA-F / R expands sgRNA based on the synthetic gRNA scaffold fragment agsA , sequence. Then, two rounds of amplification were performed by fusion PCR to fuse the 5SrRNA fragment with the above sgRNA fragment to obtain 5SrRNA-sgRNA, and the fused fragment was connected to the backbone vector pFC322 / Cas9 by homologous recombinase to obtain sgRNA agsA Gene editing expression vector. After transforming it into E. coli DH5α, single colonies were selected for PCR verification to obtain positive transformants. sgRNA agsB , sgRNA sph3 , sgRNA uge3 The expression vector construction operation is the same as above. All primers are shown in Table 1:

[0055] Table 1 Primer sequences used for gene knockout

[0056]

[0057]

[0058] Example 3

[0059] Obtaining Aspergillus niger mutants:

[0060] The plasmid of the positive Escherichia coli obtained in Example 2 was extracted and introduced into Aspergillus niger by PEG-mediated protoplast transformation to obtain candidate gene-edited transformants. A simple DNA template of the candidate transformant was prepared by lysis method, and the target gene sequence was amplified from the simple template with a primer pair, and then sequencing and comparison were performed to identify the positive gene-edited mutants. Each mutant was named AnΔA , AnΔAB , AnΔABS and AnΔABSU , four mutant strains such as Fig.16 shown.

[0061] Example 4

[0062] Aspergillus niger shake flask fermentation:

[0063] The strain was inoculated on PDA solid medium and cultured for 5 days, and then 1×10 8 100 μL of the spore suspension was inoculated into 50 mL of fermentation medium and cultured on a shaker at 28°C and 180 rpm for 3 days. The fermentation medium contained 50 g / L glucose, 10 g / L ammonium sulfate, 0.7 g / L potassium chloride, 1.5 g / L anhydrous magnesium sulfate, 0.5 g / L anhydrous sodium sulfate, 2 g / L potassium dihydrogen phosphate, 0.2 g / L calcium carbonate, and 0.5 g / L corn steep liquor powder.

[0064] Example 5

[0065] Fermentation evaluation of Aspergillus niger mutants:

[0066] For the fermentation products of Example 4, the biomass, glucose consumption, crude protein and phenotype of the wild-type Aspergillus niger and the Aspergillus niger mutant were evaluated respectively. For the determination of glucose content, 1 mL of the fermentation broth was aspirated and filtered using a water filter membrane. 10 μL of the filtered fermentation broth was aspirated and added to 990 μL of deionized water, and diluted 10 times. The biosensor analyzer was calibrated using a glucose standard solution (25 μL, 1 g / L). After the calibration was completed, 25 μL of the diluted fermentation broth was taken for detection, and each fermentation sample was tested three times and the average value was taken.

[0067] The biomass was assessed using the dry weight method. First, several 9 cm filter papers were dried to a constant weight and their mass was recorded as M 1. Then take 50 mL of the fermentation liquid into a measuring cylinder and place the dried filter paper in a vacuum filtration device. After the fermentation liquid is filtered, the bacteria are enriched on the filter paper. Put the filter paper collecting the bacteria into an oven and set the temperature to 105℃ for drying. Measure the mass every 10 minutes until constant weight is reached and stop drying. Record the mass as M 2. The formula for biomass determination is as follows:

[0068]

[0069] For the determination of crude protein, the bacteria are collected and dried after fermentation. After drying, the bacteria are fully ground and mixed evenly. Then 3.2-3.8 mg of the sample is weighed and wrapped in tin foil. The crude protein content of the bacteria is determined by the Dumas combustion method. First, the prepared sample is subjected to a combustion reaction in a combustion reactor containing high-purity oxygen for a sufficient period of time. Secondly, a lot of mixed gases will be generated in this process, in addition to NO x In addition, other mixed gases and interfering substances will be absorbed by the absorption tube. x It will be reduced by helium or CO2 in the reduction furnace, and the nitrogen generated will be measured. For each group of samples, three groups are set up in parallel. The calculation formula for the protein content in the sample is as follows:

[0070]

[0071] Wherein, X: protein content in the sample (%); C: nitrogen content in the sample (%); 6.25: conversion coefficient for nitrogen content to protein content.

[0072] like Figure 7 As shown, in order to evaluate WT , AnΔA , AnΔAB , AnΔABS and AnΔABSU The growth of the strain, its biomass, and glucose consumption were measured and found to be AnΔABSU The glucose consumption of the plant was significantly higher than that of the wild type, and the biomass reached 16.5 g / L, which was 113.1% higher than that of the wild type. AnΔABSU The growth rate is significantly accelerated.

[0073] like Figure 8 To determine the effect of hyphae morphology on protein content, we measured the extracellular protein secretion and crude protein content of the wild type and each transformant. The results showed that AnΔABSU The extracellular protein secretion of the grape was significantly lower than that of the wild type, and the crude protein content increased by 107.3% compared with the wild type, reaching 47%.

[0074] Example 6

[0075] Observe the growth pattern:

[0076] Will WT , AnΔA , AnΔAB , AnΔABS and AnΔABSU Conidia of the strain (final concentration was 1×10 7 spores / mL) were inoculated into 50 mL of liquid fermentation medium in a 250 mL conical flask and rotated at 180 rpm for 36 h at 28 °C to observe the growth morphology of the strain in the liquid fermentation medium.

[0077] like Figure 3 As shown in the figure, in order to more intuitively observe the mycelial morphology of the Aspergillus niger mutant, the mycelial bodies in the 250mL shake flask were poured into a plate. The results showed that the mycelial balls formed by the wild-type strain were significantly larger than those of the strain AnΔAB , AnΔABSU The hyphae formed by the strain were completely dispersed.

[0078] Example 7

[0079] Transmission electron microscopy observation:

[0080] Will WT and AnΔABSU Conidia of the strain (final concentration was 1×10 7Spores / mL) were inoculated and cultured according to the method of Example 6, and the culture solution was centrifuged through a centrifuge tube to obtain a bacterial precipitate. Then, the sample was washed three times with PBS buffer and fixed at 4°C for 24 hours with 1 mL of 2.5% (v / v) glutaraldehyde solution. The sample was dehydrated in a graded ethanol series, replaced with ethanol in a one-to-one ratio, and embedded. Ultrathin sections were cut with an ultramicrotome Leica EM UC7, stained with uranyl acetate and lead citrate, and observed with a Hitachi HT7700 transmission electron microscope.

[0081] like Figure 5 As shown, the results found WT The cell wall is dense and uniform in composition. AnΔABSU The cell wall structure is destroyed and the cell surface is uneven. This indicates that α-1,3 glucan synthase ( agS , aiZd ) and galactosaminegalactan synthase ( sph3 , uge3 ) is essential for the normal growth of cells, participates in the biosynthesis of cell walls, and plays an important role in maintaining the integrity of cell walls.

[0082] Example 8

[0083] Manifestations of coercion:

[0084] like Figure 6 As shown, further exploration AnΔABSU Performance in different stress environments. WT , AnΔABSU 1 μL of spore suspension was taken and cultured in fermentation plates with different stress conditions (300 μg / mL CR, 150 μg / mL CFW) for three days. The fermentation plates with Congo red and calcofluor white were used for AnΔABSU There is a significant inhibition, indicating AnΔABSU It has a weak ability to maintain cell wall integrity.

[0085] Example 9

[0086] wild type and AnΔABSU Transcriptomic analysis of strains:

[0087] Select wild type and AnΔABSU Total RNA was extracted from fresh mycelium of the strain after three days of culture. After passing the quality inspection, mRNA library was constructed and transcriptome sequencing and analysis were performed. AnΔABSU Three biological replicates were set for each strain.

[0088] Specifically, the Meiji Bio-sequencing platform was used to sequence Aspergillus niger and mutants. AnΔABSUTranscriptome sequencing was performed to complete transcriptome analysis of 6 samples. After filtering the obtained data, a total of 42.98 Gb of Clean Data was obtained. The Clean Data of each sample reached more than 6.79 Gb, and the Q30 base percentage was more than 94.68%. The CleanReads of each sample were sequenced with the specified reference genome, and the alignment rates ranged from 93.95% to 94.27%. This shows that the sequencing quality is high and can be used for subsequent quality analysis, as shown in Table 2:

[0089] Table 2 Sequencing quality statistics

[0090]

[0091] like Fig. 9 As shown in Figure 2, the screening criteria for differentially expressed genes in this study were that the fold change (FC) was more than 2 times, i.e., |log2(FoldChange)|≥1 and padj≤0.05, and the parameter false discovery threshold (FDR) was <0.05. AnΔABSU Transcriptome data analysis was performed on the mycelial tissues of mutants and wild-type Aspergillus niger cultured for three days. Compared with the wild-type, a total of 2903 genes were expressed differently, of which 1015 genes were upregulated and 1888 genes were downregulated.

[0092] like Fig.10 and Fig.11 As shown, to understand AnΔABSU The differential gene expression between mutants and wild-type Aspergillus niger was analyzed by GO enrichment and KEGG analysis. GO enrichment and KEGG pathway enrichment are often used to study the functional annotation of differentially expressed genes. GO functional enrichment mainly includes molecular function, biological process and cellular component. In biological process, it is mainly concentrated in translation, peptide biosynthesis, peptide metabolism process and amide biosynthesis process, with the number of enriched genes being 70, 71, 86 and 78 respectively; in cellular components, it is mainly concentrated in cytoplasmic small ribosomal subunits, ribosomes and organelles, with the number of enriched genes being 11, 79 and 139 respectively; in molecular function, it is mainly concentrated in the structural components and structural molecular activity of ribosomes, with the number of enriched genes being 71 and 74 respectively, indicating that the mutant has a strong protein synthesis ability.

[0093] Further KEGG enrichment analysis showed that the enrichment was mainly in genetic information processing and metabolism. In genetic information processing, it was mainly concentrated in ribosome, DNA replication and mismatch repair, with the number of enriched genes being 68, 25 and 12 respectively; in metabolism, it was mainly concentrated in lysine biosynthesis and nitrogen metabolism, with the number of enriched genes being 9 and 15 respectively. These results indicate that the common destruction agS , agsB, sph3, uge3 The gene has a significant effect on the protein synthesis and bacterial growth cycle of Aspergillus niger.

[0094] Example 10

[0095] AnΔABSU Optimization of fermentation medium for strains

[0096] 1. Carbon source optimization

[0097] First, a spore suspension was prepared, and then a shake flask fermentation culture was performed. The specific process is shown in Example 4. Here, different carbon sources were selected, including 50 g / L glucose, 35 g / L xylose, 50 g / L fructose, 47.9 g / L sucrose, 38.7 g / L ethanol, 30 g / L malt extract, and 51.6 g / L glycerol. The other components remained unchanged for fermentation culture. The fermentation culture was continued for 3 days at 28°C and the rotation speed was 180 rpm. According to the method mentioned in Example 5, the biomass and protein content of the bacteria were evaluated. The results are as follows Fig.12 shown.

[0098] It can be seen from the figure that when the carbon source is glucose 50 g / L, Aspergillus niger AnΔABSU The biomass and protein content were the highest, reaching about 10.5 g / L and about 40%. Therefore, glucose was selected as the optimal carbon source.

[0099] 2. Nitrogen source optimization

[0100] Based on the results of carbon source optimization, organic nitrogen and inorganic nitrogen were optimized in turn. The inorganic nitrogen selected was ammonium sulfate 6 g / L, ammonium chloride 4.8 g / L, and urea 2.7 g / L. Fermentation was carried out at 28°C and 180 rpm for 3 days, and then the biomass and protein content were measured. The results are shown in Fig.13 As shown, when the inorganic nitrogen was ammonium sulfate 6 g / L, the biomass and protein content were the highest, reaching about 11 g / L and 40%.

[0101] The concentration of ammonium sulfate was further optimized and fermented at concentrations of 4 g / L, 6 g / L, 8 g / L, 10 g / L, and 12 g / L, respectively, with other conditions remaining unchanged. Fig.14 As shown, when the ammonium sulfate content was 10 g / L, the biomass and protein content were the highest, reaching 11 g / L and 43.7%.

[0102] Then, organic nitrogen was optimized by selecting 0.5 g / L corn steep liquor powder, 0.5 g / L peptone, and 0.5 g / L yeast powder for fermentation, while other ingredients remained unchanged. Fig.15 As shown, when the organic nitrogen content was 0.5 g / L corn steep liquor powder, the biomass and protein content were the highest, which were 16.5 g / L and 47%, respectively.

[0103] The optimized culture medium was 50 g / L glucose, 10 g / L ammonium sulfate, 0.7 g / L potassium chloride, 1.5 g / L anhydrous magnesium sulfate, 0.5 g / L anhydrous sodium sulfate, 2 g / L potassium dihydrogen phosphate, 0.2 g / L calcium carbonate, and 0.5 g / L corn steep liquor powder.

[0104] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A high-yield mycelial protein Aspergillus niger, characterized in that: The Aspergillus niger is Aspergillus niger AnΔABSU , which is classified as Aspergillus niger ( Aspergillus niger ), Aspergillus niger ( Aspergillus niger ) AnΔABSU , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short), with the deposit number CGMCC NO. 41621. The deposit date is November 8, 2024, and the address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. A fungal agent containing the Aspergillus niger according to claim 1.

3. Use of the Aspergillus niger with high mycelial protein production according to claim 1 or the bacterial agent according to claim 2 in producing mycelial protein.

4. A method for high yield of mycelial protein, characterized in that: The Aspergillus niger with high mycelial protein production according to claim 1 or the bacterial agent according to claim 2 is used for fermentation culture in a fermentation medium, wherein the nitrogen source of the fermentation medium is an inorganic nitrogen source.

5. The method for high yield of mycelial protein according to claim 4, characterized in that: The fermentation culture temperature is 26~30℃, the culture method is shaking culture, and the culture time is 3 days.

6. The method for high yield of mycelial protein according to claim 4, characterized in that: The inorganic nitrogen source is ammonium sulfate 1-12g / L.

7. The method for high yield of mycelial protein according to claim 4, characterized in that: The inorganic nitrogen source was ammonium sulfate 10 g / L.

8. The method for high yield of mycelial protein according to claim 4, characterized in that: The fermentation medium comprises: 1-80 g / L of glucose, 1-20 g / L of ammonium sulfate, 0.1-2 g / L of potassium chloride, 0.1-3 g / L of anhydrous magnesium sulfate, 0.1-1 g / L of anhydrous sodium sulfate, 0.1-4 g / L of potassium dihydrogen phosphate, 0.1-0.5 g / L of calcium carbonate, and 0.1-1 g / L of corn steep liquor powder.

9. The method for high yield of mycelial protein according to claim 4, characterized in that: After the fermentation culture is completed, the bacteria are collected and dried.

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