Process for producing microbial protein feed by multi-strain synergistic fermentation of steam-exploded corn stalks

Abstract

The present application belongs to the field of biotechnology, and specifically relates to a process for producing cell protein feed by multi-strain synergistic fermentation of steam-exploded corn stalks. The present application adopts mixed fermentation of Aspergillus niger GRJC003 with Trichoderma reesei, which has a preservation number of CCTCC NO: M 2025084, and combines steam explosion pretreatment and the addition of pH-responsive sophorolipid slow-release particles and other optimized conditions to achieve remarkable results in the preparation of cell protein feed from steam-exploded corn stalks. The process realizes complete hydrolysis of cellulose and enhanced tolerance to inhibitors through the unique enzyme complementation, metabolic mutualism and microenvironment regulation synergistic mechanism of the two strains, increases the yield of cell protein, and at the same time realizes efficient utilization of carbon and nitrogen resources and stable process control, and has outstanding industrial application value and sustainability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically a process for producing microbial protein feed from corn stalks through synergistic fermentation of multiple microorganisms. Background Technology

[0002] With the continued growth in global demand for protein and the rapid development of animal husbandry, the development of new sustainable protein feed resources has become a research hotspot. Corn stalks are rich in carbohydrates such as cellulose and hemicellulose, making them an ideal and inexpensive substrate for producing microbial spore protein (SCP). However, the complex lignocellulose structure of corn stalks makes them difficult for microorganisms to directly and effectively degrade and utilize, usually requiring pretreatment. Steam explosion, as an efficient physicochemical pretreatment method, can effectively destroy the structure of corn stalks, but it still produces some phenolic compounds that inhibit microbial growth. In the fermentation stage, utilizing fungi to convert pretreated corn stalks into microbial spore protein is key to resource utilization. *Aspergillus niger* and *Trichoderma reesei* are classic cellulase-producing strains often used to degrade cellulose. However, traditional single-strain fermentation suffers from problems such as incomplete enzyme systems, incomplete substrate conversion, and low product yield; while conventional mixed fermentation strategies often fail due to competition or lack of effective synergy between strains, sometimes even resulting in lower yields than single-strain fermentation, making it difficult to achieve the efficiency and economy required for industrial applications. Therefore, screening strain combinations with specific synergistic functions and developing innovative processes that can enhance their synergistic effects, mitigate the effects of inhibitors, and simultaneously optimize the enzymatic hydrolysis and fermentation processes have become key to breaking through the technological bottlenecks in the high-value utilization of corn straw. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a process for producing microbial protein feed from corn straw through multi-strain synergistic fermentation.

[0004] A process for producing microbial protein feed through multi-strain synergistic fermentation and steam explosion of corn straw includes the following steps:

[0005] (1) By mass, 2400-2600 parts of corn stalks with a moisture content of 25-35wt% are crushed to a particle size of 0.1-0.5cm, and then mixed with 2-4 parts of SiO2@QPEG for 1-2h. The mixture is then steam-exploded at 180-200℃ and 2-3Mpa for 4-6min. The SiO2@QPEG is then recovered by HGMS magnetic separator to obtain steam-exploded straw and recovered SiO2@QPEG.

[0006] (2) Adjust the carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) to 25:(1-2) with urea, then add potassium dihydrogen phosphate and magnesium sulfate, sterilize at 120-122℃ for 19-21 min, and finally add pH-responsive sophorolipid slow-release granules to obtain fermentation medium. Inoculate fermentation bacteria in the above fermentation medium and aerobic ferment at 25-35℃ and 4.8-5.2 pH for 36-72 h. After fermentation, dry to obtain the microbial protein feed.

[0007] The product SiO2@QPEG recovered in step (1) can be reduced by soaking in 0.08-0.12 mol / L ascorbic acid for 20-40 min, filtering, washing, vacuum drying, and reuse.

[0008] The concentration of potassium dihydrogen phosphate in step (2) is 0.08-0.12 wt% of the steam-exploded straw, and the concentration of magnesium sulfate is 0.03-0.07 wt% of the steam-exploded straw.

[0009] The fermentation bacteria in step (2) are obtained by mixing Aspergillus niger GRJC003 and Trichoderma reesei, with the inoculum size of Aspergillus niger GRJC003 being 0.4 × 10⁻⁶. 7 -0.6×10 7 Spores / g fermentation medium, the inoculum size of *Trichoderma reesei* was 0.4 × 10⁻⁶. 7 -0.6×10 7 Spores / g fermentation medium.

[0010] The pH of 4.8-5.2 in step (2) is adjusted by 1-2 mol / L sulfuric acid.

[0011] The preparation method of SiO2@QPEG in step (1) includes the following steps:

[0012] S1, by weight, dissolve 4-6 parts of quercetin and 7-8 parts of carboxyl-polyethylene glycol-carboxyl in 100-200 parts of N,N-dimethylformamide, add 0.1-0.3 parts of 4-dimethylaminopyridine and 2-4 parts of dicyclohexylcarbodiimide, and stir the reaction at 24-26℃ under nitrogen protection for 22-26 h to obtain a reaction solution; then, dropwise add the reaction solution into 400-500 parts of ice-cold diethyl ether to precipitate, centrifuge at 6000-8000 rpm for 9-11 min, wash the precipitate with diethyl ether 2-3 times, and vacuum dry at 30-40℃ for 24-48 h to obtain QPEG-COOH;

[0013] S2, by mass, dissolve 5-6 parts of ferric chloride hexahydrate and 1-3 parts of ferric chloride tetrahydrate in 90-100 parts of water, add 18-22 mL of 26-30 wt% ammonia solution dropwise under nitrogen protection, stir at 78-82℃ for 1-2 h, magnetically separate and wash with water until neutral to obtain ferric oxide; disperse the above ferric oxide in 180-200 parts of ethanol aqueous solution, add 1-2 parts of 26-30 wt% ammonia solution and 2-3 parts of tetraethoxysilane, stir at 24-26℃ for 5-7 h, centrifuge and wash the precipitate with water 1-2 times to obtain magnetic silica particles;

[0014] S3. By mass, disperse the magnetic silica particles obtained in step S2 in 90-100 parts of toluene, add 1-2 parts of γ-aminopropyltriethoxysilane, reflux at 78-82℃ for 10-14 h, centrifuge, wash and dry to obtain aminated magnetic silica particles.

[0015] S4. By mass, dissolve 1-2 parts of QPEG-COOH obtained in step S1 in 50 parts of MES buffer, add 0.1-0.2 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.07-0.08 parts of N-hydroxysuccinimide, stir at 24-26℃ for 30-40 min, then add 1-2 parts of aminated magnetic silica particles obtained in step S3, adjust the pH to 7-8 with HEPES buffer, stir at 38-42℃ for 12-14 h, separate with a magnetic field, wash and dry to obtain the SiO2@QPEG.

[0016] In the ethanol-water solution described in step S2, the volume ratio of ethanol to water is 4:(1-2).

[0017] The concentration of 2-N-morpholinoethanesulfonic acid in the MES buffer solution described in step S4 is 0.1-0.2 mol / L.

[0018] The concentration of 4-hydroxyethylpiperazine ethanesulfonic acid in the HEPES buffer solution described in step S4 is 0.1-0.2 mol / L.

[0019] The amount of pH-responsive sophorolipid sustained-release granules added in step (2) is 0.5-1 wt% of the mass of the steam-exploded straw.

[0020] The preparation method of the pH-responsive sophorolipid sustained-release granules in step (2) includes the following steps:

[0021] N1 Dissolve 2-3 parts of sodium alginate in 90-100 parts of water by weight, and stir until completely dissolved to obtain an aqueous solution of sodium alginate;

[0022] N2 is added to the sodium alginate aqueous solution obtained in step S1 by mass of 0.1-0.2 parts of sophorolipid, and stirred evenly to obtain sophorolipid-sodium alginate aqueous solution;

[0023] N3, by weight, mix 40-60 parts corn oil and 0.3-0.8 parts Span80 to obtain an emulsion; mix the sophorolipid-sodium alginate aqueous solution obtained in step N2 with the emulsion and homogenize at 10000-15000 rpm for 5-10 min to obtain a sophorolipid-sodium alginate emulsion.

[0024] N4. The sophorolipid-sodium alginate emulsion obtained in step N3 is added dropwise to a calcium chloride aqueous solution with a concentration of 1.8-2.2wt% at a rate of 1-2 ml / min. After standing for 30-40 min, the solution is filtered, washed, and dried to obtain the pH-responsive sophorolipid sustained-release particles.

[0025] The beneficial effects of this invention are:

[0026] This invention utilizes a co-fermentation process between *Aspergillus niger* GRJC003 (accession number CCTCCNO:M 2025084) and *Trichoderma reesei*, combined with optimized conditions such as steam explosion pretreatment and the addition of pH-responsive sophorolipid slow-release granules. This process has achieved significant results in preparing microbial protein feed from steam-exploded corn straw. Through the unique enzyme complementarity, metabolic synergy, and microenvironment regulation synergistic mechanism of the two strains, the process thoroughly hydrolyzes cellulose and enhances tolerance to inhibitors, resulting in a microbial protein yield of up to 432.6 g / kg, more than 100% higher than conventional co-fermentation. Simultaneously, it achieves efficient utilization of carbon and nitrogen resources and robust process control, demonstrating outstanding industrial application value and sustainability. Detailed Implementation

[0027] Quercetin, CAS No.: 117-39-5.

[0028] Carboxyl-polyethylene glycol-carboxyl, product number: YS-P6623, polyethylene glycol molecular weight is 2000, Chongqing Yusi Pharmaceutical Technology Co., Ltd.

[0029] 2-N-morpholinoethanesulfonic acid, CAS No.: 145224-94-8.

[0030] 4-Hydroxyethylpiperazine ethanesulfonic acid, CAS No.: 7365-45-9.

[0031] Rutin, CAS No.: 153-18-4.

[0032] Butylhydroxytoluene, CAS Registry No. 128-37-0.

[0033] Sophorolipid, product number: HBWS-L429, Hubei Weideli Chemical Technology Co., Ltd.

[0034] Sodium alginate, product number: S11053, Shanghai Yuanye Biotechnology Co., Ltd.

[0035] Trichoderma reesei, accession number: CGMCC3.3711, purchased from China General Microbiological Culture Collection Center.

[0036] This invention provides a *Aspergillus niger* fungus, named *Aspergillus niger* GRJC003. *Aspergillus niger* GRJC003 was deposited on January 10, 2025, at the China Center for Type Culture Collection (CCTCCNO: M2025084), located at Wuhan University, China.

[0037] Isolation and culture of Aspergillus niger GRJC003: Rumen fluid collected from adult small-tailed Han sheep was diluted with physiological saline at different gradients to a concentration of 0.9 wt%. 10... -3 10 -4 10 -5 Three dilution gradients (0.1 mL each) were plated onto PDA medium and incubated at 30°C for 3-5 days, observing colony formation. Isolates exhibiting Aspergillus culture characteristics were subcultured on fresh PDA medium to obtain pure isolates. Colony morphology was observed, and suspected strains were picked for repeated isolation and purification until pure strains were obtained. Gene sequencing results were compared using the NCBI database.

[0038] Culture conditions: The culture medium was PDA medium (3 g / L potato extract, 20 g / L glucose, 20 g / L agar), pH=6.06; culture temperature was 30℃; culture time was 3 days.

[0039] Identification: The pure strain was sent to Sangon Biotech (Shanghai) Co., Ltd. for gene identification, and the gene sequence of Aspergillus niger GRJC003 was obtained as follows:

[0040] .

[0041] Example 1

[0042] A process for producing microbial protein feed through multi-strain synergistic fermentation and steam explosion of corn straw includes the following steps:

[0043] (1) By mass, 2500 parts of corn stalks with a moisture content of 30wt% were crushed to a particle size of 0.5cm, and then mixed with 3 parts of SiO2@QPEG for 1h. The mixture was then steam-exploded at 190℃ and 2.5MPa for 5min. The SiO2@QPEG was recovered by HGMS magnetic separator to obtain steam-exploded straw and recovered SiO2@QPEG.

[0044] (2) Adjust the carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) to 25:1 with urea, then add potassium dihydrogen phosphate and magnesium sulfate, sterilize at 121℃ for 20 min, and finally add pH-responsive sophorolipid slow-release granules to obtain fermentation medium. Inoculate fermentation bacteria in the above fermentation medium and aerobic ferment at 30℃ and 5.0 pH for 72 h. After fermentation, dry to obtain the microbial protein feed.

[0045] The product SiO2@QPEG recovered in step (1) can be reduced by soaking in 0.08-0.12 mol / L ascorbic acid for 20-40 min, filtering, washing, vacuum drying, and reuse.

[0046] The concentration of potassium dihydrogen phosphate in step (2) is 0.1 wt% of the steam-exploded straw, and the concentration of magnesium sulfate is 0.05 wt% of the steam-exploded straw.

[0047] The fermentation bacteria in step (2) are obtained by mixing Aspergillus niger GRJC003 and Trichoderma reesei, with the inoculum size of Aspergillus niger GRJC003 being 0.5 × 10⁻⁶. 7 Spores / g fermentation medium, Trichoderma reesei inoculum size 0.5 × 10⁻⁶ 7 Spores / g fermentation medium.

[0048] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0049] The preparation method of SiO2@QPEG in step (1) includes the following steps:

[0050] S1, by weight, dissolve 5 parts quercetin and 8 parts carboxyl-polyethylene glycol-carboxyl in 200 parts N,N-dimethylformamide, add 0.2 parts 4-dimethylaminopyridine and 3 parts dicyclohexylcarbodiimide, and stir the mixture at 25°C under nitrogen protection for 24 h to obtain a reaction solution; then, dropwise add the reaction solution into 500 parts ice-cold diethyl ether to precipitate, centrifuge at 8000 rpm for 10 min, wash the precipitate three times with diethyl ether, and dry it under vacuum at 40°C for 24 h to obtain QPEG-COOH;

[0051] S2, by mass, dissolves 5.4 parts of ferric chloride hexahydrate and 2 parts of ferric chloride tetrahydrate in 100 parts of water, adds 20 mL of 28 wt% ammonia solution dropwise under nitrogen protection, stirs at 80°C for 1 h, magnetically separates and washes with water until neutral to obtain iron(III) oxide; disperses the above iron(III) oxide in 200 parts of ethanol aqueous solution, adds 2 parts of 28 wt% ammonia solution and 2.8 parts of tetraethoxysilane, stirs at 25°C for 6 h, centrifuges and washes the precipitate twice with water to obtain magnetic silica particles;

[0052] S3. By mass, the magnetic silica particles obtained in step S2 are dispersed in 100 parts of toluene, and 1.5 parts of γ-aminopropyltriethoxysilane are added. The mixture is refluxed at 80°C for 12 hours, centrifuged, washed and dried to obtain aminated magnetic silica particles.

[0053] S4. By mass, dissolve 1 part of QPEG-COOH obtained in step S1 in 50 parts of MES buffer, add 0.15 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.075 parts of N-hydroxysuccinimide, stir at 25°C for 30 min, then add 2 parts of aminated magnetic silica particles obtained in step S3, adjust the pH to 7.4 with HEPES buffer, stir at 40°C for 12 h, separate with a magnetic field, wash and dry to obtain the SiO2@QPEG.

[0054] In step S2, the volume ratio of ethanol to water in the aqueous ethanol solution is 4:1.

[0055] The concentration of 2-N-morpholinoethanesulfonic acid in the MES buffer solution described in step S4 is 0.1 mol / L.

[0056] The concentration of 4-hydroxyethylpiperazine ethanesulfonic acid in the HEPES buffer solution described in step S4 is 0.1 mol / L.

[0057] The amount of pH-responsive sophorolipid sustained-release granules added in step (2) is 0.8 wt% of the mass of the steam-exploded straw.

[0058] The preparation method of the pH-responsive sophorolipid sustained-release granules in step (2) includes the following steps:

[0059] N1 Dissolve 2 parts of sodium alginate in 100 parts of water by weight, and stir until completely dissolved to obtain an aqueous solution of sodium alginate;

[0060] N2, by mass, adds 0.1 parts of sophorolipid to the sodium alginate aqueous solution obtained in step N1, and stirs until homogeneous to obtain a sophorolipid-sodium alginate aqueous solution;

[0061] N3, by weight, 50 parts corn oil and 0.5 parts Span80 are mixed to obtain an emulsion; the sophorolipid-sodium alginate aqueous solution obtained in step N2 is mixed with the emulsion and homogenized at 12000 rpm for 10 min to obtain a sophorolipid-sodium alginate emulsion.

[0062] N4 The sophorolipid-sodium alginate emulsion obtained in step N3 was added dropwise to a 2wt% calcium chloride aqueous solution at a rate of 1 ml / min. After standing for 30 min, the solution was filtered, washed, and dried to obtain the pH-responsive sophorolipid sustained-release particles.

[0063] Example 2

[0064] A process for producing microbial protein feed through multi-strain synergistic fermentation and steam explosion of corn straw includes the following steps:

[0065] (1) By weight, 2500 parts of corn stalks with a moisture content of 30wt% were crushed to a particle size of 0.5cm and steam-exploded at 190℃ and 2.5Mpa for 5min to obtain steam-exploded straw;

[0066] (2) Adjust the carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) to 25:1 with urea, then add potassium dihydrogen phosphate and magnesium sulfate, sterilize at 121℃ for 20 min, and finally add pH-responsive sophorolipid slow-release granules to obtain fermentation medium. Inoculate fermentation bacteria in the above fermentation medium and aerobic ferment at 30℃ and 5.0 pH for 72 h. After fermentation, dry to obtain the microbial protein feed.

[0067] The concentration of potassium dihydrogen phosphate in step (2) is 0.1 wt% of the steam-exploded straw, and the concentration of magnesium sulfate is 0.05 wt% of the steam-exploded straw.

[0068] The fermentation bacteria in step (2) are obtained by mixing Aspergillus niger GRJC003 and Trichoderma reesei, with the inoculum size of Aspergillus niger GRJC003 being 0.5 × 10⁻⁶. 7 Spores / g fermentation medium, Trichoderma reesei inoculum size 0.5 × 10⁻⁶ 7 Spores / g fermentation medium.

[0069] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0070] The amount of pH-responsive sophorolipid sustained-release granules added in step (2) is 0.8 wt% of the mass of the steam-exploded straw.

[0071] The preparation method of the pH-responsive sophorolipid sustained-release granules in step (2) includes the following steps:

[0072] N1 Dissolve 2 parts of sodium alginate in 100 parts of water by weight, and stir until completely dissolved to obtain an aqueous solution of sodium alginate;

[0073] N2, by mass, adds 0.1 parts of sophorolipid to the sodium alginate aqueous solution obtained in step N1, and stirs until homogeneous to obtain a sophorolipid-sodium alginate aqueous solution;

[0074] N3, by weight, 50 parts corn oil and 0.5 parts Span80 are mixed to obtain an emulsion; the sophorolipid-sodium alginate aqueous solution obtained in step N2 is mixed with the emulsion and homogenized at 12000 rpm for 10 min to obtain a sophorolipid-sodium alginate emulsion.

[0075] N4 The sophorolipid-sodium alginate emulsion obtained in step N3 was added dropwise to a 2wt% calcium chloride aqueous solution at a rate of 1 ml / min. After standing for 30 min, the solution was filtered, washed, and dried to obtain the pH-responsive sophorolipid sustained-release particles.

[0076] Example 3

[0077] A process for producing microbial protein feed through multi-strain synergistic fermentation and steam explosion of corn straw includes the following steps:

[0078] (1) By mass, 2500 parts of corn stalks with a moisture content of 30wt% were crushed to a particle size of 0.5cm, and then mixed with 3 parts of SiO2@QPEG for 1h. The mixture was then steam-exploded at 190℃ and 2.5MPa for 5min. The SiO2@QPEG was recovered by HGMS magnetic separator to obtain steam-exploded straw and recovered SiO2@QPEG.

[0079] (2) Adjust the carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) to 25:1 with urea, then add potassium dihydrogen phosphate and magnesium sulfate, sterilize at 121℃ for 20 min to obtain fermentation medium, inoculate fermentation bacteria in the above fermentation medium, and aerobic ferment at 30℃ and 5.0 pH for 72 h. After fermentation, dry to obtain the microbial protein feed.

[0080] The product SiO2@QPEG recovered in step (1) can be reduced by soaking in 0.08-0.12 mol / L ascorbic acid for 20-40 min, filtering, washing, vacuum drying, and reuse.

[0081] The concentration of potassium dihydrogen phosphate in step (2) is 0.1 wt% of the steam-exploded straw, and the concentration of magnesium sulfate is 0.05 wt% of the steam-exploded straw.

[0082] The fermentation bacteria in step (2) are obtained by mixing Aspergillus niger GRJC003 and Trichoderma reesei, with the inoculum size of Aspergillus niger GRJC003 being 0.5 × 10⁻⁶. 7 Spores / g fermentation medium, Trichoderma reesei inoculum size 0.5 × 10⁻⁶ 7 Spores / g fermentation medium.

[0083] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0084] The preparation method of SiO2@QPEG in step (1) includes the following steps:

[0085] S1, by weight, dissolve 5 parts quercetin and 8 parts carboxyl-polyethylene glycol-carboxyl in 200 parts N,N-dimethylformamide, add 0.2 parts 4-dimethylaminopyridine and 3 parts dicyclohexylcarbodiimide, and stir the mixture at 25°C under nitrogen protection for 24 h to obtain a reaction solution; then, dropwise add the reaction solution into 500 parts ice-cold diethyl ether to precipitate, centrifuge at 8000 rpm for 10 min, wash the precipitate three times with diethyl ether, and dry it under vacuum at 40°C for 24 h to obtain QPEG-COOH;

[0086] S2, by mass, dissolves 5.4 parts of ferric chloride hexahydrate and 2 parts of ferric chloride tetrahydrate in 100 parts of water, adds 20 mL of 28 wt% ammonia solution dropwise under nitrogen protection, stirs at 80°C for 1 h, magnetically separates and washes with water until neutral to obtain iron(III) oxide; disperses the above iron(III) oxide in 200 parts of ethanol aqueous solution, adds 2 parts of 28 wt% ammonia solution and 2.8 parts of tetraethoxysilane, stirs at 25°C for 6 h, centrifuges and washes the precipitate twice with water to obtain magnetic silica particles;

[0087] S3. By mass, the magnetic silica particles obtained in step S2 are dispersed in 100 parts of toluene, and 1.5 parts of γ-aminopropyltriethoxysilane are added. The mixture is refluxed at 80°C for 12 hours, centrifuged, washed and dried to obtain aminated magnetic silica particles.

[0088] S4. By mass, dissolve 1 part of QPEG-COOH obtained in step S1 in 50 parts of MES buffer, add 0.15 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.075 parts of N-hydroxysuccinimide, stir at 25°C for 30 min, then add 2 parts of aminated magnetic silica particles obtained in step S3, adjust the pH to 7.4 with HEPES buffer, stir at 40°C for 12 h, separate with a magnetic field, wash and dry to obtain the SiO2@QPEG.

[0089] In step S2, the volume ratio of ethanol to water in the aqueous ethanol solution is 4:1.

[0090] The concentration of 2-N-morpholinoethanesulfonic acid in the MES buffer solution described in step S4 is 0.1 mol / L.

[0091] The concentration of 4-hydroxyethylpiperazine ethanesulfonic acid in the HEPES buffer solution described in step S4 is 0.1 mol / L.

[0092] Comparative Example 1

[0093] The difference from Example 1 is that the fermentation bacteria in step (2) is Aspergillus niger GRJC003, and the inoculum amount of Aspergillus niger GRJC003 is 1×10⁻⁶. 7 Spores / g fermentation medium.

[0094] Comparative Example 2

[0095] The difference from Example 1 is that the fermentation bacteria in step (2) is *Trichoderma reesei*, and the inoculum size of *Trichoderma reesei* is 1 × 10⁻⁶. 7 Spores / g fermentation medium.

[0096] Comparative Example 3

[0097] The difference from Example 1 is that the fermentation bacteria in step (2) are obtained by mixing Aspergillus niger and Trichoderma reesei, and the inoculum amount of Aspergillus niger is 0.5 × 10⁻⁶. 7 Spores / g fermentation medium, Trichoderma reesei inoculum size 0.5 × 10⁻⁶ 7 spores / g fermentation medium; the aforementioned Aspergillus niger, Aspergillus niger Accession number: CICC40273, purchased from China Industrial Microbial Culture Collection Center.

[0098] Comparative Example 4

[0099] The difference from Example 1 is that the fermentation bacteria in step (2) are obtained by mixing Aspergillus niger and Trichoderma reesei, and the inoculum amount of Aspergillus niger is 0.5 × 10⁻⁶. 7 Spores / g fermentation medium, Trichoderma reesei inoculum size 0.5 × 10⁻⁶ 7 spores / g fermentation medium; the Aspergillus niger strain, Aspergillus niger Accession number: CGMCC3.3926, purchased from China General Microbiological Culture Collection Center.

[0100] Comparative Example 5

[0101] Using 1500g of corn stalks as raw material, the microbial protein content in the microbial protein feeds obtained in Examples 1-3 and Comparative Examples 1-4 was determined, and the results are shown in Table 1.

[0102] After fermentation, add water at three times the mass of the microbial protein feed, stir and extract for 1 hour. Then, separate the solid residue from the liquid containing microorganisms through coarse filtration. The liquid containing microorganisms is then separated and collected through a plate and frame filter press. The microorganisms are washed with water and dried to obtain the microbial protein.

[0103] Table 1: Bacterial Protein Content

[0104]

[0105] Table 1 shows that the microbial protein feed obtained in Example 1 of this invention has the highest microbial protein content. Example 2 lacks SiO2@QPEG in the steam explosion process. SiO2@QPEG uses magnetic silica particles as a carrier to load quercetin-polyethylene glycol conjugates. The magnetic silica particles enable the recyclability of SiO2@QPEG and improve fermentation efficiency. PEG separates the active site (phenolic hydroxyl group) of quercetin from the SiO2 surface, reducing steric hindrance and improving the dispersibility of hydrophobic quercetin during fermentation. It also further prevents the catalytic oxidation of quercetin by SiO2 at high temperatures. The resulting SiO2@QPEG, when applied to the preparation process of microbial protein feed produced by steam explosion fermentation of corn straw, combines high microbial protein yield with recyclability.

[0106] In Example 3, the fermentation process lacked sophorolipid slow-release particles. As *Trichoderma reesei* grew and metabolized, producing acid, the environmental pH gradually decreased. This caused the sodium alginate gel in the sophorolipid slow-release particles to decompose, resulting in the slow and continuous release of sophorolipids. The released sophorolipids, through their amphiphilic structure, significantly improved the wettability of the hydrophobic, vaporized straw, leading to a more uniform distribution of moisture and mycelia. They also disrupted the hydrophobic barrier of lignocellulose, releasing bound fermentable sugars and improving substrate accessibility and saccharification efficiency. Simultaneously, sophorolipids effectively encapsulated or adsorbed fermentation inhibitors such as furfural, reducing their toxic effects on *Trichoderma reesei*. Through the synergistic effect of these three mechanisms, the growth environment and metabolic efficiency of *Trichoderma reesei* were fundamentally improved, ultimately resulting in a significant increase in mycelial protein production.

[0107] Comparative Example 1, using only *Aspergillus niger* GRJC003 for fermentation, showed a decrease in microbial protein yield. Comparative Example 2, using only *Trichoderma reesei* for fermentation, also showed a decrease in microbial protein yield. *Aspergillus niger* GRJC003, isolated from the rumen fluid of adult small-tailed Han sheep and obtained through targeted domestication, possesses excellent cellulose and hemicellulose degradation capabilities, enabling efficient fermentation of steam-exploded straw to produce microbial protein feed. The *Aspergillus niger* GRJC003 strain exhibited high enzyme activity and growth rate during fermentation, significantly increasing microbial protein yield. In mixed fermentation, *Aspergillus niger* GRJC003 and *Trichoderma reesei* formed a "division of labor and cooperation" model: *Trichoderma reesei* preferentially attached to the straw substrate, breaking down the fiber structure through enzymatic hydrolysis to provide fermentable sugars; *Aspergillus niger* GRJC003 utilized these sugar sources for rapid growth and protein synthesis, and may also secrete coenzymes (such as amylase or phenol-degrading enzymes) to further optimize substrate utilization.

[0108] By comparing the data from Comparative Examples 3 and 4, a key phenomenon can be further revealed: the conventional mixed fermentation of Aspergillus niger and Trichoderma reesei not only failed to achieve the expected synergistic effect, but its yield was also significantly lower than that of fermentation with a single dominant strain. This result clearly indicates that in the fermentation system of this invention (including steam explosion pretreatment, use of adjuvants, and specific conditions), the mixing of conventional strains may lead to reduced efficiency or even adverse effects due to incompatibility between strains, insufficient enzyme matching, or metabolic competitive inhibition, thus lacking a mutually promoting effect.

[0109] In contrast, Example 1, using a co-fermentation of Aspergillus niger GRJC003 and Trichoderma reesei, achieved a high yield of 432.6 g, far superior to all comparative examples. This significant difference highlights the unique properties of strain GRJC003 and its specific synergistic mechanism with Trichoderma reesei. This synergy is not easily achieved by conventional strain combinations because it depends on the screened and optimized genetic background of GRJC003.

Claims

1. A process for the production of cell protein feed from steam exploded corn stover by multi-strain synergistic fermentation, characterized by, The method comprises the following steps: (1) crushing 2400-2600 parts of corn straw with a water content of 25-35 wt% to a particle size of 0.1-0.5 cm by mass fraction, then mixing and stirring with 2-4 parts of SiO2@QPEG for 1-2 h, steam explosion at 180-200 ℃ and 2-3 MPa for 4-6 min, recovering SiO2@QPEG by HGMS magnetic separator, and obtaining steam-exploded straw and recovered SiO2@QPEG; (2) adjusting the carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) to 25: (1-2) by urea, then adding potassium dihydrogen phosphate and magnesium sulfate, sterilizing at 120-122 ℃ for 19-21 min, and finally adding pH-responsive sophorolipid slow-release particles to obtain a fermentation medium, inoculating the fermentation medium with fermentation bacteria, and aerobically fermenting at 25-35 ℃ and 4.8-5.2 pH for 36-72 h, and drying after fermentation to obtain the bacterial protein feed; In step (1), the SiO2@QPEG is obtained by loading quercetin-polyethylene glycol conjugates on magnetic silica particles as carriers; The fermentation bacteria in step (2) are obtained by mixing Aspergillus niger GRJC003 and Trichoderma reesei. The preservation number of Aspergillus niger GRJC003 is CCTCC NO: M 2025084, and the inoculation amount is 0.4×10 7 -0.6×10 7 spores / g fermentation medium. The preservation number of Trichoderma reesei is CGMCC 3.3711, and the inoculation amount is 0.4×10 7 -0.6×10 7 spores / g fermentation medium.

2. The process for the production of cell protein feed from steam-exploded corn stover by multi-strain synergistic fermentation as claimed in claim 1, wherein, In step (1), the recovered SiO2@QPEG can be reduced by soaking in 0.08-0.12 mol / L ascorbic acid for 20-40 min, filtering, washing, and vacuum drying, to realize reuse.

3. The process for the production of cell protein feed from steam-exploded corn stover by multi-strain synergistic fermentation as claimed in claim 1, wherein, In step (2), the concentration of potassium dihydrogen phosphate is 0.08-0.12 wt% of the steam-exploded straw, and the concentration of magnesium sulfate is 0.03-0.07 wt% of the steam-exploded straw.

4. The process for the production of cell protein feed from steam-exploded corn stover by multi-strain synergistic fermentation as claimed in claim 1, wherein, In step (2), the 4.8-5.2 pH is adjusted by 1-2 mol / L sulfuric acid.

5. The process for the production of cell protein feed from steam-exploded corn stover by multi-strain synergistic fermentation as claimed in claim 1, wherein, In step (1), the preparation method of SiO2@QPEG comprises the following steps: S1: dissolving 4-6 parts of quercetin and 7-8 parts of carboxyl-polyethylene glycol-carboxyl in 100-200 parts of N,N-dimethylformamide by mass fraction, adding 0.1-0.3 parts of 4-dimethylaminopyridine and 2-4 parts of dicyclohexyl carbodiimide, stirring and reacting at 24-26 ℃ under nitrogen protection for 22-26 h to obtain a reaction solution; then dropping the reaction solution into 400-500 parts of ice ether for precipitation, centrifuging at 6000-8000 rpm for 9-11 min, washing the precipitate with ether for 2-3 times, and vacuum drying at 30-40 ℃ for 24-48 h to obtain QPEG-COOH; S2: dissolving 5-6 parts of iron trichloride hexahydrate and 1-3 parts of dichloride iron tetrahydrate in 90-100 parts of water, adding 18-22 mL of ammonia water with a concentration of 26-30 wt% under nitrogen protection, stirring at 78-82 ℃ for 1-2 h, magnetically separating and washing to neutral to obtain magnetite; dispersing the above magnetite in 180-200 parts of ethanol aqueous solution, adding 1-2 parts of ammonia water with a concentration of 26-30 wt% and 2-3 parts of tetraethoxysilane, stirring at 24-26 ℃ for 5-7 h, centrifuging and washing the precipitate 1-2 times to obtain magnetic silica particles; S3: 1-2 parts of the magnetic silica particles obtained in step S2 were dispersed in 90-100 parts of toluene, and 1-2 parts of γ-aminopropyltriethoxysilane was added, and the mixture was refluxed at 78-82°C for 10-14 h, and then centrifuged, washed and dried to obtain aminated magnetic silica particles; S4: 1-2 parts of QPEG-COOH obtained in step S1 were dissolved in 50 parts of MES buffer, 0.1-0.2 parts of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.07-0.08 parts of N-hydroxysuccinimide were added, and the mixture was stirred at 24-26°C for 30-40 min, then 1-2 parts of the aminated magnetic silica particles obtained in step S3 were added, and the pH was adjusted to 7-8 with HEPES buffer, and the mixture was stirred at 38-42°C for 12-14 h, and then separated by magnetic field, washed and dried to obtain the SiO2@QPEG.

6. The process for the production of cell protein feed from steam-exploded corn stover by multi-strain synergistic fermentation as claimed in claim 5, wherein, In the aqueous ethanol solution of step S2, the volume ratio of ethanol to water was 4: (1-2).

7. The process as claimed in claim 1, wherein the multi-strain synergistic fermentation of steam-exploded corn stover for production of cell protein feed is characterized by, The addition amount of the pH-responsive sophorolipid slow-release particles in step (2) was 0.5-1 wt% of the mass of the steam-exploded straw. ​ 8. The process for the production of cell protein feed from steam-exploded corn stover by multi-strain synergistic fermentation as claimed in claim 1, wherein, The preparation method of the pH-responsive sophorolipid slow-release particles in step (2) comprises the following steps: N1: 2-3 parts of sodium alginate were dissolved in 90-100 parts of water, and the mixture was stirred until completely dissolved to obtain a sodium alginate aqueous solution; N2: 0.1-0.2 parts of sophorolipid were added to the sodium alginate aqueous solution obtained in step S1, and the mixture was stirred to obtain a sophorolipid-sodium alginate aqueous solution; N3: 40-60 parts of corn oil and 0.3-0.8 parts of Span 80 were mixed to obtain an emulsion; the sophorolipid-sodium alginate aqueous solution obtained in step N2 and the emulsion were mixed and homogenized at a speed of 10000-15000 rpm for 5-10 min to obtain a sophorolipid-sodium alginate emulsion; N4: The sophorolipid-sodium alginate emulsion obtained in step N3 was added dropwise into a 1.8-2.2 wt% calcium chloride aqueous solution at a rate of 1-2 ml / min, and the mixture was allowed to stand for 30-40 min, and then filtered, washed and dried to obtain the pH-responsive sophorolipid slow-release particles.

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