Aspergillus niger and application thereof in producing mycoprotein feed from fermented steam-exploded straws

Abstract

The invention relates to the field of microbial technology and feed production, in particular to Aspergillus niger and application of the Aspergillus niger in fermentation of steam-exploded straw to produce mycoprotein feed. Compared with an existing standard strain, the aspergillus niger GRJC003 strain shows excellent mycoprotein yield under the same process condition, the strain is suitable for producing mycoprotein through fermentation of various agricultural wastes (such as wheat, rice and corn straw), the raw material source is widened, the production cost is reduced, meanwhile, through steam explosion pretreatment and controllable fermentation, the production efficiency is improved, and the production cost is reduced. The crude protein content and nutritional value of the feed are effectively improved, and an efficient and environment-friendly solution is provided for sustainable development of animal husbandry.

Description

Technical Field

[0001] This invention relates to the fields of microbial technology and feed production, specifically to a type of Aspergillus niger and its application in fermentation and steam-explosion straw to produce microbial protein feed. Background Technology

[0002] Straw, as a major agricultural waste, is produced in huge quantities globally each year, and its treatment and resource utilization have always been important issues for sustainable agricultural development. Straw is rich in cellulose, hemicellulose, and lignin, which together form a stubborn fiber structure, resulting in low digestibility, poor palatability, and limited nutritional value when used directly as feed for ruminants, making it difficult to meet the requirements of modern animal husbandry for feed protein content and digestibility.

[0003] Microbial fermentation, through pretreatment and biotransformation of straw, can effectively break down its fiber structure, degrade complex polysaccharides into monosaccharides or oligosaccharides that can be utilized by animals, and simultaneously increase the synthesis of microbial protein, thereby improving the overall protein content of feed and enhancing digestibility. Among the many microorganisms that can be used for straw fermentation, *Aspergillus niger* has attracted considerable attention due to its remarkable ability to secrete extracellular enzymes. This fungus can produce a variety of hydrolytic enzymes, including cellulase, hemicellulase, and lignin-modifying enzymes, showing great potential in breaking down plant cell wall structures.

[0004] However, despite the widespread use of Aspergillus niger in industrial enzyme production, its wild-type or commonly used industrial strains still have significant limitations in straw fermentation: limited enzyme activity, long fermentation cycles, and low overall degradation efficiency of lignocellulose in straw, ultimately leading to low protein conversion rates and making large-scale, economically feasible feed production difficult. Furthermore, the environmental resistance and enzyme production stability of natural strains need further improvement.

[0005] Therefore, addressing the shortcomings of existing Aspergillus niger strains in straw degradation and protein conversion, research on genetic improvement and efficient screening of strains is crucial to constructing engineered Aspergillus niger strains with stronger fiber degradation capabilities, higher protein production efficiency, and suitability for industrial production. This has significant scientific research and practical application value for promoting the high-value utilization of straw resources, reducing environmental pollution, developing circular agriculture, and ensuring feed food security. Developing a new generation of high-performance Aspergillus niger strains using modern gene editing technology, adaptive evolution, and fermentation process optimization has become a promising direction for in-depth exploration in the field of agricultural biotechnology. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for producing mycelial protein feed from fermented steam-exploded straw using Aspergillus niger.

[0007] In a first aspect, this invention provides a *Aspergillus niger* strain, named *Aspergillus niger* GRJC003, which was deposited on January 10, 2025, at the China Center for Type Culture Collection (CCTCCNO: M 2025084), located at Wuhan University, China. This strain was isolated from the rumen fluid of adult small-tailed Han sheep and obtained through directed domestication. It possesses excellent cellulose and hemicellulose degradation capabilities, enabling efficient fermentation of steam-exploded rice straw to produce microbial protein feed. The *Aspergillus niger* GRJC003 strain exhibits high enzyme activity and growth rate during fermentation, significantly increasing microbial protein yield.

[0008] Morphological characteristics of Aspergillus niger strain GRJC003: On PDA medium, the colonies are black, fluffy, and pale yellow on the reverse side; the conidiophores are spherical, the conidiophores are smooth, and the spores are brown. After PCR amplification and sequencing using ITS universal primers, sequence homology comparison using the NCBI database revealed that strain GRJC003 showed the highest sequence homology (100%) with Aspergillus niger (accession number: MG228414.1) and Aspergillus niger (accession number: MN818620.1).

[0009] Secondly, the present invention also provides a method for preparing a microbial protein feed from straw fermented with Aspergillus niger GRJC003.

[0010] A method for preparing a microbial protein feed from straw fermented with Aspergillus niger GRJC003 includes the following steps: (1) By weight, crush 2400-2600 parts of straw with a moisture content of 25-35wt% to a particle size of 0.1-0.5cm, and steam-explode it at 180-200℃ and 2-3Mpa for 4-6min to obtain steam-exploded straw; (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 to obtain fermentation medium, inoculate Aspergillus niger GRJC003 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.

[0011] The straw mentioned in step (1) is at least one of wheat straw, rice straw, and corn straw.

[0012] 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.

[0013] In step (2), the inoculum size of Aspergillus niger GRJC003 is 0.8 × 10⁻⁶. 7 -1.2×10 7 Spores / g fermentation medium.

[0014] The pH of 4.8-5.2 in step (2) is adjusted by 1-2 mol / L sulfuric acid.

[0015] The beneficial effects of this invention are: Compared to existing standard strains, the *Aspergillus niger* strain GRJC003 exhibits superior microbial protein yield under the same processing conditions. This is attributed to the enhanced cellulose degradation capacity and protein synthesis efficiency of the GRJC003 strain, thereby improving resource utilization and feed yield. Secondly, this invention achieves precise control of process parameters by optimizing inoculum size and straw type compatibility, ensuring the stability and scalability of the fermentation process. Furthermore, this method is applicable to various agricultural wastes (such as wheat, rice, and corn straw), broadening raw material sources and reducing production costs. Simultaneously, through steam explosion pretreatment and controlled fermentation, it effectively improves the crude protein content and nutritional value of feed, providing an efficient and environmentally friendly solution for sustainable livestock development. Overall, this invention not only improves the production efficiency of microbial protein but also enhances the practicality and economic viability of the technology. Attached Figure Description

[0016] Figure 1 Macroscopic morphology of Aspergillus niger GRJC003; Figure 2 Microscopic morphology of Aspergillus niger GRJC003. Detailed Implementation

[0017] This invention provides a *Aspergillus niger* fungus, named *Aspergillus niger* GRJC003, which was deposited on January 10, 2025, at the China Center for Type Culture Collection (CCTCCNO: M2025084) at Wuhan University, China.

[0018] 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 -5Three 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.

[0019] 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.

[0020] 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: .

[0021] Example 1

[0022] A method for preparing a microbial protein feed from straw fermented with Aspergillus niger GRJC003 includes the following steps: (1) By weight, 2500 parts of wheat straw with a moisture content of 30wt% were crushed to a particle size of 0.5cm and steam-exploded at 180℃ and 2.5Mpa for 5min to obtain steam-exploded straw; (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 Aspergillus niger GRJC003 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.

[0023] 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.

[0024] The inoculum size of Aspergillus niger in step (2) is 10. 7 Spores / g fermentation medium.

[0025] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0026] Example 2

[0027] A method for preparing a microbial protein feed from straw fermented with Aspergillus niger GRJC003 includes the following steps: (1) By weight, 2500 parts of wheat straw with a moisture content of 30wt% were crushed to a particle size of 0.5cm and steam-exploded at 180℃ and 2.5Mpa for 5min to obtain steam-exploded straw; (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 Aspergillus niger GRJC003 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.

[0028] 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.

[0029] The inoculum size of Aspergillus niger in step (2) is 2 × 10⁻⁶. 7 Spores / g fermentation medium.

[0030] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0031] Example 3

[0032] A method for preparing a microbial protein feed from straw fermented with Aspergillus niger GRJC003 includes the following steps: (1) By weight, 2500 parts of wheat straw with a moisture content of 30wt% were crushed to a particle size of 0.5cm and steam-exploded at 180℃ and 2.5Mpa for 5min to obtain steam-exploded straw; (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 Aspergillus niger GRJC003 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.

[0033] 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.

[0034] The inoculum size of Aspergillus niger in step (2) is 0.5 × 10⁻⁶. 7 Spores / g fermentation medium.

[0035] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0036] Example 4

[0037] A method for preparing a microbial protein feed from straw fermented with Aspergillus niger GRJC003 includes the following steps: (1) By weight, 2500 parts of rice straw with a moisture content of 30wt% were crushed to a particle size of 0.5cm and steam-exploded at 180℃ and 2.5Mpa for 5min to obtain steam-exploded straw; (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 Aspergillus niger GRJC003 in the above fermentation medium, and aerobic ferment at 30℃ and 5.0 pH for 72 h. After fermentation, dry to obtain the Aspergillus niger mycelial protein feed.

[0038] 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.

[0039] The inoculum size of Aspergillus niger in step (2) is 10. 7 Spores / g fermentation medium.

[0040] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0041] Example 5

[0042] A method for preparing a microbial protein feed from straw fermented with Aspergillus niger GRJC003 includes the following steps: (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 180℃ and 2.5Mpa for 5min to obtain steam-exploded straw; (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 Aspergillus niger GRJC003 in the above fermentation medium, and aerobic ferment at 30℃ and 5.0 pH for 72 h. After fermentation, dry to obtain the Aspergillus niger mycelial protein feed.

[0043] 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.

[0044] The inoculum size of Aspergillus niger in step (2) is 10. 7 Spores / g fermentation medium.

[0045] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0046] Example 6

[0047] A method for preparing a microbial protein feed from straw fermented with Aspergillus niger GRJC003 includes the following steps: (1) By weight, 2500 parts of straw with a moisture content of 30wt% were crushed to a particle size of 0.5cm and steam-exploded at 180℃ and 2.5Mpa for 5min to obtain steam-exploded straw; (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 Aspergillus niger GRJC003 in the above fermentation medium, and aerobic ferment at 30℃ and 5.0 pH for 72 h. After fermentation, dry to obtain the Aspergillus niger mycelial protein feed.

[0048] The straw in step (1) is a mixture of wheat straw, corn straw and rice straw in a mass ratio of 1:1:1.

[0049] 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.

[0050] The inoculum size of Aspergillus niger in step (2) is 10. 7 Spores / g fermentation medium.

[0051] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0052] Comparative Example 1

[0053] A method for preparing a mycelial protein feed produced by Aspergillus niger fermentation and steam explosion of straw includes the following steps: (1) By weight, 2500 parts of wheat straw with a moisture content of 30wt% were crushed to a particle size of 0.5cm and steam-exploded at 180℃ and 2.5Mpa for 5min to obtain steam-exploded straw; (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 Aspergillus niger 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.

[0054] 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.

[0055] The Aspergillus niger strain mentioned in step (2), preservation number: CICC40273, was purchased from the China Industrial Microbial Culture Collection Center; the inoculum size of Aspergillus niger was 10. 7 Spores / g fermentation medium.

[0056] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0057] Comparative Example 2

[0058] A method for preparing a mycelial protein feed produced by Aspergillus niger fermentation and steam explosion of straw includes the following steps: (1) By weight, 2500 parts of wheat straw with a moisture content of 30wt% were crushed to a particle size of 0.5cm and steam-exploded at 180℃ and 2.5Mpa for 5min to obtain steam-exploded straw; (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 Aspergillus niger GRJC003 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.

[0059] 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.

[0060] The Aspergillus niger strain mentioned in step (2), preservation number: CGMCC3.3926, was purchased from the China General Microbiological Culture Collection Center; the inoculum size of Aspergillus niger was 10. 7 Spores / g fermentation medium.

[0061] The pH of 5.0 in step (2) is adjusted by 2 mol / L sulfuric acid.

[0062] Test Example 1

[0063] Using 2500g of straw as raw material, the content of microbial protein in the microbial protein feed strains obtained in Examples 1-6 and Comparative Examples 1-2 was determined, and the results are shown in Table 1.

[0064] The specific steps for separating the microbial protein are as follows: add water with a mass of 3 times that of the microbial protein feed, stir and extract for 1 hour, then separate the solid residue from the liquid containing the microbial cells through coarse filtration, and then separate and collect the microbial cells through a plate and frame filter press. The microbial cells are washed with clean water and dried to obtain the Aspergillus niger microbial protein.

[0065] Table 1: Bacterial Protein Content

[0066] Table 1 shows that the *Aspergillus niger* GRJC003 used in this invention has significant advantages in producing microbial protein feed from fermented straw steam explosion products. A comparison of Examples 1, 2, and 3 reveals that the inoculum size of *Aspergillus niger* GRJC003 has a significant impact on the microbial protein content in the feed produced from fermented straw steam explosion products. Increased inoculum size leads to increased microbial protein yield, but this increase is not linear. In Example 2, the inoculum size was twice that of Example 1, but the yield only increased by about 7.6g, a small increase. In Example 3, the inoculum size was half that of Example 1, and the yield decreased significantly (by 49.6g). This indicates that there is an optimal range for the inoculum size; too low a size results in insufficient microbial growth, while too high a size may lead to nutrient competition or metabolic inhibition. Considering economic factors, this invention recommends an inoculum size of 0.8 × 10⁻⁶. 7 -1.2×10 7 The optimal inoculum ratio is spores / g fermentation medium.

[0067] A comparison of Examples 1, 4, 5, and 6 shows that the yield of microbial protein varies depending on the type of straw being fermented. The different chemical compositions of the straws affect the fermentation efficiency. Corn straw usually has a high cellulose content and is easily degraded, so it has the highest yield. Rice straw may contain more silicon and is difficult to degrade, so it has a lower yield. Mixed straws may provide more balanced nutrition and have the highest yield, exhibiting a certain synergistic effect.

[0068] A comparison of Example 1 with Comparative Examples 1 and 2 reveals that the Aspergillus niger strain GRJC003 is significantly superior to the other two strains, with a yield approximately 30-35% higher. This indicates that GRJC003 has a specific advantage in degrading straw and producing mycelial protein, and may possess higher enzyme activity or better adaptability.

Claims

1. A type of Aspergillus niger, characterized in that, The fungus named *Aspergillus niger* GRJC003 was deposited at the China Center for Type Culture Collection (CCTCC) on January 10, 2025, with accession number CCTCCNO: M 2025084, located at Wuhan University, China.

2. The Aspergillus niger as described in claim 1, characterized in that, The strain exhibits the following morphological characteristics: on PDA medium, the colonies are black, fluffy, and pale yellow on the back; the conidiophores are spherical, the conidiophores are smooth, and the spores are brown.

3. The application of Aspergillus niger as described in claim 1 in fermented steam-exploded straw to produce mycelial protein feed, characterized in that, Includes the following steps: (1) By weight, crush 2400-2600 parts of straw with a moisture content of 25-35wt% to a particle size of 0.1-0.5cm, and steam-explode it at 180-200℃ and 2-3Mpa for 4-6min to obtain steam-exploded straw; (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 to obtain fermentation medium, inoculate Aspergillus niger GRJC003 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.

4. The application of Aspergillus niger as described in claim 3 in fermented steam-exploded straw to produce mycelial protein feed, characterized in that, The straw mentioned in step (1) is at least one of wheat straw, rice straw, and corn straw.

5. The application of Aspergillus niger as described in claim 3 in fermented steam-exploded straw to produce mycelial protein feed, characterized in that, 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.

6. The application of Aspergillus niger as described in claim 3 in fermented steam-exploded straw-based mycelial protein feed, characterized in that, The inoculum size of Aspergillus niger GRJC003 in step (2) is 0.8 × 10⁻⁶. 7 -1.2×10 7 Spores / g fermentation medium.

7. The application of Aspergillus niger as described in claim 3 in fermented steam-exploded straw-based mycelium protein feed, characterized in that, The pH of 4.8-5.2 in step (2) is adjusted by 1-2 mol / L sulfuric acid.

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