A Ganoderma lucidum strain and its application in the preparation of mycelial protein from fermented pectin residue.

Abstract

This invention relates to the field of comprehensive utilization technology of industrial solid waste, specifically to a Ganoderma lucidum strain and its application in the preparation of mycelial protein from fermented pectin residue. The invention provides a Ganoderma lucidum strain DCNX0001, with accession number CGMCC NO.42380. This invention establishes that using industrial acid-hydrolyzed pectin residue to prepare a culture medium, and inoculating it with Ganoderma lucidum strain DCNX0001, can not only effectively solve the environmental pollution problem caused by acid-hydrolyzed pectin residue, but also allows strain DCNX0001 to utilize cellulose and hemicellulose from the pectin residue as carbon sources and plant protein and NO3- as nitrogen sources. This results in rapid mycelial growth, high protein conversion efficiency and yield, and the obtained mycelial protein raw material has a protein content of approximately 30%, possesses a pleasant Ganoderma lucidum aroma, and has good palatability. It can be used as a substitute for soybean meal in the feed industry, providing a new approach for the high-value and comprehensive utilization of industrial solid waste.

Description

Technical Field

[0001] This invention relates to the field of comprehensive utilization technology of industrial solid waste, specifically to a Ganoderma lucidum strain and its application in the preparation of mycelial protein from fermented pectin residue. Background Technology

[0002] Pectin, as a healthy food additive, possesses excellent gelling and emulsifying stabilizing properties and is widely used in the food, pharmaceutical, daily chemical, and textile industries. Statistics show that my country's pectin production exceeds 10,000 tons per year. Most pectin is composed of large molecules made up of galacturonic acid, exhibiting excellent water solubility. It is an important component of cell walls and is abundant in raw materials such as apples and citrus fruits, accounting for 15% to 20% of their dry weight. To reduce raw material costs, pectin is primarily extracted from fruit pomace, such as apple and citrus pomace, which are byproducts of fruit juice processing plants.

[0003] Pectin exists in two states: free pectin and bound pectin. The latter is mainly linked to components such as cellulose and hemicellulose in fruit fibers via covalent and ionic bonds. Hot water extraction can only yield free pectin, while bound pectin is significantly lost. Chinese patents CN 108715620, CN 112010994, and CN 114835832 disclose methods for converting bound pectin into free pectin using acid hydrolysis, thereby increasing the pectin extraction rate. Among these, the dilute nitric acid method has gradually become the main method for industrial pectin extraction due to its low equipment corrosion rate and good extraction effect. The solid residue produced after pectin extraction using this method is called acid-hydrolyzed pectin residue, which has a water content of about 80%. This means that my country's annual production of acid-hydrolyzed pectin residue exceeds 200,000 tons. This acid-hydrolyzed pectin residue is characterized by high water content, strong acidity (pH 1.5-2.0), easy perishability, and the generation of volatile nitric acid and soil leachate that pollute the air and soil, making it difficult to utilize. It is classified as hazardous industrial waste and is often directly discarded as waste. This not only wastes resources but also seriously pollutes the environment. Chinese patents CN 111171956, CN 112075350, and CN 11117109 disclose some methods for utilizing pectin residue. However, these methods only utilize some components of the pectin residue and still fail to completely eliminate the environmental impact of the waste. Therefore, how to achieve high-value and full utilization of pectin residue has become crucial for improving the raw material utilization rate of enterprises and overcoming the bottleneck of hazardous waste harmless treatment technology.

[0004] Conventional chemical analysis revealed that, excluding moisture, acid-hydrolyzed pectin residue contains approximately 70% cellulose, 12% hemicellulose, 3% lignin, about 6% protein, 1% reducing sugar, 1% ash, and 0.01M nitrate nitrogen. It is an excellent source of cellulose, hemicellulose, carbon, and protein, and can be used as feed for multigastric animals. However, the acid-hydrolyzed pectin residue is highly acidic, making it difficult for animals to ingest and thus unsuitable for direct feed formulation. Furthermore, the low pH (1.5–2.0) inhibits the growth of yeast and beneficial bacteria during silage fermentation, while promoting the growth of filamentous fungi such as Aspergillus niger and Penicillium, leading to mold growth and severely impacting the safety and quality of silage. Therefore, acid-hydrolyzed pectin residue can only be used as a feed ingredient in silage. Finally, the cellulose and hemicellulose in acid-hydrolyzed pectin residue are only suitable for multigastric animals; monogastric animals lack the corresponding hydrolytic enzymes and cannot be fed it, significantly limiting its application as feed. Therefore, there is an urgent need to develop a method for producing safe, high-quality, and widely adaptable animal feed using acid-hydrolyzed pectin residue as raw material.

[0005] Chinese patent CN115918846A discloses a fermentation method and application of white-rot fungus degrading wheat bran. It uses the white-rot fungus Auricularia auricula-judae CGMCC 5.584 to deeply ferment wheat bran fiber to produce fermented wheat bran dietary fiber and fermentation products, and applies the fermented wheat bran dietary fiber and fermentation products to different products.

[0006] Chinese patent CN116376724A discloses a solid culture medium based on monk fruit residue and its use in culturing white-rot fungi. It proposes a technical approach of culturing the white-rot fungus Proteobacterium chrysosporium BKM-F-1716 from monk fruit residue and using the obtained strain to degrade monk fruit residue.

[0007] It is evident that fermenting acid-hydrolyzed pectin residue with microbial strains or microorganisms can solve the problem of its development and utilization. However, the fermentation conditions and fermentation products of different strains or microorganisms vary greatly. In the development and utilization of acid-hydrolyzed pectin residue by fermentation, it is urgent to solve the problem of which specific microbial strains or microorganisms can be used to obtain high-quality and widely adaptable animal feed. At present, there are no relevant scientific research reports. Summary of the Invention

[0008] To address the above problems, this invention proposes a Ganoderma lucidum strain and its application in the preparation of mycelial protein from fermented pectin residue.

[0009] This invention provides a Ganoderma lucidum strain DCNX0001, which is classified and named Ganoderma lucidum. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.42380 and deposit date of November 24, 2025.

[0010] The Ganoderma lucidum strain DCNX0001 provided by this invention is used in the preparation of mycelial protein raw materials from fermented acid hydrolyzed pectin residue.

[0011] This invention also provides a method for preparing mycelial protein raw materials using Ganoderma lucidum strain DCNX0001 fermented acid hydrolysis pectin residue. The preparation method steps are as follows:

[0012] S1. Preparation of acid hydrolysis pectin residue culture medium;

[0013] S2. Preparation of Ganoderma lucidum strain: A small amount of mycelium of the Ganoderma lucidum strain DCNX0001 was transferred from the preservation tube and domesticated to obtain a second-level strain;

[0014] S3. The second-level strain obtained in S2 is inoculated into the acid-hydrolyzed pectin residue culture medium obtained in S1 at a weight ratio of 1:10~100. The culture is fermented in a sterile fermentation chamber at a temperature of 25~35℃ for 7~28 days to obtain the mycelial protein raw material.

[0015] Furthermore, the method for preparing the acid-hydrolyzed pectin residue culture medium is as follows: one or more acid-hydrolyzed pectin residues are mixed evenly with an alkaline substance to adjust the pH value to 3-9, and then mixed evenly with air-dried rice husks at a weight ratio of 1:0.1-1.0. 15-50% water by weight is added to the mixture, and after stirring evenly, it is placed in an autoclave and sterilized at a temperature of 121°C for 15-25 minutes to obtain the acid-hydrolyzed pectin residue culture medium.

[0016] Furthermore, the pH value of the culture medium is 3 to 6.

[0017] Furthermore, the particle size of the acid-hydrolyzed pectin residue is 1-200 mesh.

[0018] Furthermore, the alkaline substance is one of sodium hydroxide, potassium hydroxide, calcium hydroxide, or ammonia solution with a concentration of 0.1% to 28% by volume.

[0019] Furthermore, the air-dried rice husks mentioned are one or more of wheat husks, barley husks, sorghum husks, or rice husks.

[0020] Furthermore, the mycelial protein raw material is used as a protein feed ingredient to partially replace soybean meal in animal diets, with a replacement rate of 30-80%.

[0021] Furthermore, the mycelial protein raw material is used as a protein feed ingredient to partially replace soybean meal in animal diets, with a replacement rate of 20-30%.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. This invention establishes a method for preparing a culture medium using industrial acid-hydrolyzed pectin residue, inoculated with Ganoderma lucidum strain DCNX0001. This not only effectively solves the environmental pollution problem caused by acid-hydrolyzed pectin residue, but also allows strain DCNX0001 to utilize cellulose and hemicellulose from the pectin residue as a carbon source, along with plant protein and NO... 3- As a nitrogen source, the mycelium grows rapidly, with high protein conversion efficiency and yield. The obtained mycelium protein raw material has a protein content of about 30%, has a Ganoderma lucidum aroma, and is palatable. It can be used as a substitute for soybean meal in the feed industry, providing a new way for the high-value and full utilization of industrial solid waste.

[0024] 2. The method of the present invention is low in cost and simple in process, requires no expensive equipment, has low energy consumption and high efficiency, and the obtained mycelial protein raw material product is of stable quality and safe. It is not only suitable for multigastric animals, but also for the feeding of monogastric animals (such as pigs), thus broadening the applicability of feed.

[0025] 3. This invention aims to respond to the national call for "environmental protection" and the development of low-carbon and green industries, promote the resource utilization of enterprise waste residue, and achieve a double harvest of enterprise operation and social benefits.

[0026] 4. The Ganoderma lucidum strain DCNX0001 used in this invention can survive in a relatively acidic environment (pH>3), preventing the infection of pectin residue bacteria. After a certain population density is formed, it inhibits the growth of mold, thereby ensuring the safety of feed.

[0027] 5. The particle size of the industrial acid-hydrolyzed pectin residue used in this invention is 1-200 mesh. If the particle size of the industrial acid-hydrolyzed pectin residue is less than 1 mesh, the particles are too large, resulting in low fermentation efficiency of the Ganoderma lucidum strain DCNX0001 and difficulty in utilizing the nutrients inside the residue; if the particle size of the industrial acid-hydrolyzed pectin residue is greater than 200 mesh, the mechanical energy required for crushing the pectin residue is extremely high, leading to high costs; therefore, a particle size of 1-200 mesh for the industrial acid-hydrolyzed pectin residue is suitable.

[0028] 6. The pH range of the culture medium of this invention is wide and can be adjusted to 3-9. If the pH of the culture medium is lower than 3 or higher than 9, the growth of Ganoderma lucidum strain DCNX0001 will be slow, or even result in death. In particular, the pH of the acidic culture medium is 3-6, which can be achieved by adding a small amount of alkaline substance. Ganoderma lucidum strain DCNX0001 can then colonize and grow. During the growth process, the pH of the culture medium is gradually neutralized, and the resulting mycelial protein raw material is neutral and can be directly used as feed.

[0029] 7. This invention uses air-dried rice husks in the culture medium, with a weight ratio of pectin residue to air-dried rice husks of 1:0.1~1.0. If the weight ratio of acid-hydrolyzed pectin residue to air-dried rice husks is greater than 1:0.1, the fermentation substrate is relatively dense, resulting in low internal oxygen content, which leads to low fungal fermentation efficiency and decreased protein yield. If the weight ratio is less than 1:1.0, the air-dried rice husks dilute the acid-hydrolyzed pectin residue, reducing nitrate nitrogen concentration and decreasing protein production efficiency. Therefore, a weight ratio of 1:0.1~1.0 for acid-hydrolyzed pectin residue to air-dried rice husks is appropriate. The main function of the air-dried rice husks is to provide sufficient oxygen aeration channels for the mycelial growth of Ganoderma lucidum strain DCNX0001. The dried husks used in this invention are wheat husks, barley husks, sorghum husks, or rice husks. These plant husks degrade slowly and can continuously provide sufficient oxygen aeration channels for mycelial growth during the 7-28 days of mycelial growth in this invention. When applied to feed later, these plant husks are rich in cellulose and lignin and will not cause any side effects.

[0030] 8. The mixture obtained during the preparation of the culture medium of the present invention shall be prepared into a slurry with a concentration of 15-50% by weight using water. If the concentration of the slurry is less than 15% by weight, the Ganoderma lucidum strain DCNX0001 will only grow on the surface of the culture medium due to the excessive water content of the slurry; if the concentration of the slurry is greater than 50% by weight, the Ganoderma lucidum strain DCNX0001 will not be able to grow due to the excessive water content of the culture medium. Therefore, a slurry concentration of 15-50% by weight is appropriate, preferably 20-45%. Attached Figure Description

[0031] Figure 1 This is a comparative diagram showing the fermentation of corn straw by the three strains in Example 2. Detailed Implementation

[0032] The present invention will be further described below with reference to the embodiments.

[0033] Example 1: Screening dominant strains of fermented acid-hydrolyzed pectin residue from edible fungi:

[0034] 1.1 Sampling: Samples were collected from edible fungi products purchased from the market, including Ganoderma lucidum, oyster mushroom, morel mushroom, lion's mane mushroom, schizophyllum commune, wood ear fungus, milkweed, Phellinus linteus, button mushroom, shiitake mushroom, king oyster mushroom, button mushroom, and hydrangea.

[0035] 1.2 Separation: After sample pretreatment, the samples were spread on acidic separation plates containing chloramphenicol and streptomycin with pectin as the sole carbon source and incubated at 28°C in the dark for 5 days.

[0036] 1.3 Purification: Select 20 fast-growing strains with different colony morphologies and purify them on PDA plates.

[0037] 1.4 Initial Screening: Current technology generally suggests that strains with a (D / d) ratio greater than 2.5 have greater fermentation potential and faster fermentation speed, and can quickly produce pectinase to ferment and decompose pectin. Therefore, the purified strains from 1.3 were spotted onto seed pectin Congo red plates, and three strains with a clear zone diameter ratio (D / d) greater than 2.5 were screened from 20 strains (as shown in Table 1). These are strain 4 (D / d = 4.3 ± 0.2), strain 11 (D / d = 2.7 ± 0.2), and strain 18 (D / d = 2.8 ± 0.1). Strain 4 is derived from Ganoderma lucidum; strain 11 from Pleurotus ostreatus; and strain 18 from Schizophyllum commune.

[0038] Table 1. Results of hydrolysis zone assay of the initial screening strains on pectin Congo red agar plates:

[0039]

[0040] Note: Data are expressed as mean ± standard deviation (n=3).

[0041] 1.5. Secondary Screening: Strains 4, 11, and 18 were compared with the commercially available Aspergillus niger NRRL 330 (purchased from the China General Microbiological Culture Collection Center, accession number AS3.795), which currently dominates the feed additive market, in a comparative experiment on the synthesis of microbial protein and the degradation rate of pectin in apple pectin pomace. The results are shown in Table 2.

[0042] Table 2. Comparative results of the synthesis of bacterial cell protein and pectin degradation rate of four strains:

[0043]

[0044] As shown in Table 2, strain 4 had the highest cell dry weight, crude protein content, crude protein yield, and pectin degradation rate among the three selected strains, and was significantly higher than Aspergillus niger NRRL 330.

[0045] Example 2: Fermentation experiment of corn straw using the three strains screened in Example 1:

[0046] Strains: Strains 4, 11, and 18 screened in Example 1;

[0047] Fermentation substrate: naturally air-dried corn stalks, which are crushed and passed through a 40-mesh sieve;

[0048] Culture medium: Potato glucose agar (PDA) medium, used for strain activation.

[0049] Test method:

[0050] Raw material pretreatment: Accurately weigh 3.0g of crushed corn stalks and place them in a 250mL Erlenmeyer flask. Add 7mL of deionized water and stir well to fully wet the raw material.

[0051] Sterilization: Seal the bottle opening with breathable sealing film, sterilize in a high-pressure steam sterilizer at 121℃ for 15 minutes, remove and cool to room temperature;

[0052] Inoculation: In a clean bench, the mycelia of each strain activated to the logarithmic growth phase were rinsed with sterile physiological saline and homogenized in a sterile homogenizer for 30 seconds (300W) to prepare a homogenized bacterial suspension. 0.15 mL (5% inoculum, v / w) of the bacterial suspensions of strains 4, 11, and 18 were accurately pipetted into the corresponding sterile conical flasks.

[0053] Fermentation culture: The inoculated conical flasks were placed in a constant temperature shaker and cultured in the dark for 5 days at 28±1℃ and 150 rpm. Fermentation comparison diagram is shown below. Figure 1 As shown, Figure 1 Bottle A was inoculated with strain 4; bottle B was inoculated with strain 18; and bottle C was inoculated with strain 11.

[0054] Sample processing and testing: After the culture was completed, the fermentation product was dried at 65℃ to constant weight, pulverized and the crude protein increase was measured: The crude protein content of the material before and after fermentation was determined and calculated by the Kjeldahl method (GB / T 6432).

[0055] The experimental results are shown in Table 3. Strain 4 (Ganoderma lucidum strain) showed significant advantages in corn straw fermentation:

[0056] Table 3. Comparative experiment on the synthesis of microbial protein from corn straw by three strains:

[0057]

[0058] It is evident that fermentation by strain 4 increased the crude protein content of corn stalks by 32.7%, a much larger increase than that of strains 11 and 18. This confirms that strain 4 has a remarkable ability to utilize plant fiber carbon sources for cell protein synthesis.

[0059] Example 3: Comparative experiment on the enrichment of amino acids in apple pectin residue using screened strains:

[0060] A comparative experiment was conducted on the enrichment of amino acids in apple pectin pomace using strains 4, 11, and 18 from Example 1, and Aspergillus niger NRRL 330. Specifically, strains 4, 11, and 18, as well as Aspergillus niger NRRL 330, were inoculated from their stored state into suitable solid slant or liquid culture media and activated at a suitable temperature (e.g., 28-30°C) for 1-2 days to ensure vigorous growth and no contamination. The apple pectin pomace was dried, pulverized, and sieved. To provide sufficient nitrogen for amino acid synthesis, the pectin pomace was mixed with appropriate amounts of organic nitrogen sources (e.g., soybean meal, wheat bran) and inorganic salts, and adjusted to suitable moisture content and initial pH. The four bacterial suspensions were inoculated into their respective fermentation substrate groups at a ratio of 5%, and thoroughly mixed. All groups were subjected to solid-state fermentation under the same conditions. The temperature was 28-30°C, and fermentation lasted 5-7 days. The amino acid content was determined using an amino acid analyzer (AminoSAAYA).

[0061] The samples were divided into five groups: unfermented pomace, pomace fermented with strain 4, pomace fermented with strain 11, pomace fermented with strain 18, and pomace fermented with Aspergillus niger NRRL 330. The results are shown in Table 4.

[0062] Table 4. Comparative experimental results of amino acid enrichment in apple pectin residue by four bacterial strains:

[0063]

[0064] As shown in Table 4, the amino acid content of most of the fruit pomace groups fermented with strain 4 was significantly higher than that of the fruit pomace group without bacterial fermentation or the experimental groups fermented with other bacteria.

[0065] Example 4: Comparative experiment on the enrichment of amino acids in corn straw using screened strains:

[0066] A comparative experiment was conducted on the enrichment of amino acids from corn stalks using strains 4, 11, and 18 from Example 1, and Aspergillus niger NRRL 330. Specifically, the corn stalks were dried, pulverized (passed through a 1-2 mm sieve), and the contact area was increased. The moisture content was adjusted to 60%. The stalks were then sterilized using a high-pressure steam sterilizer to destroy the lignin fiber structure. Necessary nitrogen sources (such as ammonium sulfate, urea, or soybean meal) were added to adjust the carbon-to-nitrogen ratio to a suitable range of 25:1 to 30:1, and a small amount of inorganic salts (such as phosphates and magnesium sulfate) were added. Strains 4, 11, 18, and Aspergillus niger NRRL 330 were activated to prepare spore suspensions or bacterial suspensions of consistent concentration. These were inoculated into the sterilized fermentation substrate at a 5% inoculum and thoroughly mixed. The inoculated materials were placed in a temperature-controlled incubator and fermented in a solid-state environment at 28-30°C for 7 days. The amino acid content was determined using an amino acid analyzer (AminoSAAYA).

[0067] The samples were divided into five groups: unfermented corn stalks, corn stalks fermented with strain 4, corn stalks fermented with strain 11, corn stalks fermented with strain 18, and corn stalks fermented with Aspergillus niger NRRL 330. The results are shown in Table 5.

[0068] Table 5. Comparative experimental results of amino acid enrichment in corn straw by four strains:

[0069]

[0070] As shown in Table 5, the selected strains had lower amino acid content after fermenting corn stalks. This is due to the difference in nutrients contained in pectin residue and corn stalks themselves. However, it can be seen that strain 4 had a higher amino acid content in fermented corn stalks than the other strains.

[0071] Example 5: Comparative experiment on the enrichment of amino acids in apple pectin residue by strain 4 and other white rot fungi:

[0072] A comparative experiment was conducted on the enrichment of amino acids in apple pectin pomace using strain 4 and strain 3 (also a white-rot fungus, i.e., strain 3 from Example 1), both belonging to the Hericium erinaceus family. Specifically, strains 4 and 3 were inoculated from their storage state into suitable solid slant or liquid culture media and activated at a suitable temperature (e.g., 28-30°C) for 1-2 days to ensure vigorous growth and no contamination. The apple pectin pomace was dried, pulverized, and sieved. To provide sufficient nitrogen for amino acid synthesis, the pectin pomace was mixed with appropriate amounts of organic nitrogen sources (e.g., soybean meal, wheat bran) and inorganic salts, and adjusted to suitable moisture content and initial pH. The four prepared bacterial suspensions were inoculated into the corresponding fermentation substrate groups at a ratio of 5%, and thoroughly mixed. All groups were subjected to solid-state fermentation under the same conditions. The temperature was 28-30°C, and fermentation lasted for 5-7 days. The amino acid content was determined using an amino acid analyzer (AminoSAAYA).

[0073] The samples were divided into two groups: group 4, fermented fruit pomace with strain 4, and group 4, fermented fruit pomace with Hericium erinaceus strain 4. The results are shown in Table 6.

[0074] Table 6. Comparative experimental results of two white-rot strains on the enrichment of amino acids in apple pectin residue:

[0075]

[0076] As shown in Table 6, the content of each amino acid in apple pectin residue fermented by strain 4 was significantly higher than that in Hericium erinaceus.

[0077] Following the above experiments, strain 4 was selected as the dominant strain for fermenting and acid-hydrolyzing pectin residue, and named Ganoderma strain DCNX0001. Based on ITS sequence analysis, it was classified as Ganoderma lucidum and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42380.

[0078] Example 6: Preparation of mycelial protein raw material from apple pectin residue:

[0079] 6.1 Preparation of acid-hydrolyzed pectin residue culture medium:

[0080] Mix 50-mesh apple pectin residue with a 2.0M sodium hydroxide alkaline solution until homogeneous, and adjust the pH of the mixture to 5.5. Then, mix the mixture with wheat hulls at a weight ratio of 1:0.8 until homogeneous. The resulting mixture is then prepared into a slurry with water to a concentration of 28% by weight. The slurry is then placed in an autoclave and sterilized at 121°C for 18 minutes to obtain an acid-hydrolyzed pectin residue culture medium.

[0081] 6.2 Preparation of Ganoderma lucidum strains:

[0082] (1) Preparation of bacterial culture medium: According to the weight ratio of culture medium to distilled water of 26:900, potato glucose water culture medium produced by Qingdao Haibo Biotechnology Co., Ltd. was dissolved in distilled water. The resulting solution was then sterilized at a temperature of 121°C and a pressure of 1.15 MPa for 17 minutes to obtain the bacterial culture medium.

[0083] (2) Inoculation: Use an inoculation loop to transfer a small amount of Ganoderma lucidum strain DCNX0001 mycelium from the culture tube and inoculate it into the culture medium using the immersion inoculation method;

[0084] (3) Second-level strain culture: The culture medium inoculated with Ganoderma lucidum strain DCNX0001 was cultured in a shaker at 28℃ and 180 rpm for 3 days to obtain the first-level strain; then, 3 mL of the first-level strain was taken and inoculated into another culture medium, and cultured for 3 days under the same culture conditions to obtain the second-level strain.

[0085] 6.3 Preparation of mycelial protein raw materials:

[0086] The second-level strain obtained in step 6.2 was inoculated into the acid-hydrolyzed pectin residue culture medium obtained in step 6.1 at a weight ratio of 1:46. The culture was then fermented in a sterile fermentation chamber with ultraviolet sterilization function of wavelength 240-280nm at a temperature of 28℃ for 23 days to obtain the mycelial protein raw material.

[0087] According to the analytical method of GB / T 6432-2018, the crude protein content of the mycelial protein raw material is 34.69% by weight. According to the analytical method of DB15 / T 2600—2022, the crude fiber content of the mycelial protein raw material is 30.04% by weight.

[0088] Example 7: Preparation of mycelial protein raw material from citrus pectin residue:

[0089] 7.1 Preparation of acid-hydrolyzed pectin residue culture medium:

[0090] Mix 1-mesh citrus pectin residue with a 0.01 M potassium hydroxide alkaline solution until homogeneous, and adjust the pH of the fermentation substrate to 9.0. Then, mix the mixture with barley hulls at a weight ratio of 1:1.0 until homogeneous. The resulting mixture is then prepared into a slurry with water to a concentration of 15% by weight. The slurry is then placed in an autoclave and sterilized at 121°C for 20 minutes to obtain the acid hydrolyzed pectin residue culture medium.

[0091] 7.2 Preparation of Ganoderma lucidum strain: Same as in Example 6;

[0092] 7.3 Preparation of mycelial protein raw materials:

[0093] The secondary strain obtained in step 7.2 was inoculated into the culture medium obtained in step 7.1 at a weight ratio of 1:64. The culture was then fermented in a sterile fermentation chamber with ultraviolet sterilization function at a temperature of 25°C for 28 days to obtain the mycelial protein raw material.

[0094] According to the analytical method of GB / T 6432-2018, the crude protein content of the mycelial protein raw material is 35.02% by weight. According to the analytical method of DB15 / T 2600—2022, the crude fiber content of the mycelial protein raw material is 52.03% by weight.

[0095] Example 8: Preparation of mycelial protein raw material from sunflower pectin residue:

[0096] 8.1 Preparation of Acid-hydrolyzed Pectin Residue Culture Medium

[0097] Sunflower pectin residue with a particle size of 80 mesh was mixed evenly with an ammonia alkaline solution with a concentration of 4.5M and a volume of 14.2% to adjust the pH value of the fermentation substrate to 6.8. Then, the mixture was mixed evenly with sorghum husks at a weight ratio of 1:0.4. The resulting mixture was then prepared into a slurry with a concentration of 32% by weight using water. The slurry was then placed in an autoclave and sterilized at a temperature of 121°C for 15 minutes to obtain the acid hydrolyzed pectin residue culture medium.

[0098] 8.2 Preparation of Ganoderma lucidum strain: Same as in Example 6;

[0099] 8.3 Preparation of mycelial protein raw materials:

[0100] The secondary strain obtained in step 8.2 was inoculated into the culture medium obtained in step 8.1 at a weight ratio of 1:10. The culture was then fermented in a sterile fermentation chamber with ultraviolet sterilization function at a temperature of 30°C for 7 days to obtain the mycelial protein raw material.

[0101] According to the analytical method of GB / T 6432-2018, the crude protein content of the mycelial protein raw material is 28.78% by weight. According to the analytical method of DB15 / T 2600—2022, the crude fiber content of the mycelial protein raw material is 66.72% by weight.

[0102] Example 9: Preparation of mycelial protein raw material from tofu leaf pectin residue:

[0103] 9.1 Preparation of acid-hydrolyzed pectin residue culture medium:

[0104] Mix 200-mesh tofu and vegetable pectin residue with calcium hydroxide solid powder at a weight ratio of 1:0.001 until homogeneous, and adjust the pH of the fermentation substrate to 8.0. Then, mix the mixture with rice husks at a weight ratio of 1:0.1 until homogeneous, and then use water to prepare a slurry with a concentration of 42% by weight. Then, put it into an autoclave and sterilize it at 121°C for 25 minutes to obtain an acid hydrolyzed pectin residue culture medium.

[0105] 9.2 Preparation of Ganoderma lucidum strain: Same as in Example 6;

[0106] 9.3 Preparation of mycelial protein raw materials

[0107] The second-level strain obtained in step 9.2 was inoculated into the culture medium obtained in step 9.1 at a weight ratio of 1:28. The culture was then fermented in a sterile fermentation chamber with ultraviolet sterilization function at a temperature of 35°C for 10 days to obtain the mycelial protein raw material.

[0108] According to the analytical method of GB / T 6432-2018, the crude protein content of the mycelial protein raw material is 25.01% by weight. According to the analytical method of DB15 / T 2600—2022, the crude fiber content of the mycelial protein raw material is 70.03% by weight.

[0109] Example 10: Preparation of mycelial protein raw material from sweet potato pectin residue:

[0110] 10.1 Preparation of acid-hydrolyzed pectin residue culture medium:

[0111] Mix 120-mesh sweet potato pectin residue with calcium hydroxide solid powder at a weight ratio of 1:0.01 until homogeneous, and adjust the pH of the fermentation substrate to 3.0. Then, mix the mixture with a mixture of wheat husks and barley husks (weight ratio 1:1) at a weight ratio of 1:0.2 until homogeneous. Use water to prepare a slurry with a concentration of 50% by weight. Then, put it into an autoclave and sterilize it at 121°C for 22 minutes to obtain acid hydrolyzed pectin residue culture medium.

[0112] 10.2 Preparation of Ganoderma lucidum strain: Same as in Example 6;

[0113] 10.3 Preparation of mycelial protein raw materials

[0114] The second-level strain obtained in step 10.2 was inoculated into the culture medium obtained in step 10.1 at a weight ratio of 1:82. The culture was then fermented in a sterile fermentation chamber with ultraviolet sterilization function at a temperature of 29°C for 14 days to obtain the mycelial protein raw material.

[0115] According to the analytical method of GB / T 6432-2018, the crude protein content of the mycelial protein raw material is 29.12% by weight. According to the analytical method of DB15 / T 2600—2022, the crude fiber content of the mycelial protein raw material is 41.25% by weight.

[0116] Application of the mycelial protein raw materials prepared in Examples 11 and 6 in feeding pigs:

[0117] Control group diet: The corn-soybean meal basal diet was formulated according to the "Swine Feeding Standard" (NY / T 65-2004) to meet the nutritional needs of growing and finishing pigs weighing 60-100kg.

[0118] Experimental diet: The mycelial protein raw material prepared in Example 6 was used to replace 25% of the soybean meal protein in the control group diet with isonitrogenous protein to ensure that the two diets maintained relative consistency in the main nutritional indicators such as digestible energy, crude protein, calcium, and phosphorus.

[0119] Experimental animals: Forty castrated boars from Rongchang, Sichuan Province, with similar weight (60±2 kg), good health, and consistent genetic background were selected.

[0120] Experimental design and feeding management: A single-factor completely randomized block design was adopted, and 40 pigs were randomly divided into two groups (control group and experimental group) according to their weight and origin, with 5 replicates (pens) in each group and 4 pigs in each pen.

[0121] Pre-trial period: 7 days. All pigs were fed the control diet to acclimatize to the experimental environment and grouping.

[0122] The trial period was 60 days. Both the control and experimental groups had free access to their respective daily rations and water. Daily feed consumption in each pen was recorded, and the health status of the pigs was observed.

[0123] Measurement indicators and methods:

[0124] Growth performance: Each pig was weighed on an empty stomach before morning feeding at the start of the experiment (day 0), day 30, and day 60, and the average daily gain (ADG) was calculated. The average daily feed intake (ADFI) and feed conversion ratio (F / G) were calculated based on feed consumption records for each pen.

[0125] Nitrogen metabolism experiment: During days 45 to 50 of the trial period, six pigs (1-2 per pen) with near-average body weight were selected from each group and transferred to a dedicated metabolic cage. Feces and urine were collected continuously for five days using the total fecal-urine collection method. The nitrogen content in feed, feces, and urine was determined (Kjeldahl method, GB / T 6432). Apparent nitrogen digestibility, nitrogen deposition rate, and apparent biological value (BV) of protein were calculated.

[0126] Carcass quality determination (at the end of the experiment): Five pigs close to the average weight were selected from each group for slaughter determination. Backfat thickness and eye muscle area were measured, and samples were taken to analyze the crude fat and crude protein content of the muscle.

[0127] Experimental Results: The experimental data were statistically processed, and the results are shown in Table 7.

[0128] Table 7. Experimental results of feeding pigs with mycelial protein raw material prepared in Example 6 as a substitute for 25% soybean meal:

[0129]

[0130] Conclusions and Analysis:

[0131] This embodiment systematically verifies the application value of the mycelial protein raw material of the present invention in pig production. After replacing 25% of soybean meal protein with the product of the present invention, the average daily weight gain of fattening pigs increased significantly by 8.9%, while the feed conversion ratio decreased significantly by 7.1%. This indicates that the feed of the present invention can not only maintain the animal's feed intake, but also efficiently promote growth and save feed.

[0132] The experimental group of pigs showed a fundamental improvement in the utilization efficiency of dietary protein, with the biological value (BV) of protein jumping from 60.3% to 74.1%. This demonstrates that the Ganoderma lucidum strain DCNX0001 provided by this invention optimizes the amino acid balance of protein during the acid hydrolysis pectin residue fermentation process, making it more in line with the physiological needs of pigs and reducing nitrogen waste and emissions.

[0133] The experimental group of pigs exhibited lower backfat thickness and larger eye muscle area, while also showing increased muscle protein content. This indicates that the feed of the present invention helps improve carcass lean meat percentage and muscle quality, and has potential added-value benefits.

[0134] While improving production performance, the use of low-cost fruit pomace fermented protein replaced part of the expensive soybean meal, directly reducing feed costs. Higher nitrogen deposition rates mean lower nitrogen emissions, aligning with the current requirements for sustainable development in the livestock industry.

[0135] Example 12: Application of the mycelial protein raw material prepared according to the present invention in feeding laying hens:

[0136] Control group diet: The basic laying hen feed was formulated with corn-soybean meal according to the "Chicken Feeding Standard" (NY / T 33-2004).

[0137] The experimental group diet consisted of mycelial protein raw material prepared in Example 6, which replaced 25% of the soybean meal protein in the control group diet with isonitrogenous alternatives. The composition and nutritional level of other raw materials remained consistent.

[0138] Experimental animals: 200 34-week-old Jingbai laying hens with similar egg production rate, weight, and health status were selected.

[0139] Experimental methods and feeding management: A single-factor completely randomized design was used to randomly divide 200 laying hens into two groups (control group and experimental group), with 5 replicates in each group and 20 hens in each replicate, and they were cage-housed.

[0140] Pre-trial period: 7 days. All chickens were fed the control group diet to acclimatize to the group and environment.

[0141] The trial period was 42 days. The control and experimental groups were fed their respective diets. Feed and water were provided freely, and a 16L:8D lighting regime was used. Daily records were kept for each replicate, including egg production, egg weight, number of broken / soft eggs, number of dead eggs, and feed consumption.

[0142] Measurement indicators and methods:

[0143] Egg production performance: Egg production rate (%) = (Total number of eggs produced during the statistical period / (Number of chickens raised × Number of days in the statistical period)) × 100

[0144] Average egg weight (g): The average weight of all eggs is calculated by weighing them repeatedly each day.

[0145] Daily feed intake (g / animal·day): Accurately record the daily feed consumption.

[0146] Feed conversion ratio = Daily feed intake (g) / Average daily egg weight (g);

[0147] Egg quality determination (last 3 days of the trial period): 10 eggs were randomly selected from each replicate, and Haugh units (protein height measuring instrument), yolk color (Roche colorimeter fan), eggshell strength (eggshell strength measuring instrument) and eggshell thickness were measured.

[0148] The crude protein content of whole eggs was determined according to the national standard method (GB 5009.5).

[0149] Nitrogen metabolism and emission assessment (days 35-37 of the pilot test): Three replicates were selected for each treatment, and feces were collected continuously for 3 days using the total collection method. The nitrogen content in feed and feces was determined (Kjeldahl method, GB / T 6432), and the apparent nitrogen use efficiency (%) and nitrogen emission per kg of eggs produced were calculated.

[0150] Experimental Results: The experimental data were statistically processed, and the results are shown in Table 8.

[0151] Table 8. Results of the experiment in which the mycelial protein raw material prepared in Example 6 replaced 25% soybean meal in the feed of laying hens:

[0152]

[0153] This embodiment systematically demonstrates the comprehensive advantages of the mycelial protein raw material of the present invention in laying hen farming: after replacing 25% of soybean meal protein, the egg production rate and average egg weight were significantly improved, and the feed conversion ratio was significantly reduced, indicating improved feed conversion efficiency. Particularly noteworthy is the increase in egg protein content from 12.0% to 13.5%, with a highly significant improvement in yolk color, directly enhancing the product's nutritional value and commercial appearance. Improved nitrogen utilization and reduced nitrogen emissions per unit of egg production align with the trend of green farming. Simultaneously, the use of lower-cost fruit pomace fermentation products to replace part of the soybean meal directly reduces feed costs, resulting in significant overall economic benefits.

[0154] Example 13: Application of the mycelial protein raw material prepared according to the present invention in feeding beef cattle:

[0155] Experimental group feed: The high amino acid microbial protein feed prepared in Example 6 of this invention was used to replace 20% of the soybean meal in the basic concentrate supplement with isonitrogen.

[0156] Control group feed: a commercially available brand of basic concentrate supplement (soybean meal as the conventional protein source).

[0157] Roughage: Both groups had free access to the same whole-plant corn silage and sheep grass.

[0158] Experimental animals: 20 Qin Chuan beef cattle with similar weight (400±20 kg), age, and health status were selected.

[0159] Experimental method: A completely randomized grouped design was adopted, and 20 experimental animals were randomly divided into 2 groups of 10 animals each and housed in single pens.

[0160] Pre-trial period: 10 days. All animals were fed a basal diet to acclimate to the environment and grouping.

[0161] Trial period: 60 days. The control group was fed a basal concentrate supplement, while the experimental group was fed a concentrate supplement containing 20% ​​of the microbial protein feed of this invention. The concentrate was fed in fixed amounts according to body weight, while roughage was allowed to be consumed freely.

[0162] Measurement indicators and methods:

[0163] Growth performance: All experimental animals were weighed on an empty stomach before morning feeding at the start of the experiment (day 0) and the end of the experiment (day 60), and the initial and final weights were recorded to calculate the average daily gain (ADG). The amount of concentrate fed to each animal each day was accurately recorded to calculate the average daily feed intake (ADFI), and the feed conversion ratio (F / G) was further calculated.

[0164] Nitrogen metabolism test: During the trial period, from day 50 to 55, the total fecal and urine collection method was used for 6 consecutive days. A fixed amount of feces and urine were collected from each animal daily, mixed thoroughly, and then sampled. The nitrogen content in the feed, feces, and urine was determined according to the standard "Determination of Nitrogen in Feed" (GB / T 6432).

[0165] calculate:

[0166] Apparent nitrogen digestibility (ADN, %) = (IN - FN) / IN × 100

[0167] Nitrogen deposition rate (NR, %) = (IN - FN - UN) / IN × 100

[0168] Protein epigenetic value (BV, %) = (IN - FN - UN) / (IN - FN) × 100

[0169] Where: IN is ingested nitrogen, FN is fecal nitrogen, and UN is urinary nitrogen.

[0170] Experimental Results and Analysis:

[0171] Table 9. Results of the experiment in which mycelial protein raw material prepared in Example 6 replaced 20% soybean meal in beef cattle:

[0172]

[0173] This embodiment demonstrates that replacing 20% ​​of soybean meal with the microbial protein feed of this invention significantly increases the average daily weight gain of fattening cattle (+13.8%) and significantly reduces the feed conversion ratio (-11.1%), indicating a substantial improvement in feed conversion efficiency. The experimental group animals showed significantly improved nitrogen utilization efficiency, with a protein biological value (BV) as high as 75.2%, a 29.7% increase compared to the control group (58.0%). This confirms that fermentation with the specific strains of this invention fundamentally improves the amino acid balance and availability of pomace protein, reducing nitrogen excretion and meeting environmentally friendly farming requirements.

[0174] While improving production performance, this invention also reduces the average daily feed cost (-7.2%) because the feed cost is lower than that of soybean meal, resulting in significant overall economic benefits.

[0175] Example 14: Application of the mycelial protein raw material prepared according to the present invention in fattening sheep

[0176] Control group diet: formulated according to my country's "Standards for Feeding Meat Sheep", a corn-soybean meal type basic concentrate supplement with soybean meal as the main protein source.

[0177] Experimental diet: The mycelial protein raw material prepared in Example 6 was used to replace 30% of the soybean meal protein in the control group diet with isonitrogenous material, while other raw materials and proportions remained unchanged.

[0178] Experimental animals: 20 one-year-old Shaanbei white cashmere goats (castrated males) with similar weight (30±2 kg) and good health were selected.

[0179] Experimental method: A single-factor completely randomized experimental design was adopted, and 20 goats were randomly divided into two groups (control group and experimental group), with 10 goats in each group and kept in a single pen.

[0180] Pre-trial period: 10 days. All sheep were fed the control group diet and were dewormed and vaccinated to adapt to the experimental environment and feeding procedures.

[0181] The trial period was 60 days. The control group and the experimental group were fed their respective daily diets. They were fed twice a day at fixed times, with a fixed amount of concentrate supplement (reference amount: 1.0-1.2 kg per head per day, adjusted according to body weight), and roughage (peanut vines) was available for free access to food and water.

[0182] Measurement indicators and methods:

[0183] Growth performance: All experimental sheep were weighed on an empty stomach before morning feeding at the start of the experiment (day 0) and at the end of the experiment (day 60), and the initial and final weights were recorded. The average daily gain (ADG) was calculated.

[0184] Accurately record the daily amount of concentrate fed to each sheep and the amount remaining, and calculate the average daily feed intake (ADFI). Calculate the feed conversion ratio (F / G) based on ADG and ADFI.

[0185] Nitrogen metabolism test: During the 50th to 55th day of the trial period, for 6 consecutive days, 5 sheep with a weight close to the average weight were selected from each group, and the digestion and metabolism test was conducted using the total fecal and urine collection method. A quantitative amount of all feces and urine was collected daily, the total volume was recorded, and samples were taken after mixing. The nitrogen content in the feed, feces, and urine was determined according to the Kjeldahl method for the determination of crude protein in feed (GB / T 6432).

[0186] Calculate the following indicators:

[0187] Apparent nitrogen digestibility (ADN, %) = (IN - FN) / IN × 100

[0188] Nitrogen deposition rate (NR, %) = (IN - FN - UN) / IN × 100

[0189] Protein epigenetic value (BV, %) = (IN - FN - UN) / (IN - FN) × 100

[0190] Where: IN is ingested nitrogen, FN is fecal nitrogen, and UN is urinary nitrogen.

[0191] Experimental Results: Experimental data are expressed as mean ± standard deviation. Statistical analysis results are shown in the table below:

[0192] Table 10. Results of the experiment on replacing 30% soybean meal with mycelial protein raw material prepared in Example 6 for feeding fattening sheep:

[0193]

[0194] This embodiment scientifically demonstrates the practical application value of the mycelial protein raw material of the present invention: compared with pure soybean meal diet, using the mycelial protein raw material of the present invention to replace 30% of soybean meal protein can significantly increase the average daily weight gain of fattening goats and significantly reduce the feed conversion ratio, indicating that the feed conversion efficiency is effectively improved and the breeding cycle can be shortened.

[0195] The experimental group of goats showed a comprehensive improvement in the utilization efficiency of dietary nitrogen. The biological value (BV) of protein increased from 58.2% to 75.0%, which directly verifies that the fermentation process optimized the amino acid composition and balance, resulting in a significant increase in the efficiency of protein deposition in the animal body. This forms a strong closed loop with the previous finding of "significant increase in amino acid content".

[0196] Improved growth performance translates to higher yields within the same timeframe. Simultaneously, a higher nitrogen deposition rate means reduced nitrogen emissions (manure nitrogen, urine nitrogen) into the environment, aligning with green farming principles. This invention successfully transforms low-value fruit pomace into high-value feed, reducing reliance on soybean meal in livestock farming and yielding significant economic and social benefits.

[0197] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0198] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A Ganoderma lucidum strain DCNX0001, with the accession number CGMCC NO.42380.

2. The application of the Ganoderma lucidum strain DCNX0001 as described in claim 1 in the preparation of mycelial protein raw materials from fermented acid hydrolyzed pectin residue.

3. A method for preparing mycelial protein raw materials using the fermentation and acid hydrolysis pectin residue of the Ganoderma lucidum strain DCNX0001 as described in claim 1, characterized in that, The preparation method steps are as follows: S1. Preparation of acid-hydrolyzed pectin residue culture medium; S2. Preparation of Ganoderma lucidum strain: A small amount of mycelium of the Ganoderma lucidum strain DCNX0001 was transferred from the preservation tube and domesticated to obtain a second-level strain; S3. The second-level strain obtained in S2 is inoculated into the acid-hydrolyzed pectin residue culture medium obtained in S1 at a weight ratio of 1:10~100. The culture is fermented in a sterile fermentation chamber at a temperature of 25~35℃ for 7~28 days to obtain the mycelial protein raw material.

4. The method for preparing mycelial protein raw materials from fermented fruit residue of Ganoderma lucidum strain DCNX0001 according to claim 3, characterized in that, The method for preparing the acid-hydrolyzed pectin residue culture medium is as follows: one or more acid-hydrolyzed pectin residues are mixed evenly with an alkaline substance to adjust the pH value to 3-9, and then mixed evenly with air-dried rice husks at a weight ratio of 1:0.1-1.

0. 15-50% water by weight is added to the mixture, and after stirring evenly, it is placed in an autoclave and sterilized at a temperature of 121°C for 15-25 minutes to obtain the acid-hydrolyzed pectin residue culture medium.

5. The method for preparing mycelial protein raw materials from fermented fruit residue of Ganoderma lucidum strain DCNX0001 according to claim 4, characterized in that, The pH value of the culture medium is 3-6.

6. The method for preparing mycelial protein raw materials from fermented fruit residue of Ganoderma lucidum strain DCNX0001 according to claim 3, characterized in that, The particle size of the acid-hydrolyzed pectin residue is 1-200 mesh.

7. The method for preparing mycelial protein raw materials from fermented fruit residue of Ganoderma lucidum strain DCNX0001 according to claim 3, characterized in that, The alkaline substance is one of sodium hydroxide, potassium hydroxide, calcium hydroxide, or ammonia solution with a concentration of 0.1% to 28% by volume.

8. The method for preparing mycelial protein raw materials from fermented fruit residue of Ganoderma lucidum strain DCNX0001 according to claim 3, characterized in that, The air-dried rice husks mentioned are one or more of the following: wheat husks, barley husks, sorghum husks, or rice husks.

9. The application of the mycelial protein raw material prepared by the method of claim 3 in animal husbandry, characterized in that, The mycelium protein raw material is used as a protein feed ingredient to partially replace soybean meal in animal diets, with a replacement rate of 30-80%.

10. The application of the mycelial protein raw material according to claim 9 in animal husbandry, characterized in that, The mycelium protein raw material is used as a protein feed ingredient to partially replace soybean meal in animal diets, with a replacement rate of 20-30%.

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