A method for culturing mycelium of auricularia auricula

Abstract

The present application relates to the field of mycelium culture, and discloses a culture method of auricularia polytricha mycelium, which improves the traditional culture medium by adding sheet-shaped polylactic acid nanomaterials, chitosan nanoparticles and porous silicon dioxide nanoparticles in the traditional culture medium to form a three-dimensional porous network structure, thereby providing physical support for the mycelium, allowing the mycelium to better adhere and grow, and effectively promoting the growth and reproduction of the mycelium. Compared with the traditional culture medium, the present application solves the problems of poor physical environment for mycelium growth and low nutrient utilization efficiency.

Description

Technical Field

[0001] This invention relates to the field of mycelial culture, and more particularly to a method for culturing Sophora japonica mycelium. Background Technology

[0002] Sophora japonica is a traditional Chinese medicine with significant medicinal value. Its main active ingredients include polysaccharides and triterpenoids, possessing various effects such as hemostasis, antidiarrheal, and anticancer properties. However, due to the scarcity of its host tree species and the scarcity of wild resources, it is difficult to meet the huge market demand for Sophora japonica. Currently, the main techniques for artificially cultivating Sophora japonica include solid-state fermentation and liquid culture. While solid-state fermentation can simulate the natural growth environment, it has a long growth cycle, low biological effects, and significant resource waste. Liquid culture has advantages such as a short cultivation cycle, high biomass, and ease of industrial production, but there are still many shortcomings in the optimization of liquid culture media in existing technologies.

[0003] Traditional liquid culture media are mainly composed of corn flour, soybean meal, and other ingredients. Although they can support mycelial growth, there is still room for improvement in terms of mycelial biomass, extracellular polysaccharide yield, and quality. In addition, traditional culture media lack optimized design for mycelial growth environment, resulting in poor physical environment for mycelial growth, low nutrient utilization efficiency, and mycelial nutrient deficiency in the later stages of growth.

[0004] Therefore, to address the above problems, a new solution is needed to optimize the growth environment of Sophora japonica mycelium. Summary of the Invention

[0005] This invention overcomes the shortcomings of the prior art and provides a method for culturing Sophora japonica mycelium.

[0006] To achieve the above objectives, the technical solution provided by this invention is: a culture medium for Sophora japonica mycelium, comprising the following components: 2.00%–3.00% soluble starch;

[0007] Sheet-like polylactic acid nanomaterials: 0.03%–0.10%;

[0008] Peptone content: 1.00%–1.80%;

[0009] Chitosan nanoparticles: 0.01%–0.05%;

[0010] Potassium dihydrogen phosphate 0.15%–0.30%;

[0011] Magnesium sulfate heptahydrate 0.08%–0.15%;

[0012] Zinc sulfate 0.002%–0.010%;

[0013] L-Sodium lactate 0.03%–0.10%;

[0014] Vitamin B1 0.0005%–0.001%;

[0015] And porous silica nanoparticles 0.005%–0.020%;

[0016] The remainder is water.

[0017] In a preferred embodiment of the present invention, the specific surface area of ​​the sheet-like polylactic acid nanomaterial is 150-250 m² / g, and the sheet thickness is 10-50 nm.

[0018] In a preferred embodiment of the present invention, the soluble starch is corn starch or cassava starch with an amylopectin content of ≥50%.

[0019] In a preferred embodiment of the present invention, the chitosan nanoparticles have a particle size of 50-150 nm.

[0020] In a preferred embodiment of the present invention, the porous silica nanoparticles have a porosity of ≥80% and a particle size of 100-300 nm.

[0021] In a preferred embodiment of the present invention, the surface of the sheet-like polylactic acid nanomaterial is carboxylated to enhance its hydrogen bond adsorption capacity with starch molecules.

[0022] On the other hand, the present invention also provides a method for culturing Sophora japonica mycelium, wherein the Sophora japonica mycelium culture medium described above is used for fermentation culture, and the fermentation process includes the following parameters:

[0023] pH value: 6.5-7.0;

[0024] Rotational speed: 180 r / min;

[0025] Temperature: 30℃;

[0026] Fermentation time: 8-10 days.

[0027] In a preferred embodiment of the present invention, the pH value is dynamically adjusted using a KH2PO4 buffer.

[0028] In a preferred embodiment of the present invention, the culture medium is filtered through a 0.2-0.25 μm filter membrane for sterilization before use to ensure a sterile environment.

[0029] In a preferred embodiment of the present invention, the dissolved oxygen level is maintained at 30%-50% during the fermentation process by a dissolved oxygen controller.

[0030] This invention addresses the shortcomings of the prior art and has the following beneficial effects:

[0031] (1) This invention significantly improves the culture efficiency and product quality of Sophora japonica mycelium by adding sheet-like polylactic acid nanomaterials to the culture medium and optimizing its surface treatment and the synergistic effect of other components. The sheet-like PLA nanomaterials, together with chitosan nanoparticles and porous SiO2 nanoparticles, construct a three-dimensional porous network structure, providing physical support for the mycelium, enabling it to attach and grow better, and effectively promoting mycelial growth and reproduction. Compared with traditional culture media, this design solves the problems of poor physical environment for mycelial growth and low nutrient utilization efficiency. At the same time, through the slow-release-adsorption dual-mode nutrient supply, it ensures that nutrients are evenly dispersed in the mycelium throughout the entire growth cycle, maintaining sufficient nutrients in the mycelial community, thereby improving the growth rate and biomass of the mycelium.

[0032] (2) The present invention adopts a multi-component synergistic optimization design. By forming a hydrogen bond adsorption complex between the surface carboxyl groups of sheet-like PLA nanomaterials and starch molecules, glucose is gradually released to provide a continuous carbon source for mycelia. At the same time, PLA itself degrades to produce lactic acid monomers, which together with L-lactic acid sodium acid activate the mycelial metabolic pathway. This slow-release-adsorption dual-mode nutrient supply method, combined with the "core-shell" structure formed by the electrostatic self-assembly of chitosan and PLA, not only improves the adsorption capacity of nutrients, but also optimizes the mass transfer process, so that nutrients can be transferred to mycelial cells more efficiently.

[0033] (3) The present invention inhibits the aggregation of mycelial clusters through the three-dimensional network formed by PLA sheets and SiO2, and reduces the feedback inhibition caused by the local accumulation of metabolites. This physical barrier drag reduction effect enables the mycelia to be distributed and grow more evenly, thereby improving the metabolic efficiency of the entire culture system. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0036] The sources of the materials are shown in Tables 1 and 2:

[0037] Table 1 provides a direct access list of materials.

[0038] Material source Brand Amylopectin (amylopectin content ≥ 50%) Hefei Tianjian Chemical Co., Ltd. CAS: 9057-02-7 High in amylose (amylose content ≥ 55%) Quanyin Xiangyu (Beijing) Biotechnology Co., Ltd. Xiangyu 1946 Polylactic acid (PLA) Hefei Tianjian Chemical Co., Ltd. CAS 26100-51-6 peptone Hefei Tianjian Chemical Co., Ltd. CAS 91079-38-8 Chitosan nanoparticles Hefei Tianjian Chemical Co., Ltd. CAS 9012-76-4 Potassium dihydrogen phosphate Hefei Tianjian Chemical Co., Ltd. CAS 7778-77-0 Magnesium sulfate heptahydrate Hefei Tianjian Chemical Co., Ltd. CAS 10034-99-8 Zinc sulfate Hefei Tianjian Chemical Co., Ltd. CAS 7733-02-0 Sodium L-lactic acid Wuhan Jixin Yibang Biotechnology Co., Ltd. CAS 867-56-1 Vitamin B1 Hefei Tianjian Chemical Co., Ltd. CAS 67-03-8 Porous silica (SiO2) nanoparticles Hefei Tianjian Chemical Co., Ltd. CAS 14808-60-7 maltose Ordinary maltose purchased from the market none <![CDATA[Sodium selenite (Na2SeO3)]]> Wuhan Jixin Yibang Biotechnology Co., Ltd. CAS 10102-18-8 wheat bran Ordinary wheat bran purchased from the market none Soybean cake powder Ordinary soybean meal powder purchased from the market none

[0039] Table 2. Special Treatment Materials List

[0040] Material Special treatment wheat bran Mix wheat bran with water in a certain proportion and soak at 30-40℃ for 15-30 minutes. Filter the soaked mixture using filter cloth or filter paper to separate the solid residue and obtain wheat bran extract. Soybean cake powder Soybean meal powder and water are mixed in a certain proportion, an appropriate amount of hydrochloric acid is added, the pH value is adjusted to about 4.0, and hydrolysis is carried out at a temperature of about 110℃ for 8 hours. After filtering to remove solid residue, soybean meal powder hydrolysate is obtained. Sheet-shaped polylactic acid (PLA) nanomaterials PLA fibers are mixed with an appropriate amount of zirconia balls as grinding media. The mixture is then ball-milled at high speed in a ball mill at a speed of 500-1000 rpm for 1-3 hours. After ball milling, PLA flake particles are separated by sieving or centrifugation. After washing and drying, flake polylactic acid (PLA) nanomaterials are obtained. Example 1:

[0041] A method for culturing Sophora japonica mycelium specifically includes the following steps:

[0042] S1. Prepare the culture medium, with the following specific components and concentrations:

[0043] Soluble starch: 25 g / L (branched corn starch with amylopectin content ≥50%).

[0044] Sheet-like polylactic acid (PLA) nanomaterials: 0.8 g / L (specific surface area 200 m² / g, sheet thickness 30 nm, surface carboxylated).

[0045] Peptone: 15 g / L;

[0046] Chitosan nanoparticles: 0.3 g / L (particle size 100 nm);

[0047] Potassium dihydrogen phosphate: 2.0 g / L;

[0048] Magnesium sulfate heptahydrate: 1.2 g / L;

[0049] Zinc sulfate: 0.05 g / L;

[0050] L-Sodium lactate: 0.5 g / L;

[0051] Vitamin B1: 8 mg / L;

[0052] Porous silica (SiO2) nanoparticles: 0.1 g / L (porosity 85%, particle size 200 nm);

[0053] Dissolve the above components in deionized water, bring the volume to 1 L, and stir until homogeneous;

[0054] S2. Sterilize the prepared culture medium by filtering it through a 0.22μm filter membrane and transfer it into a 5L fermenter with a liquid volume of 3L.

[0055] S3. Inoculate with Sophora japonica mycelium at a rate of 10% (v / v). Set the fermentation parameters as follows: pH 6.8 (dynamically adjusted using KH2PO4 buffer), temperature 30℃, rotation speed 180 r / min, and dissolved oxygen level maintained at 30%-50%. Fermentation is carried out for 9 days, during which time samples are taken regularly to detect mycelial biomass, extracellular polysaccharide production, and mycelial mortality. Example 2:

[0056] A method for culturing Sophora japonica mycelium specifically includes the following steps:

[0057] S1. Culture medium preparation: Prepare the culture medium, with the specific components and concentrations as follows:

[0058] Soluble starch: 25 g / L (corn starch with amylopectin content ≥50%).

[0059] Sheet-like polylactic acid (PLA) nanomaterials: 0.6 g / L (specific surface area 200 m² / g, sheet thickness 30 nm, surface carboxylated).

[0060] Peptone: 15 g / L;

[0061] Chitosan nanoparticles: 0.3 g / L (particle size 100 nm);

[0062] Potassium dihydrogen phosphate: 2.0 g / L;

[0063] Magnesium sulfate heptahydrate: 1.2 g / L;

[0064] Zinc sulfate: 0.05 g / L;

[0065] L-Sodium lactate: 0.5 g / L;

[0066] Vitamin B1: 8 mg / L;

[0067] Porous silica (SiO2) nanoparticles: 0.1 g / L (porosity 85%, particle size 200 nm);

[0068] Dissolve the above components in deionized water, bring the volume to 1 L, and stir until homogeneous;

[0069] S2. Sterilize the prepared culture medium by filtering it through a 0.22μm filter membrane and transfer it into a 5L fermenter with a liquid volume of 3L.

[0070] S3. Inoculate with Sophora japonica mycelium at a rate of 10% (v / v). Set the fermentation parameters as follows: pH 6.8 (dynamically adjusted using KH2PO4 buffer), temperature 30℃, rotation speed 180 r / min, and dissolved oxygen level maintained at 30%-50%. Fermentation is carried out for 9 days, during which time samples are taken regularly to detect mycelial biomass, extracellular polysaccharide production, and mycelial mortality. Example 3:

[0071] A method for culturing Sophora japonica mycelium specifically includes the following steps:

[0072] S1. Prepare the culture medium, with the following specific components and concentrations:

[0073] Soluble starch: 25 g / L (corn starch with amylopectin content ≥50%).

[0074] Sheet-like polylactic acid (PLA) nanomaterials: 1.0 g / L (specific surface area 200 m² / g, sheet thickness 30 nm, surface carboxylated).

[0075] Peptone: 15 g / L;

[0076] Chitosan nanoparticles: 0.3 g / L (particle size 100 nm);

[0077] Potassium dihydrogen phosphate: 2.0 g / L;

[0078] Magnesium sulfate heptahydrate: 1.2 g / L;

[0079] Zinc sulfate: 0.05 g / L;

[0080] L-Sodium lactate: 0.5 g / L;

[0081] Vitamin B1: 8 mg / L;

[0082] Porous silica (SiO2) nanoparticles: 0.1 g / L (porosity 85%, particle size 200 nm);

[0083] Dissolve the above components in deionized water, bring the volume to 1 L, and stir until homogeneous;

[0084] S2. Sterilize the prepared culture medium by filtering it through a 0.22μm filter membrane and transfer it into a 5L fermenter with a liquid volume of 3L.

[0085] S3. Inoculate with Sophora japonica mycelium at a rate of 10% (v / v). Set the fermentation parameters as follows: pH 6.8 (dynamically adjusted using KH2PO4 buffer), temperature 30℃, rotation speed 180 r / min, and dissolved oxygen level maintained at 30%-50%. Fermentation is carried out for 9 days, during which time samples are taken regularly to detect mycelial biomass, extracellular polysaccharide production, and mycelial mortality. Example 4:

[0086] S1. Prepare the culture medium, with the following specific components and concentrations:

[0087] Soluble starch: 20 g / L (corn starch with amylopectin content ≥50%).

[0088] Sheet-like polylactic acid (PLA) nanomaterials: 0.8 g / L (specific surface area 200 m² / g, sheet thickness 30 nm, surface carboxylated).

[0089] Peptone: 15 g / L;

[0090] Chitosan nanoparticles: 0.3 g / L (particle size 100 nm);

[0091] Potassium dihydrogen phosphate: 2.0 g / L;

[0092] Magnesium sulfate heptahydrate: 1.2 g / L;

[0093] Zinc sulfate: 0.05 g / L;

[0094] L-Sodium lactate: 0.5 g / L;

[0095] Vitamin B1: 8 mg / L;

[0096] Porous silica (SiO2) nanoparticles: 0.1 g / L (porosity 85%, particle size 200 nm);

[0097] Dissolve the above components in deionized water, bring the volume to 1 L, and stir until homogeneous;

[0098] S2. Sterilize the prepared culture medium by filtering it through a 0.22μm filter membrane and transfer it into a 5L fermenter with a liquid volume of 3L.

[0099] S3. Inoculate with Sophora japonica mycelium at a rate of 10% (v / v). Set the fermentation parameters as follows: pH 6.8 (dynamically adjusted using KH2PO4 buffer), temperature 30℃, rotation speed 180 r / min, and dissolved oxygen level maintained at 30%-50%. Fermentation is carried out for 9 days, during which time samples are taken regularly to detect mycelial biomass, extracellular polysaccharide production, and mycelial mortality. Example 5:

[0100] S1. Prepare the culture medium, with the following specific components and concentrations:

[0101] Soluble starch: 30 g / L (corn starch with amylopectin content ≥50%).

[0102] Sheet-like polylactic acid (PLA) nanomaterials: 0.8 g / L (specific surface area 200 m² / g, sheet thickness 30 nm, surface carboxylated).

[0103] Peptone: 15 g / L;

[0104] Chitosan nanoparticles: 0.3 g / L (particle size 100 nm);

[0105] Potassium dihydrogen phosphate: 2.0 g / L;

[0106] Magnesium sulfate heptahydrate: 1.2 g / L;

[0107] Zinc sulfate: 0.05 g / L;

[0108] L-Sodium lactate: 0.5 g / L;

[0109] Vitamin B1: 8 mg / L;

[0110] Porous silica (SiO2) nanoparticles: 0.1 g / L (porosity 85%, particle size 200 nm);

[0111] Dissolve the above components in deionized water, bring the volume to 1 L, and stir until homogeneous;

[0112] S2. Sterilize the prepared culture medium by filtering it through a 0.22μm filter membrane and transfer it into a 5L fermenter with a liquid volume of 3L.

[0113] S3. Inoculate with Sophora japonica mycelium at a rate of 10% (v / v). Set the fermentation parameters as follows: pH 6.8 (dynamically adjusted using KH2PO4 buffer), temperature 30℃, rotation speed 180 r / min, and dissolved oxygen level maintained at 30%-50%. Fermentation is carried out for 9 days, during which time samples are taken regularly to detect mycelial biomass, extracellular polysaccharide production, and mycelial mortality.

[0114] Comparative Example 1:

[0115] A method for culturing Sophora japonica mycelium specifically includes the following steps:

[0116] S1. Culture medium preparation: Prepare liquid culture medium according to the traditional culture medium formula. The specific components and concentrations are as follows:

[0117] Soluble starch: 30 g / L (corn starch with amylopectin content ≥50%).

[0118] Maltose: 5 g / L;

[0119] Peptone: 15 g / L;

[0120] Soybean meal hydrolysate: 10 g / L;

[0121] KH2PO4: 2.0 g / L;

[0122] MgSO4·7H2O: 1.0 g / L;

[0123] ZnSO4: 0.05 g / L;

[0124] Na2SeO3: 0.02 g / L;

[0125] Wheat bran extract: 5% (v / v);

[0126] VB1: 6 mg / L;

[0127] Dissolve the above components in deionized water, bring the volume to 1 L, and stir until homogeneous;

[0128] S2. Sterilize the prepared culture medium by filtering it through a 0.22μm filter membrane and transfer it into a 5L fermenter with a liquid volume of 3L.

[0129] S3. Inoculate with Sophora japonica mycelium at a rate of 10% (v / v). Set the fermentation parameters as follows: pH 6.8 (dynamically adjusted using KH2PO4 buffer), temperature 30℃, rotation speed 180 r / min, and dissolved oxygen level maintained at 30%-50%. Fermentation is carried out for 9 days, during which time samples are taken regularly to detect mycelial biomass, extracellular polysaccharide production, and mycelial mortality.

[0130] Comparative Example 2:

[0131] S1. Culture medium preparation: Prepare liquid culture medium according to the traditional culture medium formula. The specific components and concentrations are as follows:

[0132] Soluble starch: 30 g / L (corn starch with amylopectin content ≥50%).

[0133] Maltose: 5 g / L;

[0134] Peptone: 15 g / L;

[0135] Soybean meal hydrolysate: 10 g / L;

[0136] KH2PO4: 2.0 g / L;

[0137] MgSO4·7H2O: 1.0 g / L;

[0138] ZnSO4: 0.05 g / L;

[0139] Na2SeO3: 0.02 g / L;

[0140] Wheat bran extract: 5% (v / v);

[0141] VB1: 6 mg / L;

[0142] Chitosan nanoparticles: 0.3 g / L;

[0143] Porous SiO2 nanoparticles: 0.1 g / L;

[0144] Dissolve the above components in deionized water, bring the volume to 1 L, and stir until homogeneous;

[0145] S2. Sterilize the prepared culture medium by filtering it through a 0.22μm filter membrane and transfer it into a 5L fermenter with a liquid volume of 3L.

[0146] S3. Inoculate with Sophora japonica mycelium at a rate of 10% (v / v). Set the fermentation parameters as follows: pH 6.8 (dynamically adjusted using KH2PO4 buffer), temperature 30℃, rotation speed 180 r / min, and dissolved oxygen level maintained at 30%-50%. Fermentation is carried out for 9 days, during which time samples are taken regularly to detect mycelial biomass, extracellular polysaccharide production, and mycelial mortality.

[0147] Comparative Example 3:

[0148] A method for culturing Sophora japonica mycelium specifically includes the following steps:

[0149] S1. Culture medium preparation: Prepare the culture medium, with the specific components and concentrations as follows:

[0150] Soluble starch: 30 g / L (corn starch with amylopectin content ≥50%).

[0151] Maltose: 5 g / L;

[0152] Peptone: 15 g / L;

[0153] Soybean meal hydrolysate: 10 g / L;

[0154] KH2PO4: 2.0 g / L;

[0155] MgSO4·7H2O: 1.0 g / L;

[0156] ZnSO4: 0.05 g / L;

[0157] Na2SeO3: 0.02 g / L;

[0158] Wheat bran extract: 5% (v / v);

[0159] VB1: 6 mg / L;

[0160] Sheet-like polylactic acid (PLA) nanomaterials: 0.8 g / L (specific surface area 200 m² / g, sheet thickness 30 nm, surface not carboxylated).

[0161] Dissolve the above components in deionized water, bring the volume to 1 L, and stir until homogeneous;

[0162] S2. Sterilize the prepared culture medium by filtering it through a 0.22μm filter membrane and transfer it into a 5L fermenter with a liquid volume of 3L.

[0163] S3. Inoculate with Sophora japonica mycelium at a rate of 10% (v / v). Set the fermentation parameters as follows: pH 6.8 (dynamically adjusted using KH2PO4 buffer), temperature 30℃, rotation speed 180 r / min, and dissolved oxygen level maintained at 30%-50%. Fermentation is carried out for 9 days, during which time samples are taken regularly to detect mycelial biomass, extracellular polysaccharide production, and mycelial mortality.

[0164] Comparative Example 4:

[0165] A method for culturing Sophora japonica mycelium specifically includes the following steps:

[0166] S1. Prepare the culture medium, with the following specific components and concentrations:

[0167] Soluble starch: 25 g / L (corn starch with amylose content ≥55%).

[0168] Sheet-like polylactic acid (PLA) nanomaterials: 0.8 g / L (specific surface area 200 m² / g, sheet thickness 30 nm, surface carboxylated).

[0169] Peptone: 15 g / L;

[0170] Chitosan nanoparticles: 0.3 g / L (particle size 100 nm);

[0171] Potassium dihydrogen phosphate: 2.0 g / L;

[0172] Magnesium sulfate heptahydrate: 1.2 g / L;

[0173] Zinc sulfate: 0.05 g / L;

[0174] L-Sodium lactate: 0.5 g / L;

[0175] Vitamin B1: 8 mg / L;

[0176] Porous silica (SiO2) nanoparticles: 0.1 g / L (porosity 85%, particle size 200 nm);

[0177] Dissolve the above components in deionized water, bring the volume to 1 L, and stir until homogeneous;

[0178] S2. Sterilize the prepared culture medium by filtering it through a 0.22μm filter membrane and transfer it into a 5L fermenter with a liquid volume of 3L.

[0179] S3. Inoculate with Sophora japonica mycelium at a rate of 10% (v / v). Set the fermentation parameters as follows: pH 6.8 (dynamically adjusted using KH2PO4 buffer), temperature 30℃, rotation speed 180 r / min, and dissolved oxygen level maintained at 30%-50%. Fermentation is carried out for 9 days, during which time samples are taken regularly to detect mycelial biomass, extracellular polysaccharide production, and mycelial mortality.

[0180] Performance testing:

[0181] The cultures from Examples 1-5 and Comparative Examples 1-4 were subjected to the following performance tests:

[0182] Mycelial biomass: determined by centrifugation and drying method, and the average value was taken;

[0183] Extracellular polysaccharide yield and composition: Polysaccharide content was determined by phenol-sulfuric acid method, and β-glucan ratio was analyzed by HPLC;

[0184] Mycelial mortality rate: determined by trypan blue staining method, and the average value was taken.

[0185] The test data is shown in Table 3:

[0186] Table 3 Test Data Table

[0187] Group Mycelial biomass (g / L) Extracellular polysaccharide yield (g / L) β-glucan content (%) Mycelial mortality rate (%) Example 1 30.1 2.2 72 <9 Example 2 29.5 2.1 70 <10 Example 3 28.7 2.0 68 <12 Example 4 28.9 2.2 69 <10 Example 5 29.3 2.1 70 <10 Comparative Example 1 24.5 1.5 62 21 Comparative Example 2 26.8 1.8 67 15 Comparative Example 3 27.3 1.9 65 18 Comparative Example 4 25.4 1.6 63 14

[0188] Summary and Analysis:

[0189] The comparative data from Examples 1-5 and Comparative Examples 1-4 show that the present invention significantly improves the culture efficiency and product quality of Sophora japonica mycelium by adding sheet-like polylactic acid (PLA) nanomaterials to the culture medium and optimizing its surface treatment and the synergistic effect of other components.

[0190] Comparing Examples 1-5 with Comparative Examples 1-4: In Example 1, the mycelial biomass reached 30.1 g / L, which was significantly higher than 24.5 g / L in Comparative Example 1. At the same time, the data of other examples were significantly better than those of the comparative examples. The reason for this is that: Firstly, sheet-like polylactic acid (PLA) nanomaterials are more likely to combine with starch with a high branching content. The numerous α-1,6 glycosidic bonds in amylopectin molecules form a branched structure, constructing a highly branched three-dimensional network. This structure gives it high solubility and low retrogradation, making it easy to form colloids in liquid culture media and less prone to aging and precipitation. It can be uniformly dispersed and form a stable composite slow-release carrier with PLA nanosheets. In contrast, amylose is mainly composed of linear α-1,4 glycosidic bonds, causing its molecules to be tightly coiled into a helical structure, which easily forms a semi-solid gel. In liquid fermentation, this gelation hinders oxygen dissolution and mass transfer, and the rapid retrogradation of amylose leads to a decrease in carbon source utilization and increases process complexity. Therefore, corn starch with a higher amylopectin content shows a significant advantage over high amylose corn starch in the cultivation of Sophora japonica mycelium.

[0191] Secondly, in Example 1, sheet-like PLA nanomaterials were added. In the culture medium, they synergistically interacted with chitosan nanoparticles and porous SiO2 nanoparticles to form a three-dimensional porous network structure. This structure provided physical support for the hyphae, enabling them to attach and grow better, and effectively promoting the growth and reproduction of the hyphae.

[0192] Thirdly, the sheet-like PLA nanomaterials form hydrogen-bonded adsorption complexes with starch molecules through surface carboxyl groups, gradually releasing glucose under the action of amylase, providing a continuous carbon source for the mycelium. Under the adsorption of the sheet-like PLA nanomaterials, combined with the stirring of the fermenter, the glucose is more evenly dispersed, avoiding local mycelial necrosis caused by uneven glucose distribution. At the same time, PLA itself degrades to produce lactic acid monomers, which, together with L-lactic acid sodium, activate the mycelial metabolic pathway, forming a slow-release-adsorption dual-mode nutrient supply method. This method ensures that the mycelium obtains sufficient nutrients throughout the entire growth cycle, thereby improving the mycelial growth rate and biomass.

[0193] Fourthly, chitosan (positively charged) and PLA (negatively charged) combine electrostatically to form a "core-shell" structure, enhancing the loading capacity of carbon and nitrogen sources. This surface charge regulation not only improves the adsorption capacity of nutrients but also optimizes the mass transfer process, enabling nutrients to be transferred to the hyphal cells more efficiently. In Example 1, the extracellular polysaccharide yield reached 2.2 g / L, and the β-glucan content was 72%, both of which were superior to 1.5 g / L and 62% in Comparative Example 1. This indicates that surface charge regulation and mass transfer optimization significantly improved the polysaccharide synthesis efficiency.

[0194] Fifthly, in Examples 1-5, the three-dimensional network formed by PLA sheets and SiO2 inhibits the aggregation of mycelial clusters and reduces feedback inhibition caused by the local accumulation of metabolites. This physical barrier drag reduction effect allows mycelia to be distributed and grow more evenly, improving the metabolic efficiency of the entire culture system. In Example 1, the mycelial mortality rate was less than 8% throughout the process, while in Comparative Example 1 it was as high as 21%. This shows that the physical barrier drag reduction effect effectively maintains the viability of mycelia.

[0195] Comparing Examples 1-3, where the concentrations of sheet-like PLA nanomaterials were 0.8 g / L, 0.6 g / L, and 1.0 g / L, respectively, the experimental data show that when the concentration of sheet-like PLA nanomaterials was 0.8 g / L, both mycelial biomass and extracellular polysaccharide production reached their highest values ​​(30.1 g / L and 2.2 g / L, respectively). As the concentration decreased or increased, both mycelial biomass and extracellular polysaccharide production decreased. This indicates that there is an optimal concentration range for sheet-like PLA nanomaterials; within this range, their concentrations are comparable to those of other materials. The synergistic effect of other components in the culture medium is the strongest, which can maximize their effect on promoting mycelial growth and polysaccharide synthesis. The reason for the existence of the optimal range is that when the number of PLA sheets is insufficient, they cannot form a continuous three-dimensional porous network with chitosan and SiO2 nanoparticles, resulting in room for improvement in the uniformity of mycelial attachment points and the need to improve the nutrient transfer efficiency in local areas. When the number of PLA sheets is too high, the excessive stacking of PLA sheets leads to a reduction in the network pore size, which hinders mycelial penetration and oxygen diffusion, and is also not conducive to mycelial growth.

[0196] Comparing Examples 1, 4, and 5, where the concentrations of soluble starch were 25 g / L, 20 g / L, and 30 g / L, respectively, the experimental data showed that the concentration of soluble starch had a significant impact on the growth efficiency and product synthesis of *Auricularia auricula-judae* mycelium. Similarly, there was a clear threshold for the synergistic effect with sheet-like PLA nanomaterials, requiring a balance between carbon source supply and physical structure. Amylopectin (≥50%) and carboxylated PLA formed a porous slow-release carrier through hydrogen bonding and hydrophobic interactions. When the starch content was appropriate, the carrier had a high porosity, allowing sufficient glucose to be released, promoting the growth of *Auricularia auricula-judae* mycelium. When starch was insufficient, the carrier structure became loose, resulting in high porosity and an excessively rapid glucose release rate. Conversely, excessive starch content led to over-crosslinking and blockage of the carrier pores, reducing the glucose slow-release efficiency and affecting mycelial growth.

[0197] As can be seen from the data of Example 1 and Comparative Example 1, the traditional culture medium mainly contains soluble starch, maltose, peptone, soybean meal hydrolysate, KH2PO4, MgSO4·7H2O, ZnSO4, Na2SeO3, wheat bran extract, and VB1. The culture medium of this application adds sheet-like PLA nanomaterials, chitosan nanoparticles, and porous SiO2 nanoparticles to this medium, and optimizes the concentration and ratio of each component. Through a three-dimensional network structure and a slow-release-adsorption dual-mode nutrient supply, it effectively promotes mycelial growth. Through metabolic signal cascade amplification and physical barrier drag reduction effect, it significantly improves the polysaccharide synthesis efficiency. By optimizing the physical structure and nutrient supply, it effectively reduces mycelial mortality and maintains mycelial vitality.

[0198] Comparing Example 1 with Comparative Example 2, Example 1 added chitosan nanoparticles and porous SiO2 nanoparticles, while Comparative Example 2 did not add sheet-like PLA nanomaterials, but only added chitosan and porous SiO2 nanoparticles. Experimental data showed that the mycelial biomass and extracellular polysaccharide yield of Example 1 were higher than those of Comparative Example 2. This indicates that when sheet-like PLA nanomaterials work synergistically with chitosan and porous SiO2 nanoparticles, they can more effectively promote mycelial growth and polysaccharide synthesis.

[0199] Comparing Example 1 with Comparative Example 3, the sheet-like PLA nanomaterials in Example 1 underwent carboxylation surface treatment, while the sheet-like PLA nanomaterials in Comparative Example 3 did not undergo carboxylation treatment. Experimental data showed that the mycelial biomass and extracellular polysaccharide yield of Example 1 were higher than those of Comparative Example 3. This indicates that the carboxylation surface treatment enhanced the hydrogen bond adsorption capacity between the sheet-like PLA nanomaterials and starch molecules, further optimized the slow-release-adsorption dual-mode nutrient supply, and improved the mycelial growth rate and polysaccharide synthesis efficiency.

[0200] Comparing Example 1 with Comparative Example 4, unlike Example 1, Comparative Example 4 used high-amylose corn starch with an amylose content ≥55%. Experimental data showed that the test data of Comparative Example 4, which used a higher amylose content, was significantly inferior to that of Example 1. This is because sheet-like PLA nanomaterials bind more easily to starch with a higher amylopectin content. The branched structure formed by numerous α-1,6 glycosidic bonds in amylopectin molecules creates a highly branched three-dimensional network. This structure endows it with high solubility and low retrogradation, making it easy to form colloids while resisting aging and precipitation. It can be uniformly dispersed in liquid culture media, forming a stable composite slow-release carrier with PLA nanosheets. In contrast, the linear α-1,4 glycosidic bond structure in amylose causes its molecules to tightly coil into helices, easily forming semi-solid gels. In liquid fermentation, gelation hinders dissolved oxygen and mass transfer, and the rapid retrogradation of amylose leads to decreased carbon source utilization, reducing the growth efficiency of *Auricularia auricula-judae* mycelium.

[0201] In summary, this application significantly improves the cultivation efficiency and product quality of *Auricularia auricula-judae* mycelium by adding sheet-like PLA nanomaterials to the culture medium and optimizing its surface treatment and the synergistic effect of other components. Compared with traditional culture media, the culture medium of this invention can construct a three-dimensional porous network structure, realize a dual-mode nutrient supply of slow release and adsorption, optimize the mass transfer process, and significantly improve the growth rate of mycelium and the synthesis efficiency of polysaccharides through metabolic signal cascade amplification and physical barrier drag reduction effects. This innovative technology not only provides a new technical solution for the efficient cultivation of *Auricularia auricula-judae* mycelium, but also provides important theoretical basis and practical guidance for the innovation and development of fungal fermentation processes.

[0202] Based on the preferred embodiments of the present invention described above, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A culture medium of Auricularia auricula-judae mycelium, characterized by, by mass percent, comprising the following ingredients: soluble starch 2.00-3.00%; sheet-like polylactic acid nanomaterial 0.03-0.10%; peptone 1.00-1.80%; chitosan nanoparticles 0.01-0.05%; potassium dihydrogen phosphate 0.15-0.30%; magnesium sulfate heptahydrate 0.08-0.15%; zinc sulfate 0.002-0.010%; L-sodium lactate 0.03-0.10%; vitamin B1 0.0005-0.001%; and porous silica nanoparticles 0.005-0.020%; the balance being water; wherein, the soluble starch is corn starch or cassava starch with a branched amylopectin content of ≥50%.

2. The Auricularia auricula-judae mycelium culture medium according to claim 1, characterized in that: the specific surface area of the sheet-like polylactic acid nanomaterial is 150-250 m² / g, and the sheet thickness is 10-50 nm.

3. The Auricularia auricula-judae mycelium culture medium according to claim 1, characterized in that: the particle size of the chitosan nanoparticles is 50-150 nm.

4. The Auricularia polytricha mycelium culture medium according to claim 1, characterized in that: the porosity of the porous silica nanoparticles is ≥80%, and the particle size is 100-300 nm.

5. The Auricularia mesna culture medium according to claim 1, characterized in that: the surface of the sheet-like polylactic acid nanomaterial is treated by carboxylation to enhance the hydrogen bond adsorption capacity with starch molecules.

6. A culture method of Auricularia auricula-judae mycelium, characterized by, fermenting with a Genostana panaciformis mycelium culture medium according to any one of claims 1-5, wherein the fermentation process comprises the following parameters: pH: 6.5-7.0; rotational speed: 180 r / min; temperature: 30°C; fermentation time: 8-10 days.

7. The method according to claim 6, wherein the method is characterized by: the pH is dynamically adjusted by KH2PO4 buffer.

8. The method of claim 6, wherein the method is characterized by: the Genostana panaciformis mycelium culture medium is filtered through a 0.2-0.25 μm filter to remove bacteria before use to ensure a sterile environment.

9. The method of claim 6, wherein the method is characterized by: the dissolved oxygen level is maintained at 30%-50% during the fermentation process by a dissolved oxygen controller.