A culture method of irpex lacteus

Abstract

The application discloses a culture method of Irpex laccatus, and the special mother culture medium without external vitamins is obtained through single factor screening and five-factor Box-Behnken response surface method, wherein 19.49 g of glucose, 14.48 g of yeast powder, 5.18 g of proteose peptone, 0.596 g of magnesium sulfate and 0.607 g of potassium dihydrogen phosphate are contained in one liter of distilled water; the average growth rate of mycelium can reach 0.6417 cm / d under constant temperature culture at 25 DEG C, and the relative error of model prediction is only 0.23%. The application also simultaneously establishes a composite degradation enzyme extraction process, and the culture medium formula is simple, the cost is low, the mycelium growth is excellent, and the whole culture process can be used for large-scale production of strains and high-activity degradation enzymes; the obtained strains and enzyme liquid can be widely used for degradation of agricultural and forestry wastes, decolorization of printing and dyeing wastewater, repair of heavy metal water body, fermentation of medicinal fungal polysaccharide, production of biological enzyme preparation, and have good industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for culturing *Amanita muscaria*. Background Technology

[0002] *Coriolopsistrogii*, a heat-resistant white-rot fungus belonging to the Polyporaceae family of the Basidiomycetes, naturally saprophytizes on dead broad-leaved wood in temperate and subtropical regions, and is a core degrading microorganism for forest woody residues. This strain has both medicinal and environmental value: its fruiting bodies contain active substances such as polysaccharides and polyphenols, possessing antioxidant, hepatoprotective, immunomodulatory, and antitumor effects; its hyphae can secrete various lignocellulose-degrading enzymes, which can efficiently decompose lignin, and can be used for decolorization of dye wastewater, adsorption of heavy metal ions, and resource utilization of agricultural and forestry waste.

[0003] The existing technology has four major flaws:

[0004] (1) There is a lack of domestic wild-type strains for identification. Most of the existing strains are imported, with slow mycelial growth and weak laccase production capacity. There are currently no local stable strains with complete morphology and ITS dual identification.

[0005] (2) Traditional PDA and general basic culture medium have crude nutrient ratios, and carbon, nitrogen and inorganic salts are not synergistically optimized. Both excessively high and low concentrations of a single nutrient inhibit mycelium. Existing studies often add exogenous vitamins, which reduces mycelial growth rate and increases raw material costs.

[0006] (3) Existing studies only explore the effects of a single carbon source or nitrogen source, without using a five-factor Box-Behnken response surface multi-factor coupling optimization, and there is no precise and scalable standardized mother culture formula;

[0007] (4) The temporal activity of all eight key degrading enzymes of lignin, cellulose and hemicellulose was not systematically and synchronously determined, making it impossible to selectively enrich highly active functional enzymes such as laccase based on culture time, thus limiting the development of industrialized enzyme preparations and environmental remediation materials. Summary of the Invention

[0008] To address the above problems, this invention provides a method for culturing *Phellinus styracifolius*.

[0009] To achieve the above objectives, this application provides the following technical solution:

[0010] A method for culturing A. coliformis, S1: Inoculate A. coliformis strain onto a PDA activation plate and culture at 25℃ in the dark for 5-7 days to obtain purified and activated mycelia.

[0011] S2: Prepare the culture medium, sterilize and cool it for later use;

[0012] S3: After inoculating the activated mycelium into the center of the culture medium, it is incubated at a constant temperature to obtain *Porphyra yezoensis*.

[0013] The *Porphyra yezoensis* was collected from wild fruiting bodies of broad-leaved decaying wood in the East Campus of Tianjin Agricultural College. It was purified by tissue isolation. Morphological and ITS molecular identification showed that the full-length ITS sequence was 544 bp. Under 25℃ plate culture conditions, the colonies were regular round, dense white cotton-like, and grew outwards in a uniform radial pattern, without pigment secretion.

[0014] In S1, the formula for the PDA activation plate is 200g potato, 20g glucose, 20g agar, and 1L distilled water.

[0015] In S2, the culture medium contains the following components per liter of distilled water: 19.49 g glucose, 14.48 g yeast extract, 5.18 g peptone, 0.596 g magnesium sulfate, 0.607 g potassium dihydrogen phosphate, and 18–22 g agar; the natural pH of the culture medium is 5.5–6.5.

[0016] In S2, the sterilization conditions for the culture medium are: steam sterilization at 121°C for 20 minutes.

[0017] In S3, the constant temperature incubation conditions are: 25℃, completely dark, and static incubation.

[0018] A method for preparing a crude enzyme solution of *Amanita muscaria*, comprising the following steps: Mycelial preparation using the aforementioned culture method.

[0019] Step A: Collect the stiff-haired, coarse-capillary hyphae and place them in a centrifuge tube. Freeze them in liquid nitrogen and grind them thoroughly.

[0020] Step B: Mix mycelium and deionized water at a ratio of 1:10 to obtain a mixed solution;

[0021] Step C: Centrifuge the mixed solution at 10000 r / min for 10 min and take the supernatant as the composite crude enzyme solution.

[0022] The complex crude enzyme comprises: ligninase, cellulase and hemicellulase.

[0023] The compound crude enzyme solution is used in the degradation of lignocellulose in agriculture and forestry, decolorization of dyeing and printing wastewater, remediation of heavy metal water bodies, fermentation of medicinal fungal polysaccharides, and production of enzyme preparations for the degradation of white rot fungi.

[0024] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0025] 1. Outstanding advantages of the strain: It is a native wild heat-resistant strain with significantly higher laccase yield than existing commercial strains. It has accurate classification through dual identification, is genetically stable when stored at 4℃, and is suitable for cultivation in northern regions.

[0026] 2. Significant gains from culture medium optimization: Using a precise coupled optimization with a 5-factor response surface methodology, the regression model R²=0.9906, with no significant difference in the lack-of-fit terms, and the relative error between prediction and measurement was only 0.23%, indicating that the formulation is accurate and reliable. Glucose, yeast powder, and compound inorganic salts were identified as the core growth-promoting components, and it was confirmed that VB1 / VB2 / VB6 completely inhibited mycelia, eliminating the need for vitamin raw materials and reducing production costs. Under the optimal formulation, the mycelial growth rate was much higher than that of traditional PDA basal medium, and the colonies were dense, uniform, and less prone to aging and degeneration.

[0027] 3. Simplified and scalable cultivation process: The entire process only requires a constant temperature of 25℃ and protection from light, without the need for variable temperature or special fermentation equipment, allowing edible mushroom spawn factories to produce mother cultures in batches;

[0028] 4. Complete enzyme activity time-series data supports targeted enzyme production: For the first time, the dynamic changes of 8 degradation enzymes were measured simultaneously. Laccase had the highest activity at 7 days, cellulase reached its peak at 5 days, and hemicellulase had the best activity in the early stage. The culture time can be precisely controlled according to the needs to enrich the target enzyme.

[0029] 5. Wide range of applications: One technology covers strain breeding, enzyme degradation preparation, dyeing and printing wastewater decolorization, heavy metal adsorption, agricultural and forestry straw bioconversion, and medicinal polysaccharide fermentation, with high industrialization value. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 Phylogenetic tree diagram of the mother plant of the bristle-like operculum strain;

[0032] Figure 2 This is a flowchart of the steps in Example 1;

[0033] Figure 3 Images of the front, back, and side views of the wild fruiting body of *Fodium stearens*.

[0034] Figure 4 This is a diagram showing the colony morphology of the strain on basal culture medium at 25°C.

[0035] Figure 5 Agarose gel electrophoresis image of strain ITS + screenshot of NCBI sequence alignment;

[0036] Figure 6 3D diagram of Box-Behnken multi-factor interactions;

[0037] Figure 7 This is a bar chart showing the time sequence of lignin-degrading enzyme (Lac, LiP, Mnp) activities;

[0038] Figure 8 A bar chart showing the time sequence of cellulase (CMCase, FPA, β-Gc) activity.

[0039] Figure 9 This is a bar chart showing the time sequence of hemicellulose degrading enzyme (α-Gal, BAX) activity. Detailed Implementation

[0040] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0041] Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0042] Firstly, it is proposed that the mother strain of the *Coriolopsistrogii* strain in this invention was collected from the fruiting bodies of broad-leaved decaying wood in the East Campus of Tianjin Agricultural College, and pure strains were obtained through tissue isolation and purification. Morphological characteristics: the cap is shell-shaped, chestnut brown and covered with coarse, stiff brown hairs, and the pores are fine and round. The full-length ITS sequencing is 544 bp, and the NCBI comparison showed an OR of 237085.1 with a similarity of 99.82%, confirming it as *Coriolopsistrogii*. The plate colonies are white and cottony, with uniform radiation and no discoloration.

[0043] Its ITS sequence was submitted to the NCBI GenBank database, obtaining accession number PZ899148, and its phylogenetic tree is referenced. Figure 1 .

[0044] Example 1

[0045] Reference Figures 2-8 The present invention discloses a method for culturing *Pheromus spp.*, comprising:

[0046] S1: Inoculate the bristle coarse-capped strain onto a PDA activation plate and culture it at a constant temperature of 25°C in the dark for 5-7 days to obtain purified and activated mycelia; the formula of the PDA activation plate is 200g potato, 20g glucose, 20g agar, and 1L distilled water.

[0047] S2: Prepare the culture medium, sterilize it with steam at 121℃ for 20 minutes, and then cool it for later use. The culture medium contains the following components per liter of distilled water: 19.49g glucose, 14.48g yeast powder, 5.18g peptone, 0.596g magnesium sulfate, 0.607g potassium dihydrogen phosphate, and 18-22g agar (20g is used in this example). No additional vitamins VB1, VB2, or VB6 are added to the culture medium. The natural pH of the culture medium is 5.5-6.5.

[0048] In S2, the culture medium preparation process is as follows: all solid components are added to distilled water and heated and stirred until completely dissolved. The mixture is then dispensed into culture dishes and sterilized by high-pressure steam at 121℃ for 20-30 minutes. After sterilization, the mixture is cooled and solidified in a clean bench to form plates for later use.

[0049] S3: After inoculating the activated mycelium into the center of the culture medium, the culture was carried out at a constant temperature of 25°C in the dark to obtain *Amanita muscaria*. In this example, the colony diameter was determined by the cross-cross method, and the average mycelial growth rate reached 0.6417 cm / d.

[0050] Before inoculating the activated mycelium, use a 5mm diameter sterile punch to cut off the edge of the activated mycelium and then inoculate it into the center of the culture medium plate.

[0051] Example 2

[0052] A method for preparing a crude enzyme solution of *Amanita muscaria*, comprising the following steps: Mycelial preparation using the cultivation method described in Example 1, with mycelia collected on days 3, 5, and 7 of cultivation:

[0053] Step A: Collect the stiff-haired, coarse-capillary hyphae and place them in a centrifuge tube. Freeze them in liquid nitrogen and grind them thoroughly.

[0054] Step B: Mix mycelium and deionized water at a ratio of 1:10 to obtain a mixed solution;

[0055] Step C: Centrifuge the mixed solution at 10000 r / min for 10 min and collect the supernatant, which is the composite crude enzyme solution. The composite crude enzyme contains three major degrading enzyme systems: ligninase (laccase, lignin peroxidase LiP, manganese peroxidase Mnp), cellulase (CMCase, filter paper enzyme FPA, β-glucosidase β-Gc), and hemicellulase (α-galactosidase α-Gal, basic xylanase BAX).

[0056] The peak activity and temporal characteristics of each enzyme are as follows: Lac enzyme activity was highest on day 7, at 15.2308 U / mg; LiP activity was only observed on day 3, at 2.3503 U / mg; Mnp activity was low throughout, with a peak of 0.3091 U / mg; CMCase and FPA activity were highest on day 5, at 1.345 U / mg and 0.1123 U / mg, respectively; β-Gc activity peaked on day 3 at 1.28 × 10⁻⁶. 5 U / mg; α-Gal peak on day 3 was 198.192 U / mg; BAX activity remained stable at low levels throughout the course, with a peak of 0.0467 U / mg.

[0057] Example 3

[0058] Application of a composite crude enzyme solution in the degradation of lignocellulose in agriculture and forestry, decolorization of dyeing and printing wastewater, remediation of heavy metal water bodies, fermentation of medicinal fungal polysaccharides, and production of enzyme preparations for degradation by white-rot fungi.

[0059] This invention uses Design-Expert 10.0.1 software to perform multinomial regression on the data, and then performs analysis of variance on the regression model (Table 11) to establish a regression model between glucose (A), yeast extract (B), peptone (C), MgSO4 (D), KH2PO4 (E) and mycelial growth rate. The regression equation is as follows:

[0060] Y=0.64-0.022A-0.016B+0.017C-8.863х10 -3 D+6.25х10 -6 E+8.75х10 -3 AB+2.725х10 -3 AC-0.013AD+0.012AE+0.02BC-9.4х10 -3 BD-0.044BE+0.027CD-3.25х10 -3 CE+4.3х10 -3 DE-0.11A 2 -0.079B 2 -0.091C 2 -0.057D 2 -0.039E 2 .

[0061] The analysis of variance showed that the model was highly significant (P<0.01), the lack-of-fit term P=0.7443>0.05, and the model F-value was 131.96, indicating that the regression model was reliable. The linear terms are A, B, C, and D; the interaction terms are AD, AE, BC, BE, and CD; and the quadratic term is A. 2 B 2 C 2 D 2 E2 The effect on mycelial growth rate was extremely significant (P<0.01), the interaction terms AB and BD were significant (P<0.05), and the remaining terms were not significant.

[0062] Model determination coefficient R 2 =0.9906, indicating that the regression equation fits the actual data well. The corrected coefficient of determination R2adj=0.9831, indicating that the model can explain 98.31% of the variation, and the predicted value is highly correlated with the actual value.

[0063] The factors affecting mycelial growth rate, from largest to smallest, are A>C>B>D>E, namely glucose>peptone>yeast extract>MgSO4>KH2PO4.

[0064] Table 1: Response Surface Experimental Design and Results

[0065] 1 20 15 5 0.6 0.6 0.6442 2 20 15 2.5 0.6 0.8 0.4988 3 20 15 5 0.6 0.6 0.63 4 15 15 5 0.6 0.8 0.4922 5 20 15 7.5 0.6 0.4 0.5325 6 15 15 5 0.7 0.6 0.5063 7 25 15 7.5 0.6 0.6 0.4384 8 15 20 5 0.6 0.6 0.4567 9 25 15 5 0.5 0.6 0.4721 10 20 15 5 0.7 0.8 0.5496 11 20 15 5 0.5 0.4 0.5492 12 25 10 5 0.6 0.6 0.4383 13 20 20 5 0.5 0.6 0.5029 14 20 10 5 0.6 0.8 0.5883 15 20 10 5 0.5 0.6 0.5283 16 20 15 2.5 0.6 0.4 0.485 17 20 15 5 0.6 0.6 0.64 18 20 15 5 0.6 0.6 0.6388 19 20 15 7.5 0.6 0.8 0.5333 20 25 20 5 0.6 0.6 0.4317 21 20 10 5 0.6 0.4 0.4925 22 20 15 2.5 0.7 0.6 0.4383 23 15 15 7.5 0.6 0.6 0.4738 24 20 20 5 0.6 0.4 0.5496 25 20 15 2.5 0.5 0.6 0.5171 26 20 15 7.5 0.5 0.6 0.4971 27 15 15 5 0.5 0.6 0.4988 28 20 20 5 0.7 0.6 0.4591 29 20 15 5 0.6 0.6 0.6375 30 20 15 5 0.5 0.8 0.5433 31 20 15 5 0.7 0.4 0.5383 32 20 20 7.5 0.6 0.6 0.4913 33 20 15 7.5 0.7 0.6 0.5254 34 15 10 5 0.6 0.6 0.4983 35 15 15 5 0.6 0.4 0.5338 36 20 10 2.5 0.6 0.6 0.4871 37 25 15 5 0.6 0.8 0.4742 38 20 10 7.5 0.6 0.6 0.4775 39 25 15 5 0.6 0.4 0.4683 40 20 15 5 0.6 0.6 0.6588 41 25 15 5 0.7 0.6 0.4279 42 20 20 5 0.6 0.8 0.4696 43 20 20 2.5 0.6 0.6 0.4217 44 25 15 2.5 0.6 0.6 0.4016 45 20 10 5 0.7 0..6 0.5221 46 15 15 2.5 0.6 0.6 0.4479

[0066] Table 2: Analysis of Variance for Regression Models

[0067] Model 0.19 20 <![CDATA[9.295х10 -3 ]]> 131.96 <0.0001 ** A <![CDATA[7.890х10 -3 ]]> 1 <![CDATA[7.890х10 -3 ]]> 112.01 <0.0001 ** B <![CDATA[3.900х10 -3 ]]> 1 <![CDATA[3.900х10 -3 ]]> 55.37 <0.0001 ** C <![CDATA[4.617х10 -3 ]]> 1 <![CDATA[4.617х10 -3 ]]> 65.55 <0.0001 ** D <![CDATA[1.257х10 -3 ]]> 1 <![CDATA[1.257х10 -3 ]]> 17.84 0.0003 ** E <![CDATA[6.250х10 -10 ]]> 1 <![CDATA[6.250х10 -10 ]]> <![CDATA[8.873х10 -6 ]]> 0.9976 - AB <![CDATA[3.063х10 -4 ]]> 1 <![CDATA[3.063х10 -4 ]]> 4.35 0.0474 * AC <![CDATA[2.970х10 -5 ]]> 1 <![CDATA[2.970х10 -5 ]]> 0.42 0.5220 - AD <![CDATA[6.682х10 -4 ]]> 1 <![CDATA[6.682х10 -4 ]]> 9.49 0.0050 ** AE <![CDATA[5.641х10 -4 ]]> 1 <![CDATA[5.641х10 -4 ]]> 8.01 0.0091 ** BC <![CDATA[1.568х10 -3 ]]> 1 <![CDATA[1.568х10 -3 ]]> 22.26 <0.0001 ** BD <![CDATA[3.534х10 -4 ]]> 1 <![CDATA[3.534х10 -4 ]]> 5.02 0.0342 * BE <![CDATA[7.726х10 -3 ]]> 1 <![CDATA[7.726х10 -3 ]]> 109.69 <0.0001 ** CD <![CDATA[2.868х10 -3 ]]> 1 <![CDATA[2.868х10 -3 ]]> 40.71 <0.0001 ** CE <![CDATA[4.225х10 -5 ]]> 1 <![CDATA[4.225х10 -5 ]]> 0.60 0.4459 - DE <![CDATA[7.396х10 -5 ]]> 1 <![CDATA[7.396х10 -5 ]]> 1.05 0.3153 - <![CDATA[A 2 ]]> 0.10 1 0.10 1462.09 <0.0001 ** <![CDATA[B 2 ]]> 0.055 1 0.055 774.66 <0.0001 ** <![CDATA[C 2 ]]> 0.073 1 0.073 1035.55 <0.0001 ** <![CDATA[D 2 ]]> 0.029 1 0.029 407.34 <0.0001 ** <![CDATA[E 2 ]]> 0.013 1 0.013 186.33 <0.0001 ** residual <![CDATA[1.761х10 -3 ]]> 25 <![CDATA[7.044х10 -5 ]]> - - - Missing item <![CDATA[1.297х10 -3 ]]> 20 <![CDATA[6.483х10 -5 ]]> 0.70 0.7443 - Pure error <![CDATA[4.644х10 -4 ]]> 5 <![CDATA[9.287х10 -5 ]]> - - - Total variation 0.19 45 - - - - <![CDATA[R 2 ]]> 0.9906 R2 adj 0.9831 R2 pred 0.9688 -

[0068] Comparative Example 1

[0069] The culture medium composition was the same as in Example 1, except that VB1 10 mg / L + VB2 10 mg / L + VB6 10 mg / L were added; all other conditions were exactly the same as in Example 1.

[0070] Experimental results: The average mycelial growth rate decreased to 0.4231 cm / d, and the mycelia were sparse; this confirmed that VB1, VB2, and VB6 significantly inhibited the mycelial proliferation of the strain.

[0071] Comparative Example 2

[0072] The remaining components remained unchanged from Example 1; all glucose was replaced with an equal mass of lactose; and the remaining culture conditions were exactly the same.

[0073] Table 3: Effects of different carbon sources on mycelial growth

[0074] fructose <![CDATA[0.373±0.048 c ]]> + Soluble starch <![CDATA[0.426±0.013 bc ]]> ++ maltose <![CDATA[0.462±0.056 b ]]> ++ glucose <![CDATA[0.566±0.010 a ]]> +++ lactose <![CDATA[0.205±0.013 d ]]> + sucrose <![CDATA[0.459±0.008 b ]]> ++

[0075] Table 3 shows that (Note: "++" indicates dense hyphae, "++" indicates relatively dense hyphae, and "+" indicates sparse hyphae) different carbon sources had significant effects on mycelial growth (P<0.05). Glucose was the optimal carbon source, resulting in the fastest mycelial growth rate (0.566 cm / d), significantly higher than other carbon sources (P<0.05), and also producing dense hyphae. Maltose and sucrose were the next most suitable carbon sources, with no significant difference between the two groups. When lactose was used as the carbon source, the mycelial growth rate was the slowest and the mycelium was the sparsest.

[0076] Experimental results: The mycelial growth rate was only 0.205 cm / d, and the mycelia were extremely sparse; this proves that lactose significantly inhibits the mycelial growth of this strain and is not suitable as a carbon source.

[0077] Comparative Example 3

[0078] The remaining components remained unchanged from those in Example 1; the yeast extract and peptone nitrogen source were replaced with an equal amount of urea; the remaining culture conditions were exactly the same.

[0079] Table 4: Effects of different nitrogen sources on mycelial growth

[0080] yeast powder <![CDATA[0.557±0.031 a ]]> +++ Yeast paste <![CDATA[0.514±0.011 ab ]]> +++ ammonium sulfate <![CDATA[0.410±0.001 c ]]> + Urea <![CDATA[0.000±0.000 d ]]> + Beef extract <![CDATA[0.450±0.031 bc ]]> ++

[0081] Table 4 shows that different nitrogen sources had significant effects on mycelial growth (P<0.05). Organic nitrogen sources were more effective than inorganic nitrogen sources in promoting growth. Yeast powder was the most suitable nitrogen source, with a mycelial growth rate of 0.557 cm / d, which was significantly higher than other treatments (P<0.05); yeast extract was the second most effective; and urea was the slowest nitrogen source for mycelial growth.

[0082] Experimental results: almost no mycelial growth, growth rate ≈ 0 cm / d; proving that urea completely inhibits the mycelial growth of the *Amanita muscaria* strain and cannot be used as a nitrogen source.

[0083] Comparative Example 4

[0084] Option A: Add only KH2PO4, without adding MgSO4;

[0085] Option B: Add only MgSO4, without adding KH2PO4;

[0086] The remaining components and culture conditions are the same as in Example 1.

[0087] Table 5: Effects of different inorganic salts on mycelial growth

[0088] NaCl <![CDATA[0.541±0.052 bc ]]> ++ <![CDATA[CaCl2]]> <![CDATA[0.573±0.030 abc ]]> +++ <![CDATA[FeSO4]]> <![CDATA[0.595±0.024 ab ]]> +++ <![CDATA[CaSO4]]> <![CDATA[0.530±0.080 bc ]]> ++ <![CDATA[KH2PO4]]> <![CDATA[0.544±0.012 bc ]]> ++ <![CDATA[MgSO4]]> <![CDATA[0.501±0.061 c ]]> + <![CDATA[KH2PO4-MgSO4]]> <![CDATA[0.661±0.046 a ]]> +++

[0089] Table 5 shows that different inorganic salts had significant effects on mycelial growth (P<0.05). The KH2PO4-MgSO4 composite inorganic salt was the optimal inorganic salt, with a mycelial growth rate of 0.661 cm / d, which was significantly better than the single salt treatment (P<0.05). FeSO4 and CaCl2 were the next best, but there was no significant difference between them and the composite inorganic salt.

[0090] Experimental results: KH2PO4 alone: ​​mycelial growth rate 0.544 cm / d; MgSO4 alone: ​​mycelial growth rate 0.501 cm / d;

[0091] This invention discloses a strain of *Coriolopsistrogii*, its optimal mother culture medium, standardized culture process, and a method for enriching lignocellulose-degrading enzymes, belonging to the field of edible fungi and environmental biotechnology. The strain was isolated from wild broadleaf decaying wood in Tianjin. It was identified by both morphological and ITS sequencing, with the ITS sequence showing 99.82% similarity to *Coriolopsistrogii*. A dedicated mother culture medium without exogenous vitamins was obtained through single-factor screening combined with a five-factor Box-Behnken response surface methodology. Each liter of distilled water contained 19.49 g glucose, 14.48 g yeast extract, 5.18 g peptone, 0.596 g magnesium sulfate, and 0.607 g potassium dihydrogen phosphate. The average mycelial growth rate during constant temperature culture at 25℃ reached 0.6417 cm / d, with a model prediction relative error of only 0.23%.

[0092] This invention simultaneously establishes a composite degrading enzyme extraction process, systematically measuring the sequential activities of eight lignocellulose-degrading enzymes. Laccase reached its highest activity on day 7, making it the core functional degrading enzyme. The culture medium formulation of this invention is simple, low-cost, and produces excellent mycelial growth. The entire cultivation process allows for large-scale production of strains and highly active degrading enzymes. The resulting strains and enzyme solutions can be widely used for the degradation of agricultural and forestry waste, decolorization of dyeing and printing wastewater, remediation of heavy metal-containing water bodies, fermentation of medicinal fungal polysaccharides, and production of biological enzyme preparations, demonstrating promising industrialization prospects.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing *Pheromus spp.*, characterized in that, include: S1: Inoculate the *Pterocarya stearata* strain onto a PDA activation plate and culture at 25°C in the dark for 5–7 days to obtain purified and activated mycelia; S2: Prepare the culture medium, sterilize and cool it for later use; S3: Inoculate the activated mycelia into the center of the culture medium and culture at a constant temperature to obtain *Pterocarya stearata*.

2. The method for culturing *Amanita muscaria* according to claim 1, characterized in that, The *Porphyra yezoensis* was collected from wild fruiting bodies of broad-leaved decaying wood in the East Campus of Tianjin Agricultural College. It was purified by tissue isolation. Morphological and ITS molecular identification showed that the full-length ITS sequence was 544 bp. Under 25℃ plate culture conditions, the colonies were regular round, dense white cotton-like, and grew outwards in a uniform radial pattern, without pigment secretion.

3. The method for culturing *Amanita muscaria* according to claim 1, characterized in that, In S1, the formula for the PDA activation plate is 200g potato, 20g glucose, 20g agar, and 1L distilled water.

4. The method for culturing *Amanita muscaria* according to claim 1, characterized in that, In S2, the culture medium contains the following components per liter of distilled water: 19.49 g glucose, 14.48 g yeast extract, 5.18 g peptone, 0.596 g magnesium sulfate, 0.607 g potassium dihydrogen phosphate, and 18–22 g agar; the natural pH of the culture medium is 5.5–6.

5.

5. The method for culturing *Amanita muscaria* according to claim 1, characterized in that, In S2, the sterilization conditions for the culture medium are: steam sterilization at 121°C for 20 minutes.

6. The method for culturing *Amanita muscaria* according to claim 1, characterized in that, In S3, the constant temperature incubation conditions are: 25℃, completely dark, and static incubation.

7. A method for preparing a crude enzyme solution of *Amanita muscaria*, comprising mycelial preparation using the culture method described in any one of claims 1-5, characterized in that... The steps include: Step A: Collect the stiff-haired, coarse-capped mycelium and place it in a centrifuge tube, then freeze it with liquid nitrogen and grind it thoroughly; Step B: Mix the mycelium and deionized water at a ratio of 1:10 to obtain a mixed solution; Step C: Centrifuge the mixed solution at 10,000 r / min for 10 min and take the supernatant, which is the composite crude enzyme solution.

8. The method for culturing *Amanita muscaria* according to claim 6, characterized in that, The complex crude enzyme comprises: ligninase, cellulase and hemicellulase.

9. The application of the composite crude enzyme solution according to claim 7 in the degradation of lignocellulose in agriculture and forestry, decolorization of dyeing and printing wastewater, remediation of heavy metal water bodies, fermentation of medicinal fungal polysaccharides, and production of enzyme preparations for degradation by white-rot fungi.

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