Method for cultivating m. palmivorus by using cordyceps militaris cultivation residues

Abstract

The application provides a method for cultivating Cordyceps militaris cultivation residues and relates to the technical field of microorganisms.The Cordyceps militaris cultivation residues are applied to the cultivation process of the Cordyceps militaris for the first time in the application, a new resource recycling method for the Cordyceps militaris cultivation residues is provided, the problems of resource waste and environmental pollution of the Cordyceps militaris cultivation residues are solved, a new low-cost high-quality cultivation resource for the Cordyceps militaris is provided, and the problem of high cultivation cost of the Cordyceps militaris is solved.The application provides a cultivation method for the Cordyceps militaris, realizes the rapid cultivation of the Cordyceps militaris, can realize full bagging in 35 days, and the cultivation period can be up to 47 days at the fastest, the method has high fruiting body yield for cultivating the Cordyceps militaris, the fruiting body yield can be up to 81.2% at the highest, and the cultivated Cordyceps militaris has good mycelium growth and good quality, contains various antioxidant active substances such as total polysaccharides, flavones and polyphenols, and has strong antioxidant capacity.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a method for culturing palmate rosea using Cordyceps militaris cultivation residues. Background Technology

[0002] Cordyceps militaris, also known as North Cordyceps, is a rare edible and medicinal fungus. It is rich in nutrients and active ingredients such as protein, amino acids, cordycepin, and polyphenols, and has been approved as a new food ingredient. Studies have shown that cultivated Cordyceps militaris residues are rich in nutrients such as protein and starch. However, due to a lack of resource utilization technology, it is usually used as fuel and discarded in the vicinity of production sites. Only a small portion is purchased by small-scale farms at low prices. Therefore, the resource utilization of Cordyceps militaris cultivation residues is urgently needed.

[0003] Palmate rose fungus, also known as netted red pleated umbrella, belongs to the Agaricaceae family and the Chlorophyllum genus. Its fruiting body is pink and gelatinous, rich in polysaccharides and polyphenols, and has effects such as liver protection, bowel lubrication, anti-oxidation, and immune enhancement. It has high ornamental and nutritional value. Currently, palmate rose fungus has been successfully cultivated artificially. The fruiting body of palmate rose fungus has a smooth texture and a unique fruity aroma, with excellent color, aroma, taste, and shape, and has a broad consumer market. However, the artificial large-scale cultivation of palmate rose fungus is still in its early stages, and there are few reports on its growth characteristics.

[0004] No reports have been found on the cultivation technology of palmate rose ear using Cordyceps militaris cultivation residues as a substitute for raw materials such as sawdust. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a culture medium for cultivating *Rosa palmata* using *Cordyceps militaris* cultivation residues, along with its cultivation method and applications. On one hand, by optimizing the *Rosa palmata* mother culture, liquid culture medium, and cultivation conditions, the mycelial growth of *Rosa palmata* is promoted, and the culture cycle is shortened. On the other hand, by using *Cordyceps militaris* cultivation residues as the main raw material, an innovative cultivation substrate is developed, the cultivation formula is simplified, production costs are reduced, and biological efficiency is improved. Simultaneously, the content of active ingredients and their correlation with antioxidant activity in the cultured *Rosa palmata* are investigated, resulting in *Rosa palmata* fruiting bodies rich in flavonoids, polyphenols, and other physiologically active substances, which can be used as raw materials for food and health products.

[0006] On the one hand, this application provides the application of Cordyceps militaris cultivation residues in the cultivation of palmate rose.

[0007] Furthermore, the Cordyceps militaris cultivation residues are obtained by culturing Cordyceps militaris in a Cordyceps militaris culture medium; preferably, the Cordyceps militaris culture medium includes wheat grains, potassium dihydrogen phosphate, and / or magnesium sulfate.

[0008] Those skilled in the art will understand that a common Cordyceps militaris culture medium can be selected to culture Cordyceps militaris, thereby obtaining Cordyceps militaris cultivation residues.

[0009] In a preferred embodiment, the Cordyceps militaris culture medium comprises wheat grains and / or nutrient solution, prepared at a mass ratio of 1:(1.6-1.7). Preferably, the nutrient solution comprises: 1.0-2.0 g potassium dihydrogen phosphate, 0.5-1.0 g magnesium sulfate, and 1000 mL water.

[0010] The cultivation of Cordyceps militaris can be carried out using general methods. The specific palmate rose ear variety, Cordyceps militaris variety, and Cordyceps militaris cultivation method have no impact on the technical effect of this application, and this application does not impose too many restrictions on them.

[0011] This application is the first to apply Cordyceps militaris cultivation residues to the cultivation of *Rosa palmatum*, providing a novel method for resource reuse and solving the problems of resource waste and environmental pollution associated with these residues. Furthermore, this application verifies that using Cordyceps militaris cultivation residues to cultivate *Rosa palmatum* can improve its growth characteristics, shorten the time to full bag formation, and increase the fruiting body yield, providing a high-quality new culture medium component for *Rosa palmatum*. In addition, this application demonstrates that using Cordyceps militaris cultivation residues to cultivate *Rosa palmatum* can effectively increase the content of antioxidant active substances in *Rosa palmatum*, such as polysaccharides, flavonoids, and polyphenols, thereby enhancing the antioxidant activity and hydroxyl radical scavenging activity of *Rosa palmatum*.

[0012] On the other hand, this application also provides a palmate rosea cultivation medium, wherein the palmate rosea cultivation medium includes Cordyceps militaris cultivation residues.

[0013] Furthermore, the Cordyceps militaris cultivation residues are obtained by culturing Cordyceps militaris in a Cordyceps militaris culture medium; preferably, the Cordyceps militaris culture medium includes wheat grains, potassium dihydrogen phosphate, and / or magnesium sulfate.

[0014] In a preferred embodiment, the Cordyceps militaris culture medium comprises wheat grains and / or nutrient solution, prepared at a mass ratio of 1:(1.6-1.7). Preferably, the nutrient solution comprises: 1.0-2.0 g potassium dihydrogen phosphate, 0.5-1.0 g magnesium sulfate, and 1000 mL water.

[0015] Further, based on the mass fraction of the culture medium, the content of the Cordyceps militaris cultivation residues is 200-600 parts; preferably, 200-500 parts; more preferably, 500 parts.

[0016] The content of the Cordyceps militaris cultivation residues can be any value among 200 parts, 300 parts, 400 parts, 500 parts, and 600 parts.

[0017] Furthermore, the culture medium further includes sawdust and / or quicklime; preferably, the sawdust is 400-800 parts; preferably, the quicklime is 8-15 parts; more preferably, the sawdust is 500 parts; even more preferably, the quicklime is 10 parts;

[0018] And / or, the culture medium further includes water; preferably, the water content of the culture medium is 55%-60% by weight.

[0019] In a preferred embodiment, the palmate rose cultivation medium comprises 500g of sawdust, 500g of Cordyceps militaris cultivation residue, 10g of quicklime, and water added to make the moisture content of the culture medium reach 55%-60%.

[0020] Those skilled in the art will understand that the palmate rose cultivation medium can be prepared using common methods.

[0021] In a preferred embodiment, the method for preparing the palmate rosehip cultivation medium includes:

[0022] Step 1: Soak the Cordyceps militaris cultivation residue in water for 3-4 hours, drain the water, and set aside.

[0023] Step 2: Mix the Cordyceps militaris cultivation residue, sawdust, quicklime, and water.

[0024] Preferably, the culture medium further includes a high-temperature sterilization step, which involves sterilizing at 121°C-125°C for 2 hours.

[0025] On the other hand, this application also provides a method for culturing palmate rosea using Cordyceps militaris cultivation residues, the method comprising: culturing palmate rosea using the palmate rosea cultivation medium.

[0026] Furthermore, the method includes the following steps:

[0027] Step 1: Inoculate palmate rose fungus into palmate rose fungus cultivation medium at an inoculation rate of 10-15 mL / bag, and manage mycelium growth in a dark environment at 18℃-22℃ and relative humidity below 70%.

[0028] Step 2: Perform bud-inducing management under the following conditions: 300-500 Lux diffused light for 12-16 hours / day, carbon dioxide concentration of 800-1000 ppm, humidity of 80%-90%, and temperature of 16℃-22℃.

[0029] Step 3: Conduct mushroom cultivation management under the following conditions: 300-500 Lux diffused light for 12-16 hours / day, carbon dioxide concentration of 800-1000 ppm, humidity of 80%-90%, and temperature of 20-22℃.

[0030] In one specific implementation, the mycelium can be spread and grown throughout the entire culture medium during the mycelium growth management to induce bud formation; fruiting body culture can begin when white to light pink palmate rose primordia appear.

[0031] Preferably, the method further includes the step of inoculating *Rhizoctonia purpurea* onto a *Rhizoctonia purpurea* mother culture medium to obtain *Rhizoctonia purpurea* mother culture; more preferably, the *Rhizoctonia purpurea* mother culture medium includes: a carbon source and / or a nitrogen source; more preferably, the carbon source is selected from one or more of sucrose, lactose, glucose, maltose, and mannitol; more preferably, the carbon source is maltose; more preferably, the nitrogen source is selected from one or more of soybean flour, cornmeal, malt powder, wheat bran, ammonium sulfate, ammonium chloride, peanut meal powder, and wheat grain powder; more preferably, the nitrogen source is peanut meal powder; more preferably, the *Rhizoctonia purpurea* mother culture medium further includes: potassium dihydrogen phosphate, magnesium sulfate, and / or vitamin B1; more preferably, the *Rhizoctonia purpurea* mother culture medium includes: 1%-5% maltose, 1%-5% peanut meal powder, 0.1%-0.5% potassium dihydrogen phosphate, 0.1%-0.5% magnesium sulfate, and vitamin B1. 0.001%-0.005%; more preferably, the culture medium for the palmate rose mother seed includes: 2% maltose, 1.5% peanut cake powder, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and 0.002% vitamin B1; preferably, the culture conditions include: 16℃-28℃, pH 4-9; more preferably, 22℃-25℃, pH 7-9, 15-20 days.

[0032] More preferably, 22°C, pH 7.

[0033] In a preferred embodiment, the palmate rose mother culture medium comprises: 20g maltose, 15g peanut cake powder, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 20g agar, 1000mL water, pH 7.0.

[0034] Preferably, the method further includes the step of inoculating the mother plant of *Rosa palmatum* into a liquid primary culture medium of *Rosa palmatum* to obtain liquid primary culture of *Rosa palmatum*; more preferably, the liquid primary culture medium of *Rosa palmatum* includes: a carbon source and / or a nitrogen source; more preferably, the carbon source is selected from one or more of sucrose, lactose, glucose, maltose, and mannitol; more preferably, the carbon source is maltose; more preferably, the nitrogen source is selected from one or more of soybean flour, cornmeal, malt powder, wheat bran, ammonium sulfate, ammonium chloride, peanut meal powder, and wheat grain powder; more preferably, the nitrogen source is peanut meal powder; more preferably, the liquid primary culture medium of *Rosa palmatum* further includes: potassium dihydrogen phosphate, magnesium sulfate, and / or vitamin B1; more preferably, the liquid primary culture medium of *Rosa palmatum* includes: 1%-5% maltose, 1%-5% peanut meal powder, 0.1%-0.5% potassium dihydrogen phosphate, 0.1%-0.5% magnesium sulfate, and vitamin B1. 0.001%-0.005%; more preferably, the liquid culture medium for palmate rose ear includes: 3% maltose, 1% peanut cake powder, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and 0.002% vitamin B1; preferably, the culture conditions include: pH 4-9, 15℃-28℃, 100-200 r / min, and 5-14 days.

[0035] More preferably, the culture conditions include: pH 7, 22°C, 150 r / min, 7 days.

[0036] In a preferred embodiment, the palmate rosehip liquid culture medium comprises: 30g maltose, 10g peanut cake powder, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 1000mL water, pH 7.0.

[0037] Preferably, the method further includes a propagation step.

[0038] The propagation process includes: inoculating the palmate rosea strain into the propagation medium, incubating it upside down and in the dark at 22°C, terminating the culture when the palmate rosea hyphae cover the surface of the plate medium, and storing it for later use.

[0039] The propagation culture medium comprises: 200g potato, 20g glucose, 3g peptone, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 10mg vitamin B1, 20g agar, and 1000mL water.

[0040] In a preferred embodiment, a method for culturing palmate rosettes using Cordyceps militaris cultivation residues includes the following steps:

[0041] Step 1: Inoculate the palmate rose fungus strain into the propagation medium and incubate it upside down and in the dark at 22℃-25℃. Stop the culture when the palmate rose fungus hyphae cover the surface of the plate medium to obtain the palmate rose fungus propagation strain.

[0042] Step 2: Inoculate the palmate rose auricula propagation strain into the mother culture medium, and incubate at 22℃-25℃, pH 7-9, upside down and in the dark for 15-20 days to obtain the palmate rose auricula mother culture.

[0043] Step 3: Inoculate the palmate rose mother culture into palmate rose liquid primary culture medium at pH 4-9, 15℃-28℃, 100-200r / min, and culture at a constant temperature with shaking for 5-14 days to obtain palmate rose liquid primary culture.

[0044] Step 4: Inoculate the liquid seed culture of *Rhizoctonia solani* into the *Rhizoctonia solani* cultivation medium at an inoculation rate of 10-15 mL / bag. Manage the mycelium growth in a dark environment at 18℃-22℃ and relative humidity below 70%.

[0045] Step 5: When the mycelium spreads and grows throughout the entire culture medium, bud induction management is carried out under the following conditions: 300-500 Lux diffused light for 12-16 hours / day, carbon dioxide concentration of 800-1000 ppm, humidity of 80%-90%, and temperature of 16-22℃.

[0046] Step 6: When pale pink palmate rose ear primordia appear, manage the fruiting process under the following conditions: 300-500 Lux diffused light for 12-16 hours / day, carbon dioxide concentration of 800-1000 ppm, humidity of 80%-90%, and temperature of 20-22℃.

[0047] On the other hand, this application also provides the palmate rosea cultivation medium or the palmate rosea obtained by the method described above; preferably, the palmate rosea contains antioxidant active substances; more preferably, the antioxidant active substances include polysaccharides, flavonoids and / or polyphenols.

[0048] On the other hand, this application also provides the palmate rosea cultivation medium or the method described therein, or the application of the palmate rosea in the preparation of antioxidant active substances and / or in antioxidation; preferably, the antioxidant active substances include polysaccharides, flavonoids and / or polyphenols.

[0049] The present invention has the following beneficial effects:

[0050] 1. This application is the first to apply Cordyceps militaris cultivation residues to the cultivation of Rhizoctonia palmata, providing a new method for resource reuse of Cordyceps militaris cultivation residues, solving the problems of resource waste and environmental pollution of Cordyceps militaris cultivation residues, and providing a new low-cost, high-quality culture resource for Rhizoctonia palmata, while also solving the problem of high cultivation cost of Rhizoctonia palmata.

[0051] 2. This application provides a cultivation method for *Rosa palmata*, which utilizes *Cordyceps militaris* culture residues to efficiently cultivate *Rosa palmata*, achieving rapid cultivation of *Rosa palmata*. The cultivated *Rosa palmata* mycelium grows well and is of high quality, achieving full bag formation in 35 days, with a cultivation cycle as fast as 47 days. Furthermore, this method for cultivating *Rosa palmata* also has a high fruiting body yield, with a maximum fruiting body yield of up to 81.2%. The fruiting bodies contain various antioxidant active substances, such as total polysaccharides, flavonoids, and polyphenols, exhibiting good antioxidant capacity. Attached Figure Description

[0052] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0053] Figure 1 This is a schematic diagram showing the growth of *Rhodotorula palmatum* on different culture media in Example 1.

[0054] Figure 2 This is a schematic diagram showing the growth of *Rosa palmata* on different carbon source culture media.

[0055] Figure 3 This is a schematic diagram showing the growth of *Rosa palmatum* on different nitrogen source culture media.

[0056] Figure 4 The growth of *Rosa palmatum* on culture media with different pH values;

[0057] Figure 5 This is a schematic diagram showing the growth of *Rhodotorula palmatum* hyphae under different culture temperature conditions.

[0058] Figure 6 This is a schematic diagram of the growth of *Rhizoctonia solani* on an optimized liquid culture medium. Figure A shows the microscopic morphology of the hyphae at a magnification of 40x, while Figures B and C show the sensory morphology of the liquid culture.

[0059] Figure 7 This is a schematic diagram showing the mycelial growth and fruiting body growth of the palmate palmate rose ear experimental group and the control group in Example 4. Detailed Implementation

[0060] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0061] Unless otherwise specified in the examples, the conditions shall be performed in accordance with the standard conditions or the conditions recommended by the manufacturer.

[0062] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0063] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0064] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0065] In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage.

[0066] Among them, Rhodotus palmatus belongs to the family Agaricaceae and the genus Rhodotus.

[0067] Cordyceps militaris culture medium formula: Wheat grains and nutrient solution are prepared at a mass ratio of 1:1.6-1.7. The nutrient solution includes: 1.0-2.0g potassium dihydrogen phosphate, 0.5-1.0g magnesium sulfate, and 1000mL water.

[0068] Measurement method:

[0069] Polysaccharide content determination method: Phenol-sulfuric acid method was used. Following the determination method for polysaccharides in edible fungi, the spectrophotometric method in NY / T1676-2023 "Determination of Crude Polysaccharides in Edible Fungi" was employed. With glucose mass concentration as the abscissa and A490 absorbance as the ordinate, the linear regression equation was obtained as: y = 504071X + 0.0125, R0 2 =0.9972.

[0070] Polyphenol content determination method: The polyphenol content was determined by spectrophotometry. Following the method described by Deng Zhenhua et al. in "Determination and Analysis of Total Polyphenol and Polyphenol Monomer Content in Different Mulberry Leaf Varieties," the content was determined by plotting gallic acid concentration on the x-axis and the corresponding A750 absorbance value on the y-axis. The linear regression equation was obtained as y = 18.684X - 0.0266, R² = 0.9985.

[0071] Method for determining flavonoid content: Total flavonoid content was determined by aluminum nitrate colorimetric method. The flavonoid content was determined according to the method described by Ge Xinhui et al. in their paper "Analysis of Phenolic Composition and Antioxidant Activity of Wild Edible Fungi in Wutai Mountain". The linear regression equation was obtained by plotting the rutin mass concentration on the x-axis and the corresponding A510 absorbance value on the y-axis: y = 2.9284X + 0.0182, R² = 0.9974.

[0072] The method for determining hydroxyl radical scavenging rate (%) includes: analysis using a hydroxyl radical scavenging ability assay kit. The calculation method for the hydroxyl radical scavenging rate of the test sample solution is: hydroxyl radical scavenging rate / % = [A] 空白 -(A 测定 -A 对照 )]÷A 空白 ×100.

[0073] The DPPH scavenging capacity determination method includes: using a DPPH free radical scavenging capacity assay kit for determination. The calculation method for the DPPH free radical scavenging rate of the test sample solution is given by the formula: DPPH free radical scavenging rate / % = [1-(A 测定 -A 对照 )÷A 空白 ]×100; Where: A 测定 A represents the absorbance of the sample mixed with DPPH solution. 对照 A represents the absorbance of the sample mixed with anhydrous ethanol. 空白 The absorbance is the value of the DPPH and anhydrous ethanol mixture.

[0074] The method for determining total antioxidant activity includes: using the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid method, and analyzing the results using the T-AOC assay kit. The formula for calculating the total antioxidant capacity of the test sample solution is: Total antioxidant capacity ( / μmol Trolox / mL)=0.3×(△A+0.029)×D; where: △A=A 空白 -(A 测定 -A 对照 ), where A 测定 A represents the absorbance of the sample and the ABTS working solution. 对照 A represents the absorbance of the sample mixed with PBS. 空白 denoted as ABTS working solution and PBS mixture; D is the sample dilution factor.

[0075] The method for determining superoxide anion includes: using a superoxide anion reagent kit for determination. The formula for calculating the superoxide anion content in the test sample solution is: Superoxide anion scavenging capacity (μmol / g) = 237.1 ×

[0076] (△A+0.0011) / W×D; where: △A=A测定 -A 空白 ), where A 测定 A represents the absorbance of the sample and the developing solution. 空白 The absorbance of the extraction solvent and the colorimetric solution is given; W is the sample weight, and D is the sample dilution factor.

[0077] Example 1: Optimization of Mother Culture Medium and Culture Conditions for Palmar Rosehips

[0078] 1. Strain propagation and culture

[0079] Plate culture medium preparation: 200g potato, 20g glucose, 3g peptone, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 10mg vitamin B1, 20g agar, 1000mL water; after preparing the culture medium according to the above ratio, sterilize it at 121℃ for 25min, and aseptically pour it into petri dishes to prepare the propagation plate culture medium for later use;

[0080] Strain propagation: Inoculate the palmate rosea strain into the center of the propagation plate medium, invert it at 22°C and incubate in the dark. Stop the culture when the palmate rosea hyphae cover the surface of the plate medium, and store it for later use.

[0081] 2. Effects of different mother culture medium formulations on the growth of *Rosa palmatum* mycelium

[0082] Because the mycelium of *Rosa palmata* grows slowly, the components of the mother culture medium were screened in this embodiment. The formula of the mother culture medium is as follows:

[0083] Mother culture medium #1: 20g glucose, 2g peptone, 2g yeast powder, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20g agar, 1000mL water;

[0084] Mother culture medium #2: 20g glucose, 2g yeast powder, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 20g agar, 1000mL water;

[0085] Mother culture medium #3: 10g wheat bran, 20g glucose, 1.0g yeast extract, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 20g agar, 1000mL water;

[0086] Mother culture medium #4: 5g soybean flour, 2.5g peptone, 8g glucose, 2g yeast extract, 1000mL water;

[0087] Mother culture medium #5: 200g potatoes (boiled and juiced), 30g wheat grains (pre-soaked and boiled for 20 minutes, then filtered and juiced), 25g glucose, 5g soybean flour, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 20g agar, and 1000mL water.

[0088] Mother culture medium #6: 200g potato, 25g glucose, 5g soybean flour, 5g wheat flour, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 20g agar, 1000mL water;

[0089] Mother culture medium #7: 200g potato, 25g glucose, 10g soybean flour, 10g peptone, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 20g agar, 1000mL water;

[0090] Mother culture medium #8: 20g glucose, 15g soybean flour, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 20g agar, 1000mL water;

[0091] Mix the above proportions thoroughly, sterilize at 121℃ for 25 min, and pour into petri dishes to obtain the mother culture medium; use a sterile punch to divide the palmate rosehip strain propagated in step (1) into approximately 0.3 cm pieces. 2 Small pieces were aseptically inoculated into the center of different mother culture media, and incubated upside down and in the dark at 22°C until they covered the culture medium. The mycelial growth was observed and the mycelial growth rate was measured.

[0092] In this embodiment, the growth of *Rosa palmatum* on different culture media is as follows: Figure 1 As shown in Table 1.

[0093] Table 1. Effects of different culture media on the growth of *Rosa palmatum* hyphae.

[0094]

[0095] Depend on Figure 1 As shown in Table 1, *Rosa palmata* can grow on all eight culture media, but there are significant differences in the mycelial growth rate and vigor on different media. The mycelium grows fastest and most densely on medium 8. Therefore, mother culture medium 8# was selected as the basal medium for subsequent experiments.

[0096] 3. Effects of different carbon and nitrogen sources on the growth of palmate rose mycelium

[0097] The optimal culture medium formulation, Mother Culture Medium #8, was determined through experiments on the effects of different culture medium formulations on the growth of *Rosa palmatum* mycelium. This medium was then used as the basal medium for subsequent experiments. In this example, screening experiments were also conducted on the types of carbon and nitrogen sources and the pH value in the culture medium. Details are as follows:

[0098] Basic culture medium: 20g glucose, 15g soybean meal, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 20g agar, 1000mL water;

[0099] Carbon source test: The glucose in the basal medium was replaced with equal amounts of sucrose, lactose, maltose, and mannitol. The prepared culture media were poured into 250mL Erlenmeyer flasks, 100mL per flask. After autoclaving and cooling, the flasks were poured into Petri dishes. Using a sterile punch, the palmate rosehip strain propagated in step (1) was divided into segments approximately 0.3cm in size. 2 Small pieces were aseptically inoculated into the center of different solid agar plates and incubated upside down in the dark at 22°C. Mycelial growth was observed and the mycelial growth rate was measured. Specific results are as follows: Figure 2 As shown in Table 2.

[0100] Nitrogen source test: Corn flour, malt flour, wheat bran, peanut flour, wheat grain flour, ammonium sulfate, and ammonium chloride were used to replace soybean flour in the basal culture medium, respectively. The prepared culture media were poured into 250mL Erlenmeyer flasks, 100mL per flask. After autoclaving and cooling, the media were poured into Petri dishes, and the palmate rosehip strain propagated in step (1) was divided into approximately 0.3cm sections using a sterile punch. 2 Small pieces were aseptically inoculated into the center of different solid agar plates and incubated upside down in the dark at 22°C. Mycelial growth was observed and the mycelial growth rate was measured. Specific results are as follows: Figure 3 As shown in Table 3.

[0101] Table 2 Effects of carbon source on the growth of *Rosa palmatum* hyphae

[0102]

[0103] from Figure 2 Table 2 shows that *Rosa palmatum* can grow on all five carbon source media. While the average daily growth rate of mycelium did not differ significantly, the mycelial vigor showed marked differences. The mycelium grew slightly faster on the maltose-based carbon source medium than on the other carbon sources, but exhibited the most vigorous and dense mycelial growth. The mycelial vigor, from strongest to weakest, was maltose > glucose > mannitol > lactose > sucrose. Therefore, maltose is the optimal carbon source for *Rosa palmatum* mycelial growth.

[0104] Table 3 Effects of nitrogen source on the growth of *Rosa palmatum* hyphae

[0105]

[0106] Depend on Figure 3 Table 3 shows that *Rosa palmatum* can grow on all six organic nitrogen source media. While the daily average growth rate of mycelium did not differ significantly, the growth vigor varied considerably. No growth was observed on media using ammonium sulfate or ammonium chloride as nitrogen sources. The fastest daily average growth rate and best growth were observed on peanut meal media. Therefore, peanut meal is the optimal nitrogen source for *Rosa palmatum* mycelial growth.

[0107] 4. Effects of different pH values ​​on the growth of *Rosa palmatum* hyphae

[0108] The pH of the basal culture medium was adjusted to six gradients: 4, 5, 6, 7, 8, and 9, respectively, using 1.0N HCl and 1.0N NaOH. The prepared culture media were then poured into 250mL Erlenmeyer flasks, 100mL per flask. After autoclaving and cooling, the flasks were poured into Petri dishes, and the palmate rosehip strain propagated in step (1) was divided into segments approximately 0.3cm in size using a sterile punch. 2 Small pieces were aseptically inoculated into the center of different solid agar plates and incubated upside down in the dark at 22°C. Mycelial growth was observed and the mycelial growth rate was measured. Specific results are as follows: Figure 4 As shown in Table 4.

[0109] Table 4. Effects of pH on the growth of *Rosa palmatum* hyphae.

[0110]

[0111] Depend on Figure 4 Table 4 shows that *Rosa palmatum* can grow on six different pH media, with significant differences in daily average mycelial growth rate and vigor. Specifically, regarding growth rate, the daily average mycelial growth rate gradually increased with increasing pH. When the pH of the media ranged from 7.0 to 9.0, the mycelial growth rate was relatively uniform and reached a high level, with pH 7.0 showing the best mycelial growth. Therefore, pH 7.0 was selected as the optimal pH for *Rosa palmatum* mycelial growth.

[0112] Based on the comprehensive experimental results, the culture medium formula for palmate rose mother culture was determined to be: 20g maltose, 15g peanut cake powder, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, 20g agar, 1000mL water, pH 7.0.

[0113] 5. Effects of different temperatures on the growth of palmate rose mycelium

[0114] This embodiment also investigated the effects of different temperature conditions on the growth of palmate rose mycelium.

[0115] Using a sterile punch, the palmate rosehip strain propagated in step (1) was divided into segments approximately 0.3 cm in size. 2 Small pieces were aseptically inoculated into the center of the optimized solid plate culture medium from step (4), and incubated upside down and in the dark at 16℃-30℃. The mycelial growth was observed and the mycelial growth rate was measured. The results are as follows: Figure 5 As shown in Table 5.

[0116] Table 5. Effects of temperature on the growth of *Rosa palmatum* hyphae.

[0117]

[0118] Depend on Figure 5 Table 5 shows that temperature has a significant impact on the growth vigor and growth rate of *Rosa palmatum* mycelium. Specifically, regarding growth rate, the growth rate of *Rosa palmatum* mycelium initially increases and then decreases with increasing culture temperature. Mycelial growth ceases at 30℃, with the fastest growth rates at 22℃ and 25℃, reaching daily growth rates of 0.358 cm / d and 0.352 cm / d, respectively. As for mycelial vigor, *Rosa palmatum* mycelium grows well within a culture temperature range of 16℃-28℃, exhibiting vigorous growth and dense colonies. Considering the overall mycelial growth rate, a culture temperature of 22℃-25℃ is optimal.

[0119] Example 2: Optimization of the Liquid Original Culture Medium Formula for Palmar Rosehips

[0120] 1. The propagation and mother culture of *Rosa palmata* strain were carried out under the optimized conditions of Example 1.

[0121] 2. Preparation of liquid primary culture medium for different palmate rose ears:

[0122] Based on the experimental results of Example 1, the formulation of the liquid primary culture medium for *Rhizoctonia solani* was optimized. Using the optimized mother culture medium formulation as the liquid basal medium, the effects of different amounts of added maltose and peanut cake powder on the liquid culture of mycelium were investigated.

[0123] Liquid basal culture medium: 20g maltose, 15g peanut cake powder, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, pH 7.0, 1000mL water.

[0124] Maltose addition test: 10g, 20g, 30g and 40g of maltose were added to each liter of liquid basal culture medium to prepare liquid primary culture medium for palmate rose ear.

[0125] Peanut cake powder addition test: Based on the optimal addition of maltose test, the addition of peanut cake powder was tested. 5g, 10g, 15g and 20g of peanut cake powder were added to each liter of liquid basal culture medium to prepare palmate rose ear liquid primary culture medium.

[0126] After preparing the culture medium according to the above proportions, dispense it into 250mL Erlenmeyer flasks, with each flask containing 100mL. Sterilize at 121℃ for 25 minutes and set aside.

[0127] 3. Culture of *Rosa palmata* strain:

[0128] Five pieces of each of the palmate rose spore strains were inoculated into different culture media and cultured at 22℃ and 150r / min for 7 days with constant temperature shaking. 100mL of fermentation broth was taken into a 100mL graduated cylinder and allowed to stand for 30min to investigate the volume ratio of mycelia in the fermentation broth in different culture media.

[0129] The effect of maltose addition on mycelial biomass in this embodiment is shown in Table 6, and the effect of peanut cake powder addition on mycelial biomass is shown in Table 7.

[0130] Table 6. Effects of maltose addition on the growth of mycelium in liquid culture of *Rosa palmatum*

[0131] Maltose addition (g) 10 20 30 40 Mycelial volume ratio in fermentation broth / (%) 24.5 28.6 40.3 39.6

[0132] As shown in Table 6, different concentrations of maltose in the liquid primary culture medium of *Rhizoctonia solani* have a significant effect on mycelial growth. With the increase of maltose addition, the amount of mycelium gradually increases. When the addition amount is 30g, the amount of mycelium reaches the maximum. Further increases in the amount of mycelium do not increase the amount of mycelium. Therefore, the optimal addition amount of maltose is 30g.

[0133] Table 7. Effect of peanut cake powder addition on the growth of mycelium in liquid culture of *Rosa palmatum*

[0134] Peanut cake powder addition amount (g) 5 10 15 20 Mycelial volume ratio in fermentation broth / (%) 35.3 41.5 28.6 28.5

[0135] As shown in Table 7, different concentrations of peanut cake powder in the liquid original culture medium of *Rhizoctonia solani* have a significant effect on mycelial growth. With the increase of peanut cake powder, the amount of mycelium gradually increases and then decreases. When the amount added is 10g, the amount of mycelium reaches the maximum. Further increases do not increase the amount of mycelium but instead decrease it. Therefore, the optimal amount of peanut cake powder added is 10g.

[0136] Based on the experimental results of adding maltose and peanut meal, the optimal liquid culture medium formula for *Rhizoctonia solani* was determined to be: 30g maltose, 10g peanut meal, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, pH 7.0, and 1000mL water. This was then verified, and the culture effect of the liquid culture under the optimized conditions in this example is as follows: Figure 6 As shown.

[0137] Depend on Figure 6 It was found that *Rosa palmatum* exhibited dense mycelial pellets on the optimized liquid spawn medium, with a mycelial volume ratio reaching 42.9%, a significant increase compared to the previous 28.6%. Therefore, the optimal liquid spawn medium formula for *Rosa palmatum* is: 30g maltose, 10g peanut cake powder, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, pH 7.0, and 1000mL water.

[0138] Example 3: Effects of different amounts of Cordyceps militaris cultivation residues on the growth of palmate rose fruiting bodies

[0139] 1. Preparation of palmate rose mother seed

[0140] (1) The palmate rose mother culture medium is the solid slant culture medium selected in Example 1, and its formula is: 20g maltose, 10g peanut cake powder, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, pH 7.0, 15g agar, and 1000mL water.

[0141] (2) Aseptically inoculate fresh culture of *Rhizoctonia solani* strain into mother culture medium, invert and in the dark at 22°C for 15-20 days until it grows to cover the culture medium.

[0142] 2. Preparation of liquid seed of *Rosa palmata*

[0143] (1) The liquid culture medium for palmate rose ear is the liquid culture medium selected in Example 2. Its formula is: 30g maltose, 10g peanut cake powder, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 20mg vitamin B1, pH 7.0, and 1000mL water.

[0144] (2) The method for culturing liquid primary culture is the same as in Example 2.

[0145] 3. Preparation of cultivation medium

[0146] (1) Cultivation medium formula

[0147] The following are the formulations of five cultivation media designed using Cordyceps militaris cultivation residues as the main raw material:

[0148] Cultivation medium #1: 800g sawdust, 200g Cordyceps militaris cultivation residue, 10g quicklime, and the remainder is water with a moisture content of 55%-60%.

[0149] Cultivation medium #2: 700g sawdust, 300g Cordyceps militaris cultivation residue, 10g quicklime, add water to make the moisture content of the culture medium reach 55%-60%;

[0150] Cultivation medium #3: 600g sawdust, 400g Cordyceps militaris cultivation residue, 10g quicklime, add water to make the moisture content of the culture medium reach 55%-60%;

[0151] Cultivation medium #4: 500g sawdust, 500g Cordyceps militaris cultivation residue, 10g quicklime, add water to make the moisture content of the culture medium reach 55%-60%;

[0152] Cultivation medium #5: 400g sawdust, 600g Cordyceps militaris cultivation residue, 10g quicklime, add water to make the moisture content of the culture medium reach 55%-60%.

[0153] (2) Method for preparing cultivation materials

[0154] Before use, the Cordyceps militaris cultivation residues need to be soaked in water for 3-4 hours, drained, and then mixed with other materials according to the specified ratio. Water is then added and stirred to make the moisture content of the culture medium reach 55%-60%. The mixture is then packed into 17cm×33cm polypropylene bags, with a material height of 10cm and a filling amount of 260g (dry material). The bags are then sealed tightly and sterilized at 121℃-125℃ for 2 hours.

[0155] 4. Vaccination

[0156] Place the sterilized polypropylene cultivation bags in a sterile environment. When the material temperature drops below 22℃, place them in a sterilized inoculation box and inoculate them with the pre-cultured palmate rosehip liquid culture from the sealed end according to the aseptic operation procedure. Inoculate 10mL of liquid culture into each cultivation bag and seal the bag after inoculation.

[0157] 5. Spawn Management

[0158] Wipe and disinfect the cultivation room and cultivation rack with disinfectant. After inoculation, transfer the polypropylene bags to the cultivation rack for mycelial growth. Ventilate the room daily during the entire mycelial growth period. Maintain a constant temperature of 18℃-22℃ and a relative humidity of less than 70%. Cultivate in the dark and observe the mycelial growth. Once the mycelium has spread and grown throughout the entire culture medium, you can start fruiting management.

[0159] 6. Mushroom Management

[0160] After the mycelium has developed, transfer the polypropylene bags to the fruiting room, place them upright, remove the bag stoppers, open the bags, and manage bud induction and fruiting under conditions of 300-500 Lux diffused light for 12-16 hours / day, carbon dioxide concentration of 800-1000 ppm, and humidity of 80%-90%. During the bud induction stage, the culture temperature should be controlled between 16℃ and 22℃. Once light pink palmate rose bud primordia appear, adjust the culture temperature to 20℃-22℃ for fruiting management. Harvest the fruiting bodies promptly when the caps have unfolded. Harvest one batch per bag and assess the fruiting body yield.

[0161] The mycelial growth and time to full coverage of the bag were monitored during the cultivation process, and the fruiting body yield was recorded. The specific results are shown in Table 8.

[0162] Table 8. Mycelial growth on different cultivation media

[0163] formula mycelial growth Time to fill the bag (days) Fruit body yield (%) Cultivation medium #1 The mycelium is white and relatively vigorous. 40 50.4 Cultivation medium #2 The mycelium is white and vigorous. 38 55.8 Cultivation medium #3 The mycelium is white and vigorous. 35 71.5 Cultivation medium #4 The mycelium is white and vigorous. 35 80.8 Cultivation medium #5 The mycelium is white and relatively vigorous. 37 61.2

[0164] Table 8 shows that both the mycelium and fruiting bodies of *Rosa palmatum* can grow well on a culture medium containing 200-600g of *Cordyceps militaris* cultivation residues. This proves that *Cordyceps militaris* cultivation residues can be used to cultivate *Rosa palmatum*.

[0165] Among them, the content of Cordyceps militaris cultivation residues ranged from 200-500g. With the increase of the amount added, the mycelial growth rate and fruiting body yield significantly improved. Then, with the increase of the addition ratio, the mycelial growth rate and fruiting body yield decreased. Among them, the effect of adding 500g of Cordyceps militaris cultivation residues to cultivation medium #4 was the best. At this time, the shortest time for the mycelium to fully consume the cultivation medium was 35 days, and the fruiting body yield reached more than 80.8%, which was the best cultivation effect.

[0166] Example 4: Cultivation effect and activity evaluation of palmate rose ear under optimized conditions

[0167] 1. Prepare palmate rose mother culture and liquid primary culture according to the method and culture medium in Example 3.

[0168] 2. Palmate rose auricles were cultivated using the optimized culture medium from Implementation Case 3.

[0169] 3. Preparation of cultivation medium:

[0170] (1) Cultivation medium formula: 500g sawdust, 500g Cordyceps militaris cultivation residue, 10g quicklime, add water to make the moisture content of the culture medium reach 55%-60%;

[0171] In this embodiment, a culture medium without Cordyceps militaris cultivation residues was used as a control culture medium. The formula of the control culture medium was: 680g sawdust, 100g cottonseed hulls, 150g wheat bran, 50g soybean meal, 10g gypsum, and 10g lime. Water was added to make the moisture content of the culture medium reach 55%-60%.

[0172] (2) The method for preparing the cultivation material is the same as in Example 3.

[0173] 4. Inoculation is performed in the same manner as in Example 3.

[0174] 5. The management of the inoculum growth is the same as in Example 3.

[0175] 6. Mushroom management is the same as in Example 3.

[0176] In this embodiment, the growth of palmate roselle and the content of active substances were measured, and the results are shown in Table 9. Figure 7 As shown in Tables 10 and 11.

[0177] Table 9. Growth of palmate rose ears in Example 5

[0178] mycelial growth Full Bag Time Primordial formation time Cultivation cycle Fruiting body yield % Optimized cultivation medium strong and vigorous 35 days 38-39 days 47 days 81.2 Control culture medium strong and vigorous 50 days 54-57 65 48.7

[0179] Table 10. Content of active substances in the fruiting bodies of palmate rosehips and the control group in Example 5.

[0180]

[0181] Table 11. Content of active substances and antioxidant activity in the fruiting bodies of *Rosa palmata* cultured in the optimized cultivation medium of Example 5.

[0182]

[0183] from Figure 7 Table 9 shows that, under optimized conditions, the culture medium containing 50% Cordyceps militaris cultivation residues provided good growth conditions for palmate mushrooms. The palmate mushroom mycelium grew vigorously and densely, filling the bags in 35 days. Under conditions of 15℃-22℃, light pink palmate mushroom primordia were formed in 3-4 days. After 7-8 days of continued cultivation in the range of 20-22℃, the fruiting bodies matured, and the yield of single-flush mushroom fruiting bodies reached 81.2%.

[0184] This technology not only features a simple and easy-to-prepare cultivation medium, but also innovates the cultivation substrate for palmate rose ear fungus. It replaces 50% of raw materials such as sawdust and bran with Cordyceps militaris cultivation residues, saving forestry resources and reducing the cost of cultivation raw materials by more than 13%. It achieves a one-time burst of fruiting, with a single-flush fruiting body yield of over 80%, significantly improving the cultivation efficiency of palmate rose ear fungus and making it easier for industrial production. At the same time, it realizes the large-scale secondary utilization of Cordyceps militaris cultivation residues, turning waste into treasure, reducing resource waste and environmental pollution, and is of great significance to the efficient, green and sustainable development of the rare edible fungi industry.

[0185] Further, as shown in Table 10, compared with the control group, using Cordyceps militaris cultivation residues to replace bran, sawdust, and other components in the culture medium for the cultivation of *Rhizoctonia solani* increased the fruiting body yield from 48.7% to 81.2%. The contents of total polysaccharides, polyphenols, and flavonoids in the fruiting bodies increased by 1.39, 1.33, and 1.02 times, respectively. This is mainly because the Cordyceps militaris cultivation residues are waste wheat grains after the harvest of Cordyceps militaris fruiting bodies, which increases the aeration capacity of the culture medium. At the same time, the cultivation residues obtained after Cordyceps militaris cultivation contain abundant nutrients such as starch, protein, and cordycepin compounds, providing a rich nutritional environment for the growth of *Rhizoctonia solani* fruiting bodies and promoting the growth of fruiting bodies and the synthesis of polysaccharides and flavonoids.

[0186] As can be seen from Table 11, the fruiting body of *Rosa palmata* has good antioxidant activity and is a rare renewable resource for antioxidant foods and health products.

[0187] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A method for culturing palmate rosettes using Cordyceps militaris cultivation residues, characterized in that, The method includes the following steps: Step 1: Inoculate the palmate rose agar strain onto the propagation medium and incubate upside down in the dark at 22°C. Stop the culture when the palmate rose agar hyphae cover the surface of the agar plate to obtain the palmate rose agar propagation strain. The propagation medium comprises: 200 g potato, 20 g glucose, 3 g peptone, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 20 g agar, and 1000 mL water. Step 2: Inoculate the palmate rose auricula propagation strain into the mother culture medium, and incubate at 22℃, pH 7.0, upside down, in the dark for 15-20 days, until it grows to cover the culture medium, thus obtaining the palmate rose auricula mother culture; the palmate rose auricula mother culture medium includes: 2% maltose, 1.5% peanut cake powder, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and 0.002% vitamin B1; Step 3: The palmate rosehip mother culture is inoculated into a palmate rosehip liquid primary culture medium at pH 7.0, 22℃, 150 r / min, and incubated with constant temperature shaking for 7 days to obtain the palmate rosehip liquid primary culture. The palmate rosehip liquid primary culture medium includes: 3% maltose, 1% peanut cake powder, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and 0.002% vitamin B1. Step 4: Inoculate the liquid spawn of *Rosa palmata* into the *Rosa palmata* cultivation medium at an inoculation rate of 10 mL / bag. Manage the disseminated mycelium in a dark environment at 18℃-22℃ and relative humidity below 70%. The *Rosa palmata* cultivation medium includes *Cordyceps militaris* cultivation residues, obtained by culturing *Cordyceps militaris* in a *Cordyceps militaris* culture medium. The *Cordyceps militaris* culture medium comprises wheat grains and nutrient solution, prepared at a mass ratio of 1:(1.6-1.7). The nutrient solution comprises: 1.0-2.0 g potassium dihydrogen phosphate, 0.5-1.0 g magnesium sulfate, and 1000 mL water. The content of the *Cordyceps militaris* cultivation residues is 500 parts by mass of the *Rosa palmata* cultivation medium. The *Rosa palmata* cultivation medium also includes sawdust, quicklime, and water. The water content of the *Rosa palmata* cultivation medium is 55%-60% by mass. Step 5: When the mycelium spreads and grows throughout the entire culture medium, bud induction management is carried out under the following conditions: 300-500 Lux diffused light for 12-16 h / day, carbon dioxide concentration of 800-1000 ppm, humidity of 80%-90%, and temperature of 16℃-22℃. Step 6: When the pale pink palmate rose ear primordia appear, manage the fruiting process under the following conditions: 300-500 Lux diffused light for 12-16 h / day, carbon dioxide concentration of 800-1000 ppm, humidity of 80%-90%, and temperature of 20℃-22℃. The content of total polysaccharides, polyphenols, and flavonoids in the fruiting bodies of *Rosa palmata* is increased.

2. The application of palmate rosea obtained by the method of culturing palmate rosea using Cordyceps militaris cultivation residues as described in claim 1 in the preparation of antioxidant active substances and / or in antioxidation; wherein the antioxidant active substances include polysaccharides, flavonoids and polyphenols.

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