A method for detecting the maturity of *Agaricus esculentus* mycelium

Abstract

This invention discloses a method for detecting the maturity of *Pleurotus ostreatus* mycelium, belonging to the field of edible mushroom cultivation technology. This invention provides a marker metabolite of physiological maturity in *Pleurotus ostreatus* mycelium—glutamine. This invention detects the glutamine content in *Pleurotus ostreatus* mycelium during the post-ripening stage. When the glutamine content in the post-ripening stage reaches 0.42 μmol / g, the *Pleurotus ostreatus* mycelium has reached physiological maturity and can be managed for fruiting. Different post-ripening times of *Pleurotus ostreatus* mycelium can significantly affect the yield and quality of *Pleurotus ostreatus*. This invention, through the determination of the physiological maturity of *Pleurotus ostreatus*, can increase the yield per bottle of *Pleurotus ostreatus* by 15%-32%, and the obtained *Pleurotus ostreatus* fruiting bodies have a higher number of Grade A mushrooms, higher amino acid and total flavonoid content, and better overall characteristics.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi cultivation technology, specifically relating to a method for detecting the maturity of *Pleurotus ostreatus* mycelium. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Deer antler mushroom, scientifically known as *Lyophyllum decastes*, is named for its cap morphology, which resembles slices of deer antler, a valuable traditional Chinese medicine. It is a large fungus used for both food and medicinal purposes. The cultivation of deer antler mushrooms mainly involves three stages: mycelial culture, catalytic primordium formation, and fruiting body growth and development. During the mycelial culture stage, the mycelium absorbs lignin, cellulose, and hemicellulose from the culture medium and converts them into its own nutrients to meet the nutritional needs of the reproductive growth stage (fruiting body growth and development). The mycelial culture time varies considerably depending on the type of edible fungus, such as deer antler mushroom, shiitake mushroom, and shiitake mushroom. After the mycelium has fully colonized the substrate, it needs to continue cultivation for a period of time, known as the post-ripening stage. Only after the mycelium reaches physiological maturity does it enter the fruiting period. The post-ripening period for deer antler mushroom mycelium is typically 10-20 days, and the length of this period directly affects the yield and quality of the mushroom. If the after-ripening period is too short, and fruiting management begins as soon as the bags are fully covered, the mycelium will not have reached physiological maturity, resulting in insufficient nutrient accumulation, which will lead to reduced yield and decreased fruiting body quality. If the after-ripening period is too long, firstly, the mycelium of *Pleurotus ostreatus* will age prematurely, and secondly, the longer the mycelium cultivation time, the higher the probability of contamination in the containers, while also increasing the energy consumption of industrialized mushroom cultivation. Since *Pleurotus ostreatus* mycelium is entirely white and filamentous during the cultivation stage without obvious morphological changes, there are currently no corresponding morphological or physiological indicators to determine whether the mycelium has reached physiological maturity. Therefore, choosing the after-ripening time for fruiting management in industrialized production of *Pleurotus ostreatus* is somewhat arbitrary and cannot guarantee the yield and quality of each batch. Developing a rapid method to detect the physiological maturity of *Pleurotus ostreatus* mycelium could greatly reduce the arbitrariness in industrialized production of *Pleurotus ostreatus*, thereby ensuring the yield and quality of *Pleurotus ostreatus*. Therefore, researching and developing a rapid method for detecting the physiological maturity of *Agaricus esculentus* mycelium is of great significance for the production and cultivation of *Agaricus esculentus*. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting the maturity of *Pleurotus ostreatus* mycelium. This invention detects the main differential metabolite, glutamine, at different post-ripening stages of *Pleurotus ostreatus* mycelium, thereby determining whether the mycelium has reached physiological maturity. This allows for the selection of appropriate post-ripening times for fruiting management, thus ensuring the yield and quality of *Pleurotus ostreatus*.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the invention provides the use of glutamine as a marker metabolite of physiological maturation of *Agaricus esculentus* mycelium during the culture stage of *Agaricus esculentus*.

[0007] This invention conducted fruiting experiments on the yield and quality of *Pleurotus ostreatus* at different post-ripening times. The results showed that when the glutamine content of *Pleurotus ostreatus* mycelium reached 0.42 μmol / g, the mycelium had reached physiological maturity and could be managed for the next stage of fruiting. When the glutamine content was lower than this concentration, the yield and quality of *Pleurotus ostreatus* per bottle decreased significantly, thus affecting the commercial value of *Pleurotus ostreatus*.

[0008] Furthermore, in addition to determining the glutamine content in mycelia at different after-ripening stages, this invention also conducted metabolomic analysis on *Agaricus esculentus* mycelia at different after-ripening times. From the differential expression analysis of the metabolome, glutamine was again identified as a differential metabolite between 55 days and 45 days. Therefore, it can be determined that the glutamine content can serve as a marker metabolite for the physiological maturity of *Agaricus esculentus* mycelia.

[0009] In some embodiments of the present invention, the application is as follows: detecting the glutamine content in the mycelium of *Pleurotus ostreatus* during the post-ripening stage, and determining whether the mycelium has reached physiological maturity based on the glutamine content. The present invention uses sampling at different post-ripening stages, mainly because the mycelium at different post-ripening stages is morphologically white and lacks obvious morphological characteristics.

[0010] In some embodiments of the present invention, when the glutamine content in the mycelium of *Pleurotus ostreatus* during the post-ripening stage reaches 0.42 μmol / g, the mycelium of *Pleurotus ostreatus* reaches physiological maturity.

[0011] A second aspect of the present invention provides a method for detecting the maturity of *Pleurotus ostreatus* mycelium, comprising the following steps: detecting the glutamine content in *Pleurotus ostreatus* mycelium during the post-maturation stage; when the glutamine content in *Pleurotus ostreatus* mycelium during the post-maturation stage reaches 0.42 μmol / g, the *Pleurotus ostreatus* mycelium reaches physiological maturity.

[0012] This invention does not limit the detection method of glutamine content in the mycelium of *Agaricus esculentus* during the post-ripening stage. The glutamine content in the mycelium of *Agaricus esculentus* at different post-ripening stages can be determined by spectrophotometry, or the glutamine content can be detected by a corresponding kit, as long as the purpose of accurately detecting the glutamine content can be achieved.

[0013] A third aspect of the present invention provides a method for cultivating antler fungus to improve its quality and yield, comprising the following steps:

[0014] (1) Prepare a culture medium for *Pleurotus ostreatus* and inoculate it with *Pleurotus ostreatus* mycelium;

[0015] (2) Cultivate mycelium;

[0016] (3) The content of glutamine in the mycelium of *Pleurotus ostreatus* during the post-maturation period was detected. When it reached 0.42 μmol / g, the mycelium of *Pleurotus ostreatus* reached physiological maturity, and the mushroom bottles were managed for fruiting.

[0017] (4) Harvest the fruiting bodies after they have matured.

[0018] In some embodiments of the present invention, the solid raw materials of the *Pleurotus ostreatus* culture medium are: 31-33 wt% poplar sawdust, 20-22 wt% corn cob, 4-6 wt% cottonseed hull, 16-18 wt% wheat bran, 6-8 wt% soybean hull, 14-16 wt% corn flour, and 2-4 wt% soybean meal.

[0019] The water content of the *Deer Antler Mushroom* culture medium is 65-70 wt%.

[0020] In some embodiments of the present invention, the solid raw materials of the *Pleurotus ostreatus* culture medium are: 32 wt% poplar sawdust, 21 wt% corn cob, 5 wt% cottonseed hulls, 17 wt% wheat bran, 7 wt% soybean hulls, 15 wt% corn flour, and 3 wt% soybean meal.

[0021] In some embodiments of the present invention, in step (2), the culture temperature of the cultured mycelium is 21-23°C, and the culture is carried out in the dark.

[0022] In some embodiments of the present invention, after the mycelium of *Pleurotus eryngii* reaches physiological maturity, the mushroom bottles are managed for fruiting. The mycelium is stimulated to twist and form primordia by scratching. After scratching, the relative humidity is increased to 90-95%, the temperature to 16-18℃, and the light intensity to 1500-2000 lux, with ventilation 2-3 times daily. For 1-6 days after scratching, the light duration is 6-8 hours; for 7-11 days after scratching, the light duration is 3-5 hours; for 12-16 days after scratching, the light is turned off to promote primordia formation; for 17-18 days after scratching, the light duration is 3-5 hours; for 19-24 days after scratching, the light duration is 19-21 hours; and for 25-30 days after scratching, the light duration is 9-11 hours.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention provides the application of glutamine as a marker metabolite of physiological maturity in *Agaricus esculentus* mycelium during the mycelial culture stage. Specifically, in the post-ripening stage of *Agaricus esculentus* mycelium, when the glutamine content reaches 0.42 μmol / g, the mycelium has reached physiological maturity and can proceed to the next stage of fruiting management. When the glutamine content is lower than this concentration, the yield and quality of *Agaricus esculentus* per bottle decrease significantly, thus affecting the commercial value of the mushroom. Glutamine is a crucial link in the synthesis of various amino acids, participating in the synthesis of amino acids such as phenylalanine, lycine, arginine, and proline. Simultaneously, glutamine is also an important precursor in the biosynthesis of secondary metabolites, regulating the biosynthesis of secondary metabolites in *Agaricus esculentus*. Through metabolomics data analysis at different post-ripening stages, the inventors found that the main changes in *Agaricus esculentus* mycelium at different post-ripening stages were primarily in amino acid synthesis and secondary metabolic pathways. Therefore, glutamine, as a central metabolite of various amino acids and secondary metabolic pathways, can be used to evaluate the physiological maturity of mycelium. This invention also discovered that different after-ripening times of *Pleurotus ostreatus* mycelium can significantly affect the yield and quality of *Pleurotus ostreatus*. When the mycelium reaches its optimal physiological maturity of 55 days (glutamine content reaches 0.42 μmol / g), the yield per bottle is 321.99 ± 3.43 g. However, when the after-ripening time of the mycelium is too short (45 days), the yield per bottle is only 280.33 g. Furthermore, as the after-ripening time increases from 55 days to 65 days, the yield per bottle also decreases. At a ripening time of 65 days, the yield per bottle is 244.55 ± 6.42 g, significantly lower than the yield per bottle at a ripening time of 55 days. This invention can increase the yield of *Pleurotus ostreatus* by 15%-32% by accurately determining the after-ripening time of the mycelium, and the obtained *Pleurotus ostreatus* fruiting bodies exhibit better overall characteristics. Secondly, when the mycelium of *Pleurotus ostreatus* reached physiological maturity at 55 days, the content of flavonoids, a secondary metabolite produced in its fruiting bodies (2.94 mg / g), was significantly higher than that at 45 days (2.67 mg / g) and 65 days (2.61 mg / g), with the total flavonoid content increasing by 10%-13%. Simultaneously, when the mycelium of *Pleurotus ostreatus* reached physiological maturity at 55 days, the content of its five flavor amino acids reached its highest level, with glutamic acid being the most abundant at 177.90 μg / g, followed by aspartic acid (95.74 μg / g), phenylalanine (31.68 μg / g), tyrosine (13.79 μg / g), and alanine (10.99 μg / g). Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 A statistical chart showing the glutamine content in the mycelium of *Flammulina velutipes* at different post-ripening times;

[0027] Figure 2 The effects of different post-ripening times on the yield and biomass of *Flammulina velutipes*.

[0028] Figure 3 The effect of different post-ripening times on the number of primordia formed in *Pleurotus ostreatus*.

[0029] Figure 4 The effect of different post-ripening times on the amino acid content of *Pleurotus ostreatus*.

[0030] Figure 5 The effect of different post-ripening times on the grading of *Pleurotus ostreatus* is shown in Figure A, where A represents the morphological images of Grade A, Grade B, and Grade C mushrooms, and B represents the statistical chart of the number of Grade A, Grade B, and Grade C mushrooms produced under different post-ripening times.

[0031] Figure 6 To investigate the effect of different post-ripening times on the total flavonoid content of *Agaricus esculentus*. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1: Preparation of a sample for detecting mycelial maturity

[0034] The samples were taken from mycelia at different stages of cultivation (post-ripening stage). The mycelia were scraped from the surface of the culture substrate, pre-cooled with liquid nitrogen, and stored at -80°C.

[0035] Example 2: A method for detecting the maturity of *Agaricus esculentus* mycelium

[0036] The method includes the following steps:

[0037] (1) Preparation of culture medium for *Pleurotus ostreatus* and inoculation of *Pleurotus ostreatus* mycelium:

[0038] The solid raw materials and their weight percentages for the *Pleurotus ostreatus* culture medium are as follows: poplar sawdust 32%, corn cob 21%, cottonseed hulls 5%, wheat bran 17%, soybean hulls 7%, corn flour 15%, and soybean meal 3%.

[0039] The culture medium has a water content of 65%–70%, is sterilized at 121°C for 2 hours, and inoculated with deer antler mycelium after cooling.

[0040] (2) Culture of mycelium: The culture temperature of mycelium is 22±1℃, and culture is carried out in the dark.

[0041] (3) After cultivating mycelia at different maturation stages for 45 days, 55 days and 65 days respectively, the mushroom bottles were managed for fruiting. The mycelia of *Pleurotus ostreatus* were stimulated to form primordia by scratching. The light duration was 7 hours for 1-6 days after scratching; 4 hours for 7-11 days after scratching; and closed light treatment was applied for 12-16 days after scratching to promote primordia formation. The light duration was 4 hours for 17-18 days after scratching; 20 hours for 19-24 days after scratching; and 10 hours for 25-30 days after scratching.

[0042] (4) On the first day after the fungal infection, increase the relative humidity to 90-95%, the temperature to 16-18℃, the light intensity to 1500-2000 lux, and ventilate 2-3 times a day;

[0043] (5) Harvest the fruiting bodies after they have matured.

[0044] The method for detecting glutamine content in the mycelium of *Pleurotus ostreatus* during the post-ripening stage is as follows:

[0045] Weigh the *Agaricus esculentus* mycelium sample and Extract 1 (it is recommended to weigh 0.1 g of tissue and add 1 mL of Extract 1 (distilled water)) at a ratio of 1:5 to 10 (g / mL), mix, and homogenize in an ice bath; centrifuge at 12000 g at 4℃ for 5 min, take the supernatant and add 500 μL of Extract 2 (chloroform), shake vigorously for 5 min, centrifuge at 12000 g at 4℃ for 5 min, take the upper liquid (clear state) and place it on ice for testing.

[0046] React at 37℃ in the dark for 1 hour, centrifuge at 12000g at room temperature for 5 minutes, collect 200 μL of the supernatant, and measure the absorbance at 450 nm. Record this value as A. 测定 A 0.4 μmol / mL glutamine solution (using distilled water as solvent) was used as a standard solution (prepared fresh before use). The absorbance was measured at 450 nm and denoted as A. 标准 A control was prepared by adding only the sample without the reaction solution (an aqueous solution of glutaminase). The absorbance was measured at 450 nm and denoted as A. 对照 Using distilled water as a blank instead of the sample, the absorbance value was measured at 450 nm and denoted as A. 空白 Calculate ΔA separately. 测定 =A 测定 -A 对照 ΔA 标准 =A 标准 -A 空白 (A control tube must be included with each test tube.) ΔA 测定 The measurement range is between 0.005 and 0.7.

[0047] Glutamine content (μmol / g) = ΔA 测定 ×C 标准 ×V 样总 ÷W=ΔA 测定 ×0.4÷ΔA 标准 -W

[0048] C 标准 V represents the concentration of the standard solution, i.e., 0.4 μmol / mL; 样总 denoted as the total volume of the sample; W represents the mass of the sample in grams.

[0049] Experimental results:

[0050] (1) Glutamine content in the mycelium of *Agaricus esculentus* at different post-ripening times

[0051] After testing, such as Figure 1 As shown, the glutamine content in *Pleurotus ostreatus* mycelium after 45 days of post-ripening was 0.19 μmol / g; the glutamine content in *Pleurotus ostreatus* mycelium after 50 days of post-ripening was 0.23 μmol / g; the glutamine content in *Pleurotus ostreatus* mycelium after 55 days of post-ripening was 0.42 μmol / g; the glutamine content in *Pleurotus ostreatus* mycelium after 60 days of post-ripening was 0.36 μmol / g; the glutamine content in *Pleurotus ostreatus* mycelium after 65 days of post-ripening was 0.37 μmol / g; and the glutamine content in *Pleurotus ostreatus* mycelium after 70 days of post-ripening was 0.23 μmol / g.

[0052] (2) Effects of different post-ripening times on yield and biomass of *Flammulina velutipes*

[0053] This embodiment investigated the effects of different after-ripening times of 45d, 55d and 65d on the yield and biomass of *Pleurotus ostreatus*.

[0054] The test results are as follows Figure 2 As shown, the yield per bottle of *Pleurotus eryngii* fruiting bodies follows a normal distribution with increasing after-ripening days. The highest yield is achieved when the mycelium reaches its optimal physiological maturity (glutamine content of 0.42 μmol / g) at 55 days, with a yield of 321.99 ± 3.43 g per bottle. However, when the after-ripening time is too short (45 days), the yield is only 280.33 g per bottle. Furthermore, as the after-ripening time increases from 55 to 65 days, the yield per bottle also decreases. At 65 days, the yield is 244.55 ± 6.42 g, significantly lower than the yield at 55 days. This indicates that both excessively short and excessively long after-ripening periods lead to a decrease in both the yield per bottle and the biological conversion rate of *Pleurotus eryngii*. The method of this invention can increase the yield of *Pleurotus eryngii* by 15%-32% by accurately determining the after-ripening time of the mycelium.

[0055] Based on the above experiments, this invention further explored the effect of different post-ripening times on the number of primordia formed in *Pleurotus ostreatus*.

[0056] Fruiting experiments were conducted on *Pleurotus ostreatus* spawn bottles with different after-ripening periods (45d, 55d, and 65d). The appearance time and number of primordia were recorded. The study found that primordia appeared on the 6th day after mycelial scratching at all after-ripening periods. The number of primordia formed initially increased and then decreased with increasing after-ripening time. The highest number of primordia was observed at 55d (515.00±7.57 per bottle), but the number decreased significantly with further extension of the after-ripening time, reaching a minimum of 236.67±11.46 per bottle at 65d. The experimental results are shown below. Figure 3 As shown.

[0057] Furthermore, this invention further investigated the effects of different post-ripening times on the amino acid content of *Flammulina velutipes*.

[0058] The contents of 20 known amino acids in the fruiting bodies of *Flammulina velutipes* cultivated at different post-ripening stages were determined by high performance liquid chromatography as described below.

[0059] High-performance liquid chromatography (HPLC) for the determination of free amino acids:

[0060] 1. Sample preparation

[0061] Weigh 0.1g of sample, add 1.5mL of ultrapure water, vortex mix, sonicate for 30min, add 1mL of dichloromethane, mix at 4℃ for 30min, centrifuge at 13000rpm at 4℃ for 5min, take the upper aqueous phase, filter through a 0.22μm filter membrane, and perform instrumental analysis.

[0062] 2. Liquid Chromatography Conditions

[0063] Chromatographic column: Hypersil GOLD C18 column, 100mm × 2.1mm, 3μm;

[0064] Column temperature: 40℃;

[0065] Mobile phase A: 0.1% (v / v) formic acid aqueous solution;

[0066] Mobile phase B: Methanol;

[0067] Flow rate: 0.2 mL / min;

[0068] Injection volume: 3 μL;

[0069] Gradient elution program: 0–0.5 min, 4% B; 0.5–2.5 min, 4% B–35% B; 2.5–4.5 min, 35% B–77% B; 4.5–6.5 min, 77% B–100% B; 6.5–6.6 min, 100% B–4% B; 6.6–10 min, 4% B.

[0070] 3. Mass spectrometry conditions

[0071] Ion source: ESI+;

[0072] Spray voltage: 3500V;

[0073] Sheath gas: 35 Arb;

[0074] Auxiliary gas: 10 Arb;

[0075] Atomization temperature: 275℃;

[0076] Ion transfer tube temperature: 325℃.

[0077] Testing revealed that all 19 free amino acids except cysteine ​​were detectable. The results showed that the content of the five flavor amino acids reached its highest level when the mycelium of *Pleurotus ostreatus* reached physiological maturity (55 days). Glutamic acid had the highest content at 177.90 μg / g, followed by aspartic acid (95.74 μg / g), phenylalanine (31.68 μg / g), tyrosine (13.79 μg / g), and alanine (10.99 μg / g). The experimental results are as follows... Figure 4 As shown.

[0078] Based on the above experiments, this invention further explored the effects of different post-ripening times on the grading of *Agaricus esculentus*.

[0079] The effects of different post-ripening stages on the grading of *Flammulina velutipes* fruiting bodies were studied by statistically analyzing indicators such as cap diameter, stipe diameter, and stipe length. Figure 5 A). The results showed that when the after-ripening period was 55 days, the number of Grade A mushrooms increased significantly compared to after-ripening periods of 45 days and 65 days, while the number of Grade C mushrooms decreased significantly. Figure 5 (B) As the post-ripening period gradually lengthens, the number of Grade B mushrooms shows a trend of first increasing and then decreasing, but reaches its lowest point at 65 days of mycelial age. These results indicate that different post-ripening periods have a significant impact on the grade of *Pleurotus eryngii*. The highest number of Grade A mushrooms were obtained at a post-ripening period of 55 days, and the overall characteristics of the fruiting bodies obtained at 55 days were also better. Furthermore, the total flavonoid content in the fruiting bodies formed at different post-ripening periods of *Pleurotus eryngii* was also measured. Figure 6As shown, the results indicate that when the mycelium of *Agaricus esculentus* reaches physiological maturity at 55 days, the total flavonoid content of secondary metabolites produced in its fruiting bodies (2.94 mg / g) is significantly higher than that at 45 days (2.67 mg / g) and 65 days (2.61 mg / g), increasing by 10%-13%.

[0080] Based on the above experimental results, the mechanism of action can be analyzed as follows: Glutamine is a crucial hub in the synthesis of various amino acids, participating in the synthesis of amino acids such as phenylalanine, lycine, arginine, and proline. Simultaneously, glutamine is also an important precursor in the biosynthesis of secondary metabolites, regulating the biosynthesis of secondary metabolites in *Agaricus esculentus*. Metabolomics data analysis at different after-ripening stages revealed that the main changes in mycelia at different after-ripening stages were primarily in amino acid synthesis and secondary metabolic pathways. Therefore, as a central metabolite of various amino acids and secondary metabolic pathways, the content of glutamine can be used to evaluate the physiological maturity of mycelia.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of glutamine as a marker metabolite of physiological maturity of Cordyceps militaris mycelium in evaluating the maturity of Cordyceps militaris mycelium. When the content of glutamine in the post-mature Cordyceps militaris mycelium reaches 0.42 μmol / g, the Cordyceps militaris mycelium reaches physiological maturity.

2. A method for detecting the maturity of the mycelium of Cervi Corni Colla, characterized in that, The method comprises the following steps: detecting the content of glutamine in the post-mature Cordyceps militaris mycelium, and when the content of glutamine in the post-mature Cordyceps militaris mycelium reaches 0.42 μmol / g, the Cordyceps militaris mycelium reaches physiological maturity.

3. The method of claim 2, wherein, The content of glutamine in the post-mature Cordyceps militaris mycelium is determined by spectrophotometry.

4. A culture method for improving the quality and yield of deer antler, characterized in that, The method comprises the following steps: (1) preparing Cordyceps militaris culture medium and inoculating Cordyceps militaris mycelium; (2) culturing the mycelium; (3) detecting the content of glutamine in the post-mature Cordyceps militaris mycelium, and when the content of glutamine reaches 0.42 μmol / g, the Cordyceps militaris mycelium reaches physiological maturity, and the mycelium bottle is managed for fruiting; (4) picking the fruiting bodies after the fruiting bodies mature.

5. The culture method according to claim 4, wherein The solid raw material of the Cordyceps militaris culture medium comprises 31-33 wt% of poplar sawdust, 20-22 wt% of corn cob, 4-6 wt% of cottonseed hull, 16-18 wt% of bran, 6-8 wt% of soybean hull, 14-16 wt% of corn flour and 2-4 wt% of soybean meal. The Cordyceps militaris culture medium contains 65-70 wt% of water.

6. The culture method as claimed in claim 5, wherein The solid raw material of the Cordyceps militaris culture medium comprises 32 wt% of poplar sawdust, 21 wt% of corn cob, 5 wt% of cottonseed hull, 17 wt% of bran, 7 wt% of soybean hull, 15 wt% of corn flour and 3 wt% of soybean meal.

7. The culturing method according to claim 4, wherein In step (2), the culture temperature for culturing the mycelium is 21-23℃, and the culture is carried out in the dark.

8. The culturing method according to claim 4, wherein When the Cordyceps militaris mycelium reaches physiological maturity, the mycelium bottle is managed for fruiting, the mycelium is stimulated to form primordia by scratching, the relative humidity of air is increased to 90-95% after scratching, the temperature is 16-18℃, the light intensity is 1500-2000 lux, and the air is ventilated 2-3 times a day; the light time is 6-8 hours 1-6 days after scratching; the light time is 3-5 hours 7-11 days after scratching; the light is turned off 12-16 days after scratching to promote the formation of primordia; the light time is 3-5 hours 17-18 days after scratching; the light time is 19-21 hours 19-24 days after scratching; and the light time is 9-11 hours 25-30 days after scratching.

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