Application of liquid culture medium for fermentation of phlebopus portentosus liquid strain

Abstract

The invention relates to application of a liquid culture medium for fermentation of a phlebopus portentosus liquid strain, and belongs to the technical field of edible fungus strain fermentation culture media. The liquid culture medium comprises the following components in parts by weight: 10-30 parts of molasses, 10-30 parts of soluble starch, 2-5 parts of yeast extract, 1-4 parts of monopotassium phosphate, 1-3 parts of magnesium sulfate, 0.1-1 part of adenine, 0.3-0.5 part of a defoaming agent and 1000 parts of water, and the pH value of the liquid culture medium is 5.0-5.8. Hypha blocks of phlebopus portentosus are inoculated and cultured to obtain mother strains, then the mother strains are inoculated into the liquid culture medium to be fermented to obtain seed liquid, the seed liquid is transferred into the liquid culture medium in a fermentation tank to be deeply fermented, and the liquid strains are obtained. The liquid culture medium can be fermented in a short time to obtain a large number of phlebopus portentosus liquid strains, the submerged fermentation time of the phlebopus portentosus liquid strains is greatly shortened, the quality of the liquid strains is excellent, and a new way is provided for industrial application of the phlebopus portentosus liquid strains.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi fermentation culture medium technology, specifically relating to the application of a liquid culture medium in the liquid inoculum fermentation of Boletus thunbergii. Background Technology

[0002] Dark brown thread-stalked Boletus ( Phlebopus portentosus Phleboporus is a large fungus with significant edible and medicinal value, belonging to the family Boletaceae and the genus Phleboporus. Its cap is dark brown to brownish-brown, and its stipe has a prominent reticulate structure. It is mainly distributed in the broad-leaved forests of subtropical regions such as southern China and Southeast Asia. Its fruiting body is fleshy and thick, with a unique flavor, rich in protein, polysaccharides, and various bioactive components, making it highly popular and in high market demand. However, due to its demanding requirements for specific natural habitats, artificial cultivation is difficult, and relying mainly on wild harvesting cannot meet market demand. Therefore, researchers have begun to study artificial cultivation techniques.

[0003] Existing artificial cultivation techniques for *Boletus edulis* typically use solid-state inoculum. However, solid-state inoculum suffers from drawbacks such as long cultivation cycles, high costs, and difficulty in automated inoculation. Liquid-state inoculum, on the other hand, can shorten cultivation time, increase inoculum yield, and is suitable for automated industrial inoculation, making its development a superior solution. However, in practical applications, the integration technology between liquid-state inoculum preparation and solid-state cultivation substrates is still immature. Problems such as poor permeability of the fermentation broth and low colonization rates hinder industrial application. Furthermore, common challenges such as inoculum degradation, risk of contamination by other microorganisms, and the lack of standardized fermentation parameter systems further exacerbate the difficulty of technology promotion. Therefore, further optimization of liquid-state inoculum production processes is needed to meet industrialization requirements.

[0004] Chinese patent CN113234605A discloses a liquid culture medium for *Boletus thunbergii*, prepared using glucose, soluble starch, yeast powder, potassium dihydrogen phosphate, and magnesium sulfate. The liquid culture is obtained by shaking culture at 22-32℃ for 5-12 days. However, the liquid culture obtained by this method is still in the shaker culture stage, not a liquid culture obtained through submerged fermentation. Furthermore, there is no specific data on the obtained liquid culture, and no fruiting verification has been conducted, lacking evidence to support its industrial application. Chinese patent CN101381684A, which discloses a method for cultivating a liquid culture of *Boletus thunbergii*, and Chinese patent CN106929432A, which discloses a new method for submerged fermentation of a liquid culture of *Boletus thunbergii*, also suffer from the same problems.

[0005] Chinese patent CN109097288A discloses another liquid culture medium for *Boletus edulis*, whose main components include molasses, sucrose, yeast extract, potassium dihydrogen phosphate, and magnesium sulfate. Liquid spawn is obtained by culturing under suitable conditions for 7 days. The patent claims that its culture medium and method can be used for industrial cultivation; however, it lacks supporting data on the liquid spawn and fruiting results from industrial production.

[0006] In summary, existing technologies are mostly in the laboratory stage, with few examples of industrial-scale production. Summary of the Invention

[0007] The purpose of this invention is to address the problems existing in the prior art by providing a liquid culture medium for the fermentation of Boletus thunbergii liquid spawn, which can produce a large quantity of high-quality Boletus thunbergii liquid spawn in a short time.

[0008] The technical solution adopted in this invention is as follows: The liquid culture medium is used for the fermentation of Boletus thunbergii liquid spawn, wherein the liquid culture medium is prepared by weight as follows: molasses 10-30 parts, soluble starch 10-30 parts, yeast extract 2-5 parts, potassium dihydrogen phosphate 1-4 parts, magnesium sulfate 1-3 parts, adenine 0.1-1 parts, antifoaming agent 0.3-0.5 parts, and water 1000 parts, and the pH of the liquid culture medium is 5.0-5.8; The culture medium is used for the liquid inoculum fermentation of Boletus thunbergii, comprising the following steps: a. Preparation of mother culture medium: The mother culture medium is prepared by weight as follows: 100-300 parts soil, 10-30 parts glucose, 1-3 parts yeast extract, 1-3 parts potassium dihydrogen phosphate, 1-3 parts magnesium sulfate, and 15-25 parts agar. Add 1000 parts water to the soil, stir and let stand. Take the soil supernatant as a solvent, then add the remaining ingredients, heat to dissolve and boil. After dispensing, autoclave at 121℃ for 40 min, pour into plates and cool to solidify to obtain the mother culture medium. b. Cultivating the mother culture: Take a 5mm diameter mycelial block of Boletus edulis and inoculate it into the mother culture medium described in step a. Incubate at a constant temperature of 28-30℃ for 14-18 days to obtain the mother culture. c. Cultivating the seed culture liquid strain: Prepare the liquid culture medium, with a volume of 1.5L / 3L-2L / 3L, sterilize at 121℃ for 40min, and cool; then chop the mother culture prepared in step b and inoculate it into the liquid culture medium, and incubate at 28-30℃ on a magnetic stirrer at 150-250r / min for 5-7 days to obtain the seed culture liquid strain; d. Submerged fermentation liquid culture medium: Prepare the liquid culture medium in a fermenter, filling it to 60-80% volume, v / v, sterilize at 121℃ for 60-80 min, and cool it with circulating water to the inoculation temperature; then inoculate the seed liquid liquid culture obtained in step c, culture at 28-30℃ with sterile air, and ferment for 3-5 days to obtain a flocculent liquid culture with a biomass of 9-15 g / L.

[0009] Further, the flocculent liquid inoculum obtained in step d is inoculated into the cultivation medium at an inoculation rate of 3-6% and cultured at 28-30℃ for 45-55 days to obtain mature fruiting bodies; the cultivation medium is prepared by weight in the following proportions: 2-4 parts wheat grains, 2-4 parts sawdust, 1-2 parts cottonseed hulls, 1-2 parts wheat bran, and 1-2 parts corn flour.

[0010] Furthermore, in step d, during the deep fermentation of the liquid culture medium, the sterilization process after preparing the liquid culture medium in the fermenter adopts dynamic pressure sterilization, that is: the pressure is controlled at 0.12 MPa from 0 to 20 minutes, the pressure is controlled at 0.15 MPa from 20 to 60 minutes, and the pressure is controlled at 0.12 MPa from 60 to 80 minutes; when the seed liquid culture obtained in step c is circulated with sterile air for culture, the aeration adopts a dynamic flow mode, that is: the aeration flow rate is 1-1.5 vvm from 0 to 48 hours, the aeration flow rate is 1.5-2 vvm from 48 to 120 hours, and the pressure is controlled at 0.03-0.05 MPa.

[0011] Furthermore, the soil used for the mother culture medium is mountain red soil.

[0012] The present invention has the following beneficial effects: 1. By using a composite carbon source (molasses + soluble starch) for synergistic energy supply and adenine growth factor enhancement, the deep fermentation cycle of liquid microbial cultures is compressed to 3–5 days. Combining the mother culture (14–18 days) and seed culture (5–7 days) process, the entire cycle from mother culture inoculation to industrial-scale microbial production takes only 23–25 days, which is more efficient than traditional solid microbial culture preparation (≥60 days), and significantly reduces time and economic costs.

[0013] 2. This invention employs a multifunctional culture medium design. The mineral components and humic acid in the mother culture medium, derived from the red soil of the mountain plateau, simulate the natural habitat, which can significantly improve the mycelial proliferation rate and provide a high-quality mother culture. The liquid culture medium has an optimized carbon source, with a gradient of fast-acting carbon source molasses and slow-release carbon source soluble starch to meet the metabolic needs of mycelia at different growth stages. The addition of adenine, an auxiliary growth factor, as a precursor for nucleic acid synthesis, is beneficial for promoting rapid mycelial proliferation.

[0014] 3. The optimized liquid culture medium of this invention enables deep fermentation of *Boletus edulis* liquid spawn. The carbon source is derived from a molasses-soluble starch complex, and the growth factor is adenine. Under controlled aerobic fermentation conditions, the mycelium forms uniformly sized flocculent aggregates with high mycelial density. The low viscosity of the liquid spawn and the uniform mycelial ball structure allow for effective compatibility with automated inoculation equipment (such as centrifugal atomization systems and pneumatically driven spray guns). This feature eliminates the cumbersome processes of crushing, weighing, and manual dispersing required by traditional solid spawn, simplifying the inoculation process to a unidirectional fluid transport operation. In industrial applications, it possesses production capabilities that are easily standardized and controlled, making it particularly suitable for small-scale cultivation and industrialized deep fermentation. Attached Figure Description

[0015] Figure 1 The mother culture of *Boletus edulis* (dark brown reticulate stalk); Figure 2 The diagram shows the state of the liquid microbial culture in the submerged fermentation equipment. In the diagram, A represents the submerged fermentation equipment and B represents the state of the liquid microbial culture placed in the submerged fermentation equipment. Figure 3 This is the state of the liquid microbial culture after the deep fermentation is completed; Figure 4 The microscopic morphology of liquid bacterial strains; Figure 5 This is the fruiting state of the liquid culture in the example. Detailed Implementation

[0016] Based on the above description of the invention, the following embodiments will further explain and illustrate the present invention. It should be particularly noted that the following embodiments are only for reference as some application examples and do not constitute a limitation on the scope of protection of the present invention. All embodiments within the scope of the technical solutions defined in the claims of this invention are within the scope of protection of this invention.

[0017] Example 1

[0018] A deep fermentation medium for *Boletus edulis* liquid spawn comprises the following ingredients: 30g molasses, 30g soluble starch, 5g yeast extract, 3g magnesium sulfate, 4g potassium dihydrogen phosphate, 1g adenine, 0.5g silicone-free defoamer, and 1000mL deionized water. The molasses, soluble starch, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, adenine, and silicone-free defoamer are added to the deionized water, dissolved, and stirred until homogeneous to prepare the liquid culture medium. The pH of the liquid culture medium is 5.8.

[0019] The liquid culture medium for *Boletus thunbergii* was used to prepare the liquid culture of *Boletus thunbergii* through submerged fermentation, including the following steps: a. Preparation of mother culture medium: The mother culture medium, by weight, is prepared as follows: 200g of mountain red soil, 20g of glucose, 2g of yeast extract, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, and 20g of agar. Thoroughly mix the soil with 1000mL of water, let it stand for 1 hour, filter to obtain the soil supernatant, then add glucose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate, and agar to the supernatant, dissolve and boil, sterilize at 121℃ for 40 minutes, pour into petri dishes and allow to cool naturally to obtain the mother culture medium.

[0020] The mountain red soil mentioned is a type of red soil distributed in the central part of the Yunnan Plateau. Its main components are about 30-40% organic matter, an average total nitrogen content of 1.15%, an average total phosphorus content of 0.72%, a total potassium content of about 10-15%, and a pH value of 5.0-5.5.

[0021] b. Cultivating the mother culture: Take a 5mm diameter mycelial block of *Boletus edulis* and inoculate it into the mother culture medium described in step a for activation culture. Incubate at 29℃ for 15 days to obtain... Figure 3 The mother culture of *Boletus glomeratus* in good physiological condition is shown. This *Boletus glomeratus* was collected from the wild in Xishuangbanna Dai Autonomous Prefecture, Yunnan Province. This strain was deposited on April 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 5.2272, strain number Ph5-2, and classified as follows: Phlebopus portentosus .

[0022] c. Cultivating the seed culture liquid strain: Take the prepared liquid culture medium, fill it to a volume of 2L / 3L, sterilize it at 121℃ for 40min, and cool it. Then, use a sterile scalpel to chop the mother culture from step b and inoculate it into the liquid culture medium. Incubate it at 28℃ on a magnetic stirrer at 150r / min for 7 days to obtain the seed culture liquid strain.

[0023] d. Submerged fermentation liquid culture: The liquid culture medium is loaded into the fermenter to 70% capacity (v / v), and sterilized at 121℃ for 60-80 min. Dynamic pressure sterilization is used during the sterilization process: 0.12 MPa for 0-20 min, 0.15 MPa for 20-60 min, and 0.12 MPa for 60-80 min. After sterilization, the mixture is cooled to the inoculation temperature using circulating water. The seed culture obtained in step c is then inoculated, and sterile air is introduced at 28℃ for incubation. Aeration is performed using a dynamic flow rate mode: 1.5 vvm for 0-48 h, and 2 vvm for 48-96 h, with the pressure controlled at 0.03-0.05 MPa. The fermentation cycle is 4 days, yielding a high-density liquid culture (biomass approximately 10 g / L). Figure 2The diagram shows the state of the liquid microbial culture in the submerged fermentation equipment. In the diagram, A represents the commonly used submerged fermentation equipment in the industry, and B represents the state of the liquid microbial culture placed in the submerged fermentation equipment. Figure 3 This is the state of the liquid culture after the deep fermentation is completed. Figure 4 The microscopic morphology of the liquid culture medium is shown. It can be seen that the hyphae are dense and uniform, the mycelia are highly active and small in diameter, exhibiting a flocculent appearance. Table 1 lists the data of the liquid culture medium obtained after submerged fermentation.

[0024] Table 1. Data on liquid microbial strains obtained from submerged fermentation using liquid culture medium.

[0025] e. Verification of fruiting from liquid spawn: Inoculate the flocculent liquid spawn obtained in step d into the cultivation medium at a 3% inoculum rate and incubate at 28℃ for 50 days to obtain mature fruiting bodies. See [link to relevant documentation]. Figure 5 The fruiting body has a dark brown cap, yellow gills, and a yellowish-brown stipe. Each fruiting body weighs an average of 70-90g and exhibits good commercial characteristics. The cultivation medium, by weight, is composed of: 2 parts wheat grain, 2 parts sawdust, 1 part cottonseed hulls, 1 part wheat bran, and 1 part corn flour. Table 2 lists the fruiting data for the liquid spawn.

[0026] Table 2. Fruiting data of liquid spawn

[0027] Example 2 A culture medium for fermentation of Boletus thunbergii liquid strain comprises the following components: 10g molasses, 10g soluble starch, 2g yeast extract, 1g magnesium sulfate, 1g potassium dihydrogen phosphate, 0.1g adenine, 0.3g silicone-free defoamer, and 1000mL water. Add the molasses, soluble starch, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, adenine, and silicone-free defoamer to deionized water, dissolve, and stir until homogeneous to obtain a liquid culture medium with a pH of 5.0.

[0028] The liquid culture medium for *Boletus thunbergii* was used to prepare the liquid culture of *Boletus thunbergii* through submerged fermentation, including the following steps: a. Preparation of mother culture medium: The mother culture medium, by weight, is prepared as follows: 200g of mountain red soil, 20g of glucose, 2g of yeast extract, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, and 20g of agar. Thoroughly mix the soil with 1000mL of water, let it stand for 1 hour, filter to obtain the soil supernatant, then add glucose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate, and agar to the supernatant, dissolve and boil, sterilize at 121℃ for 40 minutes, pour into petri dishes and allow to cool naturally to obtain the mother culture medium.

[0029] b. Cultivating the mother culture: A 5mm diameter mycelial block of *Boletus glomeratus* was inoculated into the mother culture medium described in step a for activation culture. The culture was maintained at 29℃ for 15 days to obtain a physiologically healthy *Boletus glomeratus* mother culture. The *Boletus glomeratus* source was the same as in Example 1.

[0030] c. Cultivating the seed culture in liquid form: Take the prepared liquid culture medium, fill it to a volume of 1.5L / 3L, sterilize it at 121℃ for 40 minutes, and cool it. Then, use a sterile scalpel to chop the mother culture from step b and inoculate it into the liquid culture medium. Incubate it at 29℃ on a magnetic stirrer at 250 rpm for 5 days to obtain the seed culture in liquid form.

[0031] d. Submerged fermentation liquid culture: Prepare the liquid culture medium (80% capacity, v / v) in a fermenter, sterilize at 121℃ for 70 min, using dynamic pressure sterilization (0-20 min: 0.13 MPa, 20-50 min: 0.15 MPa, 60-70 min: 0.12 MPa), and cool with circulating water to the inoculation temperature. Then inoculate with the seed culture obtained in step c, and culture at 28℃ with sterile air. Aeration uses a dynamic flow mode: 1 vvm for 0-48 h, 1.5 vvm for 48-120 h, with pressure controlled at 0.03-0.05 MPa. The fermentation cycle is 5 days, yielding a high-density liquid culture. The mycelium is dense and uniform, the mycelial pellets are highly active and small in diameter, exhibiting a flocculent appearance. Table 3 lists the data of the liquid culture obtained after submerged fermentation using the liquid culture medium.

[0032] Table 3. Data on liquid microbial strains obtained from submerged fermentation using liquid culture medium.

[0033] e. Verification of fruiting growth from liquid spawn: The flocculent liquid spawn obtained in step d was inoculated into the cultivation medium at a 6% inoculation rate and cultured at 28℃ for 55 days to obtain mature fruiting bodies. The fruiting bodies have dark brown caps, yellow gills, and yellowish-brown stipes, with an average weight of approximately 80-95g per fruiting body, exhibiting good commercial characteristics. The cultivation medium, by weight, is composed of: 4 parts wheat grains, 3 parts sawdust, 2 parts cottonseed hulls, 1 part wheat bran, and 1 part corn flour. Table 4 lists the fruiting data from the liquid spawn.

[0034] Table 4. Fruiting data of liquid spawn

[0035] Example 3 A culture medium for fermentation of *Boletus edulis* liquid strain comprises the following components: 20g molasses, 15g soluble starch, 3g yeast extract, 1.5g magnesium sulfate, 3.5g potassium dihydrogen phosphate, 0.2g adenine, 0.4g silicone-free defoamer, and 1000mL water. Molasses, soluble starch, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, adenine, and silicone-free defoamer are added to deionized water, dissolved, and stirred until homogeneous to obtain the liquid culture medium with a pH of 5.4.

[0036] The liquid culture medium for *Boletus thunbergii* was used to prepare the liquid culture of *Boletus thunbergii* through submerged fermentation, including the following steps: a. Preparation of mother culture medium: The mother culture medium, by weight, is prepared as follows: 200g of mountain red soil, 20g of glucose, 2g of yeast extract, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, and 20g of agar. Thoroughly mix the soil with 1000mL of water, let it stand for 1 hour, filter to obtain the soil supernatant, then add glucose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate, and agar to the supernatant, dissolve and boil, sterilize at 121℃ for 40 minutes, pour into petri dishes and allow to cool naturally to obtain the mother culture medium.

[0037] b. Cultivating the mother culture: A 5mm diameter mycelial block of *Boletus glomeratus* was inoculated into the mother culture medium described in step a for activation culture. The culture was maintained at 29℃ for 15 days to obtain a physiologically healthy *Boletus glomeratus* mother culture. The *Boletus glomeratus* source was the same as in Example 1.

[0038] c. Cultivating the seed culture in liquid form: Take the prepared liquid culture medium, fill it to a volume of 1.5L / 3L, sterilize it at 121℃ for 40 minutes, and cool it. Then, use a sterile scalpel to chop the mother culture from step b and inoculate it into the liquid culture medium. Incubate it at 29℃ on a magnetic stirrer at 250 rpm for 5 days to obtain the seed culture in liquid form.

[0039] d. Submerged fermentation liquid culture: Prepare the liquid culture medium (80% capacity, v / v) in a fermenter, sterilize at 121℃ for 60 min, using dynamic pressure sterilization (0-20 min: 0.13 MPa, 20-40 min: 0.15 MPa, 50-60 min: 0.12 MPa), and cool with circulating water to the inoculation temperature. Then inoculate with the seed culture obtained in step c, and culture at 28℃ with sterile air. Aeration uses a dynamic flow mode: 1.1 vvm for 0-48 h, 1.4 vvm for 48-120 h, with pressure controlled at 0.03-0.05 MPa. The fermentation cycle is 5 days, yielding a high-density liquid culture. The mycelium is dense and uniform, the mycelial pellets are highly active and small in diameter, exhibiting a flocculent appearance.

[0040] e. Verification of fruiting from liquid spawn: The flocculent liquid spawn obtained in step d is inoculated into the cultivation medium at a 5% inoculation rate and cultured at 28℃ for 50 days to obtain mature fruiting bodies. The fruiting bodies have dark brown caps, yellow gills, and yellowish-brown stipes, with an average weight of approximately 75-90g per fruiting body, exhibiting good commercial characteristics. The cultivation medium is prepared by weight in the following proportions: 2 parts wheat grains, 3 parts sawdust, 2 parts cottonseed hulls, 1 part wheat bran, and 1 part corn flour.

[0041] The raw materials used in this invention, such as molasses, soluble starch, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, adenine, and silicone-free defoamer, are all commercially available.

[0042] Unless otherwise stated, all percentages mentioned in this invention are mass percentages.

[0043] The culture medium and fermentation system for the liquid spawn of *Boletus thunbergii* of this invention can significantly shorten the deep fermentation time of the liquid spawn, and the liquid spawn has excellent quality, high uniformity, and stable yield. Actual fruiting verification has yielded very good fruiting results, and the obtained fruiting bodies have good commercial characteristics, providing a new avenue for the industrial application of *Boletus thunbergii* liquid spawn.

Claims

1. The application of a liquid culture medium for the fermentation of Boletus thunbergii liquid inoculum, characterized in that, The liquid culture medium, by weight, is prepared in the following proportions: molasses 10-30 parts, soluble starch 10-30 parts, yeast extract 2-5 parts, potassium dihydrogen phosphate 1-4 parts, magnesium sulfate 1-3 parts, adenine 0.1-1 parts, antifoaming agent 0.3-0.5 parts, and water 1000 parts. The pH of the liquid culture medium is 5.0-5.

8. The culture medium is used for the liquid inoculum fermentation of Boletus thunbergii, comprising the following steps: a. Preparation of mother culture medium: The mother culture medium is prepared by weight as follows: 100-300 parts soil, 10-30 parts glucose, 1-3 parts yeast extract, 1-3 parts potassium dihydrogen phosphate, 1-3 parts magnesium sulfate, and 15-25 parts agar. Add 1000 parts water to the soil, stir and let stand. Take the soil supernatant as a solvent, then add the remaining ingredients, heat to dissolve and boil. After dispensing, autoclave at 121℃ for 40 min, pour into plates and cool to solidify to obtain the mother culture medium. b. Cultivating the mother culture: Take a 5mm diameter mycelial block of Boletus edulis and inoculate it into the mother culture medium described in step a. Incubate at a constant temperature of 28-30℃ for 14-18 days to obtain the mother culture. c. Cultivating the seed culture liquid strain: Prepare the liquid culture medium, with a volume of 1.5L / 3L-2L / 3L, sterilize at 121℃ for 40min, and cool; then chop the mother culture prepared in step b and inoculate it into the liquid culture medium, and incubate at 28-30℃ on a magnetic stirrer at 150-250r / min for 5-7 days to obtain the seed culture liquid strain; d. Submerged fermentation liquid culture medium: Prepare the liquid culture medium in a fermenter, filling it to 60-80% capacity, v / v, sterilize at 121℃ for 60-80 min, and cool it with circulating water to the inoculation temperature; then inoculate the seed liquid liquid culture obtained in step c, culture at 28-30℃ with sterile air, and ferment for 3-5 days to obtain a flocculent liquid culture with a biomass of 9-15 g / L.

2. The application of the liquid culture medium for fermentation of *Boletus globosum* liquid inoculum as described in claim 1, characterized in that, The flocculent liquid inoculum obtained in step d is inoculated into the cultivation medium at an inoculation rate of 3-6%, and cultured at 28-30℃ for 45-55 days to obtain mature fruiting bodies; the cultivation medium is prepared by mass parts as follows: 2-4 parts wheat grains, 2-4 parts sawdust, 1-2 parts cottonseed hulls, 1-2 parts wheat bran, and 1-2 parts corn flour.

3. The application of the liquid culture medium for fermentation of *Boletus globosum* liquid inoculum as described in claim 1, characterized in that, In step d, during the deep fermentation of the liquid culture medium, the sterilization process after loading the liquid culture medium into the fermenter adopts dynamic pressure sterilization, that is: the pressure is controlled at 0.12 MPa from 0 to 20 minutes, the pressure is controlled at 0.15 MPa from 20 to 60 minutes, and the pressure is controlled at 0.12 MPa from 60 to 80 minutes; when the seed liquid culture obtained in step c is circulated with sterile air for culture, the aeration adopts a dynamic flow mode, that is: the aeration flow rate is 1-1.5 vvm from 0 to 48 hours, the aeration flow rate is 1.5-2 vvm from 48 to 120 hours, and the pressure is controlled at 0.03-0.05 MPa.

4. The application of the liquid culture medium for fermentation of *Boletus globosum* liquid inoculum as described in claim 1, 2, or 3, characterized in that, The soil used for the mother culture medium is mountain red soil.

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