Liquid culture medium and method for preparing Auricularia auricula-judae spawn in high-altitude areas

Abstract

This invention provides a liquid culture medium for the preparation of *Auricularia auricula-judae* spawn in high-altitude areas, comprising a primary fermentation liquid culture medium, a secondary fermentation liquid culture medium, and a fermenter liquid culture medium. The primary fermentation liquid culture medium has a pH of 6-7 and comprises: 5-10g soluble starch, 0-5g soybean meal, 10-15g glucose, 1-3g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, and 10mg vitamin B1 per liter of medium. The secondary fermentation liquid culture medium also has a pH of 6-7 and comprises: 5-10g soluble starch, 0-5g soybean meal, 10-15g glucose, 1-3g peptone, 2g potassium dihydrogen phosphate, and 0.5g magnesium sulfate per liter of medium. The fermenter liquid culture medium is prepared by adding 1.5%-2.0% by weight of sawdust powder to the primary fermentation liquid culture medium. This invention also provides a method for preparing *Auricularia auricula-judae* spawn in high-altitude areas. The method of this invention can produce the original strain of golden ear fungus in about 20 days, with a short production cycle, and is suitable for the rapid production of original strain of golden ear fungus in high-altitude areas.

Description

Technical Field

[0001] This invention relates to the field of Auricularia auricula-judae strain technology, and more specifically to a liquid culture medium and a method for preparing Auricularia auricula-judae strains in high-altitude areas. Background Technology

[0002] Golden ear fungus (scientific name: *Naematelia aurantialba* (Bandoni & M. Zang) Millanes & Wedin) is a fungus belonging to the family Auriculariaceae and the genus *Naematelia*. The golden ear fungus spores are composed of two types of hyphae: *Naematelia aurantialba* hyphae and *Naematelia pubescens* hyphae, forming a dimorphic hyphae complex. Golden ear fungus is rich in fat, protein, and trace elements such as phosphorus, sulfur, manganese, iron, magnesium, calcium, and potassium, making it a nutritious tonic and also useful for medicinal purposes.

[0003] The production of auricularia auricula-judae spawn generally includes three steps: mother culture activation, primary culture production, and spawn propagation. In current technology, most of the traditional solid-state spawn production methods are used for primary culture production. The general process is as follows: sawdust, wheat bran, cornmeal, rice husks, cottonseed hulls, white sugar, etc. are mixed in different proportions, bagged and sterilized, then inoculated with mother culture, and cultured at a temperature of 18-25℃ to allow the mycelium to fully grow in the culture bottle, thoroughly consume the culture medium, and form white fleshy primordia when the mycelium twists and forms primordia with a diameter of 4-5cm. These primordia are then used as primary cultures.

[0004] However, because solid spawn uses raw materials such as sawdust, it takes a certain amount of time for the mycelium to fully absorb the culture medium and grow into the spawn bag. The slow growth rate of the mycelium makes the production cycle of the original spawn as long as 45-50 days, which leads to the long production cycle of auricularia auricula var. auricula. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a liquid culture medium and a method for producing original strains of *Auricularia auricula-judae* in high-altitude areas. By changing the traditional steps of producing original strains of *Auricularia auricula-judae*, the method changes the use of liquid fermentation culture instead of solid culture, which greatly reduces the production cost of original strains of *Auricularia auricula-judae*, shortens the production cycle of original strains of *Auricularia auricula-judae*, and consequently shortens the production cycle of cultivated strains of *Auricularia auricula-judae*.

[0006] The first aspect of this invention relates to a liquid culture medium for producing original strains of Auricularia auricula-judae in high-altitude areas, comprising a primary fermentation liquid culture medium, a secondary fermentation liquid culture medium, and a fermenter liquid culture medium;

[0007] The pH of the primary fermentation liquid culture medium is 6-7, and the components include: 5-10g of soluble starch, 0-5g of soybean meal, 10-15g of glucose, 1-3g of peptone, 2g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, and 10mg of vitamin B1 per liter of culture medium.

[0008] The secondary fermentation liquid culture medium has a pH of 6-7 and its components include: 5-10g of soluble starch, 0-5g of soybean meal, 10-15g of glucose, 1-3g of peptone, 2g of potassium dihydrogen phosphate, and 0.5g of magnesium sulfate per liter of culture medium.

[0009] The fermenter liquid culture medium is made by adding 1.5%-2.0% by weight of sawdust powder to the primary fermentation liquid culture medium.

[0010] In an optional embodiment, the primary fermentation liquid culture medium has a pH of 6-7 and comprises: 10g of soluble starch, 15g of glucose, 3g of peptone, 2g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, and 10mg of vitamin B1 per liter of culture medium.

[0011] In an optional embodiment, the primary fermentation liquid culture medium has a pH of 6-7 and comprises the following components per liter: 5g soluble starch, 10g glucose, 1g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, and 10mg vitamin B1.

[0012] In an optional embodiment, the pH of the primary fermentation liquid culture medium is 6-7, and the components include: 10g of soluble starch, 5g of soybean meal, 15g of glucose, 1g of peptone, 2g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, and 10mg of vitamin B1 per liter of culture medium.

[0013] The second aspect of this invention relates to a method for producing original strains of golden ear fungus from high-altitude areas, comprising the following steps:

[0014] S1. Inoculate the solid mother culture of Auricularia auricula-judae into the primary fermentation liquid culture medium, and culture it in a constant temperature shaker at the preset temperature and speed to obtain the primary liquid culture of Auricularia auricula-judae.

[0015] S2. The primary liquid culture of *Auricularia auricula-judae* obtained in step S1 is inoculated into the secondary fermentation liquid culture medium and cultured in a constant temperature shaker at a preset temperature and speed to obtain the secondary liquid culture of *Auricularia auricula-judae*.

[0016] S3. The secondary liquid strain of *Auricularia auricula-judae* obtained in step S2 is inoculated into the liquid culture medium of the fermenter, filtered sterile air is continuously introduced, and cultured at a preset temperature to obtain the original strain of *Auricularia auricula-judae*.

[0017] In an optional embodiment, in step S1, 3.0-5.0 cm of *Auricularia auricula-judae* solid mother culture is inoculated into every 100 mL of primary fermentation liquid culture medium. 2 .

[0018] In an optional implementation, the culture conditions in step S1 are as follows:

[0019] Temperature 22-26℃, rotation speed 140-200 r / min, incubation time 84-96 h.

[0020] In an optional embodiment, in step S2, the inoculation amount of the primary liquid culture medium for *Auricularia auricula-judae* is 5%-15% of the total volume of the secondary fermentation liquid culture medium.

[0021] In an optional implementation, the culture conditions in step S2 are as follows:

[0022] Temperature 22-26℃, rotation speed 200-240r / min, incubation time 84-96h (a range can be given).

[0023] In an optional embodiment, in step S3, the inoculation amount of the secondary liquid strain of Auricularia auricula-judae is 5%-10% of the total volume of the liquid culture medium in the fermenter.

[0024] The cultivation conditions are as follows:

[0025] The aeration rate is 3-5 L / min, the temperature inside the fermenter is 22-26℃, and the culture time is 96-120 h.

[0026] Through the implementation of the above scheme, the liquid culture medium proposed in this invention for the preparation of auricularia auricula var. ...

[0027] The method for producing *Auricularia auricula-judae* spawn of this invention involves three biological fermentations. The first fermentation is carried out at high temperature and low speed with rich nutrients to promote the activation of dormant mycelia and accelerate germination. Then, a second fermentation is carried out at low temperature and high speed to promote the rapid propagation of mycelia and the formation of mycelial balls. Finally, the fermentation is carried out in a fermenter to expand the culture and achieve the optimal mycelial vitality and biomass. In this way, *Auricularia auricula-judae* spawn can be obtained in 17-20 days, and the dry weight of mycelia can reach 7.16-9.23 mg / mL, and the diameter of the mycelial balls is less than 2 mm. This greatly shortens the production cycle of *Auricularia auricula-judae* spawn, and thus shortens the production cycle of *Auricularia auricula-judae* cultivars. Attached Figure Description

[0028] Figure 1 This is a flowchart of the method for producing high-altitude golden ear fungus strains according to the present invention.

[0029] Figure 2 This is a physical diagram of the primary fermentation process in Example 1 of the present invention.

[0030] Figure 3 This is a physical diagram of the secondary fermentation culture process in Example 1 of the present invention.

[0031] Figure 4 This is a physical image of the original strain of *Auricularia auricula-judae* mycelial balls obtained in Example 1 of the present invention. Detailed Implementation

[0032] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0033] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0034] Linxia is a high-altitude, cold region with an average altitude of 2000 meters and an average annual temperature of 5.6–9.7℃. The experiments of this invention were conducted at the Beiyuan Experimental Station in Linxia County, which is at an altitude of about 2000 meters. Therefore, the high-altitude region involved in this invention refers to the region with an altitude of 1900–2300 meters. The formula adjustment conditions of this invention are suitable for high-altitude regions, but are not limited to high-altitude regions.

[0035] The auricularia auricula spawn is a mixture of auricularia auricula mycelium and sclerosis coarse hairs mycelium. In the process of cultivating auricularia auricula spawn, it is necessary to ensure that both auricularia auricula mycelium and sclerosis coarse hairs mycelium are growing normally before they can be used for cultivation.

[0036] Based on this, the present invention provides a liquid culture medium that utilizes the principle of biological fermentation to provide optimal growth conditions for mycelia, enabling them to grow rapidly and multiply quickly. The original strain of *Auricularia auricula-judae* can be obtained in 17-20 days, shortening the original strain production cycle and greatly reducing the original strain production cost.

[0037] In an exemplary embodiment of the present invention, a liquid culture medium for preparing original strains of *Auricularia auricula-judae* from high-altitude areas is provided, comprising:

[0038] Primary fermentation liquid culture medium: per liter of culture medium, there are 5-10g of soluble starch, 0-5g of soybean meal, 10-15g of glucose, 1-3g of peptone, 2g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 10mg of vitamin B1, and pH 6-7.

[0039] Secondary fermentation liquid culture medium: per liter of culture medium, there are 5-10g of soluble starch, 0-5g of soybean meal, 10-15g of glucose, 1-3g of peptone, 2g of potassium dihydrogen phosphate, and 0.5g of magnesium sulfate.

[0040] Fermentation tank liquid culture medium: Add 1.5%-2.0% by weight of sawdust powder to the primary fermentation liquid culture medium.

[0041] The main components of sawdust powder are crude fiber and carbohydrates, which can provide carbon sources and "parasitic nuclei" for mycelial balls, significantly promoting the formation and development of mycelial balls, and can also produce liquid inoculum that can be rapidly colonized in the cultivation medium.

[0042] It should be understood that soluble starch is a common substance in the field, such as corn starch and potato starch. Those skilled in the art can choose according to the actual situation, and there is no further limitation on its types here.

[0043] In a preferred embodiment of the present invention, the C / N ratio (i.e., the mass ratio of carbon source to nitrogen source) of the liquid culture medium is mainly adjusted by changing the proportion of soluble starch, soybean meal, glucose, and peptone in the formula. After adjustment, the C / N range of the primary fermentation liquid culture medium of the present invention is between 20:1 and 30:1, which meets the nutritional requirements for the growth of golden ear fungus.

[0044] In one preferred embodiment of the present invention, the amounts of soluble starch, soybean meal, glucose, and peptone are adjusted by controlling the C / N ratio of the formula. The C / N ratio is set to 20:1, and the formula is as follows:

[0045] 10g soluble starch, 5g soybean meal, 15g glucose, 3g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 10mg vitamin B1, pH 6-7.

[0046] In another preferred embodiment of the present invention, the amounts of soluble starch, soybean meal, glucose, and peptone are adjusted by controlling the C / N ratio of the formula, with the C / N ratio set to 25:1, and the formula is as follows:

[0047] 5g soluble starch, 5g soybean meal, 10g glucose, 1g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 10mg vitamin B1, pH 6-7.

[0048] In another preferred embodiment of the present invention, the amounts of soluble starch, soybean meal, glucose, and peptone are adjusted by controlling the C / N ratio of the formula, with the C / N ratio set to 30:1, and the formula is as follows:

[0049] Soluble starch 10g, soybean meal 5g, glucose 15g, peptone 1g, potassium dihydrogen phosphate 2g, magnesium sulfate 0.5g, vitamin B1 10mg, pH 6-7.

[0050] Preferably, the difference between the secondary fermentation liquid culture medium and the primary fermentation liquid culture medium is that vitamin B1 is removed from the primary fermentation liquid culture medium.

[0051] In another preferred embodiment of the present invention, a method for preparing a primary fermentation liquid culture medium is also provided, comprising:

[0052] 1. Boil purified water to 40-60℃, add soluble starch and soybean meal powder while stirring, and stir until well mixed;

[0053] 2. Dissolve glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate, and vitamin B1 in purified water, add them to the solution from step 1 while stirring, heat over low heat for 1 minute, and add purified water until sufficient.

[0054] 3. Use precision pH test paper to check; pH 6-7 is sufficient. Otherwise, adjust with 1 mol / L NaOH or HCl solution.

[0055] 4. Sterilize the prepared liquid culture medium at 115℃ for 30 minutes, and then cool it for later use.

[0056] Understandably, the preparation method of the secondary fermentation liquid culture medium is the same as that of the primary fermentation liquid culture medium, except that vitamin B1 needs to be removed.

[0057] In another preferred embodiment of the present invention, a method for preparing a liquid culture medium for a fermenter is also provided, comprising:

[0058] Preparation of wood chip powder: Select fresh, mold-free broad-leaved wood chips, soak them in pure water for 24-36 hours, sterilize them at 121℃ for 30 minutes, drain the water, place them in an electric constant temperature oven and dry them at 65℃ until constant weight, crush them using a pulverizer, and pass them through a 120-mesh sieve (0.125mm aperture) for later use.

[0059] To prepare the primary fermentation liquid culture medium as described above, in step 2, simply take glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1, and 1.5% of the aforementioned sawdust powder (by weight of the primary fermentation liquid culture medium), dissolve them in purified water, add them to the solution from step 1 while stirring, heat over low heat for 1 minute, and then add purified water to the required volume.

[0060] In another exemplary embodiment of the present invention, a method for producing original strains of golden ear fungus from high-altitude areas is provided, comprising the following steps:

[0061] S1. Inoculate the solid mother culture of Auricularia auricula-judae into the primary fermentation liquid culture medium, and culture it in a constant temperature shaker at the preset temperature and speed to obtain the primary liquid culture of Auricularia auricula-judae.

[0062] S2. The primary liquid culture of *Auricularia auricula-judae* obtained in step S1 is inoculated into the secondary fermentation liquid culture medium and cultured in a constant temperature shaker at a preset temperature and speed to obtain the secondary liquid culture of *Auricularia auricula-judae*.

[0063] S3. The secondary liquid strain of *Auricularia auricula-judae* obtained in step S2 is inoculated into the liquid culture medium of the fermenter, filtered sterile air is continuously introduced, and cultured at a preset temperature to obtain the original strain of *Auricularia auricula-judae*.

[0064] In an optional embodiment, in step S1, 3.0-5.0 cm of *Auricularia auricula-judae* solid mother culture is inoculated into every 100 mL of primary fermentation liquid culture medium. 2 .

[0065] In a more preferred embodiment, 4.0 cm of *Auricularia auricula-judae* solid mother culture is inoculated into every 100 mL of primary fermentation broth. 2 During inoculation, cut the mother plant of the golden ear fungus into 1cm pieces. 2 Re-inoculate small pieces of different sizes.

[0066] In an optional implementation, the culture conditions in step S1 are as follows:

[0067] Temperature 20-26℃, rotation speed 140-200 r / min, incubation time 84-96 h.

[0068] In a more preferred embodiment, the temperature was 24°C, the rotation speed was 180 r / min, and the incubation time was 96 h.

[0069] In an optional embodiment, in step S2, the inoculation amount of the primary liquid culture of *Auricularia auricula-judae* is 5%-15% of the total volume of the secondary fermentation liquid culture medium, that is, 50-150 mL of primary liquid culture of *Auricularia auricula-judae* needs to be inoculated in each liter of secondary fermentation liquid culture medium.

[0070] In a more preferred embodiment, the primary liquid culture is inoculated into a 1L shake flask containing secondary fermentation liquid culture medium at an inoculation rate of 15%, with a liquid volume of 400-500mL, particularly preferably 400mL. The dissolved oxygen content is optimal when the liquid volume of the shake flask is between 40% and 50%, which can provide sufficient oxygen for mycelial growth.

[0071] In an optional implementation, the culture conditions in step S2 are as follows:

[0072] Temperature 22-26℃, rotation speed 200-240 r / min, incubation time 84-96 h.

[0073] In a more preferred embodiment, the temperature was 24°C, the rotation speed was 240 r / min, and the incubation time was 96 h.

[0074] In an optional embodiment, in step S3, the inoculation amount of the secondary liquid culture of Auricularia auricula-judae is 5%-10% of the total volume of the liquid culture medium in the fermenter, that is, 50-150 mL of secondary liquid culture of Auricularia auricula-judae needs to be inoculated in each liter of liquid culture medium in the fermenter.

[0075] The cultivation conditions are as follows:

[0076] The aeration rate is 3-5 L / min, the temperature inside the fermenter is 22-26℃, and the culture time is 96-120 h.

[0077] In a more preferred embodiment, the secondary liquid strain is inoculated into a fermenter containing the fermentation tank liquid culture medium at an inoculation rate of 15%, and cultured at 24°C with an aeration rate of 4.5 L / min for 48 h; then, at 24°C, the aeration rate is adjusted to 3 L / min and cultured for 48-72 h to obtain the original strain of Auricularia auricula-judae.

[0078] The fermenter is a deep fermentation tank. In the early stage, high oxygen flow ensures a high O2 concentration inside the tank, promoting rapid mycelial growth and establishing dominance to reduce contamination. In the later stage, oxygen flow is reduced to minimize mechanical damage to the mycelium, maintain mycelial integrity, and ensure viability. Under the above conditions, the obtained *Auricularia auricula-judae* spawn has a mycelial dry weight of 7.16-9.23 mg / mL, and the diameter of the mycelial balls is all below 2 mm.

[0079] The present invention will be further described below with reference to specific embodiments.

[0080] The following experiments were all conducted in the laboratory of the Beiyuan Experimental Station of the Linxia Hui Autonomous Prefecture Academy of Agricultural Sciences, at an altitude of 2010–2035 meters.

[0081] Example 1

[0082] I. Activation of Golden Ear Mother Culture

[0083] 1. Activation medium formula: 200g potato (peeled), 20g glucose, 2g potassium dihydrogen phosphate, 2g peptone, 1g magnesium sulfate, 1 tablet (10mg) of vitamin B1, 15g agar, natural pH, 1000mL distilled water.

[0084] 2. Preparation of activation culture medium:

[0085] ① Peel 200g of potatoes and cut them into approximately 1cm pieces. 3 Add potato chunks to a cube, boil in 400mL of purified water for 15 minutes (cooked but not mushy), filter twice through 6 layers of gauze, and reserve the filtrate for later use;

[0086] ② Weigh 20g of glucose, 2g of potassium dihydrogen phosphate, 2g of peptone, 1g of magnesium sulfate, and 1 vitamin B1 tablet. Dissolve them in 200mL of purified water and add them to the filtrate from the previous step while stirring. Set aside.

[0087] ③ Weigh 20g of agar powder, dissolve it in 200mL of purified water, add it to the filtrate from the previous step while stirring. After stirring evenly, heat over low heat, boil for 2 minutes, turn off the heat, add water to 1000mL, stir evenly, and dispense into containers; sterilize at 121℃ for 20 minutes.

[0088] ④ Plate preparation: Sterilize 10cm diameter glass petri dishes in advance and irradiate them with ultraviolet light in a clean bench for 30 minutes before use. Pour approximately 15mL of culture medium into each plate, which should be about 0.5cm thick.

[0089] ⑤ Testing: After the condensate in the petri dish has evaporated, seal it and place it at 37°C for 1 day. If no bacteria grow, it is ready for use.

[0090] 3. Activation: Take 1cm of auricularia auricula-judae spawn. 2 The sized cubes were inoculated in the center of the activation medium prepared in step 2, inverted, and cultured at a constant temperature of 22℃ for 9 days to obtain the activated golden ear mother seed.

[0091] II. Preparation of Golden Ear Seed Culture (Liquid Culture)

[0092] 1. Liquid culture medium formulation:

[0093] Primary fermentation liquid culture medium: per liter of culture medium, there are 10g corn starch, 5g soybean meal, 15g glucose, 3g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 10mg vitamin B1, and pH 6.5.

[0094] Secondary fermentation liquid culture medium: 10g soluble starch, 5g soybean meal, 15g glucose, 3g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, pH 6.5.

[0095] Fermentation tank liquid culture medium: Add 1.5% by weight of sawdust powder to the primary liquid culture medium containing microorganisms.

[0096] 2. Preparation of liquid culture medium

[0097] Preparation of primary fermentation liquid culture medium:

[0098] ① Boil purified water to 60℃, add cornstarch and soybean meal powder while stirring until well mixed.

[0099] ② Dissolve glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate, and vitamin B1 in purified water, add them to the solution from the previous step while stirring, heat over low heat for 1 minute, and add purified water until sufficient.

[0100] ③ Use precision pH test paper to test; pH 6.5 is sufficient. Otherwise, adjust with 1 mol / L NaOH or HCl solution.

[0101] ④ Sterilize the prepared liquid culture medium at 121℃ for 20 minutes, and then cool it for later use.

[0102] The difference between the preparation of secondary fermentation liquid culture medium and the preparation of primary fermentation liquid culture medium is that vitamin B1 is not included in step ②.

[0103] The difference between the preparation of liquid culture medium for fermentation tanks and the preparation of liquid culture medium for primary fermentation is that step ② requires the addition of 1.5% sawdust powder by weight.

[0104] The process of obtaining wood chip powder is as follows: Select fresh, mold-free broad-leaved wood chips, soak them, sterilize and dry them to constant weight, crush them using a pulverizer, and pass them through a 120-mesh sieve (0.125mm aperture) for later use.

[0105] 3. Cultivation of Golden Ear Strawberry Seeds

[0106] Obtaining the primary liquid culture: Inoculate 4cm of the *Auricularia auricula-judae* mother culture into every 100mL of the above primary liquid culture medium. 2 Cut the mother plant of the golden ear fungus into 1cm pieces. 2 Inoculate with cubes of the desired size, incubate at 24℃ and 180 rpm for 96 hours to obtain primary liquid spawn of *Auricularia auricula-judae*. The fermentation process is as follows: Figure 2 As shown.

[0107] Obtaining the secondary liquid culture: The primary liquid culture was inoculated at a 15% inoculum rate into a 1L shake flask containing the secondary fermentation liquid medium (400mL). The flask was incubated at 22℃ and 240r / min for 96 hours to obtain the secondary liquid culture for *Auricularia auricula-judae*. The fermentation process is as follows: Figure 3 As shown.

[0108] Obtaining the liquid culture medium in the fermenter: The secondary liquid culture medium was inoculated into the fermenter containing the above-mentioned fermentation liquid culture medium at an inoculation rate of 15%. The culture was carried out at 24°C and an aeration rate of 4.5 L / min for 48 h; then at 24°C and an aeration rate of 3 L / min for 72 h to obtain the liquid culture medium for the Auricularia auricula-judae fermenter, which is the original culture.

[0109] like Figure 3 As shown, the original strain of Auricularia auricula-judae obtained under the above conditions can reach a dry weight of 9.23 mg / mL of mycelium after 20 days of cultivation, and the diameter of the mycelial balls is less than 2 mm.

[0110] Example 2

[0111] The difference between this embodiment and Embodiment 1 lies in the raw material ratios of the primary fermentation liquid culture medium for *Auricularia auricula-judae* in step two, as well as the raw material ratios of the secondary fermentation liquid culture medium. In this embodiment, the raw material ratio of the primary fermentation liquid culture medium is 5g corn starch, 5g soybean meal, 10g glucose, 1g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 10mg vitamin B1, and pH 7; the raw material ratio of the secondary fermentation liquid culture medium is 5g corn starch, 5g soybean meal, 10g glucose, 1g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, and pH 7.

[0112] The remaining steps and parameters are the same as in Example 1.

[0113] The original strain of Auricularia auricula-judae obtained under the above conditions can reach a dry weight of 7.76 mg / mL of mycelium after 20 days of cultivation, and the diameter of the mycelial balls is less than 2 mm.

[0114] Example 3

[0115] The difference between this embodiment and Embodiment 1 lies in the raw material ratios of the primary fermentation liquid culture medium for *Auricularia auricula-judae* in step two, as well as the raw material ratios of the secondary fermentation liquid culture medium. In this embodiment, the raw material ratio of the primary fermentation liquid culture medium is 10g corn starch, 5g soybean meal, 15g glucose, 1g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 10mg vitamin B1, and pH 7; the raw material ratio of the secondary fermentation liquid culture medium is 10g corn starch, 5g soybean meal, 15g glucose, 1g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, and pH 7.

[0116] The remaining steps and parameters are the same as in Example 1.

[0117] Under the above conditions, the original strain of Auricularia auricula-judae obtained can reach a dry weight of 7.16 mg / mL of mycelium after 20 days of cultivation, and the diameter of the mycelial balls is less than 2 mm.

[0118] Comparative Example 1

[0119] The difference between Comparative Example 1 and Example 1 is that the raw materials in the liquid culture medium of the fermenter in step two are different. No sawdust powder is added to the liquid culture medium of the fermenter in Comparative Example 1.

[0120] The remaining steps and parameters are the same as in Example 1.

[0121] Under the above conditions, the original strain of Auricularia auricula-judae obtained can reach a dry weight of 6.69 mg / mL of mycelium after 20 days of cultivation, and the diameter of the mycelial balls is less than 2 mm.

[0122] Comparative Example 2

[0123] The difference between Comparative Example 2 and Comparative Example 1 is that the raw material ratio of the liquid culture medium for secondary fermentation of Auricularia auricula-judae in step two is different. The raw material ratio of the secondary fermentation culture medium in this comparative example is: 25g corn starch, 5g soybean meal, 3g peptone, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, pH 6.5.

[0124] The remaining steps and parameters are the same as in Example 1.

[0125] After fermentation, the original strain of Auricularia auricula-judae in the fermentation tank was tested for indicators: the experiment found that the fermentation effect of a single carbon source was not as good as that of a compound carbon source. Without the addition of sawdust powder, the mycelial biomass of the original strain of Auricularia auricula-judae after secondary fermentation (mycelial biomass is expressed as mycelial dry weight, in mg / mL) was only 4.89 mg / mL.

[0126] Golden Ear Cultivation Seed Inoculation and Culture

[0127] The original seed culture of Auricularia auricula-judae obtained in Examples 1, 2 and 3 were used respectively. Commercially available Auricularia auricula-judae cultivation material was selected as the cultivation material. 6 mL of Auricularia auricula-judae original seed culture was inoculated for every 1200 g of cultivation material, with two holes in each hole, and cultured at 22℃.

[0128] After inoculation with liquid microbial culture, the culture medium began to germinate within 48 hours. White fuzz could be seen around the inoculation holes, and the medium could be fully covered in as little as 20 days at 22°C.

[0129] As can be seen from the above process, the present invention can cultivate golden ear fungus strains in about 20 days, and the entire golden ear fungus cultivation strain production cycle is only about 40 days. The short cultivation strain production cycle greatly reduces the production cost of golden ear fungus original strains.

[0130] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for producing a high-altitude area gold ear original seed, characterized in that, The method comprises the following steps: S1, inoculating the solid mother strain of Tremella aurantiaca into a first-stage fermentation liquid medium, and performing shock culture in a constant-temperature shaker at a preset temperature and rotating speed to obtain a first-stage liquid strain of Tremella aurantiaca; The pH of the first-stage fermentation liquid medium is 6-7, and each liter of the medium contains 5-10 g of soluble starch, 0-5 g of soybean meal, 10-15 g of glucose, 1-3 g of protein peptone, 2 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 10 mg of vitamin B1. The culture conditions are as follows: a temperature of 22-26℃, a rotating speed of 140-200 r / min, and a culture time of 84-96 h. S2, inoculating the first-stage liquid strain of Tremella aurantiaca obtained in step S1 into a second-stage fermentation liquid medium, and performing shock culture in a constant-temperature shaker at a preset temperature and rotating speed to obtain a second-stage liquid strain of Tremella aurantiaca; The pH of the second-stage fermentation liquid medium is 6-7, and each liter of the medium contains 5-10 g of soluble starch, 0-5 g of soybean meal, 10-15 g of glucose, 1-3 g of protein peptone, 2 g of potassium dihydrogen phosphate, and 0.5 g of magnesium sulfate. The culture conditions are as follows: a liquid volume in a shaking flask of 40%-50%, a temperature of 22-26℃, a rotating speed of 200-240 r / min, and a culture time of 84-96 h. S3, inoculating the second-stage liquid strain of Tremella aurantiaca obtained in step S2 into a fermentation tank liquid medium, continuously introducing filtered sterile air, and performing culture at a preset temperature to obtain a primary strain of Tremella aurantiaca; The fermentation tank liquid medium is the first-stage fermentation liquid medium to which 1.5%-2.0% of wood chip powder by weight of the first-stage fermentation liquid medium is added. The culture conditions are as follows: a temperature of 24℃, an air flow of 4.5 L / min, a culture time of 48 h, then a temperature of 24℃, an air flow adjusted to 3 L / min, and a culture time of 48-72 h. The wood chip powder is prepared as follows: fresh wood chips of broad-leaved trees without mildew are soaked in pure water for 24-36 h, sterilized at 121℃ for 30 min, then drained and dried in an electric constant-temperature oven at 65℃ until the weight is constant, crushed by a crusher, and sieved through a 120-mesh sieve for standby use.

2. The method of claim 1, wherein the high altitude region gold ear original seed production method is characterized by, The pH of the first-stage fermentation liquid medium is 6-7, and each liter of the medium contains 10 g of soluble starch, 15 g of glucose, 3 g of protein peptone, 2 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 10 mg of vitamin B1.

3. The method of claim 1, wherein the high altitude area gold ear original seed production method is characterized by, The pH of the first-stage fermentation liquid medium is 6-7, and each liter of the medium contains 5 g of soluble starch, 10 g of glucose, 1 g of protein peptone, 2 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 10 mg of vitamin B1.

4. The method of claim 1, wherein the high altitude production of the original inoculum of the Korean fungus is characterized by, The pH of the first-stage fermentation liquid medium is 6-7, and each liter of the medium contains 10 g of soluble starch, 5 g of soybean meal, 15 g of glucose, 1 g of protein peptone, 2 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 10 mg of vitamin B1.

5. The method of claim 1, wherein the high altitude production of the original inoculum of the Korean fungus is characterized by, In the step S1, 3.0-5.0 cm of the solid Chondrostereum purpureum mother culture is inoculated per 100 mL of the primary fermentation liquid medium 2 .

6. The method of claim 1, wherein the high altitude production of the original inoculum of the Korean fungus is characterized by, In step S2, the inoculation amount of the first-stage liquid strain of Tremella aurantiaca is 5%-15% of the total volume of the second-stage fermentation liquid medium.

7. The method of claim 1, wherein the high altitude production of the gold ear original seed is characterized by, In step S3, the inoculation amount of the second-stage liquid strain of Tremella aurantiaca is 5%-10% of the total volume of the fermentation tank liquid medium.

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