A rapid submerged liquid fermentation culture method for Hirsutella sinensis

Abstract

The invention discloses a rapid deep liquid fermentation culture method of Chinese hairy spore, comprising primary seed shake flask culture, secondary seed liquid culture and seed liquid fermentation tank culture, wherein betaine, choline chloride, L-carnitine and cobalamin are added to the fermentation medium used in the fermentation tank culture. The invention adds functional fermentation regulating substances in the fermentation process, so that the yield of artificially cultured Chinese hairy spore cells, the yield of cordycepin, adenosine and other substances are higher, and the fermentation cycle is shortened, the fermentation efficiency is higher, the fermentation cost is reduced, the use of water and electricity is reduced, and the method is environmentally friendly. The product can replace wild Chinese hairy spores, and has good economic benefits and market prospects in the pharmaceutical market.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for rapid deep liquid fermentation of Hirsutella sinensis. Background Art

[0002] The Chinese medicine Cordyceps sinensis is a fungus (C. sinensis (Berk.) Sacc.) of the genus Cordyceps in the family Clvaieipiacteae of the kingdom Fungi. It is a complex of the stroma and larval corpses that parasitize the larvae of the insects of the family Bat Moth. It is often found in plateau meadows and alpine shrub vegetation on the shady and semi-shady slopes of mountains, especially in alpine meadows with snow and good drainage. It is mainly distributed in Tibet, Qinghai, Gansu, Sichuan, Yunnan and other places in my country. Due to its special growth mode and unique growth environment as well as its important medicinal value, Cordyceps sinensis has become a rare and precious Chinese medicinal material in my country. In recent years, studies have found that the mycelium obtained by artificial fermentation of the asexual form of Cordyceps sinensis is basically consistent with the chemical composition and pharmacological effects of natural Cordyceps sinensis, and can be used to replace natural Cordyceps sinensis to produce Cordyceps products and make up for the shortage of natural resources.

[0003] Taking into account the scarcity and high price of natural Cordyceps sinensis, obtaining Cordyceps sinensis by artificial fermentation has a higher cost-effectiveness ratio. However, the traditional artificial Cordyceps sinensis fermentation method has the disadvantages of long fermentation cycle, low bacterial yield, low content of important substances such as adenosine, etc. Therefore, it is extremely important to optimize the nutrients required for fermentation and improve the fermentation efficiency of Chinese Hirsutella serrata. The present invention aims to propose a fermentation method of Chinese Hirsutella serrata, further improve the fermentation efficiency of Chinese Hirsutella serrata, and increase the bacterial yield and the content of medicinal substances such as adenosine, uridine, mannitol and the like. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a liquid fermentation culture method of Hirsutella sinensis, aiming to solve the problems of long fermentation cycle, low bacterial yield and low yield of medicinal substances in traditional Hirsutella sinensis fermentation.

[0005] In order to solve the above technical problems, the technical solution of the present invention is:

[0006] A rapid deep liquid fermentation culture method for Hirsutella sinensis comprises primary seed shake flask culture, secondary seed liquid culture and seed liquid fermentation tank culture, characterized in that betaine, choline chloride, L-carnitine and cobalamin are added to the fermentation medium used in the fermentation tank culture.

[0007] Preferably, the betaine content in the fermentation medium is ≤0.3%, the choline chloride content is ≤0.3%, the L-carnitine content is ≤0.1%, and the cobalamin content is ≤0.02%. It is further preferred that the betaine content in the fermentation medium is 0.1-0.3%, the choline chloride content is 0.1-0.3%, the L-carnitine content is 0.05-0.2%, and the cobalamin content is 0.002-0.006%. It is further preferred that the betaine content in the fermentation medium is 0.1%, the choline chloride content is 0.1%, the L-carnitine content is 0.05%, and the cobalamin content is 0.003-0.005%. It is most preferred that the betaine content in the fermentation medium is 0.1%, the choline chloride content is 0.1%, the L-carnitine content is 0.05%, and the cobalamin content is 0.005%.

[0008] Preferably, the fermentation medium comprises: 1-2.5% glucose, 0.2-1.5% millet flour, 0.5-1% peptone, 0.3% yeast powder, 1-2% bran, 0.3-0.5% potassium dihydrogen phosphate, 0.2-0.3% magnesium sulfate heptahydrate, 0.03-0.06% defoamer, betaine content ≤0.3%, choline chloride content ≤0.3%, L-carnitine content ≤0.1%, cobalamin content ≤0.02%, and the pH value is adjusted to 6.0-6.5.

[0009] The most preferred fermentation medium is 2% glucose, 0.6% millet flour, 0.5% peptone, 0.3% yeast powder, 2% bran, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.03% defoamer, 0.1% betaine, 0.1% choline chloride, 0.05% L-carnitine, and 0.005% cobalamin, and the pH value is adjusted to 6.0.

[0010] The preferred inoculation amount is 20%, and the glucose concentration in the tank is controlled at ≤0.5%.

[0011] The first-level seed shake flask culture is to inoculate the mycelium of Chinese Hirsutella spp. on the solid culture medium into the first-level seed shake flask, culture at 180rpm and 18-20°C for 22-28h. When the culture temperature is lower than 18°C, the culture time is close to 28h, and the higher the temperature, the shorter the culture time, but the culture temperature should not exceed 22°C.

[0012] The secondary seed liquid culture is to put all the obtained primary seed liquid into the seed tank for seed liquid culture, and culture at 20°C for 16-22h to obtain the secondary seed liquid. The volume of the primary seed shake flask culture is much smaller than the volume of the secondary seed tank culture (volume ratio is 3-5%). At this time, after all the primary seed liquid is put into the secondary seed tank, the bacteria can quickly enter the logarithmic growth phase, speed up the seed culture, and reduce the cost. The culture temperature shall not be lower than the culture temperature of the primary seed, but should not exceed 22°C.

[0013] Preferably, the seed liquid fermentation tank culture is to introduce the obtained secondary seed liquid into the fermentation tank and culture it at 18-22° C. for 48-55 hours.

[0014] The culture medium used in the first-level seed shaking flask culture is: 1.25% glucose, 0.5% milk powder, 1% millet powder, 1.4% bran, 0.3% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, 0.1% betaine, 0.002% cobalamin, 0.03% defoaming agent, and the pH value is adjusted to 6.2; organic nitrogen sources such as rice powder, peptone, yeast powder, etc. can also be used.

[0015] The secondary seed liquid culture uses an airlift fermentation tank with a ventilation ratio of 1:0.5vvm and a tank pressure of 0.03Mpa; the culture medium used is: 1.25% glucose, 0.5% milk powder, 1% millet powder, 1.4% bran, 0.4% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, 0.1% betaine, 0.1% choline chloride, 0.0025% cobalamin, 0.03% defoaming agent, and the pH value is adjusted to 6.2.

[0016] The preferred seed liquid fermentation tank culture uses an airlift fermentation tank with a ventilation ratio of 1:0.3-1.5vvm and a tank pressure of 0.02-0.06Mpa. The seed liquid inoculation amount is 15-20% (volume ratio). Therefore, it is necessary to determine the seed liquid culture scale according to the final fermentation scale. The fermentation temperature is 18-22°C, and 20°C is preferably used. The fermentation can be carried out under double-stage temperature control, that is, the temperature in the early stage of fermentation is controlled at 20-22°C, and the temperature in the later stage of fermentation is controlled at 18-20°C. The fermentation culture is carried out for 48-55h, and it is determined whether to continue the fermentation according to the growth of the bacterial cell. If the growth of the bacterial cell stops and the bacterial cell amount is stable, the fermentation is terminated.

[0017] After the initial sugar in the fermentation medium is exhausted, 80% glucose solution is fed to maintain the glucose concentration in the fermentation broth at 0.3-1% during the fermentation process.

[0018] Preferably, the fermentation culture uses an airlift fermenter with a ventilation ratio of 1:0.3-1.5vvm and a tank pressure of 0.02-0.06Mpa.

[0019] Preferably, after the initial sugar in the fermentation medium is exhausted, 80% glucose solution is added to maintain the glucose concentration in the fermentation liquid at 0.3-1% during the fermentation process, and the glucose concentration in the tank is detected using an SBA biosensor.

[0020] It is particularly noted that the culture media have all been sterilized (121° C., 20 min). The “%” in the present invention refers to mass percentage unless otherwise specified.

[0021] Beneficial effects:

[0022] 1. Compared with natural Cordyceps sinensis, the use of biological fermentation to produce Chinese Cordyceps sinensis has greatly increased the yield and solved the problems of resource shortage, poor growth environment, high price and insufficient supply.

[0023] 2. The traditional Chinese Hirsutospore fermentation process has a long fermentation cycle (usually 1 month), low equipment utilization, high cost and low efficiency. The entire cultivation process of this process is only 5 days and the fermentation process is only 50 hours, which greatly saves time costs.

[0024] 3. The traditional fermentation process has a low yield of medicinal products such as adenosine, which is one of the substances with the greatest medicinal value in Chinese Hirsutospore. In addition to adenosine, the yield levels of substances such as mannitol and amino acids are also low. This process greatly increases the content of medicinal substances and the yield of the bacteria is also greatly improved, which undoubtedly improves the efficiency of fermentation.

[0025] 4. Betaine can be used as a regulator of cell osmotic pressure, which can alleviate the damage to cells caused by fermentation broth under high sugar and high salt conditions; it can also be used as a protective group for intracellular components, including stress-sensitive proteins and enzymes; it can also be used as a methyl donor to promote rapid cell growth and accelerate cell metabolism.

[0026] 5. Choline is a component of all biological membranes. The addition of choline chloride can greatly increase the growth and metabolic rate of bacteria. At the same time, as a precursor of acetylcholine, it plays an important regulatory role in the growth and development of many organisms. In addition, it can also be used as a methyl donor. One molecule of choline chloride contains 3 active methyl groups, which can promote the biosynthesis of organic acids.

[0027] 6. L-carnitine greatly increases the maximum oxygen consumption of bacteria, reduces the excessive oxidation of bacterial cells by dissolved oxygen in the fermentation liquid, reduces the damage of superoxide and peroxyl free radicals to bacteria, and slows down the aging of bacteria. It can also promote the metabolism of lipids by bacteria and indirectly promote energy generation, accelerating the growth and reproduction of bacteria.

[0028] 7. As a cofactor of methyltransferase, cobalamin can participate in the synthesis of amino acids, thymidine and other substances, and can also promote protein biosynthesis and increase the maximum bacterial mass. Cobalt ions can also act as a cofactor of some enzymes, participate in cell metabolism, and increase the growth and reproduction rate of bacteria.

[0029] The fermentation method can achieve the purposes of accelerating bacterial growth and reproduction, increasing biomass, increasing the yield of products such as adenosine, shortening fermentation time, improving equipment utilization, saving costs, etc., and has the advantages of being environmentally friendly and having high economic benefits. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art.

[0031] The Chinese Hirsutella strain used in the following examples is Cordyceps sinensis, which was deposited at the General Microbiological Center of China Microorganism Culture Collection Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on October 29, 2020, and its deposit number is CGMCC No. 20759.

[0032] Example 1

[0033] The present invention aims to illustrate the fermentation process of Hirsutella sinensis in the present invention.

[0034] First, the seed shake flask culture is carried out to obtain the first-level seed liquid. Specifically, the Chinese Hirsutosporium mycelium on the solid culture medium is scratched with an inoculation loop, a blade or other tools, and then placed in a first-level seed shake flask for shaker shaking culture in a sterile environment. The shaker speed is 180rpm, the culture temperature is 18-20℃, and the culture time is 22-28h. At this time, attention should be paid to the relationship between the culture temperature and the culture time. When the culture temperature is low (18℃), the culture time is close to 28h. The higher the temperature, the shorter the culture time. The culture temperature should not exceed 22℃, and finally the first-level seed liquid is obtained.

[0035] Then, the seed tank culture is carried out to obtain the secondary seed liquid. Specifically: all the first-level seed liquid is transferred into the seed tank for seed liquid culture, and cultured at 20°C for 16-22 hours to obtain the secondary seed liquid. The volume of the first-level seed shake flask culture should be much smaller than the volume of the second-level seed tank culture (the volume ratio is 3-5%). At this time, after all the first-level seed liquid is transferred into the second-level seed tank, the bacteria can quickly enter the logarithmic growth phase, speeding up the seed culture and reducing costs. The culture temperature must not be lower than the culture temperature of the first-level seed, but should not exceed 22°C.

[0036] Finally, the fermentation tank is used for fermentation culture, and the secondary seed liquid is connected to the fermentation tank, and the inoculation amount is 15-20% (volume ratio). The fermentation temperature is 18-22°C, preferably 20°C, and the temperature can be controlled at two stages, that is, the temperature is controlled at 20-22°C in the early and middle stages of fermentation, and the temperature is controlled at 18-20°C in the late stage of fermentation. The fermentation culture is carried out for 48-55 hours, and whether to continue the fermentation is determined according to the growth of the bacteria. If the growth of the bacteria stops and the amount of bacteria is stable, the fermentation is terminated.

[0037] The primary seed shake flask culture medium described in the above steps is: 1.25% glucose, 0.5% milk powder, 1% millet powder, 1.4% bran, 0.3% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, 0.1% betaine, 0.002% cobalamin, 0.03% defoamer, and the pH value is adjusted to 6.2. Organic nitrogen sources such as rice flour, peptone, and yeast powder can also be used.

[0038] The secondary seed liquid culture uses an airlift fermentation tank with a ventilation ratio of 1:0.5vvm, a tank pressure of 0.03Mpa, and a seed culture medium comprising: 1.25% glucose, 0.5% milk powder, 1% millet flour, 1.4% bran, 0.4% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, 0.1% betaine, 0.1% choline chloride, 0.0025% cobalamin, 0.03% defoaming agent, and the pH value is adjusted to 6.2.

[0039] The fermentation culture uses an airlift fermentation tank with a ventilation ratio of 1:0.3-1.5vvm, a tank pressure of 0.02-0.06Mpa, a fermentation medium comprising: 1-2.5% glucose, 0.2-1.5% millet flour, 0.5-1% peptone, 0.2-0.3% yeast powder, 1-2% bran, 0.3-0.5% potassium dihydrogen phosphate, 0.2-0.3% magnesium sulfate heptahydrate, 0.03-0.06% defoamer, betaine content ≤0.3%, choline chloride content ≤0.3%, L-carnitine content ≤0.1%, cobalamin content ≤0.02%, and a pH value of 6.0-6.5 is adjusted.

[0040] During the fermentation process, when the initial sugar in the culture medium is exhausted, 80% glucose solution is added to maintain the glucose concentration in the fermentation liquid at 0.3-1% during the fermentation process, and the glucose concentration in the tank is detected using an SBA biosensor.

[0041] Example 2

[0042] The present examples are intended to illustrate the fermentation of Hirsutella sinensis without adding functional substances such as betaine, choline chloride, L-carnitine, and cobalamin.

[0043] Specifically, the fermentation operation method in Example 1 is used, the shake flask medium and the seed tank medium are the same as in Example 1, and the fermentation temperature is 20° C. The difference is that the fermentation medium is 2% glucose, 0.6% millet powder, 0.5% peptone, 0.3% yeast powder, 2% bran, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.03% defoamer, the pH value is adjusted to 6.0, the inoculation amount is 20%, and the glucose concentration in the tank is controlled at ≤0.5%.

[0044] Taking the fermentation cycle, bacterial yield, adenosine content, uridine content, guanosine content, mannitol content and amino acid content as indicators, the results are shown in Table 1:

[0045] Table 1 Fermentation results of Hirsutella chinensis without adding functional substances

[0046]

[0047] Example 3

[0048] The present examples are intended to illustrate the effect of betaine on the fermentation of Hirsutella chinensis.

[0049] Specifically, the fermentation operation method in Example 1 is used, the shake flask medium and the seed tank medium are the same as in Example 1, and the fermentation temperature is 20° C. The difference is that the fermentation medium is 2% glucose, 0.6% millet flour, 0.5% peptone, 0.3% yeast powder, 2% bran, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.03-0.06% defoamer, 0-0.2% betaine (specifically 0%, 0.05%, 0.1%, 0.2%), 0.1% choline chloride, 0.02% L-carnitine, 0.003% cobalamin, the pH value is adjusted to 6.0, the inoculation amount is 20%, and the glucose concentration in the tank is controlled at ≤0.5%.

[0050] The usefulness of the present invention was tested using the fermentation period, bacterial yield, adenosine content, uridine content, guanosine content, mannitol content, and amino acid content as indicators. The results are shown in Table 2:

[0051] Table 2 Effect of betaine on fermentation of Hirsutella chinensis

[0052]

[0053]

[0054] It can be seen from the table that betaine has a significant positive effect on cordycepin and amino acid content, and also has a promoting effect on other indicators. A comprehensive analysis shows that the 0.1% addition amount is the best addition amount for the promotion effect. When the addition amount reaches 0.2%, the indicators do not show a significant improvement.

[0055] Example 4

[0056] The present examples are intended to illustrate the effect of choline chloride on the fermentation of Hirsutella chinensis.

[0057] Specifically, the fermentation operation method in Example 1 is used, the shake flask medium and the seed tank medium are the same as in Example 1, and the fermentation temperature is 20° C. The difference is that the fermentation medium is 2% glucose, 0.6% millet flour, 0.5% peptone, 0.3% yeast powder, 2% bran, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.03% defoamer, 0.1% betaine, 0-0.2% choline chloride (specifically 0%, 0.05%, 0.1%, 0.2%), 0.02% L-carnitine, 0.003% cobalamin, the pH value is adjusted to 6.0, the inoculation amount is 20%, and the glucose concentration in the tank is controlled at ≤0.5%.

[0058] The usefulness of the present invention was tested using the fermentation period, bacterial yield, adenosine content, uridine content, guanosine content, mannitol content, and amino acid content as indicators. The results are shown in Table 3:

[0059] Table 3 Effect of choline chloride on the fermentation of Hirsutella chinensis

[0060]

[0061] As shown in Table 3, choline chloride has a positive effect on the bacterial yield and adenosine content, and the contents of uridine and cordycepin also increase to a certain extent. Other indicators also improve to a certain extent. Comprehensive analysis shows that the addition of 0.1% is more competitive.

[0062] Example 5

[0063] The present examples are intended to illustrate the effect of L-carnitine on the fermentation of Hirsutella chinensis.

[0064] Specifically, the fermentation operation method in Example 1 is used, the shake flask medium and the seed tank medium are the same as in Example 1, and the fermentation temperature is 20° C. The difference is that the fermentation medium is 2% glucose, 0.6% millet flour, 0.5% peptone, 0.3% yeast powder, 2% bran, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.03% defoamer, 0.1% betaine, 0.1% choline chloride, 0-0.1% L-carnitine (specifically 0%, 0.02%, 0.05%, 0.1%), 0.003% cobalamin, the pH value is adjusted to 6.0, the inoculation amount is 20%, and the glucose concentration in the tank is controlled at ≤0.5%.

[0065] The usefulness of the present invention was tested by taking the fermentation period, bacterial yield, adenosine content, uridine content, guanosine content, mannitol content and amino acid content as indicators. The results are shown in Table 4:

[0066] Table 4 Effect of L-carnitine on the fermentation of Hirsutella chinensis

[0067]

[0068] As shown in Table 4, L-carnitine has a positive effect on promoting the uridine content and adenosine content. The appropriate addition amount (0.05%) also significantly improves the bacterial yield, and other indicators are also improved to a certain extent. It is worth noting that when the addition amount is too high (0.1%), the bacterial yield is reduced. Therefore, a comprehensive analysis shows that the optimal addition amount is 0.05%.

[0069] Example 6

[0070] The present examples are intended to illustrate the effects of cobalamin on the fermentation of Hirsutella chinensis.

[0071] Specifically, the fermentation operation method in Example 1 is used, the shake flask medium and the seed tank medium are the same as in Example 1, and the fermentation temperature is 20° C. The difference is that the fermentation medium is 2% glucose, 0.6% millet flour, 0.5% peptone, 0.3% yeast powder, 2% bran, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.03% defoamer, 0.1% betaine, 0.1% choline chloride, 0.05% L-carnitine, 0-0.1% cobalamin (specifically 0%, 0.002%, 0.005%, 0.01%), the pH value is adjusted to 6.0, the inoculation amount is 20%, and the glucose concentration in the tank is controlled at ≤0.5%.

[0072] The usefulness of the present invention was tested using the fermentation period, bacterial yield, adenosine content, uridine content, guanosine content, mannitol content, and amino acid content as indicators. The results are shown in Table 5:

[0073] Table 5 Effect of cobalamin on the fermentation of Hirsutella chinensis

[0074]

[0075] As shown in Table 5, cobalamin did not show an extremely significant improvement on a single product, but it promoted the indicators shown in different degrees, which was particularly evident in the bacterial yield and adenosine content. Therefore, the effect of cobalamin on the fermentation of Chinese Hirsutella is global, making the metabolism of the bacteria more active and the content of medicinal substances increased. Comprehensive analysis shows that adding 0.005% is more suitable for the fermentation production of Chinese Hirsutella.

[0076] Example 7

[0077] Based on the results obtained in Examples 3-6, the experimental results of Example 2 were used as a control group to verify the effects of the functional substances on the fermentation of Hirsutella chinensis when they were added in the optimal amounts.

[0078] Specifically, the fermentation operation method in Example 1 is used, the shake flask medium and the seed tank medium are the same as in Example 1, and the fermentation temperature is 20°C. The difference is that the fermentation medium is 2% glucose, 0.6% millet powder, 0.5% peptone, 0.3% yeast powder, 2% bran, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.03% defoamer, 0.1% betaine, 0.1% choline chloride, 0.05% L-carnitine, 0.005% cobalamin, the pH value is adjusted to 6.0, the inoculation amount is 20%, and the glucose concentration in the tank is controlled at ≤0.5%. The results are shown in Table 6:

[0079] Table 6: Fermentation results of Hirsutella chinensis when functional substances

[0080]

[0081] Compared with the control group, the fermentation cycle was shortened by 7 hours, the cell yield increased by 45.5%, adenosine increased by 149.6%, uridine increased by 17.7%, cordycepin increased by 433.3%, mannitol increased by 7%, and the amino acid content increased by 14.0%. Therefore, it can be seen that the addition of betaine, choline chloride, L-carnitine and cobalamin has a positive promoting significance for the fermentation production of Chinese Hirsutella spore, and has a positive effect on expanding the production scale, reducing fermentation costs, shortening the fermentation cycle, and improving the economic benefits of the industry.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. Any improvements and modifications made by a person skilled in the art based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. A rapid submerged liquid fermentation culture method for Hirsutella sinensis, comprising primary seed shake flask culture, secondary seed liquid culture and seed liquid fermentation tank culture, characterized in that The fermentation medium used in the fermentation tank culture is added with betaine, choline chloride, L-carnitine and cobalamin; the first-level seed shake flask culture is to inoculate the Chinese Hirsutosporium mycelium on the solid culture medium into the first-level seed shake flask, culture at 180r / min, 18-20°C for 22-28 hours; the second-level seed liquid culture is to inoculate all the first-level seed liquid into the seed tank for seed liquid culture, culture at 20°C for 16-22 hours, and obtain the second-level seed liquid; the seed liquid fermentation tank culture is to inoculate the obtained second-level seed liquid into the fermentation tank, culture at 18-22°C for 48-55 h; the fermentation medium is: glucose 1-2.5%, millet flour 0.2-1.5%, peptone 0.5-1%, yeast powder 0.3%, bran 1-2%, potassium dihydrogen phosphate 0.3-0.5%, magnesium sulfate heptahydrate 0.2-0.3%, defoamer 0.03-0.06%, betaine content ≤0.3%, choline chloride content ≤0.3%, L-carnitine content ≤0.1%, cobalamin content ≤0.02%, and the pH value is adjusted to 6.0-6.5; the medium used for the first-level seed shake flask culture is: glucose 1.25%, milk powder 0.5%, millet flour 1%, bran 1.4%, potassium dihydrogen phosphate 0.3%, magnesium sulfate heptahydrate 0.3%, betaine 0.1%, cobalamin 0.002%, defoamer 0.03%, and the pH value is adjusted to 6.2; the second-level seed liquid culture uses an airlift fermenter with a ventilation ratio of 1:0.5 vvm, tank pressure 0.03 Mpa; the culture medium used is: 1.25% glucose, 0.5% milk powder, 1% millet powder, 1.4% bran, 0.4% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, 0.1% betaine, 0.1% choline chloride, 0.0025% cobalamin, 0.03% defoamer, and the pH value is adjusted to 6.2; the seed liquid fermentation tank culture uses an airlift fermentation tank with a ventilation ratio of 1:0.3-1.5 vvm and a tank pressure of 0.02-0.06 Mpa; the seed liquid inoculation amount is 15-20%, and after the initial sugar in the fermentation medium is exhausted, 80% glucose solution is added to maintain the glucose concentration in the fermentation liquid at 0.3-1% during the fermentation process.

2. The rapid submerged liquid fermentation culture method of Hirsutella sinensis according to claim 1, characterized in that The fermentation medium contains 0.1% betaine, 0.1% choline chloride, 0.05% L-carnitine and 0.002-0.005% cobalamin.

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