A high-efficiency liquid fermentation production method of antrodia cinnamomea mycelium

Abstract

The application discloses a kind of high-efficiency liquid fermentation production methods of Antrodia cinnamomea mycelium, it is related to microbial fermentation technical field, including strain activation and seed culture, mycelium proliferation stage, metabolite induction stage and harvest;Proliferation stage is added to the proliferation culture medium 20~60 μm of micro-particle 0.5~3.0 g / L, the mycelium ball diameter is controlled at 0.5~2.0 mm, and using dissolved oxygen-supplement linkage control flow carbon source, make residual glucose concentration maintain at 0.1~1.0 g / L;When mycelium dry weight reaches 15~20 g / L, enter induction stage, flow and add methyl jasmonate and chitinous oligosaccharide containing synergist solution, and control dissolved oxygen to be 5~15% low dissolved oxygen stress level, the present application is improved mass transfer by micro-particle mediated mycelium ball shape control, combined with double-inductor synergistic activation triterpenoid synthesis pathway and low dissolved oxygen stress enhances cell response, so that fermentation cycle is shortened, mycelium dry weight biomass reaches 28~35 g / L, and triterpenoid content reaches 25~35 mg / g dry weight.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and more specifically, to a method for the efficient liquid fermentation production of Antrodia camphorata mycelium. Background Technology

[0002] Antrodia camphorata is a rare medicinal fungus endemic to Taiwan, my country. It parasitizes the inner wall of the trunk of the Cinnamomum kanehirae tree. Its fruiting body is rich in triterpenoids, polysaccharides, adenosine, and other active ingredients, and has effects such as liver protection, anti-tumor, anti-inflammation, and immune regulation. Due to the slow growth of wild Antrodia camphorata, its host specificity, and the scarcity of wild resources, artificial cultivation has a long cycle. Liquid deep fermentation has become the main method to achieve large-scale and standardized production of Antrodia camphorata mycelium.

[0003] The existing liquid fermentation technology for Antrodia camphorata has the following drawbacks: (1) Antrodia camphorata is a wood-rotting filamentous fungus. In liquid culture, it is easy to form large and dense mycelial balls with a diameter of more than 5 mm. The mycelial balls are prone to forming hypoxic zones, which leads to mass transfer obstruction and low secondary metabolic activity. The liquid fermentation cycle is usually as long as 15 to 25 days. (2) Existing technologies mostly adopt one-step fermentation under constant conditions. When the target is mycelial biomass, the triterpenoid content is low, usually only 5~10 mg / g dry weight. When the accumulation of triterpenoids is promoted by stress methods such as nutrient restriction, high osmotic pressure, and hydrogen peroxide, the stress also inhibits mycelial proliferation, resulting in a decrease in biomass and a limited increase in overall triterpenoid yield. (3) When chemical inducers such as methyl jasmonate are used alone, the dense structure of the mycelial ball of filamentous fungi prevents the inducer from penetrating into the ball. The dosage of inducer is large and the effect is slow. Furthermore, high concentrations of inducer are toxic to cells. (4) Existing technologies lack quantitative analysis of the expression of key enzyme genes in the triterpenoid synthesis pathway, making it difficult to verify the activation effect of the fermentation process on the triterpenoid synthesis pathway at the molecular level. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the technical defects of existing liquid fermentation of Antrodia camphorata, such as low mass transfer efficiency and long fermentation cycle due to excessively dense mycelial balls, contradiction between cell proliferation and triterpenoid accumulation, and low utilization rate of inducers. The invention provides a high-efficiency liquid fermentation production method of Antrodia camphorata with high mycelial biomass, high triterpenoid content, short fermentation cycle, and easy industrial scale-up.

[0005] To achieve the above objectives, an embodiment of the present invention provides the following technical solution: A method for high-efficiency liquid fermentation production of Antrodia camphorata mycelium includes the following steps: (1) Activation and seed culture of fungi: After the Antrodia camphorata fungi were activated by inoculating them on PDA slant, they were successively cultured in a primary shake flask and a secondary seed tank to obtain seed liquid; (2) Mycelial proliferation stage: The seed liquid was inoculated into the proliferation medium of the fermenter at 5-10% (v / v) and cultured at 25-28℃, aeration rate of 1.0-1.5 vvm and dissolved oxygen ≥30%. At the same time, 0.5-3.0 g / L of microparticles with a particle size of 20-60 μm was added to the culture medium to control the diameter of mycelial balls to 0.5-2.0 mm. The dissolved oxygen-feed linkage control of the fed carbon source was used to maintain the residual glucose concentration in the fermentation broth at a low level of 0.1-1.0 g / L. The culture was carried out for 4-7 days until the mycelial dry weight reached 15-20 g / L. (3) Metabolite induction stage: When the mycelial dry weight reaches 15~20 g / L, switch to induction conditions: add a co-inducer solution containing methyl jasmonate 50~150 μmol / L and chitin oligosaccharide 50~200 mg / L, while controlling dissolved oxygen at a low dissolved oxygen stress level of 5~15%, reducing the stirring speed, and continuing to culture for 3~5 days; (4) Harvesting: Place in a tank, separate the solid and liquid to obtain mycelium, and dry to obtain Antrodia camphorata mycelium product rich in triterpenoids.

[0006] As a further improvement of the present invention, the microparticles are one or more of talc, diatomaceous earth, titanium dioxide, and calcium carbonate, with a particle size of 20-60 μm and an addition amount of 0.5-3.0 g / L. The microparticles act as nucleating agents and mycelial growth sites, causing mycelia to form numerous small, loose, and uniform mycelial balls around the particles. The diameter of the mycelial balls is stably controlled at 0.5-2.0 mm, expanding the gas-liquid-solid three-phase interface and improving the oxygen transfer coefficient.

[0007] As a further improvement of the present invention, the dissolved oxygen-feed linkage control is as follows: a sudden increase in dissolved oxygen is used as a signal of carbon source depletion, automatically triggering the start of the carbon source feed pump; when the dissolved oxygen is below 30%, the stirring speed or aeration rate is increased in linkage. This control maintains the residual glucose at a low level of 0.1~1.0 g / L, avoiding the accumulation of byproducts from high concentrations of glucose, and ensuring that the cells continue to proliferate at a high specific growth rate.

[0008] As a further improvement of the present invention, the chitin oligosaccharide is obtained by chitinase-directed degradation of the cell wall of *Antrodia camphorata* fruiting body or mycelium, with a degree of polymerization of 2-6, and forms a synergistic inducer composition with methyl jasmonic acid. Methyl jasmonic acid acts as a signaling molecule to activate the transcriptional expression of key enzymes in triterpenoid synthesis, while chitin oligosaccharide acts as a pathogen-associated molecular pattern to stimulate cellular defensive secondary metabolic responses. The two synergistically activate triterpenoid synthesis through different signaling pathways.

[0009] As a further improvement of the present invention, the low dissolved oxygen stress control is as follows: the ventilation rate is reduced to 0.3~0.6 vvm, and the dissolved oxygen is maintained at 5~15%. Low dissolved oxygen acts as an environmental stress signal to induce the cells to produce an antioxidant defense response, which synergistically promotes the synthesis of triterpenes with chemical inducers.

[0010] As a further improvement of the present invention, the proliferation medium formula is as follows: glucose 30-50 g / L, yeast extract 5-10 g / L, peptone 3-5 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.5 g / L, pH 5.0-5.5; the nitrogen source concentration in the medium added during the induction phase is 40-60% of the nitrogen source concentration during the proliferation phase, so as to create a moderately elevated carbon-nitrogen ratio environment, which is conducive to the shift of secondary metabolism.

[0011] Compared with the prior art, the advantages of this invention are: (1) Microparticle-mediated precise control of mycelial ball morphology and dissolved oxygen-feed linkage control reduce the diameter of mycelial balls from 3~8 mm in conventional technology to 0.5~2.0 mm, with no obvious hypoxic necrosis zone inside the balls, increasing the oxygen transfer coefficient by more than 40%, shortening the fermentation cycle from conventional 15~25 days to 7~12 days, and increasing the mycelial dry weight biomass from conventional 12~15 g / L to 28~35 g / L; (2) The two-stage induction strategy of dual inducer synergy and low dissolved oxygen stress increased the content of triterpenoids from the conventional 5~10 mg / g dry weight to 25~35 mg / g dry weight; since the induction was carried out after the high-density proliferation of bacteria, the inhibition of biomass by stress was avoided, and the total yield of triterpenoids was increased to 6~9 times that of conventional technology. (3) The uniform and fine mycelial ball structure constructed by microparticle morphology regulation improves oxygen and material transfer efficiency, creates mass transfer conditions for rapid infiltration of inducers and efficient cell response, shortens the induction response time by about 50%, and reduces the amount of inducer to less than 1 / 3 of the conventional amount of single induction to achieve higher induction intensity. (4) The two inducers synergistically activate triterpenoid synthesis through different signaling pathways. Low dissolved oxygen stress enhances the cell’s response sensitivity to the inducers. The expression level of the key enzyme in triterpenoid synthesis, squalene epoxygenase, is upregulated by more than 4.5 times, and the expression level of mevalonate pyrophosphate decarboxylase is upregulated by more than 3.8 times. (5) This invention combines microparticle morphology regulation, dual inducer synergy and low dissolved oxygen stress, and simultaneously increases biomass and triterpenoid content. The total triterpenoid yield is higher than the sum of the effects of each technology used alone. Moreover, each step can be achieved using conventional industrial fermentation equipment, which is easy to scale up for production. Attached Figure Description

[0012] Figure 1This is a process flow diagram of the present invention; Figure 2 This is a comparative schematic diagram of the mycelial ball morphology of the present invention; Figure 3 This is a timing diagram of the two-stage dissolved oxygen-feeding linkage control of the present invention; Figure 4 This is a comparison chart of total triterpenoid yields in this invention; Figure 5 This is a comparative diagram of the expression of key enzyme genes in this invention. Detailed Implementation

[0013] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0014] Example 1 The strain is Antrodia cinnamomea, which can be any Antrodia cinnamomea strain disclosed in this field (such as Antrodia cinnamomea strains purchased from the China General Microbiological Culture Collection Center or the China Agricultural Microbiological Culture Collection Center, or strains isolated from fruiting bodies).

[0015] (1) Activation of strains: Inoculate fresh PDA slant culture with 4℃ stored Antrodia camphorata slant culture and incubate at 26℃ for 10 days.

[0016] (2) Primary seed culture: Take a block of mycelium with a 5 mm diameter punch and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of seed culture medium. The seed culture medium is: 20 g / L glucose, 4 g / L yeast extract, 3 g / L peptone, 1.0 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, pH 5.2; cultured at 26℃ and 150 r / min in a shaker for 7 days.

[0017] (3) Secondary seed culture: Inoculate 5% (v / v) into a 10 L airlift seed tank containing 5 L of seed culture medium and culture at 26℃ with aeration of 1.2 vvm for 5 days.

[0018] (4) Mycelial proliferation stage: Inoculate 8% (v / v) into a 100 L mechanically stirred fermenter containing 70 L of proliferation medium. The proliferation medium consisted of: 40 g / L glucose, 8 g / L yeast extract, 4 g / L peptone, 1.0 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, pH 5.2; simultaneously, 1.5 g / L talc powder with a particle size of 40 μm was added. Cultivation was carried out at 26℃, with initial stirring at 150 r / min and aeration at 1.2 vvm. Dissolved oxygen-feed linkage control was adopted: when the online dissolved oxygen probe showed a sudden increase to above 60%, 50% (w / v) glucose was automatically added to maintain residual glucose at 0.1~1.0 g / L, and the stirring speed was controlled at 200~300 r / min under dissolved oxygen low-limit linkage. After 5 days of cultivation, the mycelial dry weight reached 18.2 g / L. g / L, microscopic examination revealed mycelial balls with diameters of 0.8~1.6 mm, uniform in size and loosely structured.

[0019] (5) Metabolite induction stage: Starting from day 6, the induction mother liquor was added. The induction mother liquor contained 100 μmol / L methyl jasmonate and 120 mg / L chitin oligosaccharide. The chitin oligosaccharide was prepared by the directional degradation of the cell wall of Antrodia camphorata mycelium by chitinase. The degree of polymerization was 2-6. The preparation method was as follows: chitinase was used to enzymatically hydrolyze the cell wall of Antrodia camphorata mycelium at pH 5.5 and 45℃ for 48 hours. The enzymatic hydrolysate was separated by ultrafiltration to obtain the chitin oligosaccharide component with a degree of polymerization of 2-6. At the same time, the aeration rate was reduced to 0.4 vvm and the stirring was reduced to 120 r / min. The dissolved oxygen was controlled at 8-12%. The induction culture lasted for 4 days.

[0020] (6) Harvesting: The mycelium was collected by plate and frame filter press and dried with hot air at 50℃ to obtain Antrodia camphorata mycelium powder. The mycelium dry weight biomass was measured to be 32.5 g / L, the triterpenoid content was 30.8 mg / g dry weight, and the fermentation cycle was 9 days.

[0021] Molecular-level validation: Samples were taken on day 5 at the end of the proliferation phase and day 9 at the end of the induction phase. Total RNA was extracted from mycelium, reverse transcribed into cDNA, and β-actin was used as an internal reference gene. The expression levels of key triterpenoid synthesis enzyme genes AcSE and AcMvd were detected by qRT-PCR. The results showed that the expression level of AcSE gene was upregulated by 5.6 times at the end of the induction phase and the expression level of AcMvd gene was upregulated by 4.3 times at the end of the proliferation phase.

[0022] Example 2 Except for the use of 2.0 g / L diatomaceous earth with a particle size of 30 μm during the proliferation stage, the process was the same as in Example 1. Results: Mycelial ball diameter was 0.6–1.4 mm, biomass was 31.8 g / L, triterpenoid content was 28.6 mg / g dry weight, AcSE gene expression was upregulated by 5.1-fold, and the cycle lasted 9 days.

[0023] Example 3 Except for adjusting the dosage of methyl jasmonate to 60 μmol / L and chitin oligosaccharide to 200 mg / L during the induction phase, the rest of the results were the same as in Example 1. Results: Biomass was 32.2 g / L, triterpenoid content was 29.5 mg / g dry weight, and AcSE gene expression was upregulated by 4.8 times.

[0024] Example 4 Except for replacing the microparticles with 1.0 g / L calcium carbonate with a particle size of 50 μm and adjusting the temperature during the proliferation stage to 28℃, the rest was the same as in Example 1. Results: Mycelial ball diameter was 1.0~1.8 mm, biomass was 30.6 g / L, triterpenoid content was 27.2 mg / g dry weight, and fermentation cycle was 10 days.

[0025] Comparative Example 1 Conventional one-step fermentation: Seed culture was conducted as in Example 1, inoculated into a 100 L fermenter, and the culture medium was the same as the proliferation medium in Example 1. No microparticles or inducers were added throughout the process, and the culture was kept constant at dissolved oxygen ≥30% for 18 days. Results: Mycelial balls were 3-8 mm in diameter and uneven in size, with a biomass of 13.6 g / L, a triterpenoid content of 7.2 mg / g dry weight, and a total triterpenoid yield of approximately 98 mg / L. The expression level of the AcSE gene gradually decreased after day 7 of fermentation.

[0026] Comparative Example 2 Microparticle morphology regulation was used only, without two-stage induction: the microparticle and feed-in-place technology of step (4) in Example 1 was used only, and the culture was continued under the same proliferation conditions for a total cycle of 9 days without induction. Results: biomass was 33.1 g / L, triterpenoid content was 11.2 mg / g dry weight, total triterpenoid yield was about 371 mg / L, and AcSE gene expression did not show significant upregulation in the later stage of fermentation.

[0027] Comparative Example 3 Induction strategy only, without microparticle regulation: No microparticles were added during the proliferation stage, forming 3-8 mm large mycelial balls. From day 6 onwards, dual inducers and low dissolved oxygen stress were applied as in Example 1. Results: Biomass 12.4 g / L, triterpenoid content 17.5 mg / g dry weight, total triterpenoid yield approximately 217 mg / L.

[0028] Comparative Example 4 Microparticle morphology regulation + methyl jasmonate as a single inducer, without chitin oligosaccharides: The proliferation stage was the same as in Example 1, with the addition of 1.5 g / L talc; the induction stage was performed with only 100 μmol / L methyl jasmonate added, without chitin oligosaccharides, under the same low dissolved oxygen stress conditions as in Example 1. Results: Biomass 30.5 g / L, triterpenoid content 19.8 mg / g dry weight, total triterpenoid yield approximately 604 mg / L, and AcSE gene expression upregulated 2.3-fold.

[0029] Comparative Example 5 Microparticle morphology regulation + dual inducers, no low dissolved oxygen stress: The proliferation stage was the same as in Example 1. During the induction stage, dual inducers were added, with methyl jasmonate 100 μmol / L and chitin oligosaccharide 120 mg / L, but dissolved oxygen was maintained at ≥30% without applying low dissolved oxygen stress. Results: Biomass 31.2 g / L, triterpenoid content 22.6 mg / g dry weight, total triterpenoid yield approximately 705 mg / L.

[0030] Comparative Example 6 Microparticle-free + methyl jasmonate as a single inducer + low dissolved oxygen stress: No microparticles were added during the proliferation stage to form large mycelial balls. During the induction stage, only 100 μmol / L of methyl jasmonate was added to apply low dissolved oxygen stress. Results: Biomass was 14.2 g / L, triterpenoid content was 20.1 mg / g dry weight, total triterpenoid yield was approximately 285 mg / L, and AcSE gene expression was upregulated by 1.9-fold.

[0031] Effect verification The results for each experimental group are shown in the table below: The data above show that the total triterpenoid yield of Comparative Example 4 was 604 mg / L, and that of Comparative Example 3 was 217 mg / L, totaling 821 mg / L. The total triterpenoid yield of Example 1 was 1001 mg / L, higher than the sum of the yields of Comparative Examples 4 and 3. In Example 1, the AcSE gene expression level was upregulated by 5.6-fold, higher than the sum of 2.3-fold in Comparative Example 4 and 2.0-fold in Comparative Example 3. These results indicate that the combination of microparticle morphology regulation, dual-inducer synergy, and low dissolved oxygen stress simultaneously increases biomass and triterpenoid content, shortens the fermentation cycle, and results in superior total triterpenoid yield and key enzyme gene expression levels.

[0032] It should be noted that this invention addresses the shortcomings of the prior art from multiple levels: First, *Antrodia camphorata* readily forms large and dense mycelial balls in liquid culture. The presence of anoxic zones within these balls not only limits the growth of the mycelium itself but also hinders the penetration of inducers into the balls. This invention introduces microparticles of a specific size as nucleating agents and mycelial growth sites during the proliferation stage. This causes the mycelium to form numerous small, loose, and uniform mycelial balls around the particles, reducing the diameter of the mycelial balls from the conventional 3-8 mm to a uniform 0.5-2.0 mm. This morphological regulation fundamentally improves the gas-liquid-solid three-phase mass transfer conditions, increasing the oxygen transfer coefficient by more than 40%, while simultaneously creating the necessary mass transfer basis for the subsequent rapid penetration of inducers.

[0033] Second, existing technologies typically use chemical inducers such as methyl jasmonate alone, activating triterpenoid synthesis solely through the JA signaling pathway, resulting in limited activation intensity. This invention combines methyl jasmonate with chitin oligosaccharides: methyl jasmonate acts as a signaling molecule to activate the transcriptional expression of key enzymes in triterpenoid synthesis (such as squalene epoxidase and mevalonate pyrophosphate decarboxylase); chitin oligosaccharides, as a pathogen-associated molecular model, stimulate defensive secondary metabolic responses through cell surface receptors. Both act on different signaling pathways, producing a synergistic effect, with significantly greater activation intensity than single-pathway activation. Simultaneously, low dissolved oxygen stress, as an environmental signal, enhances the cell's sensitivity to the inducers, forming a synergistic effect with the chemical inducers.

[0034] Third, this invention divides the fermentation process into a mycelial proliferation stage and a metabolite induction stage: during the proliferation stage, no stress or inducers are applied, ensuring that the mycelium proliferates to a high density at the maximum specific growth rate; during the induction stage, dual inducers and low dissolved oxygen stress are applied on the basis of high mycelial density to avoid the inhibition of biomass by stress. This "growth-metabolism decoupling" strategy in the time dimension allows biomass and triterpenoid content to be maximized simultaneously.

[0035] Furthermore, microparticle morphology regulation creates mass transfer conditions for elicitor penetration, shortening the induction response time by approximately 50%. The elicitor dosage can be reduced to less than one-third of the conventional single-elicitor dosage to achieve higher induction intensity. The phased strategy eliminates elicitor toxicity and stress inhibition during the proliferation phase, and combined with microparticle morphology regulation, further accumulates biomass, providing a larger "cell factory" for triterpenoid synthesis during the induction phase. Experimental verification shows that the total triterpenoid yield of the method in this invention (1001 mg / L) is higher than the sum of the total triterpenoid yields of using only microparticle morphology regulation (371 mg / L) and only the induction strategy (217 mg / L) (588 mg / L), and also higher than the sum of the total triterpenoid yields of microparticle morphology regulation + single elicitor (604 mg / L) and no microparticles + single elicitor + low dissolved oxygen stress (285 mg / L) (889 mg / L). This indicates that the present invention is not a simple superposition of various techniques, but rather produces a significant synergistic effect. The expression level of squalene epoxidase, a key enzyme in triterpenoid synthesis, was upregulated by 5.6 times, which was also higher than the sum of Comparative Example 4 (2.3 times) and Comparative Example 3 (2.0 times) (4.3 times), confirming the existence of a synergistic effect at the molecular level.

[0036] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and not restrictive.

[0037] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style of the specification is merely for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A high-efficiency liquid fermentation method for producing Antrodia camphorata mycelium, characterized by, Includes the following steps: (1) Activation and seed culture of strain: After activation, the Antrodia camphorata strain was subjected to primary shake-flask seed culture and secondary seed culture to obtain seed liquid; (2) Mycelial proliferation stage: The seed liquid is inoculated into a proliferation medium containing microparticles at an inoculation rate of 5-10% (v / v) for deep liquid fermentation. The microparticles have a particle size of 20-60 μm and an addition amount of 0.5-3.0 g / L to control the diameter of the mycelial balls to 0.5-2.0 mm. The dissolved oxygen-feed linkage control of the fed carbon source is used to maintain the residual glucose concentration in the fermentation broth at 0.1-1.0 g / L. The culture is carried out at 25-28℃, aeration rate of 1.0-1.5 vvm, and dissolved oxygen ≥30% for 4-7 days until the mycelial dry weight reaches 15-20 g / L. (3) Metabolite induction stage: When the mycelial dry weight reaches 15~20 g / L, add a co-inducer solution containing methyl jasmonate and chitin oligosaccharide to the fermentation broth. The concentration of methyl jasmonate is 50~150 μmol / L, the mass concentration of chitin oligosaccharide is 50~200 mg / L, and the degree of polymerization is 2~6. At the same time, reduce the aeration rate to 0.3~0.6 vvm and reduce the stirring speed to control the dissolved oxygen in the fermentation broth to a low dissolved oxygen stress level of 5~15%. Continue to culture for 3~5 days. (4) Harvest the mycelium, separate the solid and liquid and dry it to obtain Antrodia camphorata mycelium rich in triterpenoids.

2. The high-efficiency liquid-state fermentation production method of the Antrodia cinnamomea mycelium according to claim 1, characterized in that, The microparticles are one or more of talc, diatomaceous earth, titanium dioxide, and calcium carbonate, with a particle size of 20-60 μm and an addition amount of 0.5-3.0 g / L.

3. The high-efficiency liquid-state fermentation method for producing the Antrodia cinnamomea mycelium according to claim 1, wherein, The dissolved oxygen-feeding linkage control in step (2) is as follows: a sudden increase in dissolved oxygen concentration in the fermentation broth is used as a signal of carbon source depletion, triggering the carbon source feed pump to start feeding carbon source; when dissolved oxygen is below 30%, the stirring speed or aeration rate is increased to maintain dissolved oxygen above 30% during the proliferation stage; the carbon source is a 50% (w / v) glucose solution.

4. The high-efficiency liquid-state fermentation production method of the Antrodia cinnamomea mycelium according to claim 1, characterized in that, The chitin oligosaccharide mentioned in step (3) is prepared by chitinase-directed degradation of the cell wall of the fruiting body or mycelium of Antrodia camphorata, with a degree of polymerization of 2 to 6.

5. The method for high-efficiency liquid fermentation production of Antrodia camphorata mycelium according to claim 1, characterized in that, The low dissolved oxygen stress level mentioned in step (3) is achieved by reducing the aeration rate to 0.3~0.6 vvm and reducing the stirring speed, and the dissolved oxygen is maintained at 5~15% during the induction stage.

6. The method for high-efficiency liquid fermentation production of Antrodia camphorata mycelium according to claim 1, characterized in that, In step (3), while switching to the induction stage, feed medium with a nitrogen source concentration of 40-60% of the nitrogen source concentration of the proliferation medium is added to the fermentation broth to increase the carbon-nitrogen ratio of the fermentation broth.

7. The method for high-efficiency liquid fermentation production of Antrodia camphorata mycelium according to claim 1, characterized in that, The formulation of the proliferation medium in step (2) is as follows: glucose 30~50 g / L, yeast extract 5~10 g / L, peptone 3~5 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.5 g / L, pH 5.0~5.5; fermentation temperature 25~28℃, aeration rate 1.0~1.5 vvm, and mycelial proliferation stage culture time 4~7 days.

8. The method for high-efficiency liquid fermentation production of Antrodia camphorata mycelium according to claim 1, characterized in that, The inoculation amount of the seed culture into the proliferation medium is 5-10% (v / v), and the secondary seed culture is carried out in an airlift seed tank for 3-6 days.

9. The method for high-efficiency liquid fermentation production of Antrodia camphorata mycelium according to claim 1, characterized in that, The fermentation cycle of the method shall not exceed 12 days, the mycelial dry weight biomass shall not be less than 28 g / L, and the triterpenoid compound content shall not be less than 25 mg / g dry weight.

10. Antrodia camphorata mycelium prepared by the method according to any one of claims 1 to 9, characterized in that, The mycelium contains no less than 25 mg / g dry weight of triterpenoids, and the expression level of squalene epoxidase gene is upregulated by no less than 4.5 times compared with the proliferation stage. The mycelium is obtained by a liquid deep fermentation method with a fermentation cycle of no more than 12 days. The mycelium is used to prepare Antrodia camphorata functional health food or biopharmaceutical preparations.

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