A fermentation method for Antrodia cinnamomea mycelium and Antrodia cinnamomea fermentation auxiliary composition

Abstract

The present invention discloses a fermentation method for Antrodia camphorata mycelium and an auxiliary composition for fermentation of Antrodia camphorata. The method provided by the present invention comprises the following steps: S1. subjecting an Antrodia camphorata strain to liquid fermentation for 60-120 hours; S2. adding precursors to the fermented Antrodia camphorata strain liquid fermentation broth, and continuing the fermentation for more than 120 hours to obtain Antrodia camphorata mycelium; the precursors being coenzyme Q0, p-hydroxybenzoic acid, linalool, and α-terpineol, and the fermentation broth after adding the precursors having a coenzyme Q0 content of 100-500 mg / L, a p-hydroxybenzoic acid content of 20-300 mg / L, a linalool content of 0.1-0.5 mg / L, and an α-terpineol content of 0.3-0.7 mg / L. This method adds precursor substances of specific composition and concentration to the liquid fermentation broth of Antrodia cinnamomea at a certain time and combines them with each other, which is beneficial to the secondary metabolism of Antrodia cinnamomea and promotes the synthesis of triterpenoid compounds, thereby increasing the yield. Compared with the existing technology, the yield of triterpenoid compounds produced by Antrodia cinnamomea mycelium is increased, the culture cycle is shortened, and it is more suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and fermentation engineering, and particularly relates to a fermentation method of Antrodia cinnamomea mycelium and an Antrodia cinnamomea fermentation auxiliary composition. Background Art

[0002] Antrodia camphorata, also known as Antrodia cinnamomea, red camphor, and Antrodia cinnamomea, belongs to the Polyporaceae family and the genus Ceratopogon. In nature, it parasitizes exclusively on Cinnamomum camphora trees. When first discovered, it was mistakenly thought to be a type of Ganoderma lucidum. It has long been used by the general public to treat difficult ailments such as alcoholism, abdominal pain, and diarrhea. In fact, the variety and content of triterpenes in Antrodia cinnamomea far surpass those in Ganoderma lucidum, making it a veritable king of medicinal herbs.

[0003] Research has shown that the pharmacological effects of Antrodia camphorata stem from its polysaccharides, triterpenes, flavonoids, benzene rings, ubiquinone derivatives, and succinic acid derivatives. Triterpenes are the core pharmacological components of Antrodia camphorata, exhibiting anti-tumor, antioxidant, hypoglycemic, and immunomodulatory properties. Several triterpenes isolated from Antrodia camphorata fruiting bodies exhibit anti-tumor effects through mechanisms such as cytotoxicity and inhibition of tumor cell metastasis. Triterpenes in Antrodia camphorata methanol extracts exhibit significant anti-inflammatory and antioxidant activities, inducing endogenous antioxidant gene expression and limiting excessive production of reactive oxygen species, thereby protecting liver cells from oxidative stress. Antrodia camphorata triterpenoid extracts can effectively improve insulin resistance and increase hepatic insulin sensitivity, showing promising therapeutic potential for diabetes and hyperlipidemia. Some triterpenes can promote T cell proliferation and differentiation, enhancing immune responses. Therefore, Antrodia camphorata triterpenoids possess significant research value and broad application prospects.

[0004] While pharmacological research on triterpenoids from Antrodia camphorata is heating up both domestically and internationally, low production has severely hampered their in-depth study and widespread application. The slow growth and host specificity of Antrodia camphorata have led to an extreme scarcity of wild Antrodia camphorata resources, making it difficult to meet market demand. Consequently, artificial cultivation techniques for Antrodia camphorata have emerged. Currently, these techniques are primarily divided into solid cultivation and liquid fermentation. The former yields fruiting bodies, which produce high triterpenoid yields, but has a long cultivation cycle and high production costs, making large-scale commercial production difficult. The latter primarily produces mycelium, which has a short cultivation cycle, low production costs, and ease of quality control. While this is the primary method for artificial cultivation of Antrodia camphorata, the resulting fermentation product, mycelium, has a low triterpenoid content. Therefore, increasing the yield of Antrodia camphorata triterpenoids in liquid fermentation through various methods has become a hot topic of research in this field.

[0005] Patent publication number CN104212726A (a liquid fermentation medium for Antrodia cinnamomi) discloses another method for improving the production of triterpenoid compounds by Antrodia cinnamomi fermentation, but it uses a large amount of submerged camphor wood chips as raw materials. On the one hand, submerged camphor wood is a precious wood and is expensive. On the other hand, submerged camphor wood grows slowly, making it difficult to achieve stable production or realize industrialization; Patent publication number CN104126412A discloses a method for adding terpineol (pinpineol) to promote the growth of Antrodia cinnamomi liquid fermentation mycelium and high yield of terpenoid compounds, and discloses a method for adding a single precursor substance to promote the growth of Antrodia cinnamomi mycelium and the accumulation of terpenoid compounds during the fermentation process of Antrodia cinnamomi. This method increases the biomass of Antrodia cinnamomi by 6% and the triterpenoid yield by 36% when the fermentation time is 12 days, but its triterpenoid yield is still not high and the culture cycle is long. Therefore, how to develop a method for a liquid fermentation medium of Antrodia cinnamomea that is more suitable for large-scale and continuous production based on existing precursor substances, so as to effectively increase the yield of triterpenoid compounds in Antrodia cinnamomea fermentation, is of great significance to Antrodia cinnamomea fermentation and its upstream and downstream industries. Summary of the Invention

[0006] The object of the present invention is to provide a fermentation method for Antrodia cinnamomea mycelia, which can achieve a higher yield of triterpenoid compounds in Antrodia cinnamomea mycelia obtained after fermentation of Antrodia cinnamomea liquid within a shorter fermentation time than the prior art.

[0007] Another object of the present invention is to provide the fermented mycelium of Antrodia cinnamomea prepared by the above method; another object of the present invention is to provide the use of the fermented mycelium of Antrodia cinnamomea as a raw material for extracting triterpenoid compounds.

[0008] Another object of the present invention is to provide an auxiliary composition for fermentation of Antrodia cinnamomea, which can be used for fermentation of Antrodia cinnamomea mycelia to obtain Antrodia cinnamomea mycelia with a higher yield of triterpenoid compounds.

[0009] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0010] A fermentation method for Antrodia cinnamomea mycelium comprises the following steps:

[0011] S1. Liquid fermentation of Antrodia cinnamomea strain for 60-120h;

[0012] S2. Adding a precursor substance to the fermented liquid fermentation broth of the Antrodia cinnamomea strain, and continuing the fermentation for more than 120h to obtain the Antrodia cinnamomea mycelium;

[0013] The precursor substances are coenzyme Q0, p-hydroxybenzoic acid, linalool and α-terpineol, and the content of coenzyme Q0 in the fermentation broth after adding the precursor substances is 100-500 mg / L, the content of p-hydroxybenzoic acid is 20-300 mg / L, the content of linalool is 0.1-0.5 mg / L, and the content of α-terpineol is 0.3-0.7 mg / L.

[0014] The present invention has found through experiments that after 60-120 hours of liquid fermentation of Antrodia cinnamomea, the precursor substances coenzyme Q0 100-500 mg / L, p-hydroxybenzoic acid 20-300 mg / L, linalool 0.1-0.5 mg / L and α-terpineol 0.3-0.7 mg / L are added together to the Antrodia cinnamomea liquid fermentation liquid and fermented for another 120 hours. The precursor substances can be mutually compatible in the fermentation of Antrodia cinnamomea mycelium, which is beneficial to the synthesis of terpenoid compounds by the secondary metabolism of Antrodia cinnamomea, and greatly promotes the synthesis and accumulation of triterpenoid compounds in the fermentation of Antrodia cinnamomea mycelium. Therefore, the specific precursor substance can be used as an elicitor to promote the production of triterpenoid compounds by fermentation of Antrodia cinnamomea, and is applied to increase the yield of triterpenoid compounds produced by fermentation of Antrodia cinnamomea mycelium.

[0015] Furthermore, in step S2, the precursor is added to the liquid fermentation liquid of the Antrodia cinnamomea strain obtained by fermentation, and the timing of adding the precursor is 72 hours after the Antrodia cinnamomea liquid is fermented in step S1.

[0016] Furthermore, in the fermentation broth after adding the precursor substance in step S2, the content of coenzyme Q0 is 300 mg / L, the content of p-hydroxybenzoic acid is 100 mg / L, the content of linalool is 0.3 mg / L, and the content of α-terpineol is 0.5 mg / L.

[0017] In the present invention, any known Antrodia camphorata strain can be used as raw material, and the existing Antrodia camphorata strains have similar physiological and biochemical properties. As a specific example, in the present invention, the Antrodia camphorata strain with the deposit number of ATCC No. 200183 is used.

[0018] Furthermore, in step S2, the precursor substances are added to the liquid fermentation liquid of the Antrodia cinnamomea strain obtained by fermentation, and the adding method is to first dissolve coenzyme Q0 in the solvent DMSO, and dissolve p-hydroxybenzoic acid, linalool, and α-terpineol in the solvent ethanol; and then add them to the liquid fermentation liquid of Antrodia cinnamomea.

[0019] In the present invention, coenzyme Q0, p-hydroxybenzoic acid, linalool, and α-terpineol are dissolved in solvents respectively, which can facilitate the sufficient diffusion of the precursor substances in the fermentation liquid when they are added to the Antrodia cinnamomea liquid fermentation liquid.

[0020] Furthermore, in the step S1, the Antrodia cinnamomea liquid fermentation is carried out by transferring the Antrodia cinnamomea seed liquid into the basic fermentation medium at an inoculation rate of 15-25% (v / v) to form an Antrodia cinnamomea liquid fermentation liquid, which is then fermented in a shake flask.

[0021] Furthermore, the basic fermentation medium is composed of: 15-25 g / L glucose, 0.5-3 g / L yeast powder, 1-5 g / L magnesium sulfate heptahydrate, 1-5 g / L potassium dihydrogen phosphate, and a pH value of 4-5.

[0022] Furthermore, the preparation method of the Antrodia cinnamomea seed liquid is as follows: inoculating the Antrodia cinnamomea strain with a preservation number of ATCC No. 200183 into a PDA slant culture medium and culturing for 20-30 days; after the cultivation is completed, taking the bacterial blocks on the slant of the slant culture medium, transferring them to a seed culture medium, and culturing them at 25-28° C. and 100-150 rpm with shaking for 12-15 days to form the Antrodia cinnamomea seed liquid.

[0023] Furthermore, the seed culture medium is composed of: 15-25 g / L glucose, 0.5-3 g / L yeast powder, 1-5 g / L magnesium sulfate heptahydrate, 1-5 g / L potassium dihydrogen phosphate, and a pH value of 4-5.

[0024] Furthermore, the fermentation time of the continued fermentation in step S2 is 120 hours.

[0025] The present invention provides the fermented mycelium of Antrodia cinnamomea prepared by the above method.

[0026] The present invention provides the use of the fermented mycelium of Antrodia cinnamomea as a raw material for extracting triterpenoid compounds.

[0027] The present invention also provides an auxiliary composition for fermentation of Antrodia cinnamomea, which comprises the following components in parts by weight: 100-500 parts of coenzyme Q0, 20-300 parts of p-hydroxybenzoic acid, 0.1-0.5 parts of linalool and 0.3-0.7 parts of α-terpineol.

[0028] Furthermore, the auxiliary composition comprises the following components in parts by weight: 300 parts of coenzyme Q0, 100 parts of p-hydroxybenzoic acid, 0.3 parts of linalool and 0.5 parts of α-terpineol.

[0029] The present invention also provides the use of the above-mentioned Antrodia cinnamomea fermentation auxiliary composition as an additive for Antrodia cinnamomea fermentation.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The fermentation method of the mycelium of Antrodia cinnamomea provided by the present invention adds precursor substances to the liquid fermentation liquid of Antrodia cinnamomea at a specific time, so that the content of the precursor substances coenzyme Q0 in the fermentation liquid is 100-500 mg / L, the content of p-hydroxybenzoic acid is 20-300 mg / L, the content of linalool is 0.1-0.5 mg / L, and the content of α-terpineol is 0.3-0.7 mg / L, so that the precursor substances are effectively compatible, which is conducive to the synthesis of triterpenoid compounds by the secondary metabolism of Antrodia cinnamomea, and the yield of triterpenoid compounds produced by fermentation of Antrodia cinnamomea is increased (the yield of triterpenoid compounds can be increased by up to 74% compared with the control group). Compared with the existing technology, the fermentation cycle is shortened (the fermentation cycle can be shortened to 192 hours) and the operation is simple. The precursor substances and precursor substance compositions used in the method provided by the present invention are raw materials that can be industrially produced, are easily available, have low production costs, and can be used to increase the yield of triterpenoid compounds produced by fermentation of Antrodia cinnamomea; the method of the present invention is a method for liquid fermentation culture medium of Antrodia cinnamomea that is more suitable for large-scale and sustainable production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram of the mycelium after filtration of Antrodia cinnamomea liquid fermentation and the liquid fermentation status in a shake flask.

[0033] In the figure, it is marked: a represents the sample of comparative example 11; b represents the sample of comparative example 1; c represents the sample of comparative example 5; d represents the sample of embodiment 6; e represents the sample of embodiment 1; and f represents the sample of embodiment 2. DETAILED DESCRIPTION

[0034] The term "precursor" as used herein refers to a class of compounds that, when added to the fermentation medium, can be directly utilized by microorganisms during biosynthesis to form part of the product's molecular structure, without significantly altering their own structure, but significantly increasing product yield. The term "elicitor" as used herein refers to substances such as polysaccharides, polypeptides, glycoproteins, and small molecules that can rapidly, specifically, and selectively stimulate the expression of specific genes in organisms, affecting secondary metabolic pathways or regulating the biosynthesis of target products. The term "auxiliary composition" as used herein refers to a composition added to the fermentation of Antrodia camphorata, which can assist in the fermentation process and influence the fermentation product.

[0035] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0036] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0037] Example 1

[0038] A fermentation method for Antrodia cinnamomea mycelium comprises the following steps:

[0039] S1. Liquid fermentation of Antrodia camphorata was performed using the Antrodia camphorata strain with ATCC accession number 200183 as the starting strain. In practice, other Antrodia camphorata strains can also be used as starting strains due to their similar physiological and biochemical properties. Liquid fermentation of Antrodia camphorata involved inoculating 20% ​​(v / v) Antrodia camphorata seed liquid into a 500 mL shake flask containing 100 mL of basal fermentation medium to form a liquid fermentation broth. Fermentation was performed at 120 rpm and 26°C for 72 hours.

[0040] Among them, the above-mentioned basic fermentation medium is composed of: 20g / L glucose, 2g / L yeast powder, 3g / L magnesium sulfate heptahydrate, 3g / L potassium dihydrogen phosphate, and a pH value of 4-5. According to actual needs, the composition of the basic fermentation medium is within the following range: 15-25g / L glucose, 0.5-3g / L yeast powder, 1-5g / L magnesium sulfate heptahydrate, 1-5g / L potassium dihydrogen phosphate, and a pH value of 4-5, which can achieve good culture effects.

[0041] The preparation method of the above-mentioned Antrodia cinnamomea seed liquid is as follows: the Antrodia cinnamomea strain is inoculated into a PDA slant culture medium and cultured for 25 days at a culture temperature of 26°C. According to actual needs, the PDA slant culture can be carried out for 20-30 days at a culture temperature of 25-28°C. After the culture is completed, the bacterial block on the slant culture medium is taken and a 1×1 cm 2 The bacterial block is transferred to the seed culture medium, specifically, it is transferred to a 500mL shaking flask containing 100mL liquid seed culture medium, and cultured at 26°C, 120rpm, and shaking for 14 days to form an antrodia cinnamomea seed liquid. According to the needs, the shaking culture conditions are 25-28°C, 100-150rpm, and cultured for 12-15 days to form antrodia cinnamomea seed liquid, thereby achieving the purpose of activating the antrodia cinnamomea strain; the composition of the seed culture medium is: 20g / L glucose, 1g / L yeast powder, 3g / L magnesium sulfate heptahydrate, Potassium dihydrogen phosphate 3g / L, pH 4.5. According to actual needs, the composition of the seed culture medium is in the following range: glucose 15-25g / L, yeast powder 0.5-3g / L, magnesium sulfate heptahydrate 1-5g / L, potassium dihydrogen phosphate 1-5g / L, pH 4-5, all of which can have good culture effects; PDA medium is a universal PDA medium. The PDA medium used in this embodiment is composed of: potato 200g / L, glucose 20g / L, agar 20g / L, pH natural.

[0042] S2. Precursors were added to the fermented liquid fermentation broth of the Antrodia camphorata strain, the precursors being coenzyme Q0, p-hydroxybenzoic acid, linalool, and α-terpineol; the precursor coenzyme Q0 was dissolved in DMSO, and p-hydroxybenzoic acid, linalool, and α-terpineol were dissolved in ethanol; and then added to the Antrodia camphorata liquid fermentation broth, such that the content of coenzyme Q0 in the fermentation broth after adding the precursors was: coenzyme Q0 100 mg / L, p-hydroxybenzoic acid 20 mg / L, linalool 0.1 mg / L, and α-terpineol 0.3 mg / L. Fermentation was continued for 120 h, with three replicates (parallel samples) per group, and the resulting fermentation broth was recorded as the sample in Example 1.

[0043] Example 2

[0044] A fermentation method for Antrodia cinnamomea mycelium comprises the following steps:

[0045] Step S1 was the same as in Example 1, except that the amount of precursor added in step S2 was different. The following steps were performed: Precursors were added to the fermented liquid broth of the Antrodia camphorata strain, where the precursors were Coenzyme Q0, p-hydroxybenzoic acid, linalool, and α-terpineol; the content of Coenzyme Q0 in the fermentation broth after the addition of the precursors was: 300 mg / L Coenzyme Q0, 100 mg / L p-hydroxybenzoic acid, 0.3 mg / L linalool, and 0.5 mg / L α-terpineol. Fermentation was continued for 120 h, with three replicates (parallel samples) performed for each group. The resulting fermentation broth was recorded as the sample in Example 2.

[0046] Example 3

[0047] A fermentation method for Antrodia cinnamomea mycelium comprises the following steps:

[0048] Step S1 was the same as in Example 1, except that the amount of precursor added in step S2 was different. The following steps were performed: Precursors were added to the fermented liquid broth of the Antrodia camphorata strain; the precursors were Coenzyme Q0, p-hydroxybenzoic acid, linalool, and α-terpineol; the content of Coenzyme Q0 in the fermentation broth after the addition of the precursors was: 500 mg / L Coenzyme Q0, 300 mg / L p-hydroxybenzoic acid, 0.5 mg / L linalool, and 0.7 mg / L α-terpineol. Fermentation was continued for 120 h, with three replicates (parallel samples) performed for each group. The resulting fermentation broth was recorded as the sample in Example 3.

[0049] Comparative Examples 1-11

[0050] A fermentation method for Antrodia cinnamomea mycelium comprises the following steps:

[0051] Step S1 is the same as Example 1, except for the type of precursor added in step S2 or the amount of the precursor in the fermentation broth after the addition of the precursor, as shown in Table 1. In Table 1, Comparative Example 1 means that the only precursor added is coenzyme Q0, and the amount of coenzyme Q0 in the fermentation broth after addition is 300 mg / L. The types of precursors and the amounts of precursors in the fermentation broth after addition for the other comparative examples are shown in Table 1. The fermentation broths obtained according to the above method are recorded as samples of Comparative Examples 1-11.

[0052] Table 1 Type and amount of added precursors in Comparative Examples 1-11

[0053]

[0054]

[0055] Examples 4-6

[0056] A fermentation method for Antrodia cinnamomea mycelium comprises the following steps:

[0057] Step S1 is the same as Example 2, except that the timing of adding the precursor in step S2 is different. For example, in Example 4, after 60 hours of liquid fermentation of Antrodia cinnamomea, the same precursor as in Example 2 is added to obtain a fermentation broth. For other examples, see Table 2 for details. The obtained fermentation broths are recorded as samples of Examples 4 to 6, respectively.

[0058] Table 2 Timing of adding precursors in Examples 4-6 and Comparative Examples 12-14

[0059] serial number Timing of adding precursors Example 4 60h Example 5 96h Example 6 120h Comparative Example 12 24h Comparative Example 13 48h Comparative Example 14 150h

[0060] Comparative Examples 12-14

[0061] A method for fermenting Antrodia cinnamomea with the addition of a precursor comprises the following steps:

[0062] Step S1 is the same as Example 1, except that the timing of adding the precursor in step S2 is different, as shown in Table 2. The obtained fermentation broths are recorded as Comparative Example 12 to Comparative Example 14 samples respectively.

[0063] Example 7

[0064] An auxiliary composition for the fermentation of Antrodia cinnamomea, comprising the following components in parts by weight: 100 parts of coenzyme Q0, 20 parts of p-hydroxybenzoic acid, 0.1 parts of linalool, and 0.3 parts of α-terpineol.

[0065] When used, the composition in the above weight ratio is added to the Antrodia cinnamomea liquid fermentation liquid to make the content of coenzyme in the Antrodia cinnamomea liquid fermentation liquid be 0.100 mg / L, the content of p-hydroxybenzoic acid be 20 mg / L, the content of linalool be 0.1 mg / L and the content of α-terpineol be 0.3 mg / L.

[0066] Example 8

[0067] An auxiliary composition for fermentation of Antrodia cinnamomea, comprising the following components in parts by weight: 300 parts of coenzyme Q0, 100 parts of p-hydroxybenzoic acid, 0.3 parts of linalool, and 0.5 parts of α-terpineol.

[0068] When used, the composition in the above weight ratio is added to the Antrodia cinnamomea liquid fermentation liquid to make the content of coenzyme in the Antrodia cinnamomea liquid fermentation liquid be 0.300 mg / L, the content of p-hydroxybenzoic acid be 100 mg / L, the content of linalool be 0.3 mg / L and the content of α-terpineol be 0.5 mg / L.

[0069] Example 9

[0070] An auxiliary composition for fermentation of Antrodia cinnamomea, comprising the following components in parts by weight: 500 parts of coenzyme Q0, 300 parts of p-hydroxybenzoic acid, 0.5 parts of linalool, and 0.7 parts of α-terpineol.

[0071] When used, the composition in the above weight ratio is added to the Antrodia cinnamomea liquid fermentation liquid to make the content of coenzyme in the Antrodia cinnamomea liquid fermentation liquid be 0.500 mg / L, the content of p-hydroxybenzoic acid be 300 mg / L, the content of linalool be 0.5 mg / L and the content of α-terpineol be 0.7 mg / L.

[0072] Experimental Example 1

[0073] 1. Experimental Methods

[0074] After the completion of the above liquid fermentation culture, an equal volume (100 mL) of the fermentation broth of the samples of Examples 1-6 and Comparative Examples 1-14 was first filtered through 8 layers of gauze, then rinsed three times with deionized water and freeze-dried under vacuum to obtain dried Antrodia cinnamomea mycelium. The weight of the dried Antrodia cinnamomea mycelium was weighed, and the biomass (mg / L) was calculated using the weight of the dried Antrodia cinnamomea mycelium and the volume of each sample, and the average biomass of each parallel sample was calculated.

[0075] 2. Experimental Results

[0076] Figure 1 The mycelium and shake flask liquid fermentation state diagram of the samples of Comparative Example 1, Comparative Example 5, Comparative Example 11, Example 1, Example 2, and Example 6 after liquid fermentation filtration of Antrodia cinnamomea. Figure 1It can be seen that the colors of the mycelia and fermentation liquid of different samples of Antrodia cinnamomea have changed. The color of the example sample is darker than that of the comparative example sample, indicating that the addition of the precursor substance has changed the fermentation process of Antrodia cinnamomea.

[0077] The average biomass measurement results of the samples of Examples 1-6 and Comparative Examples 1-14 are shown in Table 3. It can be seen from the biomass of the mycelium of Antrodia cinnamomea in the Examples and Comparative Examples that the addition of one, three, or four of the four precursor substances coenzyme Q0, p-hydroxybenzoic acid, linalool, and α-terpineol after 60 hours of fermentation does not cause a significant change in the mycelial biomass of Antrodia cinnamomea. On the contrary, if the addition time is less than 60 hours (Comparative Examples 12-13), it will affect the growth of Antrodia cinnamomea, resulting in a relatively low mycelial biomass of Antrodia cinnamomea.

[0078] Table 3 Biomass of Antrodia cinnamomea mycelium of Examples 1-6 and Comparative Examples 1-14

[0079]

[0080] Experimental Example 2

[0081] 1. Experimental Methods

[0082] The yields of triterpenoid compounds in the samples of Examples 1-6 and Comparative Examples 1-14 were determined using the following method:

[0083] Detection of triterpene production: The samples of Examples 1-6 and Comparative Examples 1-14 obtained by the above-mentioned liquid fermentation culture were filtered through 8 layers of gauze, rinsed 3 times with deionized water, and then vacuum-freeze-dried to obtain dried Antrodia cinnamomea mycelium; 50 mg of dried Antrodia cinnamomea mycelium was accurately weighed and extracted with 1 mL of methanol for 24 h. After the extraction was completed, the mixture was centrifuged at 12000 rpm for 1 min, and the supernatant was collected as the sample extract; 100 μL of the sample extract was placed in a stoppered ground-mouth test tube and evaporated in a boiling water bath; after evaporation, 300 μL of freshly prepared 5% vanillin-glacial acetic acid solution and 1 mL of perchloric acid were added to the stoppered ground-mouth test tube and the mixture was incubated in a 60°C water bath for 20 min; after the water bath was completed, the stoppered ground-mouth test tube containing the sample was placed in an ice-water mixture to cool, and after cooling, 10 mL of glacial acetic acid was added and mixed, and the absorbance was measured at a wavelength of 550 nm. Using ursolic acid as the reference substance, a standard curve of triterpenoid compounds was drawn, the content of triterpenoid compounds was calculated according to the standard curve, and the average content of triterpenoid compounds in each parallel sample was calculated.

[0084] 2. Experimental Results

[0085] The average triterpene content of the samples of Examples 1-6 and Comparative Examples 1-14 is shown in Table 4. It can be seen from Table 4 that the addition amount of the four precursor substances, coenzyme Q0, p-hydroxybenzoic acid, linalool and α-terpineol, is not the higher the better, but has an impact on each other (Comparative Examples 9-10). The effect of the addition amount on the triterpene production of Antrodia cinnamomea fermentation is not a simple linear superposition. If the ratio is unreasonable, the yield of triterpene compounds in the liquid fermentation of Antrodia cinnamomea with the addition of the four precursor substances will be similar to or even lower than that of one or three precursor substances. Compared with Comparative Example 11 (without adding any precursor substance, the yield of triterpene compounds is 60.7 mg / L), Comparative Example 1-4 (only one precursor substance is added, the yield of triterpene compounds is 64.9-70.5 mg / L), Comparative Examples 5-8 (one precursor substance is missing, the yield of triterpene compounds is 70.3-78.0 mg / L) and Comparative Examples 9-10 (the precursor substance among the four precursor substances is too high or too low, the triterpene compound yield is 69.9-70.6 mg / L), the yield of triterpene compounds in Examples 1-6 is significantly increased (85.9-105.8 mg / L). It can be seen that the specific precursor substances coenzyme Q0, p-hydroxybenzoic acid, linalool and α-terpineol greatly promote the synthesis of triterpene compounds in Antrodia cinnamomea fermentation, and increase the yield of triterpene compounds produced by Antrodia cinnamomea fermentation, which is increased by 42%-74% (Examples 1-5) compared to Antrodia cinnamomea liquid fermentation without adding precursor substances.

[0086] Meanwhile, according to the triterpenoid yield results of Example 2, Example 4-6 and Comparative Examples 12-14 in Table 4, it can be found that in Antrodia cinnamomea liquid fermentation, the timing of adding precursors also has a greater impact on the synthesis of triterpenoids. Adding precursors too early can cause serious inhibitory effects on bacterial growth, and adding the above precursors 60-120h after fermentation can effectively promote the production of triterpenoids. Therefore, the precursors coenzyme Q0 100-500mg / L, p-hydroxybenzoic acid 20-300mg / L, linalool 0.1-0.5mg / L and α-terpineol 0.3-0.7mg / L of four specific additions of the present invention are the highest when the precursors are added at 60-120h.

[0087] Table 4 Determination of triterpenoid content in Examples 1-12 and Comparative Examples 1-9

[0088]

[0089]

[0090] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fermentation method for Antrodia cinnamomea mycelium, characterized in that: The steps include: S1. Ferment the Antrodia cinnamomea strain in liquid for 60-120 h; S2. Adding a precursor to the fermented liquid fermentation broth of the Antrodia cinnamomea strain, and continuing the fermentation for more than 120 h to obtain the Antrodia cinnamomea mycelium; The precursor substances are coenzyme Q0, p-hydroxybenzoic acid, linalool and α-terpineol, and the content of coenzyme Q0 in the fermentation broth after adding the precursor substances is 100-500 mg / L, the content of p-hydroxybenzoic acid is 20-300 mg / L, the content of linalool is 0.1-0.5 mg / L, and the content of α-terpineol is 0.3-0.7 mg / L.

2. The fermentation method according to claim 1, characterized in that In step S2, the precursor is added to the fermentation liquid of the Antrodia cinnamomea strain obtained by fermentation, and the timing of adding the precursor is 72 hours after the Antrodia cinnamomea liquid is fermented in step S1.

3. The fermentation method according to claim 1, characterized in that After adding the precursor substance in step S2, the content of coenzyme Q0 in the fermentation broth was 300 mg / L, the content of p-hydroxybenzoic acid was 100 mg / L, the content of linalool was 0.3 mg / L, and the content of α-terpineol was 0.5 mg / L.

4. The fermentation method according to claim 1, characterized in that The Antrodia cinnamomea strain is Antrodia cinnamomea with a deposit number of ATCC No. 200183 ( Antrodia camphorata ) strain.

5. The fermentation method according to claim 1, characterized in that In step S2, the precursor substances are added to the fermentation liquid of the Antrodia cinnamomea strain by first dissolving coenzyme Q0 in DMSO, and dissolving p-hydroxybenzoic acid, linalool, and α-terpineol in ethanol; and then adding them to the Antrodia cinnamomea liquid fermentation liquid.

6. A fermentation auxiliary composition for Antrodia cinnamomea, characterized in that: The auxiliary composition comprises the following components in parts by weight: 100-500 parts of coenzyme Q0, 20-300 parts of p-hydroxybenzoic acid, 0.1-0.5 parts of linalool and 0.3-0.7 parts of α-terpineol.

7. The antrodia cinnamomea fermentation auxiliary composition according to claim 6, characterized in that: The auxiliary composition comprises the following components in parts by weight: 300 parts of coenzyme Q0, 100 parts of p-hydroxybenzoic acid, 0.3 parts of linalool and 0.5 parts of α-terpineol.

8. Use of the Antrodia cinnamomea fermentation auxiliary composition according to any one of claims 6 to 7 as an additive for Antrodia cinnamomea fermentation.

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