A method for preparing a taiwanofungus camphoratus enzyme

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a preparation method of a taiwanofungus castaneus enzyme Trichoderma longibrachiatum and bacillus licheniformis Bacillus licheniformis are mixed to prepare a composite fermentation seed liquid. A culture medium composed of cassava residues and sawdust is used to prepare pretreated taiwanofungus castaneus culture medium. Then, the taiwanofungus castaneus strain is inoculated in the culture medium for fermentation, and a heat stress treatment step is introduced in the fermentation process. The method significantly promotes the synthesis and dissolution of active components of taiwanofungus castaneus triterpenoids and polysaccharides by the synergistic effect of specific strains and the combination of physical stress. The present application solves the technical problem of insufficient dissolution of active substances of taiwanofungus castaneus in the prior art by a unique strain combination and process design, and has high application value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing Antrodia camphorata enzyme. Background Technology

[0002] Antrodia cinnamomea is a rare medicinal fungus that grows on the inner wall of the heartwood of fallen, dead trees of the Cinnamomum camphora, a tree species endemic to Taiwan, China. Its fruiting body is rich in over 70 active ingredients, including triterpenoids, polysaccharides, superoxide dismutase (SOD), adenosine, and ergosterol. Modern pharmacological studies have confirmed its remarkable hepatoprotective, anti-tumor, anti-inflammatory, antioxidant, and immunomodulatory effects. However, the natural growth cycle of wild Antrodia cinnamomea is 3-5 years, and the increasingly depleted Cinnamomum camphora resources result in extremely low annual yields, failing to meet the growing market demand and severely restricting the in-depth development and industrial application of related products.

[0003] To alleviate resource pressure, the artificial cultivation of Antrodia camphorata currently mainly adopts three modes: liquid fermentation, solid fermentation, and linden wood cultivation. However, all existing technologies have obvious limitations:

[0004] Liquid fermentation has a fermentation cycle of 7-14 days, and the process is highly controllable and the fermentation cycle is short. However, the active ingredients of mycelium are mainly in the intracellular bound state, which requires high-pressure homogenization or enzymatic hydrolysis to destroy the cell wall, resulting in a triterpenoid extraction rate of less than 40%. Moreover, the metabolites are mainly primary products with low content of secondary metabolites.

[0005] The linden wood cultivation technique simulates the natural environment, and its active ingredient spectrum is similar to that of wild fruiting bodies. However, it relies on camphor wood resources and is susceptible to contamination by miscellaneous fungi such as Penicillium and Aspergillus, resulting in a yield of only 30%-50%. The cultivation cycle is as long as 1-2 years, which is costly.

[0006] Traditional solid-state fermentation often uses agricultural waste such as rice husks and corn cobs. However, the imbalance of carbon-nitrogen ratio leads to slow mycelial growth and inhibits the activity of triterpenoid synthases; the lack of specific enzyme systems makes it difficult to degrade the lignin-carbohydrate complex in the cell wall of Antrodia camphorata, and the dissolution rate of active ingredients is less than 25%.

[0007] Enzymes are functional products made from one or more animal, plant, or fungal organisms through fermentation by beneficial microorganisms. They are rich in enzymes, vitamins, minerals, secondary metabolites, and a large number of probiotic metabolites. They possess various physiological activities, including assisting in maintaining normal bodily functions, promoting the digestion and absorption of nutrients, and participating in tissue repair and regeneration.

[0008] Therefore, developing a method for preparing Antrodia camphorata enzyme that can overcome the shortcomings of existing technologies, achieve efficient synthesis and dissolution of active ingredients, and is suitable for industrialization is of great research value. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention employs a specific combination of microorganisms in a culture medium composed of cassava residue and sawdust to prepare a pretreated Antrodia camphorata culture medium. Antrodia camphorata spore suspension is then inoculated into the pretreated Antrodia camphorata culture medium prepared through the aforementioned compound fermentation for fermentation. During fermentation, a heat stimulation treatment is applied followed by cooling. After fermentation, the fermentation supernatant is collected by centrifugation to obtain Antrodia camphorata enzyme. The Antrodia camphorata enzyme prepared by this method significantly improves the generation and dissolution of active ingredients in Antrodia camphorata, exhibiting extremely high application value and market prospects.

[0010] On one hand, the present invention provides a method for preparing Antrodia camphorata enzyme, the method comprising the following steps:

[0011] S1. Preparation of Antrodia camphorata culture medium: using Trichoderma longifolia... Trichoderma longibrachiatum With Bacillus licheniformis Bacillus licheniformis The compound fermentation seed liquid was prepared by mixing the Trichoderma longifolia. Trichoderma longibrachiatum The accession number is CGMCC 3.1029, and the Bacillus licheniformis described therein... Bacillus licheniformis Its accession number is CICC 10103;

[0012] S2. The compound fermentation seed liquid prepared in S1 is inoculated into the compound fermentation medium for fermentation. After fermentation, the fermentation product is sterilized to obtain the Antrodia camphorata culture medium. The compound fermentation medium contains ethanol-fermented cassava residue, starch-fermented cassava residue and sawdust.

[0013] S3. Preparation of Antrodia camphorata spore suspension: Activation of Antrodia camphorata Taiwanofungus camphoratus Preparation of Antrodia camphorata spore suspension, wherein Antrodia camphorata Taiwanofungus camphoratus Its accession number is CGMCC 5.906;

[0014] S4. Fermentation preparation of enzyme: The Antrodia camphorata spore suspension is inoculated into the Antrodia camphorata culture medium for fermentation. After fermentation, the mixture is centrifuged and the fermentation supernatant is collected to obtain Antrodia camphorata enzyme.

[0015] Furthermore, in the method, in step S2, the mass ratio of ethanol-fermented cassava residue: starch-fermented cassava residue: sawdust in the composite fermentation culture medium is 2:1:2.

[0016] Furthermore, in the method, in step S2, the fermentation conditions are: 25-30℃, fermentation for 2-4 days, and the inoculation amount of the compound fermentation seed liquid is 5%-15% of the total mass of the compound fermentation culture medium.

[0017] Furthermore, in the method, in step S2, after the fermentation is completed, the method further includes a step of heating the fermentation system to 50-60℃ and keeping it at that temperature for 1-3 days, followed by the sterilization treatment.

[0018] Furthermore, in the method, in step S4, the inoculation amount of the Antrodia camphorata spore suspension is 20%-30% of the mass of the Antrodia camphorata culture medium.

[0019] Furthermore, in the method, in step S4, the fermentation includes first culturing at 25-30°C for 3-5 days, followed by heat stress treatment, wherein the heat stress treatment is to raise the temperature to 35-38°C and maintain it for 1-3 hours.

[0020] Furthermore, in the method, after the heat stress treatment, the step of lowering the temperature to 25-30°C and continuing fermentation for 1-2 days is also included.

[0021] Secondly, a culture medium for Antrodia camphorata prepared according to the present invention is also provided.

[0022] Finally, the method described in this invention also provides Antrodia camphorata enzyme.

[0023] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0024] (1) This invention creatively employs a combination of *Trichoderma longicornis* CGMCC 3.1029 and *Bacillus licheniformis* CICC10103 for pretreatment of the culture medium through compound fermentation, while simultaneously incorporating the heat stress process during the fermentation stage of *Antrodia camphorata*, resulting in a significant synergistic effect. Experimental data show that the yield of triterpenoids in the *Antrodia camphorata* enzyme prepared by this invention reaches a maximum of 15.3%, and the yield of polysaccharides reaches a maximum of 20.19%. This is more than 5 times higher than the triterpenoid yield of only 2.8% in the untreated control medium group; and nearly twice as high as the yield of the medium pretreated with other strains. This overcomes the technical bottlenecks of traditional solid-state fermentation with an active ingredient dissolution rate of less than 25% and liquid-state fermentation with a triterpenoid extraction rate of less than 40%.

[0025] (2) The Trichoderma longifolia CGMCC 3.1029 selected in this invention can efficiently secrete cellulase, hemicellulase and lignin-modifying enzyme. It works synergistically with Bacillus licheniformis CICC 10103 to effectively degrade the lignin-carbohydrate complex (LCC) in the cell wall of Antrodia camphorata, destroy the dense structure of the cell wall, and thus allow the intracellular bound triterpenes, polysaccharides and other active ingredients to be more fully dissolved into the fermentation supernatant during the fermentation process.

[0026] (3) Heat stress treatment during the fermentation stage of Antrodia camphorata effectively activates the defense mechanism of Antrodia camphorata mycelium and significantly stimulates the synthesis pathway of its secondary metabolites such as triterpenoids and polysaccharides, resulting in a significant increase in the synthesis of the target active ingredients.

[0027] (4) This invention uses agricultural processing waste such as ethanol-fermented cassava residue and starch-fermented cassava residue as the main raw materials, replacing expensive and scarce camphor wood, realizing the resource utilization of waste, reducing production costs, and providing a reliable raw material guarantee for the large-scale and sustainable production of Antrodia camphorata.

[0028] In summary, this invention overcomes the technical problem of insufficient dissolution of active ingredients in Antrodia camphorata fermentation products in the prior art through specific strain combinations and process design, and has extremely high application value and market prospects. Detailed Implementation

[0029] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0030] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0031] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0032] Trichoderma longifolia Trichoderma longibrachiatum The accession number is CGMCC 3.1029. It was purchased from the China General Microbiological Culture Collection Center and is referred to as Trichoderma longifolia CGMCC 3.1029 below.

[0033] Bacillus licheniformis Bacillus licheniformis, Accession number: CICC 10103, purchased from China Industrial Microbial Culture Collection Center, hereinafter referred to as Bacillus licheniformis CICC 10103.

[0034] Antrodia camphorata Taiwanofungus camphoratus The accession number is CGMCC 5.906. It was purchased from the China General Microbiological Culture Collection Center and is referred to as Cinnamomum camphora CGMCC 5.906 below.

[0035] Trichoderma longifolia Trichoderma longibrachiatum The accession number is CGMCC 3.1042, and it was purchased from the China General Microbiological Culture Collection Center.

[0036] Bacillus licheniformis Bacillus licheniformis,The accession number is CICC 10096, and it was purchased from the China Industrial Microbial Culture Collection Center.

[0037] Example 1: This example is for the preparation of seed liquid of Trichoderma longifolia CGMCC 3.1029.

[0038] Trichoderma longifolia CGMCC 3.1029 was taken out of the -80℃ freezer and streaked onto the surface of PDA slant medium. It was then cultured in a constant temperature incubator at 28℃ for 4 days. One loopful of activated Trichoderma longifolia CGMCC 3.1029 was picked up with an inoculation loop and inoculated into 250 mL of PDA liquid medium. The medium was then cultured at 28℃ and 220 r / min for 2 days with shaking to obtain the Trichoderma longifolia CGMCC 3.1029 seed culture.

[0039] Example 2: This example is for preparing seed culture of Bacillus licheniformis CICC 10103.

[0040] Bacillus licheniformis CICC 10103 was taken out of the -80℃ freezer, streaked onto NB solid medium, and cultured at 30℃ for 1 day. A single colony was picked and inoculated into NB liquid medium and cultured at 30℃ and 250 rpm for 2 days to obtain the seed culture of Bacillus licheniformis CICC 10103.

[0041] Example 3: This example describes the preparation of Antrodia camphorata culture medium by compound fermentation of Trichoderma longifolia CGMCC 3.1029 and Bacillus licheniformis CICC 10103.

[0042] The seed culture of Trichoderma longifolia CGMCC 3.1029 prepared in Example 1 and the seed culture of Bacillus licheniformis CICC 10103 prepared in Example 2 were mixed at a volume ratio of 1:3 to obtain a compound fermentation seed culture.

[0043] Compound fermentation medium: Ethanol-fermented cassava residue, starch-fermented cassava residue, and sawdust are mixed in a mass ratio of 2:1:2, steamed at 100℃ for 30 minutes, dried, and then pulverized to a particle size ≤3mm to obtain a mixed powder; 100g of the mixed powder is mixed evenly with 0.6g of MgSO4 and 10g of yeast extract to obtain a compound fermentation medium.

[0044] After adding water of the same mass as the compound fermentation medium and mixing evenly, add 10% of the total mass of the compound fermentation medium and compound fermentation seed liquid. Stir once every 24 hours, ferment at 28℃ for 3 days to complete the first stage of fermentation. Then raise the temperature to 55℃ and keep warm for 2 days to complete the fermentation. Collect the fermentation product, sterilize the fermentation product with steam at 100℃ for 30 minutes, and cool to room temperature to obtain the Antrodia camphorata culture medium.

[0045] Example 4: This example is for preparing a suspension of Antrodia camphorata spores.

[0046] Cinnamomum camphora CGMCC 5.906 was removed from a -80℃ freezer and inoculated onto a PDA slant culture medium. After incubation at 25℃ for 10 days, the spores attached to the slant culture medium were rinsed off with sterile water. The spore suspension was transferred to an Erlenmeyer flask containing small glass beads to disperse it evenly, and then inoculated into PDA liquid culture medium. The flask was then incubated at 30℃ on a shaker at 110 rpm for 3 days. This yielded a Cinnamomum camphora spore suspension.

[0047] Example 5: This example is for the preparation of Antrodia camphorata enzyme.

[0048] The *Antrodia camphorata* spore suspension prepared in Example 4 was stirred and mixed thoroughly, then added to the *Antrodia camphorata* culture medium prepared in Example 3. The mixture was incubated at 28°C and 110 rpm for 5 days. After fermentation, the fermentation products were collected. The amount of *Antrodia camphorata* spore suspension added was 20 wt% of the *Antrodia camphorata* culture medium. The collected fermentation products were centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. The supernatant was concentrated under vacuum at 50°C to obtain *Antrodia camphorata* enzyme, labeled as #1 *Antrodia camphorata* enzyme.

[0049] Example 6: This example is for the preparation of Antrodia camphorata enzyme.

[0050] The *Antrodia camphorata* spore suspension prepared in Example 4 was stirred and mixed thoroughly, then added to the *Antrodia camphorata* culture medium prepared in Example 3. The medium was cultured at 28°C and 110 rpm for 3 days, followed by a heat stress at 36°C for 2 hours, and then cooled to 25°C for 2 days of fermentation. After fermentation, the fermentation product was collected. The amount of *Antrodia camphorata* spore suspension added was 30 wt% of the *Antrodia camphorata* culture medium. The collected fermentation product was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. The supernatant was concentrated under vacuum at 45°C to obtain *Antrodia camphorata* enzyme, labeled as #2 *Antrodia camphorata* enzyme.

[0051] Example 7: This example is for the preparation of Antrodia camphorata enzyme.

[0052] The *Antrodia camphorata* spore suspension prepared in Example 4 was stirred and mixed thoroughly, then added to the *Antrodia camphorata* culture medium prepared in Example 3. The medium was incubated at 25°C and 220 rpm for 4 days, followed by a heat stress at 36°C for 3 hours, and then cooled to 28°C for 1 day of fermentation. The fermentation product was collected after the fermentation was completed. The amount of *Antrodia camphorata* spore suspension added was 25 wt% of the *Antrodia camphorata* culture medium. The collected fermentation product was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. The supernatant was concentrated under vacuum at 50°C to obtain *Antrodia camphorata* enzyme, labeled as #3 *Antrodia camphorata* enzyme.

[0053] Example 8: This example is for the preparation of Antrodia camphorata enzyme.

[0054] The *Antrodia camphorata* spore suspension prepared in Example 4 was stirred and mixed thoroughly, then added to the *Antrodia camphorata* culture medium prepared in Example 3. The medium was cultured at 28°C and 110 rpm for 3 days, followed by a heat stress at 36°C for 1 hour, and then cooled to 28°C for 2 days of fermentation. After fermentation, the fermentation product was collected. The amount of *Antrodia camphorata* spore suspension added was 20 wt% of the *Antrodia camphorata* culture medium. The collected fermentation product was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. The supernatant was concentrated under vacuum at 40°C to obtain *Antrodia camphorata* enzyme, labeled as #4 *Antrodia camphorata* enzyme.

[0055] Comparative Example 1

[0056] The difference between this comparative example and Example 8 is that the Antrodia camphorata culture medium used is different.

[0057] Comparative Antrodia camphorata culture medium: Ethanol-fermented cassava residue, starch-fermented cassava residue, and sawdust were mixed in a mass ratio of 2:1:2, steamed at 100℃ for 30 minutes, dried, and pulverized to a particle size ≤3mm to obtain a mixed powder; 100g of the mixed powder was mixed evenly with 0.6g of MgSO4 and 10g of yeast extract to obtain an Antrodia camphorata culture medium mixture; the same mass of water as the Antrodia camphorata culture medium mixture was added and mixed evenly, and then steam-sterilized at 100℃ for 30 minutes to obtain the comparative Antrodia camphorata culture medium.

[0058] The *Antrodia camphorata* spore suspension prepared in Example 4 was stirred and mixed thoroughly, then added to the comparative *Antrodia camphorata* culture medium. The mixture was cultured at 28°C and 110 rpm for 3 days, followed by a heat stress at 36°C for 1 hour, and then cooled to 28°C for 2 days of fermentation. The fermentation product was collected after the fermentation was completed. The amount of *Antrodia camphorata* spore suspension added was 20 wt% of the comparative *Antrodia camphorata* culture medium. The collected fermentation product was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. The supernatant was concentrated under vacuum at 40°C to obtain *Antrodia camphorata* enzyme, labeled as #1 *Antrodia camphorata* enzyme contrast agent.

[0059] Comparative Example 2

[0060] The difference between this comparative example and Example 8 is that the Antrodia camphorata culture medium used is different.

[0061] This comparative example of *Antrodia camphorata* culture medium used *Trichoderma longifolia*. Trichoderma longibrachiatum The strain with accession number CGMCC 3.1042 replaced Trichoderma longifolia CGMCC 3.1029 in Example 3, and Bacillus licheniformis was used. Bacillus licheniformis, The strain with accession number CICC 10096 replaced Bacillus licheniformis CICC 10103 in Example 3. All other reaction conditions were the same as in Example 3. The resulting culture medium was called Comparative Example 2 Antrodia camphorata culture medium.

[0062] Antrodia camphorata enzyme was prepared using Antrodia camphorata culture medium of Comparative Example 2. The fermentation conditions were the same as in Example 8. Antrodia camphorata enzyme was obtained and labeled as Antrodia camphorata enzyme #2.

[0063] Test case

[0064] This test case analyzes the content of active ingredients in Antrodia camphorata enzymes prepared using different methods.

[0065] The contents of triterpenoids and polysaccharides in the Antrodia camphorata enzymes prepared in Examples 5-8 (1#-4#), and the Antrodia camphorata enzymes prepared in Comparative Examples 1-2 (1# and 2#) were determined.

[0066] (1) Determination of triterpenoid content

[0067] Take 5g each of the Antrodia camphorata enzymes 1#-4# prepared in Examples 5-8, and the Antrodia camphorata enzyme comparison agents 1# and 2# prepared in Comparative Examples 1-2, place them in a beaker, add 90% ethanol, stir and extract, centrifuge to collect the supernatant, filter, and make up the volume of the solution in a 250mL volumetric flask to obtain the sample to be tested.

[0068] Weigh 10.00 mg of ursolic acid and add ethanol to make up to 100 mL to obtain a ursolic acid standard solution with a concentration of 0.10 mg / mL. Take 0.10, 0.20, 0.40, 0.60, 0.80, 1.00, and 1.20 mL of ursolic acid control solution, add ethanol to each solution to make up to 2 mL, evaporate to dryness in a water bath, add 0.2 mL of 5% vanillin-glacial acetic acid solution and 1 mL of perchloric acid, incubate at 70℃ for 35 min, then in an ice bath for 10 min, add 5 mL of glacial acetic acid, shake well, and cool to room temperature. A blank control group was also set up. The absorbance (A) was measured at 548 nm using a UV spectrophotometer. A standard curve was plotted with the ursolic acid mass concentration as the x-axis and the A value as the y-axis. The test sample was diluted and measured using the above method. The concentration of triterpenes in the test sample was calculated based on the standard curve, and the triterpenes yield was calculated based on the measured triterpenes concentration. The results are shown in Table 1.

[0069] Triterpenoid yield (%) = (mass of triterpenoids / mass of Antrodia camphorata enzyme used for measurement) × 100

[0070] Table 1. Yield of triterpenes in Antrodia camphorata enzyme prepared by different methods

[0071]

[0072] As shown in Table 1, the triterpenoid yields of the Antrodia camphorata enzymes #2-#4 prepared in Examples 6-8 were 13.2%-15.3%, significantly higher than the 2.8% of the comparative agent of Antrodia camphorata enzyme #1 prepared in Comparative Example 1, the 7.5% of the comparative agent of Antrodia camphorata enzyme #2 prepared in Comparative Example 2, and the 8.9% of the Antrodia camphorata enzyme #1 prepared in Example 5. In particular, the triterpenoid yield of the Antrodia camphorata enzyme #3 prepared in Example 7 was 15.3%, a five-fold increase compared to Comparative Example 1. This indicates that the fermentation medium for Antrodia camphorata prepared by the combined fermentation of Trichoderma longifolia CGMCC 3.1029 and Bacillus licheniformis CICC10103, combined with heat stress treatment, effectively promoted the synthesis and dissolution of the active ingredients in Antrodia camphorata. This effectively overcomes the technical problem of insufficient dissolution rate of active ingredients in Antrodia camphorata fermentation products in the prior art.

[0073] (2) Determination of polysaccharide content

[0074] Take 5g each of the Antrodia camphorata enzymes 1#-4# prepared in Examples 5-8, and the Antrodia camphorata enzymes 1# and 2# prepared in Comparative Examples 1-2, place them in a beaker, add 90% double-distilled water, stir and extract, centrifuge to collect the supernatant, filter, and dilute the solution to 250mL in a volumetric flask to prepare the polysaccharide sample to be tested.

[0075] Weigh 10g of glucose dried to constant weight, dissolve and dilute to 100mL with double-distilled water to obtain a glucose standard solution with a concentration of 0.1mg / mL. Take 0.10, 0.20, 0.40, 0.60, 0.80, 1.00, and 1.20mL of the glucose standard solution respectively and place them in stoppered test tubes. Add water to 2mL for each, add 1mL of 5% phenol solution to each, mix well, add 5mL of concentrated sulfuric acid, shake to mix, and place in a 30℃ water bath for 20min. Use 2mL of distilled water as a blank control. Measure the absorbance (A') at 490nm using a UV spectrophotometer. Plot a standard curve with glucose concentration on the x-axis and A' value on the y-axis. Dilute the polysaccharide sample and measure it according to the above method. Calculate the polysaccharide concentration in the sample based on the standard curve, and calculate the polysaccharide yield based on the measured polysaccharide concentration. The results are shown in Table 2.

[0076] Polysaccharide yield (%) = (Polysaccharide mass / Mass of Antrodia camphorata enzyme used for measurement) × 100

[0077] Table 2. Polysaccharide yield in Antrodia camphorata enzyme prepared by different methods

[0078]

[0079] As shown in Table 2, the polysaccharide yields of Antrodia camphorata enzymes #2-#4 prepared in Examples 6-8 were significantly higher than those of Antrodia camphorata enzyme #1 prepared in Comparative Example 1, Antrodia camphorata enzyme #2 prepared in Comparative Example 2, and Antrodia camphorata enzyme #1 prepared in Example 5. In particular, the polysaccharide yield of Antrodia camphorata enzyme #3 prepared in Example 7 reached 20.19%, a 6-fold increase compared to Comparative Example 1. This indicates that the fermentation medium for Antrodia camphorata prepared by the combined fermentation of Trichoderma longifolia CGMCC 3.1029 and Bacillus licheniformis CICC 10103, combined with heat stress treatment, effectively promoted the synthesis and dissolution of polysaccharides in Antrodia camphorata.

[0080] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A method for preparing Antrodia camphorata enzyme, characterized in that, Includes the following steps: S1. Preparation of Antrodia camphorata culture medium: using Trichoderma longifolia... Trichoderma longibrachiatum With Bacillus licheniformis Bacillus licheniformis The compound fermentation seed liquid was prepared by mixing the Trichoderma longifolia. Trichoderma longibrachiatum The accession number is CGMCC 3.1029, and the Bacillus licheniformis described therein... Bacillus licheniformis Its accession number is CICC 10103; S2. The compound fermentation seed liquid prepared in S1 is inoculated into the compound fermentation medium for fermentation. After fermentation, the fermentation product is sterilized to obtain the Antrodia camphorata culture medium. The compound fermentation medium contains ethanol-fermented cassava residue, starch-fermented cassava residue and sawdust. S3. Preparation of Antrodia camphorata spore suspension: Activation of Antrodia camphorata Taiwanofungus camphoratus Preparation of Antrodia camphorata spore suspension, wherein Antrodia camphorata Taiwanofungus camphoratus Its accession number is CGMCC 5.906; S4. Fermentation preparation of enzyme: The Antrodia camphorata spore suspension is inoculated into the Antrodia camphorata culture medium at 25-30℃ and fermented for 3-5 days. Then, a heat stress treatment is performed, in which the temperature is raised to 35-38℃ and maintained for 1-3 hours. After the heat stress treatment, the fermentation temperature is lowered to 25-30℃ and fermentation continues for 1-2 days. After the fermentation is completed, the mixture is centrifuged and the fermentation supernatant is collected to obtain Antrodia camphorata enzyme.

2. The method according to claim 1, characterized in that, In step S2, the mass ratio of ethanol-fermented cassava residue: starch-fermented cassava residue: sawdust in the composite fermentation medium is 2:1:

2.

3. The method according to claim 1, characterized in that, In step S2, the fermentation conditions are: 25-30℃, fermentation for 2-4 days, and the inoculation amount of the compound fermentation seed liquid is 5%-15% of the total mass of the compound fermentation culture medium.

4. The method according to claim 1 or 3, characterized in that, In step S2, after the fermentation is completed, the fermentation system is heated to 50-60℃ and kept at that temperature for 1-3 days, followed by the sterilization process.

5. The method according to claim 1, characterized in that, In step S4, the inoculation amount of the Antrodia camphorata spore suspension is 20%-30% of the mass of the Antrodia camphorata culture medium.

6. The Antrodia camphorata enzyme prepared by the method according to any one of claims 1-5.

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