Method for maintaining activity of aspergillus niger used for brewing baizhuo decoction

Abstract

This invention relates to the field of Aspergillus niger activity maintenance technology, and specifically discloses an Aspergillus niger for the fermentation of Baiyao Decoction and a method for maintaining its activity. The Aspergillus niger is a domesticated engineered strain of Aspergillus niger, which is deposited at the China Center for Type Culture Collection, with the biological accession number CGMCC No. 42724.

Description

Technical Field

[0001] This application relates to the field of Aspergillus niger activity maintenance technology, and specifically discloses an Aspergillus niger for the fermentation of Baiyao Decoction and a method for maintaining its activity. Background Technology

[0002] Baiyaojian is a traditional Chinese medicine fermented decoction made from gallnut and tea leaves through microbial fermentation. It has the effects of moistening the lungs, resolving phlegm, promoting body fluid production, and quenching thirst. The hydrolyzable tannins in gallnut are hydrolyzed by microbial tannin enzymes to produce gallic acid, which is the main active ingredient in Baiyaojian. Traditional Baiyaojian uses natural mixed-culture fermentation with yeast, resulting in uncontrollable microbial composition, significant environmental influences, large batch-to-batch variations, and a risk of contamination by other microorganisms, making standardized production difficult.

[0003] Traditional natural fermentation processes have several inherent drawbacks. Firstly, the fermentation microorganisms originate from the yeast and the environment, resulting in a complex and uncontrollable microbial community. The fermentation process is greatly affected by temperature, humidity, season, and workshop conditions, making it susceptible to the introduction of contaminating microorganisms such as Aspergillus flavus. Furthermore, the gallic acid conversion rate varies significantly between batches, leading to poor batch-to-batch quality consistency and making it difficult to meet the requirements of modern standardized production of traditional Chinese medicine. Secondly, traditional processes rely on operator experience to determine the fermentation endpoint, lacking quantifiable microbial strains and process control methods. This results in long production cycles, unstable product quality, and hinders large-scale industrial production.

[0004] To overcome the drawbacks of natural mixed-culture fermentation, existing research proposes to use a single functional strain for pure-culture fermentation of Baiyaojian. Studies have confirmed that Aspergillus niger is the key functional strain in the Baiyaojian fermentation system. This strain can secrete tannins, which can efficiently hydrolyze the hydrolyzable tannins in gallnuts and directionally generate gallic acid, thus having important application value in the processing of Baiyaojian.

[0005] In practical applications, significant challenges remain: a lack of standardized protocols for the long-term preservation and subculturing of target Aspergillus niger strains. The filamentous fungus Aspergillus niger is highly susceptible to strain degeneration during repeated subculturing, manifested by decreased spore production capacity, reduced enzyme activity, and diminished tannin hydrolysis capacity, directly resulting in a decrease in gallic acid yield in the Baiyao decoction fermentation product. Ordinary slant culture at low temperatures offers short preservation times and requires frequent subculturing; conventional cryopreservation lacks a suitable protection system, leading to large fluctuations in spore survival rates after thawing and unstable strain fermentation performance, severely restricting the continuous and stable use of the strain in the standardized fermentation production of Baiyao decoction.

[0006] For example, existing methods for preserving Aspergillus niger mainly include slant culture at low temperatures, glycerol cryopreservation, and freeze-drying. Slant culture at 4℃ is simple to operate, but the preservation period is short, requiring frequent subculturing. After multiple subculturings, Aspergillus niger is prone to strain degeneration, manifested as lighter spore color, decreased sporulation, mycelial collapse, and reduced fermentation metabolic activity. High-subculturing strains are prohibited in formal experiments. Ordinary 20℃ glycerol culture, if the spore suspension preparation is not standardized or lacks protective components, results in unstable survival rates after revival, with some batches showing slow growth and easy contamination by other microorganisms. Freeze-drying offers good preservation results, but the equipment cost is high, making it difficult for ordinary laboratories to implement.

[0007] Most publicly available technologies currently focus on optimizing the fermentation process parameters of Baiyao Decoction, with less attention paid to the preservation and activity maintenance of functional Aspergillus niger strains throughout their entire life cycle. If strains are improperly preserved and degenerate or become contaminated, even with reasonable fermentation process parameters, a stable quality Baiyao Decoction product cannot be obtained. Therefore, screening for suitable Aspergillus niger strains for Baiyao Decoction fermentation and establishing a complete system for graded preservation, subculturing quality control, maintaining strain sporulation, and maintaining strain activity to ensure batch-to-batch consistency in Baiyao Decoction fermentation is of significant practical importance for promoting the modern standardized processing of Baiyao Decoction.

[0008] In view of this, this application discloses a Aspergillus niger for the fermentation of Baiyao Decoction and a method for maintaining its activity. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for maintaining the activity of Aspergillus niger, with supporting processes for pre-preservation quality control, subculture number control, and resuscitation verification, to reduce strain degradation, improve spore survival rate after preservation, reduce the risk of contamination by other microorganisms, adapt to conventional laboratory equipment, and be suitable for the domestication of engineered strains.

[0010] To achieve the above objectives, the present invention provides the following basic solution: a black Aspergillus strain for fermentation of Baiyao Decoction, wherein the black Aspergillus strain is an engineered strain of black Aspergillus niger, which is deposited at the China Center for Type Culture Collection, with accession number CGMCC No. 42724 and deposit date of June 4, 2026.

[0011] Furthermore, the sporulation performance and fermentation metabolic activity of the strain remain stable when it is passaged no more than 5 times.

[0012] Based on the aforementioned Aspergillus niger for the fermentation of Baiyao Decoction, this application also discloses a method for maintaining the activity of Aspergillus niger for the fermentation of Baiyao Decoction, comprising the following steps: Step S01: Cultivate solid culture medium and liquid culture medium. Sterilize solid culture medium by autoclaving. After sterilization, allow the pressure to drop naturally and remove the solid culture medium. Place the solid culture medium on a slant while it is still hot. After cooling and solidification, set it aside for later use. Stir the liquid culture medium to prepare it and then dispense it. Step S02: Take the frozen Aspergillus niger strain out of the -20℃ low temperature freezer, activate it in a 30℃ biochemical incubator for 1 day before use, use a sterile pipette to take 100-200μL of frozen bacterial solution, spread it evenly on the surface of a qualified solid culture medium plate, invert the plate, put it in a 30℃ constant temperature incubator, and let it stand for 3-5 days. Step S03: Select a revived and morphologically pure Aspergillus niger plate, pick up a small amount of black spores and fresh hyphae with a sterile inoculation loop, and inoculate evenly on the slant of solid culture medium using the streak method. Keep the plate close to the flame of an alcohol lamp throughout the inoculation process, and incubate at a constant temperature of 30°C inverted for 3-4 days until the slant is covered with dense black spores. Step S04: Take a qualified subculture slant culture, use a sterile pipette to draw 3-5 mL of sterile physiological saline, gently rinse the slant spores, mix well by pipetting to prepare a spore suspension, and inoculate the spore suspension into the dispensed liquid culture medium at an inoculation rate of 1%-5% to form a seed culture; inoculate the qualified seed culture medium at an inoculation rate of 5%-10% into a special fermentation medium. When the culture solution turns dark brown and a large number of spores are produced, fermentation stops.

[0013] Furthermore, the solid culture medium and the liquid culture medium are PDA potato glucose agar medium and PDB liquid culture medium, respectively. The formula of the solid culture medium is as follows: by weight, potato pieces are added to 1000mL of water and boiled for 20-30 minutes. After filtration, water is added to 1000mL. 20g of agar is added and heated to melt. Then, 20g of glucose, 10g of soybean peptone, and 10g of yeast powder are added. The formula of the liquid culture medium is as follows: by weight, 40g of glucose, 10g of soybean peptone, 10g of yeast powder, and 1000mL of pure water are added and stirred to dissolve.

[0014] Furthermore, in step S01, high-pressure steam sterilization is performed at 121°C with a constant pressure of 0.1 MPa for 20 minutes. The prepared solid culture medium is then placed at 37°C for constant temperature blank culture for 24 hours. The medium is considered qualified if there is no turbidity and no contaminating bacterial colonies.

[0015] Furthermore, in step S02, after the culture is completed, the colony morphology needs to be observed. Normal Aspergillus niger colonies are initially white fluffy mycelia, which gradually turn into black or charcoal black in the later stage. The absence of bacterial turbidity indicates that the recovery is qualified.

[0016] Furthermore, in step S04, after inoculating with the special fermentation medium, the temperature of the constant temperature shaker is controlled at 36℃ and the rotation speed is 180-220 r / min, and the shake culture is carried out for 24-36 h.

[0017] Furthermore, the qualified subculture slant strains obtained through step S04 have both short-term and long-term preservation capabilities. When short-term preservation is required, select solid culture medium slants that are full of plump spores and free of contaminants, seal the tube opening with sealing film, label the strain information, date, and number of subcultures, and store them in a refrigerator at 4°C. When long-term preservation is required, place the spore suspension obtained by rinsing the solid culture medium slants in a low-temperature storage environment at -20°C.

[0018] Based on the aforementioned Aspergillus niger strain used for fermenting Baiyao Decoction, this application also discloses a microbial agent containing the aforementioned Aspergillus niger strain.

[0019] Based on the above-mentioned Aspergillus niger used for the fermentation of Baiyao Decoction, this application also includes an application of Aspergillus niger used for the fermentation of Baiyao Decoction as a fermentation and strain testing method for Baiyao Decoction.

[0020] The principle and effect of this solution are as follows: 1. Compared with the prior art, the present invention provides a strain of Aspergillus niger that has been biologically preserved and adapted for the fermentation of Baiyaojian. This strain can efficiently secrete tannins, which can directionally convert the hydrolyzable tannins in gallnuts into the medicinal substance gallic acid. This strain can be used for the fermentation of Baiyaojian, solving the problems of mixed microbial communities, high risk of contamination, and large batch-to-batch product differences in traditional natural fermentation. It provides a stable strain resource for the standardized pure microbial fermentation of Baiyaojian.

[0021] 2. Compared with the existing technology, the fermentation of Baiyaojian using the Aspergillus niger strain preserved in this invention is controllable, the gallic acid conversion rate is stable, and the batch-to-batch variation of the finished product is effectively reduced, which is conducive to promoting the standardization and industrial application of the fermentation and processing technology of Baiyaojian.

[0022] 3. Compared with the existing technology, the present invention sets up strict pre-strain quality control and subculture number control to inhibit the subculture degeneration of Aspergillus niger from the source and reduce problems such as decreased sporulation capacity, tannin enzyme activity decay and reduced gallic acid conversion efficiency caused by strain degeneration; it is equipped with a standardized resuscitation verification process, and only strains that have passed the resuscitation can be used for the fermentation of Baiyao Decoction to ensure stable fermentation performance. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The diagram shows a flow chart of Aspergillus niger for fermentation of Baiyao Decoction and a method for maintaining its activity, as proposed in an embodiment of this application. Detailed Implementation

[0025] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0026] Implementation, for example Figure 1 As shown: A type of Aspergillus niger used for fermenting Baiyao Decoction, the Aspergillus niger being a domesticated engineered strain of Aspergillus niger is deposited at the China Center for Type Culture Collection (CGMCC) with accession number CGMCC No. 42724. Experiments have shown that the sporulation performance and fermentation metabolic activity of this biopreserved strain remain stable when it is passaged for no more than 5 generations.

[0027] Based on the aforementioned Aspergillus niger for the fermentation of Baiyao Decoction, this application also discloses a method for maintaining the activity of Aspergillus niger for the fermentation of Baiyao Decoction, comprising the following steps: Step S01: Cultivate solid and liquid culture media. Sterilize the solid culture media by autoclaving. After sterilization, allow the pressure to drop naturally and remove the solid culture media. While the solid culture media is still hot, place it on a slant. After cooling and solidification, set it aside for later use. Stir and prepare the liquid culture media and then dispense it.

[0028] Specifically: the solid culture medium and the liquid culture medium are PDA potato glucose agar medium and PDB liquid culture medium, respectively. The formula of the solid culture medium is as follows (by weight): potato pieces are added to 1000mL of water and boiled for 20-30 minutes. After filtration, water is added to 1000mL. 20g of agar is added and heated to melt. Then, 20g of glucose, 10g of soybean peptone, and 10g of yeast powder are added. The formula of the liquid culture medium is as follows (by weight): 40g of glucose, 10g of soybean peptone, 10g of yeast powder, and 1000mL of pure water are added and stirred to dissolve.

[0029] In step S01, high-pressure steam sterilization is performed at 121°C with a constant pressure of 0.1 MPa for 20 minutes. The prepared solid culture medium is then placed at 37°C for 24 hours as a blank culture. The medium is considered qualified if there is no turbidity and no contaminating bacterial colonies.

[0030] Step S02: Take the frozen Aspergillus niger strain out of the -20℃ low temperature freezer, activate it in a 30℃ biochemical incubator for 1 day before use, use a sterile pipette to draw 100-200μL of frozen bacterial solution, spread it evenly on the surface of a qualified solid culture medium plate, invert the plate, put it in a 30℃ constant temperature incubator, and let it stand for 3-5 days.

[0031] In step S02, after the culture is completed, the colony morphology needs to be observed. Normal Aspergillus niger colonies are initially white fluffy mycelia, which gradually turn black or charcoal black in the later stage. The absence of bacterial turbidity indicates that the recovery is qualified.

[0032] Step S03: Select a revived and morphologically pure Aspergillus niger plate, pick up a small amount of black spores and fresh hyphae with a sterile inoculation loop, and inoculate evenly on the slant of solid culture medium using the streak method. Keep the plate close to the flame of an alcohol lamp throughout the inoculation process, and incubate at a constant temperature of 30°C inverted for 3-4 days until the slant is covered with dense black spores.

[0033] Step S04: Take a qualified subculture slant culture, use a sterile pipette to draw 3-5 mL of sterile physiological saline, gently rinse the slant spores, and mix them by pipetting to prepare a spore suspension. Inoculate the spore suspension into the dispensed liquid culture medium at an inoculation rate of 1%-5% to form a seed culture. Inoculate the qualified seed culture into a special fermentation medium at an inoculation rate of 5%-10%. When the culture solution turns dark brown and a large number of spores are produced, the fermentation stops. In step S04, after inoculating into the special fermentation medium, control the temperature of the constant temperature shaker at 36℃ and the rotation speed at 180-220 r / min, and culture for 24-36 h.

[0034] The qualified slant culture obtained through step S04 has both short-term and long-term preservation capabilities. When short-term preservation is required, select a solid culture medium slant that is full of plump spores and free of contaminants, seal the tube opening with sealing film, label the strain information, date, and number of passages, and store it in a refrigerator at 4°C. When long-term preservation is required, place the spore suspension obtained by rinsing the solid culture medium slant in a refrigerator at -20°C.

[0035] The experimental steps are as follows: First, the test strain: domesticated engineered strain Aspergillus niger, CGMCC No. 42724; culture media: solid culture medium, liquid culture medium, and special fermentation medium for Baiyaojian (a traditional Chinese medicine decoction). The special fermentation medium for Baiyaojian consisted of 80 g / L gallnut powder, 20 g / L tea leaves, diluted with pure water, and natural pH; instruments: ultra-clean workbench, constant temperature incubator, constant temperature shaker, hemocytometer, ultraviolet spectrophotometer, and high performance liquid chromatography (HPLC). The originally deposited strain (F0 generation) is revived, streaked and subcultured on slants according to steps S02 and S03 of the present application, to successively obtain slant strains of F1, F2, F3, F4, F5, F6 and F7 generations. Each generation is strictly subjected to streak inoculation and cultured at 30°C for 34 days.

[0036] The slants of F1 to F7 generations are respectively taken, and rinsed with sterile normal saline to prepare spore suspension, and the spore concentration is measured with a hemocytometer.

[0037] The spore suspension of each generation is inoculated into PDB liquid medium at an inoculum size of 3%, and shake-cultured at 36°C and 200 r / min for 30 h to obtain a seed solution; then the seed solution is inoculated into a specific fermentation medium for Chinese Gallnut Extract at an inoculum size of 8%, and subjected to simulated fermentation of Chinese Gallnut Extract at 36°C and 200 r / min. After the fermentation is completed, two key indicators are measured: ① Tannase activity in the fermentation broth; ② The production amount of gallic acid is detected by HPLC, and the conversion rate of gallnut tannin to gallic acid is calculated. Three parallels are set for each group of experiments, and the average value is taken.

[0038] As shown in Table 1 below:

[0039] Table 1 The conclusions are as follows: For the strains of F1 to F5 generations, the spore concentration is maintained at a relatively high level, the tannase activity and gallic acid conversion rate fluctuate slightly, and the sporulation performance and the fermentation metabolic activity of Chinese Gallnut Extract are stable; when subcultured to F6 and F7 generations, the number of spores decreases significantly, the tannase activity is greatly reduced, the gallic acid conversion rate is obviously attenuated, and the phenotype of strain degeneration appears. It is thus proved that: when the subculture of the deposited strain CGMCC No. 42724 does not exceed 5 generations, the sporulation performance and the fermentation metabolic activity remain stable.

[0040] Example 1: Revival and slant subculture (F2 generation) of Aspergillus niger CGMCC No. 42724: Step S01: Prepare PDA solid medium and PDB liquid medium. PDA formula: Cut potatoes into pieces, add 1000 mL of water, boil for 25 min, filter and replenish water to 1000 mL, add 20 g of agar, 20 g of glucose, 10 g of soy peptone, and 10 g of yeast powder; perform autoclaving at 121°C and 0.1 MPa for 20 min; after sterilization, naturally reduce the pressure, and place slants while hot; perform blank culture at 37°C for 24 h, and the medium is judged to be qualified if there is no turbidity and no contaminant bacterial colonies. PDB liquid medium: 40 g of glucose, 10 g of soy peptone, and 10 g of yeast powder are stirred and dissolved in 1000 mL of pure water, and then subpackaged.

[0041] Step S02: Remove the frozen bacteria CGMCC No. 42724 from the 20℃ freezer and activate it in a 30℃ biochemical incubator for 1 day; take 150μL of the frozen bacterial solution from the clean bench and spread it onto a PDA plate; incubate at 30℃ upside down for 4 days. Colony observation: Initially, white fluffy mycelium forms, which later forms dense charcoal black spores. There are no contaminating bacteria or bacterial turbidity, indicating successful resuscitation.

[0042] Step S03: Pick black spores and fresh mycelium from the plate, streak them on the PDA slant, and inoculate them near the flame of an alcohol lamp; incubate at 30℃ upside down for 3.5 days until the slant is covered with dense black spores, thus obtaining a qualified F2 generation slant.

[0043] Step S04: Add 4 mL of sterile physiological saline to the F2 generation slant, rinse and agitate to prepare a spore suspension; inoculate with PDB liquid medium at a 3% inoculum, and culture at 36℃ and 200 rpm for 30 h to obtain the seed culture; inoculate the seed culture with Baiyaojian special fermentation medium at an 8% inoculum, and culture on a shaker at 36℃ and 200 rpm; stop fermentation when the bacterial solution turns dark brown and a large number of spores are produced. Fermentation results: Tannin activity 41.5 U / mL, gallic acid conversion rate 75.1%, fermentation effect is good.

[0044] Short-term preservation: Seal the tube opening with sealing film using qualified F2 generation slant, and label it: CGMCCNo.42724, F2 generation. Long-term preservation: Prepare spore suspension from the qualified slant and store it at 20℃.

[0045] Example 2: F4 generation resuscitation, subculturing, and fermentation with Baiyao decoction. Following the procedure in Example 1, the original preserved strain was sequentially subcultured to obtain F4 generation slant culture (≤5 generations); resuscitation, spore suspension preparation, seed culture preparation, and Baiyao decoction fermentation were then carried out. Results: Spore concentration 8.21 × 10⁻⁶. 7 The concentration of CFU / mL was high, with tannin activity of 40.2 U / mL and gallic acid conversion rate of 73.6%. Sporulation was vigorous, and fermentation performance was stable. The agar slant cultures were subjected to short-term storage at 4℃ and long-term storage at 20℃.

[0046] Example 3: Verification of strain recovery after preservation: The CGMCC No. 42724 spore culture from Example 1, which was preserved at 20℃ for 10 months, was taken out and activated in a 30℃ biochemical incubator for 1 day; 180 μL was taken and spread on a PDA plate and cultured at 30℃ for 4 days; typical black Aspergillus niger colonies grew, without contamination by other microorganisms; after further subculturing, F3 generation slant was obtained, and a simulated fermentation of Baiyao Decoction was carried out. The gallic acid conversion rate was 74.2%, and the fermentation activity was well preserved after preservation.

[0047] Comparative Example 1: F6 generation (more than 5 generations) fermentation experiment: The preserved strain was continuously subcultured to obtain F6 generation slant culture. All other culture media, recovery, inoculation, and fermentation conditions were completely consistent with Example 1. Results: Spore concentration 5.27 × 10⁻⁶. 7 The concentration of CFU / mL was low, the tannin activity was 25.8 U / mL, and the gallic acid conversion rate was 47.5%. The color of the colony spores became lighter, and some hyphae collapsed and degenerated. The conversion efficiency of the medicinal substances in the Baiyao Decoction fermentation was significantly reduced, proving that it is not suitable for fermentation after more than 5 generations.

[0048] Comparative Example 2: Without passage control, the F7 generation strain preserved at 20℃ for many years was used directly: The F7 generation Aspergillus niger CGMCC No. 42724 was preserved at 20℃ for 10 months and then revived. Fermentation of Baiyao Decoction was carried out under the same conditions as in Example 1. Results: After revival, sporulation was sparse, and the gallic acid conversion rate of the fermentation system was only 30.9%; even with low-temperature preservation, the strain degeneration defects caused by high passage could not be eliminated.

[0049] This invention includes pre-preservation quality control, subculture number management, and resuscitation verification processes, which reduce strain degradation, improve spore survival rate after preservation, reduce the risk of contamination by other microorganisms, are compatible with conventional laboratory equipment, and are suitable for the domestication of engineered strains.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A type of Aspergillus niger used in the fermentation of Baiyao Decoction, characterized in that, The Aspergillus niger strain mentioned is an engineered strain of Aspergillus niger, which is deposited at the China Center for Type Culture Collection (CGMCC) with accession number CGMCC No. 42724.

2. The Aspergillus niger for fermentation of Baiyao Decoction according to claim 1, characterized in that, When the strain is passaged for no more than 5 generations, its sporulation performance and fermentation metabolic activity remain stable.

3. A microbial agent, characterized in that, It includes the Aspergillus niger strain as described in claim 1.

4. The application of Aspergillus niger as described in claim 2 in the fermentation of Baiyao Decoction and in the detection and testing of microbial strains in Baiyao Decoction.

5. A method for maintaining the activity of Aspergillus niger for the fermentation of Baiyao Decoction, wherein the activity is maintained using the Aspergillus niger for the fermentation of Baiyao Decoction as described in claim 2, characterized in that, Includes the following steps: Step S01: Cultivate solid culture medium and liquid culture medium. Sterilize the solid culture medium by autoclaving. After sterilization, allow it to depressurize naturally and remove the solid culture medium. Place the solid culture medium on a slant while it is still hot. After cooling and solidification, set it aside for later use. Stir and prepare the liquid culture medium and dispense it. Step S02: Take the frozen Aspergillus niger strain out of the -20℃ low temperature freezer, activate it in a 30℃ biochemical incubator for 1 day before use, use a sterile pipette to take 100-200μL of frozen bacterial solution, spread it evenly on the surface of a qualified solid culture medium plate, invert the plate, put it in a 30℃ constant temperature incubator, and let it stand for 3-5 days. Step S03: Select a revived and morphologically pure Aspergillus niger plate, pick up a small amount of black spores and fresh hyphae with a sterile inoculation loop, and inoculate evenly on the slant of solid culture medium using the streak method. Keep the plate close to the flame of an alcohol lamp throughout the inoculation process, and incubate at a constant temperature of 30°C inverted for 3-4 days until the slant is covered with dense black spores. Step S04: Take a qualified subculture slant culture, use a sterile pipette to draw 3-5 mL of sterile physiological saline, gently rinse the slant spores, mix well by pipetting to prepare a spore suspension, and inoculate the spore suspension into the dispensed liquid culture medium at an inoculation rate of 1%-5% to form a seed culture; inoculate the qualified seed culture medium at an inoculation rate of 5%-10% into a special fermentation medium. When the culture solution turns dark brown and a large number of spores are produced, fermentation stops.

6. The Aspergillus niger for fermentation of Baiyao Decoction and the method for maintaining its activity according to claim 5, characterized in that, The solid culture medium and the liquid culture medium are PDA potato glucose agar medium and PDB liquid culture medium, respectively. The formula of the solid culture medium is as follows: by weight, cut potato pieces, add 1000mL of water and boil for 20-30 minutes, filter and add water to 1000mL, add 20g of agar, heat to melt, then add 20g of glucose, 10g of soybean peptone and 10g of yeast powder. The formula of the liquid culture medium is as follows: by weight, 40g of glucose, 10g of soybean peptone, 10g of yeast powder and 1000mL of pure water, and stir to dissolve.

7. The Aspergillus niger for fermentation of Baiyao Decoction and the method for maintaining its activity according to claim 6, characterized in that, In step S01, high-pressure steam sterilization is performed at 121°C with a constant pressure of 0.1 MPa for 20 minutes. The prepared solid culture medium is then placed at 37°C for 24 hours as a blank culture. The medium is considered qualified if there is no turbidity and no contaminating bacterial colonies.

8. The Aspergillus niger for fermentation of Baiyao Decoction and the method for maintaining its activity according to claim 7, characterized in that, In step S02, after the culture is completed, the colony morphology needs to be observed. Normal Aspergillus niger colonies are initially white fluffy mycelia, which gradually turn black or charcoal black in the later stage. The absence of bacterial turbidity indicates that the recovery is qualified.

9. The Aspergillus niger for fermentation of Baiyao Decoction and the method for maintaining its activity according to claim 8, characterized in that, In step S04, after inoculating with the special fermentation medium, the temperature of the constant temperature shaker is controlled at 36℃ and the rotation speed is 180-220 r / min, and the shake culture is carried out for 24-36 hours.

10. The Aspergillus niger for fermentation of Baiyao Decoction and the method for maintaining its activity according to any one of claims 5-9, characterized in that, The qualified slant culture obtained through step S04 has both short-term and long-term preservation capabilities. When short-term preservation is required, select a solid culture medium slant that is full of plump spores and free of contaminants, seal the tube opening with sealing film, label the strain information, date, and number of passages, and store it in a refrigerator at 4°C. When long-term preservation is required, place the spore suspension obtained by rinsing the solid culture medium slant in a refrigerator at -20°C.

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