An Optimization Method for the Traditional Fermentation Process of Arisaema cum Bile under Controlled Environment

Abstract

This invention relates to the field of traditional Chinese medicine fermentation, specifically disclosing an optimized method for the ancient fermentation process of Arisaema cum Bile under a controlled environment, comprising the following steps: Step 1, Raw material pretreatment: Select dried Arisaema cum Bile tubers, crush them through a 50-80 mesh sieve, and control the moisture content to ≤12%; collect fresh cattle bile, centrifuge and filter to remove impurities, and determine the total bile acid content to ≥8%, for later use; Step 2, Screening of core fermentation strains: Use a gradient dilution method to isolate microorganisms in the traditional ancient Arisaema cum Bile fermentation system, identify strains through 16S rRNA gene sequencing and metagenomic analysis, and use HPLC-ELSD to detect the bile acid conversion efficiency of different strains, screening to obtain a composite fermentation strain with *Saccharomyces de Barry*, *Penicillium*, and *Lactobacillus* as the core. This invention constructs a systematic and controllable fermentation environment, clarifies the key environmental parameters and regulation mechanisms in the early and late stages of fermentation, solves the problem of uncontrollable traditional fermentation environments, and significantly improves the stability and repeatability of the fermentation process.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine fermentation, and in particular to an optimized method for the fermentation process of Arisaema cum Bile under controlled conditions. Background Technology

[0002] Arisaema cum Bile is a commonly used fermented Chinese medicine in clinical practice. It is made by fermenting Arisaema cum Bile with ox bile. It has the effects of clearing heat and resolving phlegm, calming the nerves and relieving intractable diseases. It is widely used in the treatment of diseases such as stroke with phlegm coma, epilepsy, hyperlipidemia, and fatty liver. The traditional method of fermenting Arisaema cum Bile relies on the natural environment. The raw materials need to be put into the ox gallbladder in the twelfth lunar month and placed in a cool and ventilated place to ferment for more than 100 days, which has led to the clinical consensus that "Arisaema cum Bile after nine fermentations is better than ox gallstones".

[0003] However, traditional fermentation processes have many drawbacks: First, the fermentation environment is uncontrollable, with key parameters such as temperature, humidity, and aeration fluctuating with natural conditions, leading to instability in the fermentation process; second, the fermentation strains rely on natural inoculation, resulting in complex and highly variable microbial communities that affect bile acid conversion efficiency; third, process parameters are vague, lacking clear dynamic monitoring indicators and endpoint determination standards, leading to significant batch-to-batch quality differences; and fourth, the fermentation cycle is long, making it susceptible to contamination and resulting in a low product qualification rate. These problems severely restrict the industrialization of traditional Chinese medicine (TCM) processing and fail to meet clinical demand for high-quality TCM decoction pieces.

[0004] While some existing research exists on Arisaema cum Bile fermentation, it largely focuses on optimizing single process parameters. It fails to construct a systematic and controllable fermentation system or clarify the synergistic regulation mechanism between core fermentation strains and environmental parameters. Therefore, developing an optimization method for the traditional Arisaema cum Bile fermentation process that can precisely control the fermentation environment, identify key parameters, and ensure stable product quality is of significant practical importance. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention proposes an optimization method for the fermentation process of Arisaema cum Bile under controlled conditions. By screening core fermentation strains, constructing a multi-level environmental control system, and establishing a dynamic monitoring mechanism, the method achieves standardization and controllability of the fermentation process of Arisaema cum Bile, thus solving the problem of unstable quality in traditional processes.

[0006] To solve the above-mentioned technical problems, the basic technical solution proposed by this invention is as follows: An optimized method for the fermentation process of Arisaema cum Bile under controlled conditions includes the following steps: Step 1: Raw material pretreatment: Select dried Arisaema tubers, crush them through a 50-80 mesh sieve, and control the moisture content to ≤12%; collect fresh cattle bile, remove impurities by centrifugation and filtration, and determine the total bile acid content to ≥8% for later use; Step 2: Screening of core fermentation strains: Microorganisms in the traditional ancient method of fermenting bile arisaema were isolated using the gradient dilution method. The strains were identified by 16S rRNA gene sequencing and metagenomic analysis. The bile acid conversion efficiency of different strains was detected by HPLC-ELSD method. A complex fermentation strain with *Saccharomyces debareris*, *Penicillium*, and *Lactobacillus* as the core strains was obtained. Step 3: Constructing a Controllable Fermentation Environment: Build a fermentation device including modules for controlling temperature, humidity, dissolved oxygen, and aeration rate, and set the fermentation zone parameters: Days 1-30: Temperature 18-25℃, relative humidity 60-70%, dissolved oxygen concentration 3-5mg / L, ventilation rate 0.3-0.5vvm; 31-100 days: Temperature 22-28℃, relative humidity 65-75%, dissolved oxygen concentration 2-4mg / L, ventilation rate 0.5-0.8vvm; Step 4: Fermentation process implementation: Mix Arisaema powder and bovine bile evenly at a mass-to-volume ratio of 1:4, fill the bovine gallbladder, place it in the above-mentioned controllable fermentation device, inoculate with compound fermentation bacteria at an inoculation rate of 5-8%, start the environmental control system, and carry out 100 days of controllable fermentation. Step 5: Dynamic monitoring and endpoint determination: Samples were taken every 10 days during fermentation, and the conversion rate of free bile acids and changes in fingerprint chromatograms were detected by HPLC-ELSD method. When the conversion rate of free bile acids was ≥80% and the similarity between the fingerprint chromatogram and the control chromatogram was ≥0.90, the fermentation endpoint was determined. Step 6: Vacuum dry the fermentation product at a temperature below 60°C, pulverize it through a 60-mesh sieve, and obtain controlled fermentation of traditional Arisaema cum Bile slices.

[0007] Preferably, the Arisaema tuber mentioned in step one is processed to remove its toxicity. The processing method is as follows: soak the Arisaema tuber in clean water until there is no dry core, add a 2-3% alum solution, soak for 24-36 hours, take it out and drain it for later use.

[0008] Preferably, the ratio of the compound fermentation microorganisms in step two is: the colony count ratio of *Saccharomyces de Barry*: *Penicillium*: *Lactobacillus* is 3:2:1.

[0009] Preferably, the fermentation device in step three is also equipped with a pH control module, which controls the pH value of the system to 6.0-7.5 throughout the fermentation process by automatically adding citric acid or sodium carbonate solution.

[0010] Preferably, the HPLC-ELSD detection conditions in step five are as follows: the chromatographic column is a C18 column, 4.6 mm × 250 mm, 5 μm; the mobile phase is methanol-water: 85:15, v / v, the flow rate is 1.0 mL / min, and the column temperature is 30 °C; the drift tube temperature of the evaporative light scattering detector is 80 °C, and the carrier gas flow rate is 2.0 L / min.

[0011] Preferably, the free bile acids in step five include cholic acid, deoxycholic acid, and chenodeoxycholic acid, with a total free bile acid content ≥ 8.0 mg / g.

[0012] Preferably, the fermentation process in step four also includes regular stirring, with a stirring frequency of once every 15 days, a stirring time of 5-10 minutes each time, and a stirring speed of 30-50 r / min.

[0013] The beneficial effects of this invention are: This invention constructs a systematic and controllable fermentation environment, clarifies the key environmental parameters and control mechanisms in the early and late stages of fermentation, solves the problem of uncontrollable traditional fermentation environments, and significantly improves the stability and repeatability of the fermentation process.

[0014] This invention screens out a core complex fermentation microbial community and optimizes its ratio, replacing the traditional natural inoculation method. This ensures the consistency of the fermentation strains, improves the conversion efficiency of free bile acids, and makes the product quality more stable.

[0015] This invention establishes a dynamic monitoring and endpoint determination system based on free bile acid conversion rate and fingerprint spectrum, clarifies the quantitative standard for fermentation endpoint, avoids the subjectivity of traditional processes that rely on experience to judge the endpoint, and ensures the uniformity between product batches.

[0016] The traditional Arisaema heterophyllum slices prepared by this invention have high content of free active bile acids and stable efficacy. The heavy metals and harmful residues meet national standards. They can be used as an effective substitute for natural bezoar, meeting the needs of clinical medication. At the same time, they provide a new technical approach for the modernization and standardization of fermented Chinese medicine production. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] This invention discloses an optimized method for the ancient fermentation process of Arisaema cum Bile under controlled conditions. The raw material pretreatment, specifically the pretreatment of Arisaema cum Bile, involves selecting dried tubers free from mold and insect infestation, washing them, and soaking them in clean water for 72 hours, changing the water twice daily, until the tubers no longer have a dry core. Then, a 2-3% (w / w) alum solution is added, and the tubers are soaked for 24-36 hours. After soaking, the tubers are drained, pulverized, and passed through a 50-80 mesh sieve. The moisture content is measured to be ≤12%, and the tubers are then ready for use. This processing step effectively reduces the toxicity of Arisaema cum Bile, ensuring the safety of the subsequent fermented products.

[0019] Bovine bile pretreatment: Fresh bile from healthy cattle was collected and immediately stored at 4°C. Impurities and precipitates were removed by centrifugation (3000 r / min, 10 min) within 24 hours. The total bile acid content was determined by HPLC-ELSD method to be ≥8%. If the content was insufficient, it was concentrated to meet the standard and then used for future reference.

[0020] Screening and optimization of core fermentation strains: A sample of Arisaema cum Bile fermented for 100 days using traditional methods was aseptically ground into powder and mixed with sterile physiological saline to prepare a bacterial suspension. The suspension was then inoculated onto LB medium, Czapek's medium, and MRS medium using a gradient dilution method and cultured at 25℃, 28℃, and 37℃ for 48-72 hours, respectively. Single colonies with different morphologies were then selected for purification and culture.

[0021] Strain identification: Through morphological observation, 16S rRNA gene sequencing and metagenomic analysis, a variety of microorganisms, including Debaryomycess p., Penicillium sp., Lactobacillus sp., and Candid sp., were identified.

[0022] Strain screening: The purified strains were subjected to single-strain fermentation experiments using Arisaema powder and bovine bile as fermentation substrates. Fermentation was carried out for 30 days at 25℃ and 70% humidity. The conversion rate of free bile acids was detected by HPLC-ELSD. The results showed that the bile acid conversion efficiency of *Saccharomyces debareri*, *Penicillium*, and *Lactobacillus* was significantly higher than that of other strains, at 75.3%, 68.5%, and 62.1%, respectively.

[0023] Optimization of the compound microbial community ratio: The three core microbial strains were mixed in different ratios (3:1:1, 3:2:1, 2:2:1, 1:2:1) and compound fermentation experiments were conducted to detect the conversion rate of free bile acids. The results showed that the highest free bile acid conversion rate (82.7%) was achieved when the ratio of *Saccharomyces de Barry*: *Penicillium*: *Lactobacillus* was 3:2:1 (colony count ratio). This ratio was determined to be the optimal ratio for the compound fermentation microbial community.

[0024] Controlled fermentation environment construction and fermentation device setup: An intelligent fermentation chamber is used as the controlled fermentation device. This device integrates five major control modules: temperature, humidity, dissolved oxygen, aeration rate, and pH value, enabling precise parameter setting and automatic adjustment. The device contains multiple independent fermentation chambers, each equipped with sensors to monitor environmental parameters in real time. The data is displayed and recorded in real time through the control system.

[0025] Environmental parameter settings: Based on traditional fermentation patterns and strain growth characteristics, the fermentation process is divided into two stages: early and late stages, with differentiated parameters set accordingly. Early stage (1-30 days): Temperature 18-25℃, relative humidity 60-70%, dissolved oxygen concentration 3-5mg / L, aeration rate 0.3-0.5vvm, pH 6.0-6.5. This stage is mainly the acclimatization and proliferation period of the strain. Lower temperature and aeration rate are conducive to the colonization of the strain.

[0026] Later stage (31-100 days): Temperature 22-28℃, relative humidity 65-75%, dissolved oxygen concentration 2-4 mg / L, ventilation rate 0.5-0.8 vvm, pH 6.5-7.5. This stage is the critical period for bile acid conversion; appropriately increasing the temperature and ventilation rate can promote the activity of converting enzymes.

[0027] Parameter control mechanism: Through the automatic control system of the fermentation device, when the sensor detects that the parameters deviate from the set range, the corresponding control module is activated: the temperature is adjusted by the heating tube or the cooler, the humidity is adjusted by the humidifier or the dehumidifier, the dissolved oxygen and the aeration rate are adjusted by the aeration pump, and the pH value is adjusted by automatically adding citric acid (to lower the pH) or sodium carbonate solution (to raise the pH) to ensure a stable fermentation environment.

[0028] Fermentation process, raw material mixing and sample loading: Mix Arisaema powder and ox bile at a ratio of 1:4 (mass-volume ratio) until homogeneous. Stir at a speed of 50-80 r / min for 15-20 minutes to ensure uniform mixing. Load the mixture into cleaned and dried ox gallbladders, filling each gallbladder to 70-80% of its volume. Seal and label the gallbladders.

[0029] Inoculation with microorganisms: Prepare a bacterial suspension (concentration 10) using sterile physiological saline. 6 -10 7 Inoculate the above mixture in the bovine gallbladder at an inoculation rate of 5-8% (v / w), and gently shake to ensure even distribution of the bacteria.

[0030] Controlled fermentation: Place the inoculated bovine gallbladders neatly in the fermentation chamber of the controlled fermentation device, avoiding compression. Start the fermentation device, set the environmental parameters for the early and later stages, and activate the automatic control system for 100 days of controlled fermentation. During fermentation, regularly check the device's operating status and parameter records to ensure the equipment is working properly.

[0031] Dynamic monitoring and endpoint determination, sampling frequency: Sampling was carried out every 10 days during fermentation. Three bovine gallbladders were randomly selected each time, opened under aseptic conditions, and 5-10g of internal fermentation products were taken. The samples were then sealed, labeled and used for testing.

[0032] Detection indicators and methods: Free bile acid conversion rate: Detected using HPLC-ELSD method. The chromatographic column was an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase was methanol-water (85:15, v / v), the flow rate was 1.0 mL / min, and the column temperature was 30 °C. The drift tube temperature of the evaporative light scattering detector was 80 °C, and the carrier gas flow rate was 2.0 L / min. The conversion rate of free bile acids was calculated as the ratio of the total content of free bile acids (cholic acid, deoxycholic acid, and chenodeoxycholic acid) to the total bile acid content.

[0033] Fingerprint chromatogram: The fingerprint chromatogram of the fermentation product was recorded under the above HPLC-ELSD detection conditions. The similarity was analyzed with the preset control chromatogram (obtained by averaging the fingerprint chromatograms of 3 batches of high-quality traditional Arisaema cum bile). The similarity was calculated using similarity evaluation software.

[0034] Endpoint determination criteria: When the free bile acid conversion rate is ≥80% in two consecutive sampling tests, and the similarity between the fingerprint spectrum and the control spectrum is ≥0.90, the fermentation endpoint is determined, and fermentation is stopped. If the endpoint is not reached after 100 days, the fermentation time can be extended to 120 days. If the endpoint is still not reached, the fermentation is considered a failure.

[0035] Post-processing drying: The product that has reached the fermentation endpoint is removed from the bovine gallbladder, spread on a tray to a thickness of 2-3 cm, and placed in a vacuum drying oven. It is dried at a temperature below 60℃ and a vacuum degree of -0.08 to -0.1 MPa until the moisture content is ≤9%.

[0036] Crushing and sieving: The dried product is put into a crusher and crushed. After crushing, it is passed through a 60-mesh sieve. The material on the sieve is crushed again to ensure uniform particle size.

[0037] Packaging and storage: Pack the pulverized and sieved medicinal slices into aseptic packaging bags, seal them, and label them with information such as production date and batch number. Store them in a cool, dry place to avoid moisture and mold.

[0038] The specific implementation method is as follows:

[0039] Example 1 Optimization of the Ancient Method of Fermenting Arisaema in a Controlled Environment Raw material pretreatment: Take 10 kg of dried Arisaema tubers, wash them, soak them in water for 72 hours, changing the water twice a day, take them out, add 2.5% alum solution and soak for 30 hours, drain them, crush them and pass them through a 60-mesh sieve, and determine the moisture content to be 11.2%; collect 40 L of fresh cattle bile, centrifuge and filter (3000 r / min, 10 min) to remove impurities, and determine the total bile acid content to be 9.5% by HPLC-ELSD method.

[0040] Preparation of core fermentation strains: A compound fermentation microbial community was prepared according to the ratio of *D. de Barry*: *Penicillium*: *Lactobacillus* = 3:2:1 (colony count ratio). A bacterial suspension was prepared using sterile physiological saline to a concentration of 5 × 10⁻⁶. 6 CFU / mL.

[0041] Controllable fermentation environment settings: Start the intelligent fermentation chamber and set the parameters for the early stage (1-30 days): temperature 20-25℃, relative humidity 65-70%, dissolved oxygen concentration 3-5mg / L, aeration rate 0.4vvm, pH value 6.0-6.5; parameters for the later stage (31-100 days): temperature 25-28℃, relative humidity 70-75%, dissolved oxygen concentration 2-4mg / L, aeration rate 0.6vvm, pH value 6.5-7.5.

[0042] Fermentation process: Mix Arisaema powder and bovine bile at a ratio of 1:4, stir at 60 r / min for 20 minutes, and fill 10 bovine gallbladders (4 kg per gallbladder). Inoculate with a compound fermentation culture at an inoculation rate of 6%, shake well, place in a fermentation chamber, start the automatic control system, and ferment for 100 days.

[0043] Dynamic monitoring: Sampling and testing were conducted every 10 days. On the 90th day of fermentation, the free bile acid conversion rate was 83.2%, and the fingerprint spectrum showed a similarity of 0.92 with the control spectrum. On the 100th day, the free bile acid conversion rate was 84.5%, with a similarity of 0.93, indicating that the fermentation endpoint had been reached.

[0044] Post-processing: The fermentation product was taken out and dried at 55℃ and vacuum degree -0.09MPa until the moisture content was 8.5%. It was then pulverized and passed through a 60-mesh sieve to obtain 7.6kg of controlled fermented traditional Arisaema cum Bile slices.

[0045] Example 2 Product quality testing Content determination: The slices prepared in Example 1 were analyzed by HPLC-ELSD. The contents of bile acid were 3.6 mg / g, deoxycholic acid 1.7 mg / g, chenodeoxycholic acid 2.3 mg / g, total free bile acid 7.6 mg / g, and free bile acid conversion rate 84.5%.

[0046] Fingerprint spectrum: Analysis using similarity evaluation software showed that the similarity between the fingerprint spectrum of the medicinal slices and the control spectrum was 0.93, which met the requirements.

[0047] Safety testing: According to the 2020 edition of the Chinese Pharmacopoeia, Volume IV, the heavy metal content was: lead 2.1 mg / kg, cadmium 0.09 mg / kg, arsenic 1.0 mg / kg, mercury 0.07 mg / kg, copper 14.8 mg / kg; aflatoxin B1 content was 1.1 μg / kg, all of which met the limit requirements.

[0048] Pharmacodynamic validation: The herbal slices were prepared into an aqueous solution and administered to hyperlipidemic model mice by gavage for 30 consecutive days. The results showed that the serum total cholesterol and triglyceride levels of the treated mice were significantly reduced (P<0.05), and the fat deposition in the liver tissue was significantly reduced, indicating that it has a good lipid-regulating and liver-protecting effect.

[0049] Example 3 Comparative test Three experimental groups were set up: control group (traditional natural fermentation), experimental group 1 (controlled fermentation of this invention, without inoculation of compound microorganisms), and experimental group 2 (controlled fermentation of this invention, inoculated with compound microorganisms). Three batches of samples were prepared for each group, and relevant indicators were detected. The results are shown in the table below:

[0050] The results showed that the free bile acid conversion rate of the controlled fermentation method of the present invention (experimental group 2) was significantly higher than that of the control group and experimental group 1, the fingerprint spectrum similarity was higher, and the batch-to-batch stability was better, which fully demonstrated the technical advantages of the present invention.

[0051] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.

Claims

1. An optimized method for the fermentation process of traditional Arisaema cum Bile under controlled conditions, comprising the following steps: Step 1: Raw material pretreatment: Select dried Arisaema tubers, crush them through a 50-80 mesh sieve, and control the moisture content to ≤12%; Fresh cattle bile was collected, centrifuged and filtered to remove impurities, and the total bile acid content was determined to be ≥8% for later use. Step 2: Screening of core fermentation strains: Microorganisms in the traditional ancient method of fermenting bile arisaema were isolated using the gradient dilution method. The strains were identified by 16S rRNA gene sequencing and metagenomic analysis. The bile acid conversion efficiency of different strains was detected by HPLC-ELSD method. A complex fermentation strain with *Saccharomyces debareris*, *Penicillium*, and *Lactobacillus* as the core strains was obtained. Step 3: Constructing a Controllable Fermentation Environment: Build a fermentation device including modules for controlling temperature, humidity, dissolved oxygen, and aeration rate, and set the fermentation zone parameters: Days 1-30: Temperature 18-25℃, relative humidity 60-70%, dissolved oxygen concentration 3-5mg / L, ventilation rate 0.3-0.5vvm; 31-100 days: Temperature 22-28℃, relative humidity 65-75%, dissolved oxygen concentration 2-4mg / L, ventilation rate 0.5-0.8vvm; Step 4: Fermentation process implementation: Mix Arisaema powder and bovine bile evenly at a mass-to-volume ratio of 1:4, fill the bovine gallbladder, place it in the above-mentioned controllable fermentation device, inoculate with compound fermentation bacteria at an inoculation rate of 5-8%, start the environmental control system, and carry out 100 days of controllable fermentation. Step 5: Dynamic monitoring and endpoint determination: Samples were taken every 10 days during fermentation, and the conversion rate of free bile acids and changes in fingerprint chromatograms were detected by HPLC-ELSD method. When the conversion rate of free bile acids was ≥80% and the similarity between the fingerprint chromatogram and the control chromatogram was ≥0.90, the fermentation endpoint was determined. Step 6: Vacuum dry the fermentation product at a temperature below 60°C, pulverize it through a 60-mesh sieve, and obtain controlled fermentation of traditional Arisaema cum Bile slices.

2. The method for optimizing the ancient fermentation process of Arisaema cum Bile under controlled environment according to claim 1, characterized in that, The Arisaema tuber mentioned in step one is processed to remove its toxicity. The processing method is as follows: soak the Arisaema tuber in clean water until there is no dry core, add a 2-3% alum solution, soak for 24-36 hours, take it out and drain it for later use.

3. The method for optimizing the ancient fermentation process of Arisaema cum Bile under controlled environment according to claim 1, characterized in that, In step two, the ratio of the compound fermentation microbial community is: *Saccharomyces de Barry*: *Penicillium*: *Lactobacillus* colony count ratio is 3:2:

1.

4. The method for optimizing the ancient fermentation process of Arisaema cum Bile under controlled environment according to claim 1, characterized in that, In step three, the fermentation device is also equipped with a pH control module, which controls the pH value of the system to 6.0-7.5 throughout the fermentation process by automatically adding citric acid or sodium carbonate solution.

5. The method for optimizing the ancient fermentation process of Arisaema cum Bile under controlled environment according to claim 1, characterized in that, The HPLC-ELSD detection conditions in step five were as follows: the chromatographic column was a C18 column, 4.6 mm × 250 mm, 5 μm; the mobile phase was methanol-water: 85:15, v / v, the flow rate was 1.0 mL / min, and the column temperature was 30 °C; the drift tube temperature of the evaporative light scattering detector was 80 °C, and the carrier gas flow rate was 2.0 L / min.

6. The method for optimizing the ancient fermentation process of Arisaema cum Bile under controlled environment according to claim 1, characterized in that, The free bile acids in step five include cholic acid, deoxycholic acid, and chenodeoxycholic acid, with a total free bile acid content ≥ 8.0 mg / g.

7. The method for optimizing the ancient fermentation process of Arisaema cum Bile under controlled environment according to claim 1, characterized in that, Step four of the fermentation process also includes regular stirring, with a stirring frequency of once every 15 days, each stirring lasting 5-10 minutes, and a stirring speed of 30-50 r / min.

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