A method to shorten processing time and increase gallic acid content in Baiyao Decoction

Abstract

This invention relates to the field of traditional Chinese medicine (TCM) pharmaceutical technology, specifically a method for shortening processing time and increasing the gallic acid content in Baiyao Decoction. The method involves a filamentous fungus, FAa-2 (Aspergillus austwickii), which was deposited on June 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40708, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The filamentous fungus FAa-2 can significantly increase the content of the active ingredient gallic acid in Baiyao Decoction within a short time. This invention applies this microorganism to the processing of Baiyao Decoction (referring to SCYPBZXD2021-001 "Baiyao Decoction," a TCM standard of the Sichuan Provincial Drug Administration), which can avoid contamination by other microorganisms, shorten the fermentation and processing cycle, and significantly increase the content of the active ingredient gallic acid in Baiyao Decoction within a short time.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine pharmaceutical technology, specifically a method for shortening processing time and increasing the gallic acid content in Baiyao Decoction. Background Technology

[0002] Baiyaojian is a traditional Chinese medicine decoction made from ingredients such as gallnut, tea leaves, wine lees or yeast, which are naturally fermented, processed, and dried into a block-like form. It has the effects of clearing heat and resolving phlegm, promoting body fluid production and quenching thirst. It is used for lung heat cough, toothache due to wind-heat, oral ulcers, chronic dysentery with rectal prolapse, and conditions such as pharyngitis, asthma, bronchitis, and gastroenteritis. Gallnut contains a large amount of tannins, which can bind with proteins on the gastrointestinal tract to form large molecular precipitates, irritating the stomach and intestines and causing adverse reactions. However, after fermentation and processing, the tannins in gallnut can be hydrolyzed and converted into the active ingredient gallic acid, thereby improving efficacy and reducing irritation. Gallic acid has trypanosome-killing, anti-inflammatory, antibacterial, antitumor, and antiviral activities.

[0003] Firstly, while traditional natural fermentation in the preparation of Baiyao Decoction can convert hydrolyzable tannins into gallic acid to some extent, the gallic acid content is low and may not meet pharmaceutical quality standards. Furthermore, the remaining large amount of tannins can cause adverse reactions. Moreover, the quality of Baiyao Decoction cannot be guaranteed due to the influence of harmful microorganisms in the natural environment, potentially leading to excessive levels of these microorganisms and the introduction of other harmful components. Therefore, adding a single, pure strain of microorganism capable of hydrolyzing tannins can avoid the harm caused by other microorganisms.

[0004] Secondly, the traditional processing techniques are of low quality, have long processing cycles, and suffer from poor quality control and high costs. Summary of the Invention

[0005] The purpose of this invention is to provide a filamentous fungus FAa-2 (Aspergillus austwickii) that can be used in the fermentation and processing steps of Baiyao Decoction, which can shorten the fermentation and processing time and significantly increase the content of the active ingredient gallic acid in a short time.

[0006] A filamentous fungus FAa-2 (Aspergillus austwickii) was deposited on June 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40708. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101.

[0007] Another objective of this invention is to protect the application of the filamentous fungus FAa-2 (Aspergillus austwickii) in shortening the fermentation and processing time in Baiyao Decoction.

[0008] The third objective of this invention is to protect the application of filamentous fungus FAa-2 (Aspergillus austwickii) in significantly increasing the content of gallic acid, an active ingredient, in Baiyao Decoction within a short period of time.

[0009] Furthermore, its specific application method is to add the filamentous fungus FAa-2 (Aspergillus austwickii) to the fermentation and processing steps of the traditional Baiyao Decoction method.

[0010] A method for shortening the fermentation and processing time of Baiyao Decoction includes the following steps:

[0011] In the traditional method of preparing Baiyao Decoction, the aforementioned filamentous fungus FAa-2 (Aspergillus austwickii) is added during the fermentation process, and the medicine can be obtained after about 7 days of fermentation.

[0012] A method for shortening processing time and increasing gallic acid content in Baiyao Decoction includes the following steps:

[0013] a. Preparation of gallnut powder: After removing impurities from gallnuts, crush them, remove aphids, wash them clean, dry them, and grind them into powder (preferably through an 80-mesh sieve) for later use;

[0014] b. Green tea decoction concentrate: Take green tea, add water and decoct into juice, decoct twice, filter out the tea leaves and combine the decoction liquid, vacuum concentrate, cool and set aside;

[0015] c. Baiyao Decoction: The concentrated juice of cooled green tea is mixed evenly with the lees, and then microbial mycelial balls prepared by the aforementioned filamentous fungus FAa-2 (Aspergillus austwickii) are added and mixed evenly. Then, gallnut powder and water are added and mixed evenly. After forming a dough, it is placed in a constant temperature and humidity incubator for fermentation. After fermentation for a period of time, when the surface of the medicine block is covered with "mold", it is cut into small pieces and dried at a certain temperature to obtain the finished product.

[0016] In a preferred embodiment of this application, when green tea is boiled into a juice, the ratio of green tea to water is 31.0g:500mL; the boiling time for each batch is 15-60min (specifically, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, etc.); and the vacuum concentration conditions are 45-70℃ (specifically, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, etc.).

[0017] As a preferred embodiment of this application, when 31g of green tea is boiled twice with 500mL of water, the volume after vacuum concentration is 100-175mL.

[0018] As a preferred embodiment of this application, the ratio of the volume (mL) of the green tea decoction concentrate to the mass (g) of the distiller's grains is 150:125.

[0019] In a preferred embodiment of this application, the ratio of the mass (g) of gallnut powder to the volume (mL) of the cooled green tea decoction concentrate is 500:150; the amount of water added is 15-60% of the mass of gallnut powder (specifically, it can be 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, etc.).

[0020] In this application, the preparation of microbial mycelial balls is prior art and will not be described in detail.

[0021] As a preferred embodiment of this application, the pellet size is required to be 4cm×4cm×2cm; the conditions of the constant temperature and humidity incubator are: temperature 20-35℃, humidity 35-95% (specifically, it can be 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.).

[0022] In a preferred embodiment of this application, the ratio of the volume (mL) of the microbial mycelial balls to the mass (g) of the gallnut powder is 1:50 to 1:10.

[0023] As a preferred embodiment of this application, the fermentation and processing time is 7 days, specifically based on the time when the surface of the medicinal block is covered with "mold".

[0024] In a preferred embodiment of this application, the drying temperature in step c is 45–70°C (specifically, it can be 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, etc.); the moisture content of the finished product does not exceed 13%.

[0025] Another inventive objective of this application is to provide a decoction of medicinal herbs obtained by using any of the above methods or any combination of the above steps; the decoction of medicinal herbs can reach a gallic acid content of more than 35% on the 5th day, meeting the medicinal standard; after 7 days of processing, the gallic acid content of the decoction of medicinal herbs can be between 50% and 55%.

[0026] Compared with the prior art, the positive effects of the present invention are reflected in:

[0027] (i) Traditional processing of Baiyao Decoction results in a low gallic acid content due to natural environmental conditions, which does not meet the medicinal standard of 35-55%, and the stability is poor (traditional processing generally requires fermentation and processing for more than 15 days for the gallic acid content to exceed 35% to meet the medicinal standard); using filamentous fungus FAa-2 (Aspergillus austwickii) to process Baiyao Decoction for 5 days, the gallic acid content can meet the medicinal standard and the stability is good.

[0028] (ii) Adding filamentous fungus FAa-2 (Aspergillus austwickii) to process Baiyao Decoction for about 7 days can result in gallic acid content of 50-55%, which is far higher than the medicinal standard and has good stability. Attached image description:

[0029] Figure 1 High performance liquid chromatogram of gallic acid standard (China National Institutes for Food and Drug Control, batch number: 110831-201906).

[0030] Figure 2 The high-performance liquid chromatogram of gallic acid in the traditional Chinese medicine decoction.

[0031] Figure 3 The high-performance liquid chromatogram of gallic acid in Baiyao Decoction prepared by adding screened microorganisms and optimizing the processing conditions in this invention.

[0032] Figure 4 The fungi isolated from Baiyaojian products from different sources are the fungi of this invention, wherein A and B are from Runxintang Baiyaojian products; and C and D are isolated from Qianyuan Baiyaojian products.

[0033] Figure 5 Bacteria and yeasts isolated from Baiyaojian products from different sources, of which 1 and 2 were isolated from the solid bacteria and yeast of Qianyuan Baiyaojian; and 3 and 4 were isolated from the solid bacteria of Runxintang Baiyaojian.

[0034] Figure 6 For microscopic examination of fungi, A is identified as *Aspergillus*, with a spherical apex bearing conidia and spherical spores; B is identified as *Penicillium*, with symmetrical broom-shaped conidiophores and septate, transparent hyphae.

[0035] Figure 7 Transparent rings generated on PDA tannin plates for the numbers FAa-2, FAa-4, FAa-7, and FU-a.

[0036] Figure 8 This is a diagram showing the sequence alignment results for FAa-2.

[0037] Figure 9 The graph shows the change in gallic acid content of Baiyao Decoction with fermentation and processing time after adding microorganisms ② and optimizing the processing conditions. Detailed Implementation

[0038] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in this specification are merely for explaining the invention and are not intended to limit the invention.

[0039] Unless otherwise specified, the percentage used in this application document refers to the mass percentage, i.e., wt%.

[0040] A filamentous fungus FAa-2 (Aspergillus austwickii) was deposited on June 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40708. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101.

[0041] The gallnuts used in the experiment were from the same batch of products from Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd., the green tea was from the same batch of products from Yunnan Baoshan Sanliuwu Tea Industry Co., Ltd., and the distiller's grains (rice, wheat, sorghum, etc., used as residue after brewing) were from the same batch of products from Chongzhou Winery. The following experiments were conducted using gallnuts as a control.

[0042] Screening of strains used in the preparation of traditional Chinese medicine decoctions, such as Aspergillus austwickii

[0043] Isolation, purification and identification of dominant bacterial strains at different stages of processing of 100 medicinal decoction

[0044] 1.1 Experimental Materials

[0045] The solids of Baiyaojian (a traditional Chinese medicine formula) are derived from different fermentation stages of Shanxi Qianyuan Pharmaceutical Group Co., Ltd. (hereinafter referred to as "Qianyuan") and Chengdu Runxintang Pharmaceutical Co., Ltd. (hereinafter referred to as "Runxintang").

[0046] 1.2 Culture medium

[0047] LB medium: 10g peptone, 5g yeast extract, 10g NaCl, pH 7.0-7.2.

[0048] MRS medium: 10g peptone, 10g beef extract, 5g yeast powder, 2g K2HPO4, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.58g MgSO4·7H2O, 0.25g MnSO4·4H2O, (15-20g agar), 1L distilled water.

[0049] Gao's No. 1 medium: 2% soluble starch, 0.001% ferrous sulfate, 0.1% potassium nitrate, 0.05% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.05% sodium chloride, 2% agar, pH 7.4-7.6.

[0050] PDA medium: Wash and peel the potatoes, cut them into small pieces, and place 200g of each piece in a 2L beaker. Add 800mL of distilled water and heat on an induction cooker while stirring to prevent scorching. After the potatoes are cooked through, filter through eight layers of gauze, discard the residue, and add 17g of agar to the filtrate. Continue heating and stirring until the agar dissolves, then add 20g of glucose and stir until dissolved. After slightly cooling, add water to bring the volume to 1000mL, dispense into Erlenmeyer flasks, and sterilize at 115℃ for 20 minutes.

[0051] 1.3 Reagents

[0052] 100 μg / mL bromocresol green indicator, 50 μg / mL nystatin, 50 μg / mL streptomycin sulfate, 100 μg / mL potassium dichromate.

[0053] 1.4 Experimental Methods

[0054] 1.4.1 Microbial Isolation and Purification

[0055] Under aseptic conditions, 25g of samples from different processing stages (3d, 6d, 9d, 12d, and 15d of fermentation processing) from Runxintang and Qianyuan were weighed and added to Erlenmeyer flasks containing glass beads and 225mL of sterile water. The flasks were shaken thoroughly to disperse the bacteria and serially diluted. 100μL of the diluted sample solution was evenly spread onto different isolation plates (LB medium containing nystatin solution, MRS medium containing bromocresol green, PDA medium containing streptomycin sulfate, and Gao's No. 1 medium containing potassium dichromate). MRS and LB plates were incubated at 37℃ for 1–2 days; PDA and Gao's No. 1 plates were incubated at 28℃ for 2–3 days. Colonies with differences in morphology, size, edge, and color were picked from the isolation media and inoculated onto new isolation plates. The isolated strains were purified multiple times, inoculated onto slant agar, numbered, and stored at 4℃.

[0056] 1.4.2 Strain Identification

[0057] Bacteria were stained with Gram and yeast was stained with methylene blue and then observed under a microscope for microscopic morphology; filamentous fungi were cultured using the slide method, and the morphological characteristics of hyphae and spores on the slide were observed under a microscope after the hyphae matured.

[0058] 1.4.3 Strain Preservation

[0059] Glycerin tube preservation: Prepare a 50% glycerol aqueous solution and sterilize at 121℃ for 20 min. Prepare bacterial suspensions under aseptic conditions, and mix the bacterial suspension with glycerol in a 1:1 ratio; for fungi, rinse the lower surface spores with 0.85% physiological saline, and mix with sterile glycerol aqueous solution in a 1:1 ratio, and store at -60℃.

[0060] 1.5 Experimental Results

[0061] 1.5.1 Separation, Purification, and Identification

[0062] Microorganisms were isolated from Baiyaojian solids from different fermentation stages of Qianyuan and Runxintang using isolation media containing indicators or antibiotics. A total of 26 strains were isolated, including molds (filamentous fungi), bacteria, and yeasts. (See attached text.) Figure 4 and Figure 5 The fungi were predominantly molds, totaling 14 strains. Morphological identification revealed that they belonged to the genera *Aspergillus* and *Penicillium*. (See below) Figure 6 All isolated strains were numbered and stored, as shown in Table 1.

[0063] Table 1. Fungi isolated from Baiyaojian solids from different sources

[0064]

[0065] Analysis of the tannin degradation capacity of isolated strains from 200 herbal decoctions

[0066] 2.1 Experimental Materials

[0067] The 26 bacterial strains obtained from the finished products of Runxintang and Qianyuan Baiyaojian are shown in Table 2:

[0068] Table 2. Strains obtained from the finished products of Runxintang and Qianyuan Baiyao Decoction.

[0069]

[0070] 2.2 Reagents

[0071] 0.1% Tannin Solution: Add 0.1g of tannic acid to every 100mL of distilled water.

[0072] 1% Tannins: Add 1g of tannic acid per 100mL of distilled water.

[0073] 2.3 Experimental Methods

[0074] 2.3.1 Analysis of bacterial tannin degradation ability

[0075] Under aseptic conditions, 1 mL of 0.1% tannin was added to the surface of LB solid medium. The tannin solution reacted with the LB medium, turning it into a milky white solid. Excess tannin solution was removed, and the petri dish was tilted to allow the surface of the medium to fully absorb the liquid under aseptic conditions. Bacterial culture solutions were prepared, serially diluted, and spread. The cultures were incubated at 37°C for 2 days, and the presence of discoloration zones or hydrolytic clear zones around the colonies was observed.

[0076] 2.3.2 Analysis of fungal tannin degradation capacity

[0077] Add 1 mL of 1% tannin to the surface of PDA medium. A yellowish-white opaque layer forms on the surface of the solid plate. Remove the excess liquid and inoculate yeast and filamentous fungi in the center of the medium. Incubate the plate upside down at 30°C for 4 days and observe whether a hydrolytic transparent zone appears around the strains and whether the color of the medium surface changes.

[0078] 2.3.3 Molecular biological identification

[0079] Based on whether a clear zone is produced on the tannin culture medium as the initial screening criterion, strains that produce clear zones are selected and sent to Shanghai Paisennuo Biotechnology Co., Ltd. for strain identification.

[0080] 2.4 Experimental Results

[0081] 2.4.1 Bacterial ability to degrade tannins

[0082] The bacteria obtained through isolation and screening were serially diluted, and a dilution of 10 was selected. -5 ~10 -7 100 μL of each sample was spread onto LB plates containing 0.1% tannin solution and incubated for 2 days to check whether a hydrolytic clear zone was formed. The results are shown in Table 3 below.

[0083] Table 3. Ability of bacteria isolated from Qianyuan and Runxintang to degrade tannins.

[0084]

[0085]

[0086] Note: "—" indicates a negative test result; "+" indicates a positive test result.

[0087] As shown in the table above, it can be preliminarily determined that the bacteria BR-1, BR-2, BR-3, BR-4, BQ-1, BQ-2, and BQ-3 isolated from the solid of Baiyaojian from different sources have no effect on the degradation of tannins.

[0088] 2.4.2 The ability of yeast to degrade tannins

[0089] After serial dilution of the isolated yeast, 1 mL of the yeast was spread onto PDA medium containing 1% tannin solution and cultured at 28°C for 4 days. The clear zones on the medium are shown in Table 4.

[0090] Table 4. Degradation capacity of yeast strains isolated from Qianyuan and Runxintang for tannins.

[0091]

[0092] Note: "—" indicates a negative test result; "+" indicates a positive test result.

[0093] As shown in the table above, it can be preliminarily determined that the yeast strains SR-1, SR-2, SQ-1, SQ-2, and SQ-3 isolated from Qianyuan and Runxintang have no effect on the degradation of tannins.

[0094] 2.4.3 The ability of filamentous fungi to degrade tannins

[0095] The isolated filamentous fungi were inoculated at the center of PDA medium containing 1% tannin solution and incubated at 28°C for 4 days. Clear zones were observed on the medium. Figure 7 Tables 5 and 6.

[0096] Table 5. Degradation capacity of filamentous fungi obtained from solid separation of Qianyuan Baiyao Decoction for tannins.

[0097]

[0098] Table 6. Degradation ability of filamentous fungi obtained from solid separation of Runxintang Baiyaojian (a traditional Chinese medicine decoction) on tannins.

[0099]

[0100] Note: "—" indicates a negative test result; "+" indicates a positive test result.

[0101] Tables 5 and 6 show that the four filamentous fungi isolated from Qianyuan Baiyao Decoction (numbered FAa-2, FAa-4, and FAa-7) and from Runxintang (numbered FU-a) exhibited distinct hydrolytic clear zones on PDA tannin plates. Figure 7 The remaining 10 plants did not produce obvious hydrolysis transparent zones, and it was preliminarily determined that FAa-2, FAa-4, FAa-7, and FU-a have the ability to degrade tannins.

[0102] 2.4.4 Molecular biological identification of 18S rDNA

[0103] Four filamentous fungi, FAa-2, FAa-4, FAa-7, and FU-a, were sent to Shanghai Paisennong Biotechnology Co., Ltd. for molecular biological identification. The obtained sequences were compared with those in NCBI, and the sequence with the highest similarity was selected as the species identification result. The identification results are as follows: Figure 8 (FAa-2 sequence alignment results are shown in the figure) and Table 7.

[0104] Depend on Figure 8 It is evident that the strain with the highest homology to FAa-2 is Aspergillus austwickii. The four microorganisms capable of degrading tannins—FAa-2, FAa-4, FAa-7, and FU-a—were used in subsequent experiments and simplified as ②, ④, ⑦, and a, respectively.

[0105] Table 7 Molecular biological identification results

[0106]

[0107] The method for preparing mycelial balls described in the following examples is as follows:

[0108] 1. Prepare PDB medium (do not add agar to PDA medium).

[0109] 2. Measure 100 mL of PDB culture medium and dispense it into 500 mL Erlenmeyer flasks. Autoclave at 115 °C for 20 min for later use.

[0110] 3. The selected filamentous fungi were inoculated into PDB medium and cultured in a shaking incubator at 30°C and 100 rpm for 48 hours to obtain the final product.

[0111] The following gallic acid detection method refers to the detection method in SCYPBZXD 2021-001 "Baiyaojian".

[0112] The traditional method for preparing Baiyao Decoction (referring to SCYPBZXD2021-001 "Baiyao Decoction", Sichuan Provincial Drug Administration Standard for Traditional Chinese Medicine) involves the following steps:

[0113] 1. Gallnut powder: Remove impurities, crush gallnuts, remove aphids, wash clean, dry, and grind into powder (measured using an 80-mesh sieve in this experiment), and set aside.

[0114] 2. Green tea decoction: Take 31.0g of green tea and add 500g of water to decoct into juice. Decoction twice (each decoction time is 0.5h in this experiment). Filter out the tea leaves and combine the decoction (300mL in this experiment). Cool and set aside.

[0115] 3. Baiyao Decoction: Mix the cooled green tea decoction with 125.0g of wine lees, then add 500.0g of gallnut powder and an appropriate amount of water (15% of the gallnut powder's mass in this experiment). Stir well and form into balls (4cm×4cm×2cm in this experiment). Place them in a natural environment (room temperature) for fermentation. After a certain period of time, when the surface of the medicinal blocks is covered with a "mold coat," cut them into small pieces and dry (at 55℃ in this experiment) to obtain the finished product.

[0116] 4. Detect its gallic acid content.

[0117] The optimized preparation of Baiyao Decoction includes the following steps:

[0118] a. Gallnut powder: Pick out impurities from commercially available gallnuts, crush them, remove aphids, wash them clean, dry them, grind them into powder, and pass them through an 80-mesh sieve for later use.

[0119] b. Green tea decoction concentrate: Take 31.0g of commercially available green tea leaves, add 500mL of water and decoct to make juice. Decoction twice, each time for 0.5h. Filter out the tea leaves and combine the decoction liquid. Concentrate to 150mL under vacuum at 55℃. Cool and set aside for later use.

[0120] c. Decoction of Hundred Herbs: Mix 150mL of the concentrated juice from cooled green tea decoction with 125.0g of distiller's grains. Then add 500.0g of gallnut powder prepared in step a) and 45% (by weight of gallnut powder) of water. Mix well and form into 4cm×4cm×2cm balls. Place the balls in an incubator at a constant temperature of 30℃ and a constant humidity of 75% for fermentation. After fermentation for a period of time, when the surface of the herb blocks is covered with a "mold coat", cut them into small pieces and dry them at 55℃ to obtain the finished product.

[0121] d. Detect its gallic acid content.

[0122] Galla powder, fully hydrolyzed galla powder (hydrolyzed in a water bath with 4 mol / L hydrochloric acid for 3.5 h), Baiyao decoction prepared by traditional methods, and Baiyao decoction prepared under optimized conditions were tested for gallic acid content (to determine the gallic acid content at the same fermentation time, Baiyao decoction fermented for 7 days was selected for testing, and the moisture content of Baiyao decoction was 10%). The specific experimental results are shown in the table below:

[0123]

[0124] As shown in the table above, the gallic acid content in gallnut is relatively low. When fermented for 7 days, the traditional processing method results in a lower gallic acid content due to the influence of natural environmental conditions, failing to meet the medicinal standard of 35-55%, and is unstable. Later verification showed that extending the fermentation and processing time may meet the medicinal standard (traditional processing generally requires more than 15 days of fermentation and processing for gallic acid to exceed the medicinal standard of 35%). However, under optimized processing conditions, processing Baiyao Decoction for 7 days can meet the medicinal standard for gallic acid content and has good stability. Compared with fully hydrolyzed gallnut powder, the gallic acid conversion rate is 65.72%.

[0125] Example 1:

[0126] A method to shorten the processing time of Baiyao Decoction. The steps of this method are the same as the traditional method for processing Baiyao Decoction, except for step 3. Baiyao Decoction: Mix 300mL of cooled green tea decoction with 125.0g of wine lees evenly, then add 30mL of mycelial ball liquid made from four different microorganisms (②(FAa-2), ④(FAa-4), ⑦(FAa-7), a(FU-a)) and 500.0g of gallnut powder and 15% (by weight of gallnut powder) of water, stir evenly to form a dough. The remaining steps are completely the same.

[0127] Based on Example 1, the effect of adding microbial mycelial balls prepared with a single strain on the gallic acid content of Baiyao Decoction was investigated. All other steps were the same, and the gallic acid content was determined by comparing it with traditionally processed Baiyao Decoction (all products were selected after fermentation for 7 days and then dried, with a water content of 10%). The specific results are as follows:

[0128]

[0129] As shown in the table above, during the same fermentation time, the gallic acid content of traditionally processed Baiyao Decoction is relatively low due to the influence of natural environmental conditions, failing to meet the medicinal standard of 35-55%, and its stability is poor. The gallic acid content of Baiyao Decoction processed with the addition of a single screened microorganism is significantly increased. Among them, the gallic acid content of Baiyao Decoction processed with the addition of microorganism ② is the highest, meeting the medicinal standard, and the results show good stability. Compared with fully hydrolyzed gallnut powder, the gallic acid conversion rate is 64.11%.

[0130] Example 2:

[0131] A method for shortening processing time and increasing gallic acid content in Baiyao Decoction includes the following steps:

[0132] a. Gallnut powder: Remove impurities, crush gallnuts, remove aphids, wash clean, dry, grind into powder, pass through an 80-mesh sieve, and set aside.

[0133] b. Green tea decoction concentrate: Take 31.0g of green tea and add 500mL of water to decoct into juice. Decoction twice, each time for 0.5h. Filter out the tea leaves and combine the decoction liquid. Concentrate under vacuum at 55℃ to 150mL. Cool and set aside for later use.

[0134] c. Baiyao Decoction: Mix 150mL of cooled, concentrated green tea decoction with 125.0g of distiller's grains. Add 30mL of mycelial ball liquid prepared by filamentous fungus FAa-2 (Aspergillus austwickii) and stir well. Then add 500.0g of gallnut powder and 45% (by weight of gallnut powder) of water and mix well. Form the mixture into 4cm×4cm×2cm balls and place them in a constant temperature (30℃) and constant humidity (75%) incubator for fermentation. When the surface of the medicinal blocks is covered with a "mold coat", cut them into small pieces and dry them at 55℃ to obtain the finished product.

[0135] d. Detect its gallic acid content.

[0136] Gallic acid content was determined in the following samples: traditionally processed Baiyao Decoction, Baiyao Decoction processed under optimized conditions, Baiyao Decoction prepared with the addition of microorganism ② in Example 1, and Baiyao Decoction prepared with the addition of filamentous fungus FAa-2 and optimized processing conditions in Example 2. (All products were selected after fermentation and processing for 7 days, followed by drying, and the water content in all products was 10%). The results are shown in the table below.

[0137]

[0138] As shown in the table above, after 7 days of fermentation and processing: Traditional processing results in lower gallic acid content and poor stability due to natural environmental conditions, failing to meet the medicinal standard of 35-55%. Only by extending the fermentation and processing time can the medicinal standard be met. Under optimized processing conditions, the gallic acid content of Baiyao Decoction is between 35-40%, meeting the medicinal standard and exhibiting good stability. Adding microorganism ② significantly increases the gallic acid content of Baiyao Decoction to meet the medicinal standard while maintaining good stability. Adding microorganism ② and optimizing processing conditions results in a gallic acid content of 50-55% in Baiyao Decoction, meeting the medicinal standard. This content is significantly higher than other experimental groups and exhibits good stability. Compared with fully hydrolyzed gallnut powder, the gallic acid conversion rate is 92.73%. The gallic acid content of Baiyao Decoction processed using this method varies with fermentation and processing time as follows: Figure 9 As shown, the gallic acid content can exceed 35% by the 5th day, reaching the medicinal standard, which significantly shortens the processing cycle compared to the traditional processing of Baiyao Decoction.

[0139] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make appropriate 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. A filamentous fungus Aspergillus austwickii Its features are: This strain was deposited on June 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40708. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. The application of the filamentous fungi as described in claim 1 in shortening the fermentation and processing time during the preparation of Baiyao Decoction.

3. The application of the filamentous fungi as described in claim 1 in increasing the content of gallic acid, an effective component, during the processing of Baiyao Decoction.

4. A method for shortening the fermentation and processing time of Baiyao Decoction, characterized in that... Includes the following steps: In the traditional method of preparing Baiyao Decoction, the filamentous fungus as described in claim 1 is added during the fermentation and processing step.

5. A method for shortening processing time and increasing gallic acid content in Baiyao Decoction, characterized in that... Includes the following steps: a. Preparation of Gallnut Powder: After removing impurities from the gallnuts, crush them, remove aphids, wash them clean, dry them, grind them into powder, and pass them through an 80-mesh sieve for later use. b. Green tea decoction concentrate: Take green tea, add water and decoct into juice, decoct twice, filter out the tea leaves and combine the decoction liquid, vacuum concentrate, cool and set aside; c. Baiyao Decoction: The concentrated juice of cooled green tea is mixed evenly with the lees, and then microbial mycelial balls prepared by the filamentous fungi as described in claim 1 are added and mixed evenly. Then, gallnut powder and water are added and mixed evenly. After forming a dough, it is placed in a constant temperature and humidity incubator for fermentation. After fermentation for a period of time, when the surface of the medicine block is covered with "mold", it is cut into small pieces and dried to obtain the finished product.

6. The method for shortening processing time and increasing gallic acid content in Baiyao Decoction as described in claim 5, characterized in that: In step b, the ratio of the mass of green tea (g) to the volume of water (mL) is 31:500; the decoction time is 15–60 min each time, and the vacuum concentration temperature is 45–70℃; when 31g of green tea is added to 500mL of water and decocted twice, the volume of the concentrated juice after vacuum concentration is 100–175 mL.

7. The method for shortening processing time and increasing gallic acid content in Baiyao Decoction as described in claim 5, characterized in that: In step c, the ratio of the volume (mL) of the green tea decoction to the mass (g) of the distiller's grains is 150:125; the amount of microbial mycelium added is: the ratio of the volume (mL) of the microbial mycelium to the mass (g) of the gallnut powder is 1:50 to 1:10; the volume of the added microbial mycelium is 10 to 50 mL; and the amount of water added is 15 to 60% of the mass of the gallnut powder.

8. The method for shortening processing time and increasing gallic acid content in Baiyao Decoction as described in claim 5, characterized in that: In step c, the pellet size should be 4 cm × 4 cm × 2 cm; the conditions of the constant temperature and humidity incubator are: temperature 20–35℃, humidity 35–95%.

9. The method for shortening processing time and increasing gallic acid content in Baiyao Decoction as described in claim 5, characterized in that: The drying temperature in step c is 45–70°C; the moisture content in the finished product does not exceed 13%.

10. The Baiyao Decoction obtained by any one of the methods described in claims 4-9.

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