A compound Chinese medicine preparation fermentation liquid and its preparation method and application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a fermentation broth for a compound traditional Chinese medicine preparation, its preparation method, and its application. The preparation method of the compound traditional Chinese medicine preparation fermentation broth includes the following steps: mixing Astragalus powder, Angelica powder, Red ginseng powder, and Licorice powder and dissolving them in water; adding cellulase and pectinase to disrupt the cell walls; subsequently inactivating the enzymes and sterilizing to obtain a fermentation medium for the compound traditional Chinese medicine preparation; mixing the seed liquids of *Lactobacillus plantarum* GMNL-662 and *Lactobacillus plantarum* R1012 to obtain a composite strain seed liquid; inoculating the composite strain seed liquid into the fermentation medium for the compound traditional Chinese medicine preparation for fermentation to obtain a fermentation broth; crushing the fermentation broth and collecting the supernatant; pressing and filtering the supernatant; collecting the filtrate to obtain the fermentation broth for the compound traditional Chinese medicine preparation. This invention uses composite strain fermentation combined with mechanical extrusion to increase the content of crude polysaccharides. Using the method described in this invention, the concentration of crude polysaccharides is increased from 3.4 mg / mL to 5.1 mg / mL.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fermentation broth for a compound traditional Chinese medicine preparation, its preparation method, and its application. Background Art

[0002] Astragalus, sweet in taste and slightly warm in nature, has the effects of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, and detoxifying and promoting tissue regeneration. Clinically, astragalus is often combined with other herbs, such as Atractylodes macrocephala, Salvia miltiorrhiza, Angelica sinensis, Stephania tetrandra, Cinnamomum cassia, Cornus officinalis, Leonurus japonicus, and Bletilla striata, to enhance its clinical efficacy and expand its range of applications. The combinations of astragalus are frequently recorded in ancient medical texts. The *Synopsis of Prescriptions of the Golden Chamber* records the Astragalus Jianzhong Decoction, primarily used to treat qi deficiency and internal cold syndrome, composed of astragalus, jujube, and white peony root. The *Treatise on Differentiation of Internal and External Injuries* records the Danggui Buxue Decoction, composed of astragalus and Angelica sinensis, which can be used to tonify qi and generate blood. The Buzhong Yiqi Decoction, composed of Atractylodes macrocephala, ginseng, and astragalus, has the effect of tonifying the middle and raising yang. The Guipi Decoction in the *Jisheng Fang*, composed of ginseng, astragalus, licorice, and Atractylodes macrocephala, can achieve the effect of generating blood by tonifying the spleen and replenishing qi. In terms of active ingredients, Astragalus contains astragalus polysaccharides, astragalus flavonoids, various trace elements, and essential amino acids, and is commonly used for the prevention, treatment, and adjuvant therapy of immune diseases, blood diseases, endocrine diseases, and cardiovascular diseases. Astragalus polysaccharides are often used in combination with Lycium barbarum polysaccharides, Angelica sinensis polysaccharides, and Codonopsis pilosula polysaccharides to enhance immunity; astragalus polysaccharides are also used in combination with Poria cocos polysaccharides, Pueraria lobata extract, Momordica charantia saponins, and Dioscorea opposita to regulate blood sugar; astragalus flavonoids are often used in combination with Rhodiola rosea extract, Ganoderma lucidum triterpenoids, Epimedium brevicornu total flavonoids, and Crataegus pinnatifida flavonoids in functional products for anti-oxidation and anti-aging; proteins and amino acids from Rehmannia glutinosa polysaccharides, jujube polysaccharides, and donkey-hide gelatin are also combined with astragalus polysaccharides to develop related products; in addition, astragalus polysaccharides have also been widely developed and applied in radiation protection, anti-tumor activity, and liver protection.

[0003] Crude polysaccharides, as active ingredients in traditional Chinese medicine, have broad application prospects in the pharmaceutical and health product fields. However, the current preparation of crude polysaccharides faces the challenge of low yield, which severely restricts their large-scale production and application.

[0004] In existing microbial fermentation technologies, the utilization efficiency of microorganisms for traditional Chinese medicine raw materials is generally low. On the one hand, the complex chemical components in traditional Chinese medicine may inhibit the growth and metabolic activity of microorganisms, preventing them from fully utilizing their ability to convert crude polysaccharides. On the other hand, the growth characteristics and metabolic regulation mechanisms of microorganisms themselves also limit the yield of crude polysaccharides.

[0005] The low yield of crude polysaccharides produced by microbial fermentation leads to high production costs, resulting in expensive products that fail to meet market demand. This not only hinders the widespread application of crude polysaccharides in the pharmaceutical field but also limits their large-scale promotion in the health supplement industry. Therefore, there is an urgent need to develop a method that can significantly improve the yield of crude polysaccharides produced by microbial fermentation. Summary of the Invention

[0006] To address the problem of low yield in the preparation of crude polysaccharides by microbial fermentation, this invention provides a method for preparing fermentation broth for compound traditional Chinese medicine preparations.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides a method for preparing fermentation broth of a compound traditional Chinese medicine preparation, comprising the following steps:

[0009] Astragalus powder, Angelica powder, red ginseng powder, and licorice powder were mixed and dissolved in water. Cellulase and pectinase were added to break down the cell walls, followed by enzyme inactivation and sterilization to obtain a fermentation medium for the compound traditional Chinese medicine preparation. Seed culture of Lactobacillus plantarum GMNL-662 and Lactobacillus plantarum R1012 were prepared. The seed cultures of Lactobacillus plantarum GMNL-662 and Lactobacillus plantarum R1012 were mixed to obtain a composite strain seed culture. The composite strain seed culture was inoculated into the fermentation medium for the compound traditional Chinese medicine preparation for fermentation to obtain a fermentation broth. The fermentation broth was crushed, the supernatant was collected, and the supernatant was squeezed and filtered. The filtrate was collected to obtain the fermentation broth of the compound traditional Chinese medicine preparation.

[0010] Preferably, the mass ratio of the astragalus powder, angelica powder, red ginseng powder, and licorice powder is 5:1:2:2.

[0011] Preferably, the filter membrane used in the squeeze filtration has a pore size of 0.22 μm.

[0012] Preferably, the volume ratio of the seed culture of Lactobacillus plantarum GMNL-662 to the seed culture of Lactobacillus plantarum R1012 is 1:1, and the inoculation amount of the composite strain seed culture is 4% by volume.

[0013] Preferably, the viable count of *Lactobacillus plantarum* GMNL-662 in the seed culture is 1 × 10⁻⁶. 8 CFU / mL; the viable count of *Lactobacillus plantarum* R1012 in the seed culture was 1×10⁻⁶. 8 CFU / mL.

[0014] Preferably, the fermentation culture conditions are static culture at 37°C for 48 hours.

[0015] The present invention also provides a fermentation broth for a compound traditional Chinese medicine preparation obtained according to the preparation method described above.

[0016] The present invention also provides an application of the fermentation broth of the compound traditional Chinese medicine preparation, wherein the fermentation broth of the compound traditional Chinese medicine preparation is used to prepare antioxidants.

[0017] Preferably, the antioxidant further includes pharmaceutically acceptable excipients.

[0018] Preferably, the pharmaceutically acceptable excipient is ethanol.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a method for preparing fermentation broth of a compound traditional Chinese medicine preparation, comprising the following steps:

[0021] Astragalus powder, Angelica powder, red ginseng powder, and licorice powder were mixed and dissolved in water. Cellulase and pectinase were added to disrupt the cell walls, followed by enzyme inactivation and sterilization to obtain a fermentation medium for the compound traditional Chinese medicine preparation. Seed culture of *Lactobacillus plantarum* GMNL-662 and *Lactobacillus plantarum* R1012 were prepared. The seed cultures of *Lactobacillus plantarum* GMNL-662 and R1012 were mixed to obtain a composite strain seed culture. The composite strain seed culture was inoculated into the fermentation medium for the compound traditional Chinese medicine preparation for fermentation to obtain a fermentation broth. The fermentation broth was crushed, and the supernatant was collected. The supernatant was then squeezed and filtered, and the filtrate was collected to obtain the fermentation broth of the compound traditional Chinese medicine preparation. This invention uses composite strain fermentation combined with mechanical extrusion to increase the content of crude polysaccharides. Using the method described in this invention, the concentration of crude polysaccharides was increased from 3.4 mg / mL to 5.1 mg / mL.

[0022] Lactobacillus plantarum GMNL-662 and Lactobacillus plantarum R1012, as probiotics, can spontaneously produce bacterial exovesicles during fermentation. In this invention, the supernatant of the fermentation broth from the co-fermentation of Lactobacillus plantarum GMNL-662 and Lactobacillus plantarum R1012 is collected and then mechanically extruded to prepare a compound traditional Chinese medicine fermentation broth containing bacterial exovesicles. The bacterial exovesicles can encapsulate the crude polysaccharides in the fermentation broth, improving the absorption and utilization of the crude polysaccharides and enhancing their anti-inflammatory effects.

[0023] This invention uses Astragalus membranaceus, Angelica sinensis, red ginseng, and licorice as substrates for cultivation. During growth, probiotics produce bacterial exovesicles, and their metabolism transforms the polysaccharides in these herbs, producing highly physiologically active secondary metabolites such as Astragalus polysaccharide, Angelica sinensis polysaccharide, and saponins. During mechanical extrusion, the bacterial exovesicles enrich and encapsulate the crude polysaccharides, enabling their application in food, beverage, and cosmetic ingredients. This method simplifies the engineered process of encapsulating crude polysaccharides with bacterial exoves, effectively improving absorption and utilization.

[0024] Bacterial exovesicles act as carriers to encapsulate crude polysaccharides, effectively enhancing their antioxidant activity. Furthermore, after human ingestion, they can be precisely targeted and absorbed by small intestinal epithelial cells, improving the bioavailability of the crude polysaccharides.

[0025] This invention combines probiotic fermentation technology with bacterial exovesicle preparation technology, effectively solving the problem of probiotics merely functioning as fermentation agents. The system in this invention, where bacterial exovesicles encapsulate crude polysaccharides to form nanoemulsions, significantly improves the bioavailability of crude polysaccharides. Attached Figure Description

[0026] Figure 1 The results show the characterization of bacterial extravesicles. A and B represent bacterial extravesicles under different fields of view.

[0027] Figure 2 This is a particle size distribution diagram of bacterial extravesicles.

[0028] Figure 3 The concentration of crude polysaccharide in the fermentation broth of the compound traditional Chinese medicine preparations prepared in Example 1 and Comparative Example 1.

[0029] Figure 4 To characterize the bacterial extravesicular vesicle structure after simulating in vitro digestion, A is a transmission electron microscope image of the blank control group; B is a transmission electron microscope image of the bacterial extravesicular vesicle structure after simulating digestion with artificial saliva, artificial gastric juice, and artificial intestinal juice. Detailed Implementation

[0030] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0031] The inventive concept of this invention is as follows:

[0032] The low yield of crude polysaccharides produced through microbial fermentation leads to high production costs, resulting in expensive products that struggle to meet market demand. This not only hinders the widespread application of crude polysaccharides in the pharmaceutical field but also limits their large-scale promotion in the health supplement industry.

[0033] Based on this, the present invention provides a method for preparing fermentation broth of compound traditional Chinese medicine preparation, comprising the following steps:

[0034] Astragalus powder, Angelica powder, red ginseng powder, and licorice powder were mixed and dissolved in water. Cellulase and pectinase were added to break down the cell walls, followed by enzyme inactivation and sterilization to obtain a fermentation medium for the compound traditional Chinese medicine preparation. Seed culture of Lactobacillus plantarum GMNL-662 and Lactobacillus plantarum R1012 were prepared. The seed cultures of Lactobacillus plantarum GMNL-662 and Lactobacillus plantarum R1012 were mixed to obtain a composite strain seed culture. The composite strain seed culture was inoculated into the fermentation medium for the compound traditional Chinese medicine preparation for fermentation to obtain a fermentation broth. The fermentation broth was crushed, the supernatant was collected, and the supernatant was squeezed and filtered. The filtrate was collected to obtain the fermentation broth of the compound traditional Chinese medicine preparation.

[0035] Lactobacillus plantarum GMNL-662 and R1012, as probiotics, can spontaneously produce bioactive endothelial cells (BEVs). BEVs are spherical structures with a lipid bilayer, typically ranging from 20 nm to 400 nm in diameter. As small-molecule bioactive substances derived from bacteria, BEVs possess advantages such as structural stability and long-lasting effective circulation. They can encapsulate various bioactive molecules, including proteins, lipids, nucleic acids, and metabolites. Numerous studies have demonstrated the significant potential of BEVs in immune responses and antioxidant activity, and they hold great application potential as novel metabiotics in functional foods, disease treatment alternatives, and immunomodulatory adjuvants. Furthermore, BEVs are ideal carriers for bioactive compound nanodelivery systems due to their favorable immunological and pharmacokinetic properties, as well as their ability to penetrate physiological barriers. Therefore, high-value active ingredients can be loaded using advanced technologies to prepare delivery systems. After human consumption of related products, they can achieve precisely targeted nutritional intervention effects, serving as an important carrier for constructing food nutrient delivery systems.

[0036] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0037] The list of abbreviations for this invention is shown in Table 1.

[0038] Table 1 List of Abbreviations

[0039]

[0040] The strains and manufacturers used in this invention are shown in Table 2.

[0041] Table 2. Information on strains and manufacturers

[0042]

[0043] The cellulase and pectinase used in this invention were purchased from Novozymes. The cellulase activity was 700 U / mL and the pectinase activity was 10000 U / mL. Red ginseng powder was purchased from Jilin Hongjiu Biotechnology Co., Ltd. Astragalus powder, angelica powder and licorice powder were purchased from Bozhou Qingyan Pharmaceutical Co., Ltd. Artificial saliva, artificial gastric juice and artificial intestinal juice were purchased from Shanghai Xinfan Biotechnology Co., Ltd.

[0044] In this invention, the method for detecting crude polysaccharides is as follows:

[0045] Place 2 μL of the fermentation broth of the compound traditional Chinese medicine preparation into a 20 mL stoppered test tube, add distilled water to make up to 1 mL, then add 1 mL of 5% phenol solution, and then quickly add 5 mL of 98% concentrated sulfuric acid. Use a vortex shaker to mix the reaction solution thoroughly, let it stand for 10 min, and then place it in a 30℃ water bath for 20 min. Measure the absorbance at 490 nm.

[0046] Example 1

[0047] A method for preparing a fermentation broth for a compound traditional Chinese medicine preparation is as follows:

[0048] S1. Dissolve Astragalus powder, Angelica powder, Red ginseng powder and Licorice powder in water, add cellulase and pectinase to destroy the cell wall, then inactivate the enzymes and sterilize to obtain a fermentation culture medium for compound traditional Chinese medicine preparation.

[0049] Astragalus powder, Angelica powder, red ginseng powder, and licorice powder were mixed in a mass ratio of 5:1:2:2 to obtain a compound traditional Chinese medicine preparation powder. This powder was then mixed with distilled water in a mass ratio of 1:12, and cellulase and pectinase were added. The mixture was hydrolyzed at 100 rpm for 3 hours with shaking. The enzymes were then inactivated at 85℃ for 10 minutes, followed by centrifugation at 4200 rpm for 15 minutes. The supernatant was collected and sterilized at 121℃ for 20 minutes to obtain the fermentation medium for the compound traditional Chinese medicine preparation. The amount of cellulase and pectinase added was 0.05 mL per 1 g of the compound traditional Chinese medicine preparation powder.

[0050] S2. Prepare seed solutions of Lactobacillus plantarum GMNL-662 and Lactobacillus plantarum R1012 separately. Mix the seed solutions of Lactobacillus plantarum GMNL-662 and Lactobacillus plantarum R1012 to obtain a composite strain seed solution.

[0051] Lactobacillus plantarum GMNL-662 was dissolved in sterile water to obtain a bacterial suspension. The GMNL-662 suspension was then gently streaked onto MRS solid medium using an inoculation loop and incubated at 37°C until single colonies of Lactobacillus plantarum GMNL-662 appeared. Single colonies of Lactobacillus plantarum GMNL-662 were then picked up using an inoculation loop and transferred to sterilized test tubes containing 5 mL of MRS liquid medium for inoculum activation. The tubes were then incubated statically at 37°C for 18 hours to obtain a seed culture of Lactobacillus plantarum GMNL-662. The viable count of Lactobacillus plantarum GMNL-662 in the seed culture was 1 × 10⁻⁶. 8 CFU / mL.

[0052] Lactobacillus plantarum R1012 was dissolved in sterile water to obtain a bacterial suspension. The Lactobacillus plantarum R1012 suspension was then gently streaked onto MRS solid medium using an inoculation loop and incubated at 37°C until single colonies of Lactobacillus plantarum R1012 appeared. Single colonies of Lactobacillus plantarum R1012 were then picked up using an inoculation loop and transferred to sterilized test tubes containing 5 mL of MRS liquid medium for inoculum activation. The tubes were then incubated statically at 37°C for 18 hours to obtain a seed culture of Lactobacillus plantarum R1012. The viable count of Lactobacillus plantarum R1012 in the seed culture was 1 × 10⁻⁶. 8 CFU / mL.

[0053] The seed culture of Lactobacillus plantarum GMNL-662 and the seed culture of Lactobacillus plantarum R1012 were mixed at a volume ratio of 1:1 to obtain the composite strain seed culture.

[0054] S3. Inoculate the seed liquid of the compound strain into the fermentation medium of the compound traditional Chinese medicine preparation for fermentation culture to obtain the fermentation broth.

[0055] The seed culture of the compound strain was inoculated into the fermentation medium of the compound traditional Chinese medicine preparation at a volume fraction of 4%, and then incubated statically in an incubator at 37℃ for 48 hours. After the fermentation was completed, the fermentation broth was collected.

[0056] S4. After crushing the fermentation broth, collect the supernatant, squeeze and filter the supernatant, and collect the filtrate to obtain the fermentation broth of the compound traditional Chinese medicine preparation.

[0057] After ultrasonic disruption of the fermentation broth, the cell debris was removed by centrifugation at 4000 rpm / min for 10 min at 4℃. The supernatant was retained and manually squeezed through a needle filter to pass it through a 0.22 μm filter membrane. The filtrate was collected to obtain the fermentation broth of the compound traditional Chinese medicine preparation.

[0058] Comparative Example 1

[0059] A method for preparing a fermentation broth for a compound traditional Chinese medicine preparation is as follows:

[0060] S1~S3 are exactly the same as in Example 1. The supernatant in S4 is the fermentation broth of the compound traditional Chinese medicine preparation.

[0061] The fermentation broth of the compound traditional Chinese medicine preparation prepared in Example 1 was characterized as follows:

[0062] Bacterial exovesicles in the fermentation broth of the compound traditional Chinese medicine preparation prepared in Example 1 were observed using transmission electron microscopy, and the particle size distribution and protein concentration of the bacterial exovesicles with obvious Tyndall effect were measured.

[0063] The results of transmission electron microscopy observation are as follows Figure 1 As shown, the particle size analysis results are as follows: Figure 2 As shown. By Figure 1 It can be seen that bacterial extravesicles have a circular structure. From Figure 2 The average particle size of the bacterial extravesicles was found to be 149.8 nm. Furthermore, the vesicle concentration and protein concentration of the bacterial extravesicles were determined. The results showed that the concentration of bacterial extravesicles was 8.1 × 10⁻⁶. 9 Particles / mL, protein concentration is 10.77 mg / mL.

[0064] The crude polysaccharides in the fermentation broth of the compound traditional Chinese medicine preparations prepared in Example 1 and Comparative Example 1 were determined as follows:

[0065] Depend on Figure 3 It can be seen that the crude polysaccharide concentration in the fermentation broth of the compound traditional Chinese medicine preparation prepared in Comparative Example 1 was 3.4 mg / mL; while the crude polysaccharide concentration in the fermentation broth of the compound traditional Chinese medicine preparation prepared in Example 1 was increased to 5.1 mg / mL.

[0066] In addition, an equal volume fraction of 20% SDS was added to the fermentation broth of the compound traditional Chinese medicine preparation prepared in Example 1 to demulsify it. After demulsification, the crude polysaccharide concentration reached 7.5 mg / mL, indicating that bacterial exovesicles can effectively encapsulate crude polysaccharides.

[0067] Example 2

[0068] The application of a fermentation broth for a compound traditional Chinese medicine preparation is as follows:

[0069] 1. Determine the DPPH free radical scavenging ability.

[0070] The fermentation broth of the compound traditional Chinese medicine preparation prepared in Example 1, the fermentation medium of the compound traditional Chinese medicine preparation prepared in S1 of Example 1, and the fermentation broth prepared in S3 of Example 1 were tested.

[0071] The DPPH free radical scavenging ability test kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., and the test was performed according to the instructions of the kit.

[0072] The results showed that the DPPH free radical scavenging rates of the fermentation broth, the fermentation medium of the compound traditional Chinese medicine preparation, and the fermentation broth of the compound traditional Chinese medicine preparation were 15.34%, 13.28%, and 35.89%, respectively, indicating that the antioxidant activity was improved after fermentation. Therefore, the fermentation broth of the compound traditional Chinese medicine preparation of the present invention can be prepared into an antioxidant by adding ethanol.

[0073] 2. Simulate in vitro digestion.

[0074] The fermentation broth of the compound traditional Chinese medicine preparation prepared in Example 1 was reacted sequentially in artificial saliva, artificial gastric juice, and artificial intestinal juice to simulate the digestion process, as follows:

[0075] a. Take 500 μL of the fermentation liquid of the compound Chinese medicine preparation, add 500 μL of artificial saliva kept in a 37℃ water bath, mix thoroughly, and place in a 37℃ water bath for 5 min.

[0076] b. Add 1000 μL of artificial gastric juice kept warm in a 37℃ water bath to the fermentation broth of the compound Chinese medicine preparation after artificial saliva digestion, mix thoroughly, and place in a 37℃ water bath for 2 hours.

[0077] c. Continue to add 2000μL of artificial intestinal fluid kept warm in a 37℃ water bath, mix thoroughly, and place in a 37℃ water bath for 1 hour.

[0078] d. After completing the previous step, place it in a 90℃ water bath for 5 minutes.

[0079] Centrifuge at 8000 rpm for 10 minutes, collect the supernatant, and perform analysis.

[0080] Depend on Figure 4 It can be seen that after the fermentation broth of the compound traditional Chinese medicine preparation is digested by artificial saliva, artificial gastric juice and artificial intestinal juice in sequence, it still retains the complete vesicle structure, indicating that it can resist digestion by gastrointestinal juice. The bacterial extravesicle structure in the fermentation broth of the compound traditional Chinese medicine preparation is stable and has a good ability to retain its contents.

[0081] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0082] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a fermentation broth for a compound traditional Chinese medicine preparation, characterized in that, Includes the following steps: Astragalus powder, angelica powder, red ginseng powder and licorice powder are mixed and dissolved in water. Cellulase and pectinase are added to destroy the cell wall. Then the enzymes are inactivated and sterilized to obtain a fermentation culture medium for compound Chinese medicine preparation. Preparation of seed culture of Lactobacillus plantarum GMNL-662; Preparation of seed culture of Lactobacillus plantarum R1012; The seed culture of Lactobacillus plantarum GMNL-662 and the seed culture of Lactobacillus plantarum R1012 were mixed to obtain a composite strain seed culture. The seed culture of the compound bacterial strain was inoculated into the fermentation medium of the compound traditional Chinese medicine preparation for fermentation and culture to obtain the fermentation broth; After the fermentation broth is crushed, the supernatant is collected, and the supernatant is squeezed and filtered. The filtrate is collected to obtain the fermentation broth of the compound traditional Chinese medicine preparation. The filter membrane used in the extrusion filtration has a pore size of 0.22 μm.

2. The preparation method according to claim 1, characterized in that, The mass ratio of Astragalus powder, Angelica powder, Red ginseng powder and Licorice powder is 5:1:2:

2.

3. The preparation method according to claim 1, characterized in that, The volume ratio of the seed culture of *Lactobacillus plantarum* GMNL-662 to that of *Lactobacillus plantarum* R1012 is 1:1, and the inoculation amount of the composite strain seed culture is 4% by volume.

4. The preparation method according to claim 1, characterized in that, The viable count of *Lactobacillus plantarum* GMNL-662 in the seed culture was 1 × 10⁻⁶. 8 CFU / mL; The viable count of *Lactobacillus plantarum* R1012 in the seed culture was 1 × 10⁻⁶. 8 CFU / mL.

5. The preparation method according to claim 1, characterized in that, The fermentation culture conditions were 37℃ and static culture for 48 hours.

6. A fermentation broth for a compound traditional Chinese medicine preparation obtained by the preparation method according to claim 1.

7. The application of the fermentation broth of the compound traditional Chinese medicine preparation as described in claim 6, characterized in that, The fermentation broth of the compound traditional Chinese medicine preparation is used to prepare antioxidants.

8. The application as described in claim 7, characterized in that, The antioxidants also include pharmaceutically acceptable excipients.

9. The application as described in claim 8, characterized in that, The pharmaceutically acceptable excipient is ethanol.

Images