Akkermansia myxophilus, Bazhen fermentation broth and its preparation method, and its applications in enhancing immunity and aiding digestion.

Abstract

This invention provides a *Akkermansia muciniphila* strain, with accession number CGMCC No. 46382 and Latin name *Akkermansia muciniphila*. This invention also provides a protein secreted by the aforementioned *Akkermansia muciniphila*. This protein has the effect of repairing intestinal barrier function. This invention also provides a compound fermentation agent comprising the aforementioned *Akkermansia muciniphila* and *Lactobacillus plantarum*. This invention also provides an Eight Treasures Fermentation Liquid derived from the aforementioned compound fermentation agent. This invention also provides a method for preparing the Eight Treasures Fermentation Liquid. Finally, this invention provides the application of the Eight Treasures Fermentation Liquid in health products or pharmaceuticals that help enhance immunity and digestion.

Description

Technical Field

[0001] This invention belongs to the field of probiotic fermentation, specifically relating to *Ackermania viride* and its secreted proteins. The invention also provides a compound fermentation agent comprising *Ackermania viride*, its preparation method, an Eight Treasures fermentation broth obtained by fermentation using this compound fermentation agent, a method for preparing the Eight Treasures fermentation broth, and applications of the Eight Treasures fermentation broth. Background Technology

[0002] Akkermansia muciniphila (AKK) is an important probiotic in the gut. It uses intestinal mucosal mucosa mucosa as a carbon and nitrogen source and mainly metabolizes acetic acid and propionic acid.

[0003] Recent international studies have found that AKK plays a unique role in immune regulation: its outer membrane protein Amuc_1100 can induce the secretion of the anti-inflammatory factor IL-10 through the TLR2 pathway, maintaining the intestinal mucosal barrier; heat-inactivated Akkermansia muciniphila AKK can regulate the NF-κB / MAPK signaling pathway, improve chemotherapy-induced immunosuppression, and restore immune organ function.

[0004] However, the functional effects of Akkermansia muciniphila AKK are highly dependent on strain characteristics and host status, and there is a potential risk of excessive degradation of the mucus layer under certain conditions.

[0005] Combining probiotic fermentation technology with food-medicine homologous substances has become an important direction for enhancing the efficacy and value of food and medicinal raw materials. The polysaccharides, flavonoids, and other active ingredients abundant in food-medicine homologous substances can undergo structural modification and enhanced activity after microbial enzymatic hydrolysis and transformation. Compound strain fermentation technology, building upon the fermentation modification of single strains, combines the fermentation advantages of different strains to achieve a higher level of modification and enhancement of the fermentation substrate.

[0006] Chinese patent application CN121405772A discloses a technical solution that uses dual-strain co-fermentation of chicken gizzard lining, astragalus, hawthorn, and American ginseng, resulting in a significantly higher content of active ingredients such as total peptides, total flavonoids, and total polyphenols in the fermentation broth compared to the content of active substances in fermentation broth from a single strain. Similarly, after fermentation with a compound strain, the lipid-lowering activity of polysaccharides in Polygonatum sibiricum is also significantly enhanced compared to its water extract.

[0007] However, current research focuses on lactic acid bacteria or Bacillus, and the introduction of Akkermansia muciniphila (AKK), which has both metabolic and immune regulation functions, into the fermentation system of food and medicine is still a blank.

[0008] In existing technologies, the culture cycle of Akkermansia myxophilus (AKK) is as long as several days, resulting in poor fermentation performance and insufficient development of its metabolites. In contrast, the mainstream fermentation strain in traditional food-medicine homologous fermentation broths is Lactobacillus plantarum. Combining AKK strains with Lactobacillus plantarum for co-fermentation of food-medicine homologous substances can fully utilize the metabolites produced by AKK while ensuring thorough fermentation of the substances by Lactobacillus plantarum. Therefore, developing a fermentation composition and its preparation method that utilizes Akkermansia myxophilus (AKK) and Lactobacillus plantarum for co-fermentation of food-medicine homologous substances, possesses good sensory qualities, and can meet the daily maintenance needs of the entire population throughout their entire life cycle, is of significant practical importance. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides an Akkermansia myxophilus AKK strain screened from the intestines of long-lived elderly individuals. It also provides a protein secreted by the Akkermansia myxophilus AKK strain. Furthermore, this invention provides a compound fermentation agent combining the Akkermansia myxophilus AKK strain with Lactiplantibacillus plantarum YS-Max09 (disclosed in patent CN121555376A), which possesses high cellulase activity, enabling the fermentation of food and medicinal materials with high fiber content. This invention also provides a method for preparing the compound fermentation agent. Additionally, this invention provides an Eight Treasures Fermentation Broth obtained by fermentation with the aforementioned fermentation agent. A fermentation method for the Eight Treasures Fermentation Broth is also provided. Finally, this invention provides applications of the Eight Treasures Fermentation Broth.

[0010] This invention is achieved through the following technical solution: A strain of Akkermansia muciniphila, AKK-VITA, with accession number CGMCC NO.46382, was deposited on September 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0011] The protein secreted by the myxotrophic Akkermansia AKK-VITA has the sequence shown in SEQ ID NO.12.

[0012] A compound fermentation agent, wherein the compound fermentation agent comprises the bacterial culture of Akkermansia muciniphila AKK-VITA, the bacterial culture of Lactiplantibacillus plantarum YS-Max09, a substrate and water; The matrix includes bovine brain extract, bovine heart extract, mucin, peptone, yeast extract, disodium hydrogen phosphate, sodium chloride, glucose, L-cysteine ​​hydrochloride, and N-acetyl-D-glucosamine.

[0013] The concentration of bovine brain extract powder in the matrix is ​​8-12 g / L; The concentration of ox heart extract powder in the matrix is ​​8-12 g / L; The concentration of mucin in the matrix is ​​4-8 g / L; The concentration of peptone in the matrix is ​​4-6 g / L; The concentration of yeast extract in the substrate is 3-5 g / L; The concentration of disodium hydrogen phosphate in the matrix is ​​2-3 g / L; The concentration of sodium chloride in the matrix is ​​4-6 g / L; The concentration of glucose in the matrix is ​​6-10 g / L; The concentration of L-cysteine ​​hydrochloride in the matrix is ​​0.4-0.6 g / L; The concentration of N-acetyl-D-glucosamine in the matrix is ​​1-2 g / L; The initial pH of the matrix is ​​7.0 ± 0.2.

[0014] In the compound fermentation agent, the inoculation ratio of Akkermansia muciniphila AKK-VITA bacterial solution to Lactobacillus plantarum YS-Max09 bacterial solution is 3:1 (V / V), and the total inoculation amount is 2.5% (V / V). The viable count of *Ackermania AKK-VITA* in the compound fermentation agent was 6.1 × 10⁻⁶. 9 The CFU / mL count of *Lactobacillus plantarum* YS-Max09 was 1.4 × 10⁻⁶. 10 CFU / mL.

[0015] The preparation method of the compound fermentation agent includes the following steps: inoculating the seed liquid of Akkermansia muciniphila AKK-VITA and the seed liquid of Lactobacillus plantarum YS-Max09 into a substrate for cultivation, thereby obtaining the compound fermentation agent; The initial pH of the culture was 7.0; The fermentation time for the culture was 36 hours.

[0016] A fermented liquid containing eight treasures, which is obtained by fermenting the eight-treasure soup formula with the aforementioned compound fermenting agent; The formula of Bazhen Decoction, by weight, consists of 9 parts of Codonopsis pilosula, 12 parts of Dioscorea opposita, 10 parts of Poria cocos, 3 parts of Glycyrrhiza uralensis, 12 parts of Rehmannia glutinosa, 10 parts of Lycium barbarum, 10 parts of Longan aril, and 8 parts of Prunus persica.

[0017] The preparation method of the Eight Treasures Fermentation Broth includes the following steps: The compound fermentation agent was inoculated into the fermentation medium of Bazhentang formula at an inoculation rate of 1% (V / V) and anaerobic fermented at 37℃ for 18h. After fermentation, the mixture was boiled for 30 minutes to inactivate the bacteria, then centrifuged at 6000 rpm for 5 minutes. The supernatant was collected to obtain the fermentation broth.

[0018] The preparation method of the fermentation culture medium for the Bazhen Decoction formula includes the following steps: Soak the Bazhen Decoction formula in 10 times the volume (w / v) of purified water for 30 minutes, add 0.6wt% mucin, 2wt% glucose and 1wt% peptone, autoclave at 121℃ for 15 minutes, and cool to room temperature.

[0019] The aforementioned Eight Treasures Fermentation Liquid is used in the preparation of medicines or health products that help enhance immunity and / or digestion.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention obtained a strain of Akkermansia myxophilus AKK-VITA with excellent probiotic properties from the gut microbiota of long-lived elderly people. It has excellent probiotic functions and has a stronger ability to enhance the integrity of Caco-2 cell monolayers compared with commercially available AKK quality control strains. It provides a high-quality strain resource for the application of Akkermansia myxophilus AKK in the field of food and medicine homology fermentation.

[0021] The Akkermansia AKK-VITA strain provided by this invention can secrete a novel type I membrane protein, ECM-VITA-X1, which has potential barrier regulation capabilities and immunomodulatory activity.

[0022] This invention introduces *Ackermania mucilage* AKK into a food-medicine homology fermentation system, constructing a dual-strain synergistic co-culture system of *Ackermania mucilage* AKK-VITA and *Lactobacillus plantarum* YS-Max09, solving the problem of limited substrate utilization in single fermentation of *Ackermania mucilage* AKK. The two strains form a symbiotic relationship with complementary functions: *Ackermania mucilage* AKK-VITA degrades mucin to produce short-chain fatty acids and repairs the intestinal mucosal barrier; YS-Max09 efficiently decomposes polysaccharides, flavonoids, and other macromolecules in the food-medicine homology raw materials, achieving a dual synergistic effect.

[0023] This invention is based on the traditional Chinese medicine theory of nourishing both Qi and Blood, and features a customized formula for Bazhen Tang (Eight Treasures Decoction) that combines food and medicine. Furthermore, a dual-strain co-fermentation process significantly enhances the content and bioavailability of the product's active ingredients. Compared to the unfermented control group, the co-fermented group exhibits significantly better performance in multiple indicators than the single-strain fermentation group, demonstrating a clear synergistic effect.

[0024] The fermented broth prepared by this invention has multi-target, full-cycle health conditioning effects. By activating the TLR4 / MAPK signaling pathway, it significantly restores the spleen index and thymus index of immunodeficient mice, reduces the levels of inflammatory factors IL-6 and TNF-α, and effectively alleviates oxidative stress damage and immune dysregulation. Attached Figure Description

[0025] Figure 1 Gram-stained images of Akkermansia AKK-VITA, a myxotrophic strain, are shown.

[0026] Figure 2 An SDS-PAGE image of the ECM-VITA-X1 protein separation is shown.

[0027] Figure 3 The tertiary structure of the ECM-VITA-X1 protein is shown.

[0028] Figure 4 The antioxidant activity of Bazhen Decoction in various proportions was shown.

[0029] Figure 5 Images of spleen characterization in mice from each group are shown.

[0030] Figure 6 Images of spleen protein immunoblotting from mice in each group are shown.

[0031] Figure 7 The relative expression results of TLR4 and MAPK proteins in the spleen of mice in each group are shown. Detailed Implementation

[0032] Example 1 (1) Screening of Akkermansia myxophilus strain AKK Fecal samples were collected from healthy, long-lived elderly individuals (age > 90 years) and immediately placed in sterile anaerobic sampling tubes. The samples were then anaerobically preserved at 4°C and processed as quickly as possible. 1g of fecal sample was weighed and added to 9mL of sterile pre-reduced physiological saline (purged with high-purity nitrogen for 15 minutes before use for deoxygenation). The mixture was thoroughly vortexed in an anaerobic workstation and diluted tenfold to prepare a homogeneous fecal suspension. A tenfold serial dilution was then performed using sterile pre-reduced physiological saline. 10-fold serial dilutions were selected. -3 Up to 10 -6 100 μL of each dilution solution was spread on BHI solid medium plates containing 0.6% mucin (purchased from Guangdong Huankai Microbial Technology Co., Ltd.) and incubated at 37°C under strict anaerobic conditions (80% N2, 10% H2, 10% CO2) for 5-7 days.

[0033] Select single colonies that are round, raised, with neat edges, 0.1-0.5 mm in diameter, and colorless or translucent. Confirm that the Gram staining result is negative under a microscope. Transfer single colonies that meet the above morphological characteristics to modified BHI solid medium and streak them multiple times to obtain pure bacteria.

[0034] (2) Evaluation of the probiotic function of Akkermansia myxophilus strain AKK The screened strains were identified and their in vitro probiotic properties were evaluated: molecular identification was performed by 16S rRNA gene sequencing and PCR amplification using Akkermansia myxophilus strain AKK specific primers; their tolerance in simulated gastrointestinal fluid, cell hydrophobicity, antibiotic sensitivity, and antioxidant activity were determined using national standard methods. After comprehensive evaluation of the probiotic function and safety of each strain, a superior Akkermansia myxophilus strain (named AKK-VITA) was finally obtained and deposited at the Institute of Microbiology, Chinese Academy of Sciences on September 28, 2025, with accession number CGMCC No. 46382. Microscopic examination results are as follows: Figure 1 As shown.

[0035] Table 1. Evaluation results of the probiotic function, safety and antioxidant activity of Akkermansia myxophilus AKK-VITA

[0036] The gene sequence of the selected Akkermansia AKK-VITA is shown in SEQ ID NO. 1.

[0037] SEQ ID NO. 1

[0038] (3) Effect of AKK-VITA on polarization permeability of Caco-2 cell monolayers Preparation of AKK strain supernatant: Cryopreserved strains of AKK-VITA (screened in Example 1) and control strain AKK CICC 24917 (purchased from China Industrial Microbial Culture Collection Center) were thawed and inoculated into 0.6% mucin-modified BHI liquid medium (purchased from Guangdong Huankai Microbial Technology Co., Ltd.). The cultures were incubated at 37°C under strictly anaerobic conditions for 48 hours until the stationary phase. After incubation, the culture was centrifuged at 10,000 rpm at 4°C for 20 minutes, and the supernatant was collected. The supernatant was sterilized by filtration through a 0.22 μm filter, aliquoted, and stored at -80°C for later use. When used to treat cells, the supernatant was diluted to a 10% concentration with cell culture medium.

[0039] Caco-2 Cell Culture and Grouping: Caco-2 cells (human colorectal adenocarcinoma cell line, purchased from ATCC) were cultured in high-glucose DMEM complete medium (purchased from Thermo Fisher Scientific, USA) containing 20% ​​fetal bovine serum, 2 mmol / L L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin, and were routinely passaged in a 37°C, 5% CO2 incubator. Caco-2 cells in good growth condition and in the logarithmic growth phase were harvested and the cell density was adjusted to 8 × 10⁶ cells / year. 4 Cells / mL. Transwell 24-well cell culture plates (0.4 μm pore size, 0.33 cm² membrane area) were used. 2 200 μL of cell suspension was added to each well on the apical side of the Caco-2 cell culture medium, and 600 μL of complete culture medium was added to each well on the basolateral side. After inoculation, the cells were incubated at 37°C in a 5% CO2 incubator. The culture medium was changed every other day during the first week after inoculation, and daily from the second week onwards, for a total of 21 days to allow the formation of a dense, polarized Caco-2 cell monolayer. After day 21 of cell culture, once a dense monolayer was confirmed, the original culture medium was discarded. The following treatment groups were established (apical administration, with 3 replicates per group): ① Blank control group (C): only an equal volume of complete culture medium was added, without the bacterial supernatant; ② AKK-VITA supernatant group (AKK-VITA): 10% final concentration of AKK-VITA fermentation supernatant was added; ③ AKKCICC 24917 supernatant group (AKK CICC 24917): 10% final concentration of AKK CICC 24917 fermentation supernatant was added.

[0040] Transepithelial resistance (TEER) measurement: Using a Millicell ERS-2 cell resistance meter with an STX-2 electrode, the resistance between the top and basal sides of each Transwell chamber was measured at room temperature. Before measurement, the electrodes were preheated, and the electrode tips were disinfected by immersing in 75% ethanol for 15 min, air-dried, thoroughly washed with PBS buffer, and then equilibrated for 10 min. At each measurement time point, the cell culture plate was removed, and in a clean bench, the short arm of the STX-2 electrode was inserted into the top side of the Transwell chamber, and the long arm into the basal side. The value was recorded after the resistance meter reading stabilized. Three measurements were taken per well, and the average value was recorded. Final TEER values ​​(Ω·cm) were recorded. 2 Calculate using the following formula: TEER (Ω·cm) 2 = (Measured resistance value - Blank film resistance value) × Film area (0.33 cm²) 2 ) A higher TEER value indicates better cell monolayer integrity and lower permeability; a lower TEER value suggests that tight junctions in cells are disrupted and permeability is increased.

[0041] Table 2. Changes in TEER values ​​of Caco-2 cell monolayers in different treatment groups

[0042] The experimental results showed that the TEER value of the blank control group showed a slight decreasing trend within 24 hours (from 585.2 Ω·cm). 2 Reduced to 548.6 Ω·cm 2 This indicates a natural, slight functional decline in the tight junctions of Caco-2 cell monolayers during culture. The AKK-VITA supernatant group showed a more significant TEER-promoting effect as early as 4 h after drug administration, with a TEER value reaching 682.9 ± 32.4 Ω·cm at 24 h. 2 The concentration of AKK-VITA was 24.5% higher than that of the control group and 7.5% higher than that of the AKK CICC 24917 supernatant group. This indicates that the fermentation supernatant of the AKK-VITA strain has a stronger ability to enhance the integrity of the intestinal epithelial barrier in the Caco-2 cell model. The AKK-VITA strain showed a clear strain-specific functional difference from the control strain, suggesting that this strain may secrete special extracellular proteins. Therefore, this invention will subsequently conduct specific detection of the extracellular proteins of AKK-VITA.

[0043] Example 2 Isolation, identification and analysis of extracellular proteins of Akkermansia mycotoxinophilus AKK-VITA (1) SDS-PAGE differential protein band analysis After determining the protein concentration of AKK-VITA supernatant using the BCA method, equal amounts of the sample were subjected to SDS-PAGE electrophoresis. Figure 2 Electrophoresis results showed a clear differential protein band at approximately 35 kDa in the AKK-VITA supernatant, while the corresponding band was absent in the CICC 24917 supernatant. This difference suggests that AKK-VITA may express a specific extracellular protein not present in the control strain. Currently studied AKK membrane proteins Amuc_1100 have a molecular weight of approximately 32 kDa, and Amuc_1409 has a molecular weight of approximately 16-18 kDa. The approximately 35 kDa protein discovered in this example is inconsistent with all of the above-mentioned known proteins and should belong to a new class of AKK proteins.

[0044] (2) Mass spectrometry identification and sequence analysis of differentially expressed proteins Differential protein bands in the approximately 35 kDa region of the AKK-VITA lane were excised from SDS-PAGE gels and digested in-gel: reduction with 10 mM DTT at 56 °C for 30 min, alkylation with 55 mM iodoacetamide at room temperature in the dark for 30 min, followed by digestion with sequencing-grade trypsin (Promega) at 37 °C for 16 h, with an enzyme-to-protein ratio of 1:50. The digested peptides were desalted using a C18 ZipTip and analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system (Thermo Fisher Q Exactive HF-X). The raw mass spectrometry data were searched using software to find the corresponding Akkermansia myxophilic protein whole proteome in the UniProt database, with both peptide and protein FDR controlled below 1%. A total of 11 high-confidence unique peptides were identified, and one novel unknown protein was matched, resulting in a total sequence coverage of 42.1%.

[0045] Table 3 High-reliability peptide data

[0046] Using 11 high-confidence mass spectrometry-identified peptides as seed sequences, tBLASTn homology searches were performed on the raw whole-genome sequencing data of the AKK-VITA strain. Regions not covered by mass spectrometry were completely filled using the genomic sequence, ultimately obtaining the full-length coding sequence of the protein, totaling 245 amino acids (SEQ ID NO:12). The protein's molecular weight was calculated to be approximately 26.9 kDa, with a theoretical isoelectric point of 9.32, and it was named ECM-VITA-X1. This protein has no matching record in publicly available AKK strain proteome databases, classifying it as a novel protein. Its tertiary structure is as follows... Figure 3As shown. Using SignalP 6.0 to predict the protein's topological structure, it was found that the N-terminus is a typical type I signal peptide, the C-terminus is a transmembrane helix at positions 217-241, and the intracellular tail consists of only 4 amino acids. This protein is a typical type I single-pass transmembrane protein, with positions 23-216 completely exposed on the cell surface. This cell surface localization allows it to directly interact with intestinal epithelial cells and immune cells, providing a structural basis for its participation in host-microbe interactions. This prediction result was reflected in the Caco-2 cell monolayer polarization permeability experiment in Example 1: treatment with AKK-VITA supernatant significantly increased the transepithelial electrical resistance (TEER) value of the Caco-2 cell monolayer. This may be an important molecular basis for the superior barrier regulation ability and immunomodulatory activity of the AKK-VITA strain compared to the standard strain.

[0047] SEQ ID NO:12 MKKTLLLALTVLAAVTPAFASNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAE CGLSKFIKVYRPQLPIETPAAPWTPMPLEIAFQGDGSVLKAMNAITGMQDYLFTVNSIRVYAWGELDGKVLSGPVLGPVGKFGAPSPTAAVLKSSALAIGNLIVGLLGVVLVVVLALRKKSR.

[0048] (3) Effect of ECM-VITA-X1 protein on polarization permeability of Caco-2 cell monolayer ECM-VITA-X1 protein purification: AKK-VITA bacteria cultured to the logarithmic growth phase were resuspended in cell lysis buffer (purchased from Beyotime Biotechnology Co., Ltd.), sonicated on ice, and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected and loaded onto a pre-equilibrated Ni-NTA column. Impurities were washed with washing buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole) to remove impurities. Finally, the target protein was eluted with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole). The eluent was concentrated and transferred to PBS buffer. SDS-PAGE electrophoresis was used to verify the molecular weight and purity of the target protein. After determining the protein eluent concentration, the eluent was aliquoted and stored at -80°C. For cell intervention, the eluent was diluted to 10 μg / mL with cell culture medium.

[0049] Caco-2 cells were cultured according to the method in Example 1, and the cells were treated with 10 μg / mLECM-VITA-X1 protein for 24 h. The TEER value of the cells was measured. The results are shown in Table 4. ECM-VITA-X1 protein has a regulatory effect on the permeability of Caco-2 cell monolayer.

[0050] Table 4. Effects of ECM-VITA-X1 protein on polarization permeability of Caco-2 cell monolayers.

[0051] (4) Regulation of inflammatory response of ECM-VITA-X1 protein in RAW264.7 macrophages The activated RAW264.7 cells were then subjected to a 1×10⁻⁶ thiocyanate infusion. 6 Cells were seeded at a density of 10 μg / mL in 24-well plates and cultured at 37°C and 5% CO2 for 18-24 h. After washing and re-selection, the cells were added to cell culture medium containing 10 μg / mL LECM-VITA-X1 protein for intervention. The experiment was divided into 3 groups, with more than 6 replicate wells in each group, as follows: ① Blank control group (Group C), with an equal volume of PBS added to the culture medium; ② Inflammation model group (Group M), with 1 μg / mL LPS added to the cell culture medium to induce inflammation; ③ Experimental group (ECM-VITA-X1 group), with 10 μg / mL LECM-VITA-X1 intervention after LPS induction.

[0052] Cells were collected after intervention, washed, and then lysed using lysis buffer and sonication. The cells were centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatant was collected. The levels of IL-6 and TNF-α in the cell supernatant were detected using ELISA. The results are shown in Table 5. After ECM-VITA-X1 intervention, IL-6 levels decreased by 33.6% compared to the model group, and TNF-α levels decreased by 48.5%. These data demonstrate that ECM-VITA-X1 has significant in vitro anti-inflammatory activity and can effectively inhibit LPS-induced secretion of pro-inflammatory factors from macrophages.

[0053] Table 5. Regulatory role of ECM-VITA-X1 protein in the inflammatory response of RAW264.7 macrophages.

[0054] Example 3 Preparation of a compound fermentation agent of *Ackermania pseudomallei* and *Lactobacillus plantarum* After colonizing the intestinal mucus layer, *Akkermansia muciniphila* exhibits highly efficient growth dependent on the carbon and nitrogen sources provided by mucin, but its utilization of plant-derived polysaccharides is extremely limited. *Lactobacillus plantarum*, on the other hand, possesses a rich system of carbohydrate-active enzymes, capable of effectively degrading polysaccharide components such as cellulose, hemicellulose, and pectin in food-medicine homologous substances, releasing soluble monosaccharides, oligosaccharides, and small peptides. This provides a usable nutrient matrix for *AKK* bacteria, compensating for its metabolic limitations in plant-derived substrates. Therefore, the metabolic activity of *Lactobacillus plantarum* in the co-culture system significantly promotes the growth of *AKK* bacteria and the synthesis of short-chain fatty acids. Based on this, this invention constructs a composite co-culture system of AKK-VITA and YS-Max09, which is used for the synergistic fermentation of food-medicine homologous formulations. The aim is to achieve the full release and targeted biotransformation of plant-derived active ingredients, increase the short-chain fatty acid content of fermentation products, and enhance the intestinal epithelial barrier protection function.

[0055] (1) Source of strain Akkermansia myxophilus was strain AKK-VITA, screened and preserved in Example 1, with accession number CGMCC No. 46382; Lactobacillus plantarum YS-Max09, isolated from the intestines of a long-lived elderly person, has been published in Chinese patent application CN121555376A, with accession number CGMCC No. 35530; both strains were deposited at the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; both were viable bacteria at the time of deposit.

[0056] (2) Seed liquid preparation Preparation of Akkermansia muciniphila AKK-VITA seed culture: Remove the AKK strain cryopreservation tubes from the -80℃ freezer and thaw them rapidly in a 37℃ water bath. Inoculate the culture into modified BHI liquid medium (containing 0.6% mucin) in an anaerobic workstation (80% N2, 10% H2, 10% CO2). Incubate under strict anaerobic conditions at 37℃ for 48-72 hours, until the culture becomes significantly turbid. After 2-3 subcultures to fully restore the strain's viability, collect the culture as the AKK seed culture; the viable count is approximately 1 × 10⁻⁶. 9 CFU / mL.

[0057] Preparation of *Lactobacillus plantarum* YS-Max09 seed culture: *Lactobacillus plantarum* YS-Max09 strain was streaked onto MRS solid plates under aseptic conditions and incubated upside down at 37°C for 24-48 hours. Typical single colonies were picked from the plates and streaked again on MRS for purification. The purified single colonies were inoculated into MRS liquid medium and incubated statically at 37°C for 12-18 hours. After two subcultures to restore the strain's viability, the bacterial culture was collected as the YS-Max09 seed culture, with a viable count of approximately 1 × 10⁻⁶. 9 CFU / mL.

[0058] (3) Matrix preparation AKK uses intestinal mucin as its sole carbon and nitrogen source, while *Lactobacillus plantarum* requires abundant carbohydrates and nitrogen. To ensure the good growth of both strains in the co-culture system, this embodiment designed and screened a suitable compound fermentation agent for co-culture. The specific components and culture conditions of the culture medium are shown in Table 6. The optimal basal culture medium for the co-culture system of AKK strain and *Lactobacillus plantarum* was obtained. The culture medium components were: 10 g / L bovine brain extract, 10 g / L bovine heart extract, 6 g / L mucin, 5 g / L peptone, 4 g / L yeast extract, 2.5 g / L disodium hydrogen phosphate, 5 g / L sodium chloride, 8 g / L glucose, 0.5 g / L L-cysteine ​​hydrochloride, and 1.5 g / L N-acetyl-D-glucosamine. For solid culture medium, 15 g / L agar powder was added, with the remainder being water.

[0059] Comparative Example 1: Low mucin (1.0 g / L), low glucose (2.0 g / L), and low N-acetyl-D-glucosamine (0.2 g / L) were used to investigate the effects of insufficient carbon source and inducer on the co-culture growth rate.

[0060] Comparative Example 2: High mucoprotein (12.0 g / L), high glucose (15.0 g / L), and high N-acetyl-D-glucosamine (3.0 g / L) were used to investigate whether high concentrations of substrate produced an inhibitory effect or osmotic pressure burden.

[0061] Comparative Example 3: Low peptone (1.0 g / L) and low yeast extract (0.5 g / L) were used to investigate the effects of insufficient nitrogen source on the growth of Lactobacillus plantarum and the metabolic activity of the co-culture system.

[0062] Comparative Example 4: High peptone (10.0 g / L) and high yeast extract (8.0 g / L) were used to investigate whether excessive nitrogen sources led to excessive cell proliferation and inhibited the synthesis of short-chain fatty acids.

[0063] Comparative Example 5: Without additional glucose, relying solely on trace carbon sources from mucin, bovine brain extract, bovine heart extract, and peptone, this study verified whether *Lactobacillus plantarum* could form a symbiotic relationship with AKK in the absence of exogenous glucose.

[0064] Table 6. Components of the culture medium for each group

[0065] (4) Preparation of compound fermentation agent The seed cultures of AKK-VITA and *Lactobacillus plantarum* YS-Max09 were inoculated into a substrate and cultured at specific initial pH and temperature to obtain a composite fermentation agent. The optimal parameters, including inoculum size, inoculum ratio, initial pH, fermentation temperature, and fermentation time, were determined through the following experiments.

[0066] With a fixed total inoculum of 2% (V / V), six gradients were established for the viable cell ratios of AKK-VITA and *Lactobacillus plantarum* YS-Max09: 1:1, 1:2, 1:3, 2:1, 3:1, and 4:1. After co-culturing at an initial pH of 7.0 and a temperature of 37°C for 48 hours, the OD600nm, short-chain fatty acid (SCFA) content, and Caco-2 cell TEER values ​​were measured (the culture and operation methods were consistent with those in Example 1). The results are shown in Table 7. At an inoculum ratio of 3:1, the strain produced the highest content of SCFAs. This is attributed to a cross-feeding mechanism between strains: *Lactobacillus plantarum* YS-Max09 rapidly proliferates using the fermentation substrate and metabolizes it to produce abundant lactic acid; while *Ackermania mutans* AKK-VITA can efficiently utilize this lactic acid as a precursor, converting it into SCFAs through its unique metabolic pathway. The published paper revealed that *AKK* bacteria can efficiently utilize lactic acid produced by other strains as a precursor to synthesize short-chain fatty acids, especially propionic acid, in the gut. Similarly, in Caco-2 cell permeability experiments, the 3:1 group showed the highest TEER value at 24 hours. This was compared to a single *AKK* strain (682.9 Ω·cm). 2 It increased by 30.7 Ω·cm 2 This indicates that Akkermansia myxophilus AKK-VITA and Lactobacillus plantarum YS-Max09 achieve optimal metabolic synergy through a cross-feeding mechanism at this ratio, and the short-chain fatty acids and ECM-VITA-X1 protein produced work together to enhance the intestinal epithelial barrier function of Caco-2 cells to the greatest extent.

[0067] The test results are shown in Table 7. Based on the conclusions in Table 7, the optimal ratio of AKK-VITA to *Lactobacillus plantarum* YS-Max09 was 3:1. After 48 hours of culture, the viable count of AKK-VITA was at least 5.8 × 10⁻⁶. 9 CFU / mL, YS-Max09 viable count is at least 1.1 × 10⁻⁶. 10 CFU / mL.

[0068] Table 7. Effects of different inoculation ratios on co-culture results

[0069] Based on the optimal inoculum ratio (3:1), with a fixed initial pH of 7.0 and a culture temperature of 37℃, the total inoculum amounts were set at 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% (V / V). The results after 48 hours of co-culture are shown in Table 8. The results indicate that as the total inoculum amount increased from 1% to 2.5%, the OD600 and total SCFAs continuously increased; therefore, the optimal total inoculum amount was determined to be 2.5%.

[0070] Table 8. Effects of different inoculum sizes on co-culture results

[0071] Based on the optimal inoculation ratio (3:1) and total inoculation amount (2.5%), five initial pH gradients were set up with initial pH values ​​of 5.5, 6.0, 6.5, 7.0, and 7.5. The culture temperature was 37℃, and the results were measured after 48 hours of co-culture. The initial pH value of 7.0 was selected.

[0072] Table 9. Effects of different initial pH values ​​on co-culture efficacy.

[0073] Based on the optimal strain inoculation ratio (3:1), total inoculation amount (2.5%), and initial pH 7.0, four gradients of culture time were set: 18h, 24h, 36h, 48h, and 60h. The results are shown in Table 10, and the selected fermentation time was 36h.

[0074] Table 10 Effect of fermentation time on co-culture effect

[0075] Based on the above single-factor optimization results, the optimal strain inoculation ratio of 3:1 (AKK-VITA:YS-Max09), inoculation amount of 2.5%, initial pH of 7.0, and fermentation time of 36 h were selected as the basic culture conditions for the co-culture system. After the culture was completed, the viable count of AKK-VITA in the bacterial solution was 6.1 × 10⁻⁶. 9 CFU / mL, YS-Max09 viable count is 1.4 × 10⁻⁶. 10 CFU / mL.

[0076] Example 4 Customized Eight Treasures Soup Formula Bazhen Tang (Eight-Treasure Decoction) originates from *Zhengti Leiyao* (Essentials of Orthodox Medicine) written by Xue Ji in the Ming Dynasty. It combines the Qi-tonifying "Sijunzi Tang" (Ginseng, Atractylodes macrocephala, Poria cocos, and Licorice) with the blood-tonifying "Siwu Tang" (Rehmannia glutinosa, Paeonia lactiflora, Angelica sinensis, and Ligusticum chuanxiong), comprising eight medicinal herbs in total. It is a classic representative formula in Traditional Chinese Medicine for simultaneously tonifying both Qi and blood. Modern pharmacological research shows that Bazhen Tang has various pharmacological effects, including improving hematopoietic function, promoting immune function, improving blood rheology, and exhibiting antioxidant and anti-aging properties. However, some herbs in the traditional Bazhen Tang meet the requirements for dual use as food and medicine. Therefore, based on traditional Chinese medicine compatibility theory, some herbs have been modified and replaced, as shown in the specific formula below.

[0077] Codonopsis pilosula: replenishes qi and strengthens the spleen and lungs.

[0078] Yam: Nourishes the spleen and stomach, replenishes qi and nourishes yin.

[0079] Poria cocos: strengthens the spleen and eliminates dampness, calms the mind and soothes the nerves.

[0080] Licorice: Tonifies the spleen and replenishes qi, harmonizes the effects of other herbs.

[0081] Rehmannia: Nourishes Yin and blood, benefits essence and marrow.

[0082] Goji berries: Nourish the liver and kidneys, nourish blood and improve eyesight.

[0083] Longan pulp: Nourishes the heart and spleen, replenishes blood and calms the mind.

[0084] Peach kernels: promote blood circulation, remove blood stasis, moisten the intestines and relieve constipation.

[0085] To meet the daily nutritional needs of different groups, this embodiment, based on the original formula of Bazhen Tang, combines the recommended daily intake of each alternative medicinal material with the following proportions: Codonopsis pilosula 9 parts, Dioscorea opposita 12 parts, Poria cocos 10 parts, Glycyrrhiza uralensis 3 parts, Rehmannia glutinosa 12 parts, Lycium barbarum 10 parts, Longan aril 10 parts, Prunus persica 8 parts, totaling 74 parts.

[0086] Comparative Example 6 The difference from Example 4 is that the formula of the Eight Treasures Decoction is as follows: 12 parts Codonopsis pilosula, 10 parts Dioscorea opposita, 10 parts Poria cocos, 4 parts Glycyrrhiza uralensis, 10 parts Rehmannia glutinosa, 10 parts Lycium barbarum, 10 parts Longan aril, and 6 parts Prunus persica, totaling 72 parts.

[0087] Comparative Example 7 The difference from Example 4 is that the formula of the Eight Treasures Decoction is as follows: 8 parts Codonopsis pilosula, 12 parts Dioscorea opposita, 10 parts Poria cocos, 3 parts Glycyrrhiza uralensis, 15 parts Rehmannia glutinosa, 12 parts Lycium barbarum, 12 parts Longan aril, and 6 parts Prunus persica, totaling 78 parts.

[0088] Comparative Example 8 The difference from Example 4 is that the formula of the Eight Treasures Decoction is as follows: 8 parts Codonopsis pilosula, 10 parts Dioscorea opposita, 12 parts Poria cocos, 3 parts Glycyrrhiza uralensis, 10 parts Rehmannia glutinosa, 12 parts Lycium barbarum, 15 parts Longan aril, and 6 parts Prunus persica, totaling 76 parts.

[0089] Comparative Example 9 The difference from Example 4 is that the formula of the Eight Treasures Decoction is as follows: Codonopsis pilosula 9 parts, Dioscorea opposita 15 parts, Poria cocos 15 parts, Glycyrrhiza uralensis 3 parts, Rehmannia glutinosa 8 parts, Lycium barbarum 8 parts, Longan aril 8 parts, Prunus persica 6 parts, totaling 72 parts.

[0090] The above formula, after being weighed according to the specified proportions, was decocted with three times its weight of purified water. After decocting for 1 hour, the dregs were filtered out using gauze, and the antioxidant activity of the supernatant was tested. The results are as follows. Figure 4 As shown, the best-performing formula ratio was selected as the final ratio for Bazhen Tang.

[0091] Example 5 AKK and Bacillus plantarum co-fermented Eight Treasures Soup (1) Preparation of fermentation culture medium for Bazhen soup Weigh out the ingredients for the Bazhen Tang formula (9 g of Codonopsis pilosula, 12 g of Dioscorea opposita, 10 g of Poria cocos, 3 g of Glycyrrhiza uralensis, 12 g of Rehmannia glutinosa, 10 g of Lycium barbarum, 10 g of Longan aril, and 8 g of Prunus persica, totaling 74 g). Quickly rinse with clean water to remove surface dust and drain. Place the washed ingredients in a stainless steel extraction vessel, add 10 times the volume (w / v, i.e., 740 mL) of purified water, soak for 30 minutes, add 0.6% mucin, 2% glucose, and 1% peptone, autoclave at 121℃ for 15 minutes, and cool to room temperature for later use.

[0092] (2) Preparation of AKK-VITA and YS-Max09 compound fermentation agent Following the fermentation culture medium and conditions of the fermentation system described in Example 3, a composite starter culture of AKK-VITA and YS-Max09 was prepared, with a total viable count of approximately 2 × 10⁻⁶. 10 CFU / mL.

[0093] (3) Preparation of fermentation broth of Bazhen formula Inoculate 1% AKK-VITA and YS-Max09 compound fermentation agent into the Bazhen formula fermentation medium, ferment for 18 hours, boil to inactivate for 30 minutes after fermentation, and centrifuge at 6000 r / min for 5 minutes to obtain the Bazhen formula fermentation broth.

[0094] Comparative Example 10 The difference from Example 4 is that 1% AKK-VITA bacterial solution was inoculated into the Bazhen formula fermentation medium, while the rest of the steps are the same.

[0095] Comparative Example 11 The difference from Example 4 is that 1% YS-Max09 bacterial solution was inoculated into the Bazhen formula fermentation medium, while the rest of the steps are the same.

[0096] (4) Indicator detection After co-fermentation, the fermented broth of Bazhen Tang was sterilized, centrifuged, and the supernatant was collected. The following methods were used to determine various indicators: total acid was determined by acid-base titration (lactic acid meter); total polysaccharides were determined by the phenol-sulfuric acid method (glucose as standard); total flavonoids were determined by the sodium nitrite-aluminum nitrate colorimetric method (rutin as standard); total polyphenols were determined by the Folin-Ciocalteu colorimetric method (gallic acid as standard); short-chain fatty acids (acetic acid, propionic acid, butyric acid) were quantified by gas chromatography-mass spectrometry (GC-MS) with external standard method after acidification and ether extraction; total antioxidant capacity was determined by the ABTS free radical scavenging method, and the results were expressed as Trolox equivalents. All samples were tested in triplicate, and the results are expressed as mean ± standard deviation.

[0097] The test results are shown in Table 11. Compared with the unfermented blank control group, the single YS-Max09 fermentation group and the AKK-VITA+YS-Max09 co-fermentation group were significantly better than the single AKK fermentation group in terms of total acid, total polysaccharide, total flavonoids, total polyphenols, short-chain fatty acids and total antioxidant capacity. Moreover, multiple indicators showed synergistic effects, proving that the co-fermentation process of the present invention can effectively improve the nutritional value and bioactivity of the Bazhen Tang food and medicine homology formula. This is thanks to the synergistic effect between the two strains. Lactobacillus plantarum YS-Max09 has a strong ability to degrade fiber, which can efficiently decompose plant-derived cellulose, hemicellulose and pectin and other polysaccharides in the Bazhen Tang formula, destroy the plant cell wall structure, and thus promote the full release of intracellular active ingredients (such as flavonoids, polyphenols, saponins, etc.). Akkermansia muciniphila AKK-VITA can not only use the secondary metabolites such as lactic acid of YS-Max09 to synthesize short-chain fatty acids, but also specifically secrete a large amount of extracellular protein ECM-VITA-X1 during the fermentation process. The functional complementarity between the strains makes the co-fermentation system significantly better than the single-strain fermentation group in many activity indicators.

[0098] Table 11. Indicators of each group of fermentation broth

[0099] Example 6 Evaluation of the immunomodulatory and intestinal barrier function regulation effects of the fermentation broth of the Bazhen formula in immunodeficient mice (1) Establishment of an immunodeficient mouse model Thirty female C57BL / 6J mice (n=10) aged 6-8 weeks, weighing 18-22g, were divided into three groups: ① normal control group (Group C); ② immunodeficient group (Group M); ③ Bazhen formula fermentation broth group (Group F). Except for the normal control group, mice in the other groups were intraperitoneally injected with cyclophosphamide solution at a dose of 60mg / kg body weight once daily for 5 consecutive days to establish an immunodeficiency model. The normal control group was intraperitoneally injected with an equal volume of sterile saline. The experimental phase began 3 days after the last injection. Mice in Groups C and M were administered 50μL / day of saline by gavage, while mice in Group F were administered 50μL / day of Bazhen formula fermentation broth by gavage after modeling, with the intervention lasting 28 days.

[0100] (2) Mouse sample collection Twenty-four hours after the last administration, mice in each group were fasted for 12 hours but allowed free access to water. They were then weighed and euthanized. Blood was collected from the orbital rim, and serum was separated. Spleen, thymus, and liver were aseptically collected for the following tests.

[0101] (3) Indicator detection Immune organ index determination: Immune organ index (mg / g) = organ weight (mg) / mouse body weight (g) Serum inflammatory factor assay: The levels of IL-6 and TNF-α in serum were detected by ELISA.

[0102] Detection of serum biomarkers for intestinal mucosal permeability: The levels of diamine oxidase (DAO), D-lactic acid (D-LA), and zonalin in serum were detected by ELISA.

[0103] Measurement of liver oxidative stress indicators: A 10% liver tissue homogenate was prepared, and the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were detected.

[0104] Immunoblotting of proteins: After sacrifice, approximately 50 mg of spleen tissue and 30 mg of colon tissue were collected and placed in pre-chilled RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor), respectively, and homogenized thoroughly on ice using a tissue homogenizer. The homogenate was centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The protein concentration of each sample was determined using a BCA protein quantification kit. All samples were adjusted to the same protein concentration (10 μg / μL) with lysis buffer, and 4× loading buffer was added. The samples were then boiled at 100°C for 10 minutes to denature the proteins, aliquoted, and stored at -20°C for later use. 20 μg of each denatured protein sample was separated by 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane using a wet transfer method (constant current 250 mA, 90 minutes). After transfer, the membrane was blocked with 5% skim milk (prepared with TBST) at room temperature for 1.5 hours. After blocking, primary antibodies were added, and the membranes were incubated overnight at 4°C. The next day, the membranes were washed three times with TBST (10 minutes each time), and then HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution) was added and incubated at room temperature for 1 hour. After washing the membranes three times with TBST (10 minutes each time), ECL chemiluminescence immunoassay was performed, and images were acquired using a gel imaging system. β-actin was used as an internal control protein, and the gray values ​​of each band were quantitatively analyzed using ImageJ software. The relative expression level of the target protein was calculated as (target protein gray value / internal control protein gray value).

[0105] Based on the detection results in Table 12 and Figure 5 In mice with immunodeficiency, compared with the normal control group, the spleen and thymus indices were significantly reduced, and the spleens showed obvious dark red enlargement. Serum levels of inflammatory factors IL-6 and TNF-α were significantly increased; liver tissue SOD activity was significantly decreased while MDA content was significantly increased; and serum levels of intestinal barrier damage markers diamine oxidase (DAO), D-lactate, and zonulin were significantly increased, indicating that cyclophosphamide successfully induced immune organ atrophy, inflammatory response activation, increased intestinal barrier permeability, and oxidative stress damage. After intervention with Bazhen fermentation broth, the spleen and thymus indices of mice significantly recovered to near-normal levels, and the spleen condition was similar to that of the normal group. Serum levels of IL-6 and TNF-α were significantly decreased; liver tissue SOD activity was significantly increased and MDA content was significantly decreased; serum DAO, D-lactate, and zonulin levels were significantly decreased, suggesting that the immune organs and intestinal barrier function of mice were effectively repaired. The above results indicate that the Bazhen fermentation broth can effectively improve the immunosuppression induced by cyclophosphamide and exert anti-inflammatory, antioxidant, and intestinal barrier protective effects. Furthermore, Figure 6 and Figure 7Immunoblotting results showed that the fermentation broth of Bazhen could significantly upregulate the expression of TLR4 and MAPK proteins in immunodeficient mice, indicating that the fermentation broth can effectively activate the TLR4 receptor and p38 MAPK signaling pathway, which suggests that the fermentation broth can improve immunodeficiency.

[0106] Table 12 Results of mouse index detection in each group

[0107] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several equivalent substitutions or obvious modifications can be made without departing from the concept of the present invention, and all such modifications, achieving the same performance or purpose, should be considered within the scope of protection of the present invention.

Claims

1. A type of Akkermansia myxophilus AKK-VITA, characterized in that: Its Latin name is Akkermansia muciniphila, and its accession number is CGMCC NO.46382.

2. A protein secreted by Akkermansia AKK-VITA as described in claim 1, characterized in that: Its sequence is shown in SEQ ID NO.

12.

3. A compound fermentation agent, characterized in that: The compound fermentation agent comprises the bacterial culture of Akkermansia muciniphila AKK-VITA as described in claim 1, the bacterial culture of Lactiplantibacillus plantarum YS-Max09, a substrate, and water; The matrix includes bovine brain extract, bovine heart extract, mucin, peptone, yeast extract, disodium hydrogen phosphate, sodium chloride, glucose, L-cysteine ​​hydrochloride, and N-acetyl-D-glucosamine.

4. The compound fermentation agent as described in claim 3, characterized in that: The concentration of bovine brain extract powder in the matrix is ​​8-12 g / L; The concentration of ox heart extract powder in the matrix is ​​8-12 g / L; The concentration of mucin in the matrix is ​​4-8 g / L; The concentration of peptone in the matrix is ​​4-6 g / L; The concentration of yeast extract in the substrate is 3-5 g / L; The concentration of disodium hydrogen phosphate in the matrix is ​​2-3 g / L; The concentration of sodium chloride in the matrix is ​​4-6 g / L; The concentration of glucose in the matrix is ​​6-10 g / L; The concentration of L-cysteine ​​hydrochloride in the matrix is ​​0.4-0.6 g / L; The concentration of N-acetyl-D-glucosamine in the matrix is ​​1-2 g / L; The initial pH of the matrix is ​​7.0 ± 0.

2.

5. The compound fermentation agent as described in claim 3, characterized in that: In the composite fermentation agent, the inoculation volume ratio of Akkermansia muciniphila AKK-VITA bacterial suspension to Lactobacillus plantarum YS-Max09 bacterial suspension is 3:1, and the total inoculation amount is 2.5 vol%. The number of viable Akkermansia muciniphila AKK-VITA in the complex starter culture was 6.1 x 10 9 CFU / mL, and the number of viable Lactobacillus plantarum YS-Max09 was 1.4 x 10 10 CFU / mL.

6. The method for preparing the compound fermentation agent as described in claim 3, characterized in that: The process includes the following steps: inoculating the seed culture of Akkermansia muciniphila AKK-VITA and the seed culture of Lactobacillus plantarum YS-Max09 into a substrate for cultivation to obtain a compound fermentation agent. The initial pH of the culture was 7.0; The fermentation time for the culture was 36 hours.

7. A fermentation liquid containing eight treasures, characterized in that: The formula of Bazhen Tang was obtained by fermenting the compound fermenting agent according to claim 3. The formula of Bazhen Decoction, by weight, consists of 9 parts of Codonopsis pilosula, 12 parts of Dioscorea opposita, 10 parts of Poria cocos, 3 parts of Glycyrrhiza uralensis, 12 parts of Rehmannia glutinosa, 10 parts of Lycium barbarum, 10 parts of Longan aril, and 8 parts of Prunus persica.

8. The preparation method of the eight treasures fermentation liquor according to claim 7, characterized in that, Includes the following steps: The compound fermentation agent was inoculated into the fermentation medium of Bazhentang formula at a 1 vol% inoculation rate and anaerobic fermented at 37°C for 18 h. After fermentation, the mixture was boiled for 30 minutes to inactivate the bacteria, then centrifuged at 6000 rpm for 5 minutes. The supernatant was collected to obtain the fermentation broth.

9. The method for preparing the Eight Treasures Fermentation Liquid as described in claim 8, characterized in that: The preparation method of the fermentation culture medium for the Bazhen Decoction formula includes the following steps: Soak the Bazhen Decoction formula in water for 30 minutes, add 0.6wt% mucin, 2wt% glucose and 1wt% peptone, autoclave at 121℃ for 15 minutes, and cool to room temperature.

10. The application of the Eight Treasures Fermentation Liquid according to claim 7, characterized in that: It is used in the preparation of medicines or health products that help enhance immunity and / or digestion.

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