Structural fungus-based stone flora pharmaceutical composition and application thereof

Abstract

The invention provides a structural bacteria-based stone flora pharmaceutical composition. The structural bacteria-based stone flora pharmaceutical composition is prepared from live bacteria of bifidobacterium pseudominum aFMT-202, live bacteria of bifidobacterium longum subsp. Longum aFMT-104 and live bacteria of lactobacillus mucosa aFMT-501. The invention also provides a microbial medicinal preparation and a preparation method thereof, and also provides application of the composition in preparation of antidepressant drugs, intestinal anti-inflammatory drugs and antioxidant and anti-aging drugs. The three strains are separated from faeces of healthy adults, have acid resistance, bile salt resistance and intestinal epithelium adhesion capacity, can regulate neurotransmitter balance, enhance intestinal mucosal barrier and up-regulate endogenous antioxidant enzyme activity respectively, have complementary functions and are free of drug resistance and pathogenicity; the composition forms a composite preparation according to a specific ratio, can intervene in depression, chronic intestinal inflammation and aging-related functional decline, solves the limitation that the existing single-target medicine takes effect slowly, has large side effects and cannot intervene comprehensively, and overcomes the defect of clinical multi-target collaborative treatment.

Description

Technical Field

[0001] This invention relates to the field of microbiology and biomedicine, and in particular to a structural microbial community drug combination and its application. Background Technology

[0002] With increasing societal pressures and a rapidly aging population, the incidence of conditions such as depression, chronic inflammatory bowel diseases (e.g., ulcerative colitis, Crohn's disease), and age-related functional decline continues to rise, becoming a major medical problem seriously affecting public health. The pathogenesis of these conditions is complex and interconnected, all closely related to imbalances in the gut microbiota, immune inflammation disorders, and abnormal oxidative stress. Traditional single-target treatments are insufficient for comprehensive intervention, and clinical needs remain largely unmet.

[0003] In the field of depression treatment, existing antidepressants (such as selective serotonin reuptake inhibitors) can alleviate some symptoms, but they have limitations such as slow onset of action (usually requiring 2-4 weeks), significant side effects (such as gastrointestinal discomfort, sleep disorders, and sexual dysfunction), and high relapse rates after discontinuation. Recent studies have confirmed that the gut microbiota regulates neuroendocrine stress response and neurotransmitter balance through the "brain-gut axis," providing a new direction for depression treatment. Furthermore, existing literature shows that specific strains of *Bifidobacterium longum* can reduce hopeless behavior in chronic stress models, and *Bifidobacterium pseudosporidis* can reverse anhedonia induced by a high-fat diet and restore serotonin levels in the brain. However, current research mainly focuses on basic exploration of single strains, and multi-strain synergistic drug formulations for clinical use have not yet been developed.

[0004] In the treatment of intestinal inflammation, existing treatments for chronic inflammatory bowel diseases such as inflammatory bowel disease (IBD) (such as aminosalicylic acids and glucocorticoids) often lead to further dysbiosis of the gut microbiota and adverse reactions such as immunosuppression with long-term use, and are difficult to effectively repair the damaged intestinal mucosal barrier. Strains such as *Lactobacillus mucosa* have shown clear anti-inflammatory potential in animal experiments, reducing the expression of pro-inflammatory cytokines (IL-1β, TNF-α) in colonic tissue and upregulating the anti-inflammatory factor TGF-β2. However, current technologies lack compound drugs centered on these strains and combined with other functional strains, failing to achieve a synergistic therapeutic effect of "anti-inflammation + mucosal repair + microbiota regulation".

[0005] In interventions for age-related functional decline, the accumulation of oxidative stress and chronic inflammation (“inflammatory aging”) are key mechanisms leading to functional degeneration. Most existing antioxidants are exogenous, and long-term use may disrupt the body's oxidation-antioxidant balance; while treatments targeting age-related inflammation suffer from limitations such as single-target therapy and significant side effects. Studies have found that certain lactic acid bacteria can reduce oxidative damage by upregulating the activity of endogenous antioxidant enzymes (such as superoxide dismutase SOD and glutathione peroxidase GPx), but there are currently no reports of drug development combining these strains with other functional strains to delay age-related functional decline.

[0006] In summary, current clinical treatments for depression, intestinal inflammation, and age-related conditions have significant limitations. There is an urgent need for a compound microbial drug formulation with a clearly identified source, high safety, and synergistic function, which can comprehensively intervene in the above-mentioned multiple diseases by regulating the intestinal microecology, improving the immune inflammatory state and oxidative stress level, and making up for the shortcomings of existing drugs in multi-target synergistic therapy. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for producing Bifidobacterium pseudobulb (Bifidobacterium pseudobulb) Bifidobacterium pseudocatenulatum aFMT-202, Bifidobacterium longum subsp. ( Bifidobacterium longum subsp. longum aFMT-104 and Lactobacillus mucosa ( Limosilactobacillus mucosae This study describes a drug combination of three structural microbial community strains, namely aFMT-501, and its application. These three strains were isolated from the feces of healthy adults, clearly identified and classified, and all possess GDMCC preservation numbers. They exhibit tolerance to acid and bile salt-resistant gastrointestinal environments and the ability to adhere to intestinal epithelial cells. They function by regulating neurotransmitter balance, enhancing the intestinal mucosal barrier, and upregulating the activity of endogenous antioxidant enzymes, respectively. Their functions are complementary, and safety tests have shown no drug resistance or pathogenicity, meeting pharmaceutical standards. By combining these three strains in a specific ratio, a compound microbial drug formulation targeting depression, chronic intestinal inflammation, and age-related functional decline can be formed. This overcomes the limitations of existing single-target therapies, such as slow onset of action, significant side effects, and inability to achieve comprehensive intervention for multiple diseases, thus addressing the current clinical shortcomings in multi-target synergistic treatment of these conditions.

[0008] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A structural microbial community drug combination consists of three strains: Bifidobacterium pseudobulb aFMT-202, Bifidobacterium longum subsp. longum aFMT-104, and Lactobacillus mucosa aFMT-501. The *Bifidobacterium pseudobulbarum* aFMT-202 was isolated from the feces of healthy adults and classified as follows: Bifidobacterium pseudocatenulatum aFMT-202 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 23, 2024, and showed viability. The accession number is GDMCC No. 65183. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The *Bifidobacterium longum* subsp. *aFMT-104* was isolated from the feces of healthy adults and classified as... Bifidobacterium longum subsp. Longum aFMT-104 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 3, 2025, and showed viability. The accession number is GDMCC No. 66087. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The *Lactobacillus mucosa* aFMT-501 was isolated from the feces of healthy adults and classified as... Limosilactobacillus mucosae aFMT-501 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 3, 2025, and showed viability. The accession number is GDMCC No. 66090. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. All three strains were present in the combination in live form.

[0009] As one of the preferred embodiments of the present invention, the ratio of the number of viable bacteria of the three strains is 1:2:1, that is, Bifidobacterium pseudobulbarum aFMT-202: Bifidobacterium longum subsp. longum aFMT-104: Lactobacillus mucosa aFMT-501 = 1:2:1 (the aFMT-104 strain accounts for a higher proportion). This ratio combination is used to prepare antidepressant drugs, and the corresponding formula is named Antidepressant Formula (Formula-D).

[0010] As one of the preferred embodiments of the present invention, the ratio of the number of live bacteria of the three strains is 1:1:2, that is, Bifidobacterium pseudobulbarum aFMT-202: Bifidobacterium longum subsp. longum aFMT-104: Lactobacillus mucosa aFMT-501 = 1:1:2 (the aFMT-501 strain accounts for a higher proportion). This ratio combination is used to prepare intestinal anti-inflammatory drugs, and the corresponding formula is named anti-inflammatory formula (Formula-I).

[0011] As one of the preferred embodiments of the present invention, the ratio of the number of viable bacteria of the three strains is 2:1:1, that is, Bifidobacterium pseudobulbarum aFMT-202: Bifidobacterium longum subsp. longum aFMT-104: Lactobacillus mucosa aFMT-501 = 2:1:1 (the aFMT-202 strain accounts for a higher proportion). This ratio combination is used to prepare an antioxidant and anti-aging drug, and the corresponding formula is named Antioxidant and Anti-aging Formula (Formula-A).

[0012] A microbial pharmaceutical formulation comprising the aforementioned structural microbial community drug combination, as well as pharmaceutically acceptable protectants and excipients.

[0013] As one of the preferred embodiments of the present invention, the protective agent is selected from one or more of 5-10% skim milk powder, 5% trehalose, and 1% monosodium glutamate.

[0014] As one of the preferred embodiments of the present invention, the excipients are selected from one or more of enteric coating materials, fillers, and binders.

[0015] As one of the preferred embodiments of the present invention, the dosage form of the pharmaceutical preparation is freeze-dried powder, enteric-coated capsules, enteric-coated tablets, or granules.

[0016] As one of the preferred embodiments of the present invention, the total number of viable bacteria of the three strains in each gram of the pharmaceutical preparation is 10. 9 ~10 11 CFU.

[0017] A method for preparing the above-mentioned microbial drug preparation includes the following steps: (1) Culture expansion: Three strains of Bifidobacterium pseudosmata aFMT-202, Bifidobacterium longum subsp. longum aFMT-104 and Lactobacillus mucosa aFMT-501 were inoculated into the culture medium and cultured to the logarithmic growth phase. (2) Large-scale fermentation: The seed liquids of each strain are separately inoculated into fermentation tanks and fermented anaerobically with stirring until the viable cell count in the liquid reaches 10. 9 CFU / mL or higher; (3) Collection and mixing of bacterial cells: Collect the bacterial cell precipitates of each strain by centrifugation and mix them; (4) Addition of protective agent and freeze drying: Add a pharmaceutically acceptable protective agent to the mixed bacteria, pre-freeze to below -40°C and then freeze-dry under vacuum to obtain freeze-dried powder; (5) Formulation: The freeze-dried powder is mixed with pharmaceutically acceptable excipients to form enteric capsules, enteric tablets or granules.

[0018] The application of a combination of the above-mentioned structural bacterial matrix bacteria in the preparation of an antidepressant, said antidepressant for improving depressive-like behavior induced by chronic stress.

[0019] The application of a combination of the above-mentioned structural bacterial matrix bacteria in the preparation of an intestinal anti-inflammatory drug, said intestinal anti-inflammatory drug for relieving inflammation associated with inflammatory bowel disease or intestinal mucosal damage.

[0020] The application of the above-mentioned structural bacterial matrix drug combination in the preparation of antioxidant and anti-aging drugs, wherein the antioxidant and anti-aging drugs are used to reduce oxidative stress damage in the body and improve age-related functional decline.

[0021] As one of the preferred embodiments of the present invention, in the daily dosage of the above-mentioned drug, the total viable count of the three strains is 1×10⁻⁶. 9 ~1×10 10 CFU / day, preferably 3×10 9 ~1×10 10 CFU / day.

[0022] The advantages of this invention compared to the prior art are: (1) The source is clear and the safety meets the pharmaceutical standards. The three strains used in this invention (Bifidobacterium pseudosporidis aFMT-202, Bifidobacterium longum subsp. aFMT-104, and Lactobacillus mucosa aFMT-501) were all isolated from fecal samples from healthy adults. 16S rRNA sequence analysis, genome identification, and in vitro safety evaluation confirmed that they had no drug resistance or pathogenicity, meeting the safety requirements for microbial drugs and providing a reliable basis for clinical application.

[0023] (2) Functional synergy to achieve multi-target intervention The three strains of this invention have complementary mechanisms of action: Bifidobacterium pseudosporidis and Bifidobacterium longum can regulate neuroendocrine stress response, balance neurotransmitter levels, and improve systemic inflammatory state; Lactobacillus mucosa can enhance the integrity of the intestinal mucosal barrier and inhibit local intestinal inflammatory pathways. Through scientific combination, the strains form a synergistic effect in vivo, breaking through the limitations of single strain targets, and can simultaneously exert intervention effects on multiple pathological mechanisms related to depression, intestinal inflammation, and oxidative aging, providing a new solution for the comprehensive treatment of multiple diseases.

[0024] (3) Optimized formulation to suit different treatment needs By adjusting the ratio of live bacteria counts of the three strains, specialized formulas targeting different symptoms were created: Formula-D (aFMT-202:aFMT-104:aFMT-501=1:2:1) for depressive symptoms, Formula-I (aFMT-202:aFMT-104:aFMT-501=1:1:2) for intestinal inflammation, and Formula-A (aFMT-202:aFMT-104:aFMT-501=2:1:1) for oxidative aging-related functional decline. Each formula, through its clearly defined proportions, ensures targeted intervention for specific symptoms while maintaining a stable gut microbiota structure, avoiding antagonistic issues after mixing multiple strains, and improving the controllability of drug action.

[0025] (4) Experimental verification shows that the efficacy and safety are clear. Animal model experiments have confirmed that Formula-D can significantly improve anhedonia (increased sucrose water preference rate) and behavioral despair (shortened immobility time during forced swimming) in animals with chronic stress depression; Formula-I can alleviate colonic inflammation in animals with DSS-induced colitis (restoration of colon length and reduction of pro-inflammatory factor TNF-α); Formula-A can reduce oxidative damage (reduction of malondialdehyde (MDA) content) and enhance endogenous antioxidant capacity (increased superoxide dismutase (SOD) activity) in animals with D-galactose premature aging; and no significant adverse reactions were observed in any of the experiments, confirming the efficacy and safety of the formulation of this invention and providing experimental evidence for subsequent clinical studies. Attached Figure Description

[0026] Figure 1 This is a colony morphology diagram of the *Bifidobacterium pseudosporidis* aFMT-202 strain in Example 1; Figure 2 This is a colony morphology diagram of the long subspecies of Bifidobacterium longum aFMT-104 strain in Example 1; Figure 3 This is a colony morphology diagram of the *Lactobacillus mucosa* aFMT-501 strain in Example 1; Figure 4 These are the HE staining results of colon tissue from mice in each group in Experiment Example 3 (in the figure, "Normal control" represents the normal control group, "Colitis model" represents the colitis model group, and "Colitis model-Formula-I" represents the colitis model + Formula-I group). Figure 5 The results of HE staining of liver and kidney tissues of mice in each group in Experiment Example 4 are shown in the figure. The left figure is the liver staining result, and the right figure is the kidney staining result. "Model" represents the premature aging model group, and "Model + Formula-I" represents the model + Formula-A group. Detailed Implementation

[0027] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0028] The culture medium formulations involved in the following examples are as follows: Standard MRS medium: 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate trihydrate, 2g ammonium citrate, 2g K2HPO4, 0.2g MgSO4·7H2O, 0.05g MnSO4·H2O, 1mL Tween-80.

[0029] Modified selective culture medium for Bifidobacterium: Standard MRS medium + 15g agar + 3g sodium propionate + 0.5g L-cysteine ​​hydrochloride + 100mg mupirocin.

[0030] MRS selective medium for Lactobacillus: Standard MRS medium + 15g agar + 3g sodium propionate + 100mg cyclohexylimide.

[0031] Modified MRS medium containing 0.05% cysteine: standard MRS medium + 0.5g L-cysteine ​​hydrochloride.

[0032] Example 1: Screening, isolation, and identification of target strains: 1. Strain aFMT-202 Bifidobacterium strains were isolated from fresh fecal samples of healthy adult volunteers. Using a modified selective medium for Bifidobacterium, the cultures were anaerobic at 37°C for 48 hours. Colonies with high lactic acid production and a milky-white, convex shape were selected and named aFMT-202 (colony morphology as shown). Figure 1 (As shown). Gram-positive, immobile short rod-shaped cells. 16S rRNA gene sequencing results (SEQ ID NO.1) showed that its sequence was similar to that of the type strain. Bifidobacterium pseudocatenulatum With a homology of over 99%, it was preliminarily identified as Bifidobacterium pseudosporidis (Bifidobacterium pseudosporidis). Bifidobacterium pseudocatenulatum ).

[0033] The strain was deposited with the Guangdong Provincial Center for Microbial Culture Collection on September 23, 2024, and obtained the accession number GDMCC No. 65183. The collection center officially classified and named it... Bifidobacterium pseudocatenulatum aFMT-202.

[0034] 2. Strain aFMT-104 A strain of *Bifidobacterium longum* subsp. *longum* was isolated from fresh fecal samples of healthy adult volunteers. Anaerobic enrichment culture was performed using a modified selective medium for *Bifidobacterium*, yielding multiple colonies. Acid and gas production tests and carbon source utilization profiles were used to screen for a superior strain, which was named strain aFMT-104 (colony morphology as shown in the image). Figure 2 (As shown). 16S rRNA gene sequencing results (SEQ ID NO.2) show that this strain is similar to the type strain.Bifidobacterium longum subsp. Longum The standard strain had a sequence similarity of 99.5%, and it was preliminarily identified as *Bifidobacterium longum* subsp. *longum*. Bifidobacterium longum subsp. Longum ).

[0035] The strain was deposited with the Guangdong Provincial Center for Microbial Culture Collection on April 3, 2025, and obtained the accession number GDMCC No. 66087. The collection center officially classified and named it... Bifidobacterium longum subsp. Longum aFMT-104.

[0036] 3. Strain aFMT-501 Lactobacillus strains were isolated from fresh fecal samples of healthy adults. Lactic acid bacteria were enriched and cultured on MRS Lactobacillus selective medium under microaerophilic conditions, yielding several candidate Lactobacillus strains. Morphological observation revealed a Gram-positive short bacillus strain capable of fermenting glucose to produce large amounts of lactic acid without gas production; this strain was named aFMT-501 (colony morphology as shown). Figure 3 (As shown). Sequencing results of the 16S rRNA gene (SEQ ID NO.3) show that its sequence is similar to... Limosilactobacillus mucosae The type strain showed 98.7% homology and was preliminarily identified as *Lactobacillus mucosa*. Limosilactobacillus mucosae ).

[0037] The strain was deposited with the Guangdong Provincial Center for Microbial Culture Collection on April 3, 2025, and obtained the accession number GDMCC No. 66090. The collection center officially classified and named it... Limosilactobacillus mucosae aFMT-501.

[0038] Example 2: Preparation of an antidepressant formula (Formula-D) microbial drug preparation (enteric-coated capsule): (1) Propagation of microbial strains Take the *Bifidobacterium pseudosporidis* screened in Example 1 ( Bifidobacterium pseudocatenulatum aFMT-202, Bifidobacterium longum subsp. ( Bifidobacterium longum subsp. longum The aFMT-104 strain was inoculated into modified MRS medium (for Bifidobacterium) containing 0.05% cysteine; the mucosal lactobacilli screened in Example 1 were taken... Limosilactobacillus mucosae aFMT-501 strain was inoculated into standard MRS medium (for Lactobacillus).

[0039] Incubate at 37℃ in an anaerobic incubator (aFMT-202, aFMT-104) or a microaerophilic incubator (aFMT-501) for 24 hours until the bacterial culture OD reaches its maximum.600 A value of 1.0 ± 0.1 confirms that the bacteria have entered the logarithmic growth phase.

[0040] (2) Large-scale fermentation The seed cultures of each strain were inoculated into fermenters at a ratio of 1:100 and cultured separately. Fermentation conditions: constant temperature of 37℃, pH controlled at 6.2-6.5 (adjusted by automatic addition of alkali solution), anaerobic stirring fermentation for more than 20 hours, until the viable cell count in the culture reached 10^6. 9 CFU / mL or higher. Monitor the cell growth curve during fermentation and harvest at the appropriate time to ensure strain viability.

[0041] (3) Collection and mixing of bacterial cells After fermentation, the bacterial precipitate was collected by low-temperature centrifugation (temperature ≤4℃, 4000×g, 15 minutes). The bacterial cells were washed twice with sterile physiological saline to remove residual culture medium. Subsequently, the three bacterial strains were mixed evenly according to the wet bacterial weight ratio of “aFMT-202:aFMT-104:aFMT-501=1:2:1” (Formula-D formula), and the mixture was kept at a low temperature during the mixing process to maintain bacterial activity.

[0042] (4) Addition of preservatives and freeze drying Preservative preparation: 5% skim milk powder + 5% trehalose + 1% monosodium glutamate, dissolved in sterile water and then sterilized.

[0043] Add the preservative to the mixed bacterial sludge and stir until the sludge is completely dispersed to obtain a preservative-mixed bacterial solution. Dispense the obtained preservative-mixed bacterial solution into freeze-drying bottles, pre-freeze them in a freeze dryer to below -40°C, control the vacuum degree to below 10 Pa, sublime dry for more than 24 hours, and finally dry them under vacuum for 8 hours to obtain freeze-dried bacterial powder containing a combination of three strains.

[0044] The obtained freeze-dried bacterial powder was removed under aseptic drying conditions, and its moisture content was found to be 3.2%, with a total viable count of 1.8 × 10⁻⁶ per gram. 11 CFU / g meets quality requirements.

[0045] (5) Formulation (enteric-coated capsules) The freeze-dried bacterial powder was passed through a 100-mesh sieve and mixed thoroughly with excipients (inulin / fructooligosaccharides) and lubricants / flow aids (silica ≤0.5%, magnesium stearate ≤0.5%) to ensure uniformity of filling amount and content. Each capsule was labeled with 1.5 × 10⁻⁶ live bacteria. 9 The CFU (total of three strains) is dispensed into enteric-coated hard capsules (ensuring the release of the bacterial powder into the intestines). Finally, the capsules are sealed in aluminum foil at low temperature (2-8°C) and protected from light, with 30 capsules per bottle, resulting in the final Formula-D enteric-coated capsule formulation. Strict aseptic techniques are employed throughout the entire process to guarantee the quality and safety of the formulation.

[0046] Example 3: Preparation of Enteric-coated Tablets (Formula-I) Microbial Drug Preparation for Intestinal Anti-inflammatory Formulation: (1) Propagation of microbial strains Same as Example 2.

[0047] (2) Large-scale fermentation Same as Example 2.

[0048] (3) Collection and mixing of bacterial cells After fermentation, the bacterial precipitate was collected by low-temperature centrifugation (temperature ≤4℃, 4000×g, 15 minutes). The bacterial cells were washed twice with sterile physiological saline to remove residual culture medium. Subsequently, the three bacterial strains were mixed evenly according to the wet bacterial weight ratio of “aFMT-202:aFMT-104:aFMT-501=1:1:2” (Formula-I formula), and the mixing process was kept at a low temperature to maintain bacterial activity.

[0049] (4) Addition of preservatives and freeze drying Same as Example 2. The freeze-dried bacterial powder was taken out under aseptic drying conditions, and the total number of viable bacteria reached 2.1 × 10⁻⁶. 11 CFU / g.

[0050] (5) Formulation (enteric-coated tablets) After passing the freeze-dried bacterial powder through a 100-mesh sieve, it is mixed with excipients in the following proportions: 80g lactose, 20g microcrystalline cellulose, 5g hydroxypropyl methylcellulose (HPMC, pharmaceutical binder / forming), and 0.5g colloidal silica (flow aid). A 2.5% (w / w, based on powder) povidone K30 ethanol solution is slowly added as a binder for low-humidity "micro-moist" granulation, ensuring uniform wetting while avoiding over-wetting. The resulting soft mass is granulated through a 20-mesh sieve, dried under vacuum (or fluidized bed low-temperature) at 35°C until the moisture content meets the standard, and then granulated through an 18-mesh sieve. 1g magnesium stearate is added, and the mixture is mixed under low shear for 2.5 minutes. Low-compression tableting is used (0.5g / tablet) to achieve a viable bacteria content of 1.0 × 10⁻⁶ per tablet. 10 CFU (total of three strains). Finally, acrylic resin No. IV was used as the enteric coating material to prepare an 8% coating solution, which was used to coat the tablets. After drying, enteric tablets were obtained.

[0051] Example 4: Preparation of an antioxidant and anti-aging formula (Formula-A) microbial drug preparation (lyophilized powder): (1) Propagation of microbial strains Same as Example 2.

[0052] (2) Large-scale fermentation Same as Example 2.

[0053] (3) Collection and mixing of bacterial cells After fermentation, the bacterial precipitate was collected by low-temperature centrifugation (temperature ≤4℃, 4000×g, 15 minutes). The bacterial cells were washed twice with sterile physiological saline to remove residual culture medium. Subsequently, the three bacterial strains were mixed evenly according to the wet weight ratio of “aFMT-202:aFMT-104:aFMT-501=2:1:1” (Formula-A formula), and the mixing process was kept at a low temperature to maintain bacterial activity.

[0054] (4) Addition of preservatives and freeze drying Same as Example 2. The freeze-dried bacterial powder was taken out under aseptic drying conditions, and the total number of viable bacteria reached 2.5 × 10⁻⁶ per gram. 11 CFU / g.

[0055] (5) Formulation (lyophilized powder) The freeze-dried bacterial powder (i.e., freeze-dried powder) is directly dispensed into sterile vials (1g±2% per vial), sealed with butyl rubber stoppers, and pressed with aluminum caps; the ambient humidity is controlled to ≤20% during packaging to prevent the powder from absorbing moisture.

[0056] To verify the effectiveness of the above-described bacterial community combinations and formulations, systematic in vitro characterization tests and multiple animal model experiments were subsequently conducted.

[0057] Experimental Example 1: In vitro tolerance and adhesion ability test of the strain of this invention: I. Experimental Objective The survival ability and adhesion performance of three core strains (aFMT-202, aFMT-104, and aFMT-501) in a simulated gastrointestinal environment were tested. These properties are the basis for the strains to exert their functional effects.

[0058] II. Experimental Methods Three strains of bacteria, *Bifidobacterium pseudosporidis* aFMT-202, *Bifidobacterium longum* subsp. *longum* aFMT-104, and *Lactobacillus mucosa* aFMT-501, were cultured to the logarithmic growth phase. The bacterial cells were collected by centrifugation and washed with PBS. The bacterial suspensions were then incubated in simulated gastric fluid (pH 2.0, containing 0.3% pepsin) at 37°C for 2 hours to test acid tolerance. Separately, bacterial suspensions were added to simulated intestinal fluid containing 0.3% ox bile salts and incubated at 37°C for 4 hours to test bile salt tolerance. The survival rate was calculated using a plate count method after each treatment, and the percentage was compared to the untreated control.

[0059] For the adhesion test, the human colon adenocarcinoma epithelial cell line Caco-2 was cultured in 24-well plates until it reached monolayer confluence. After washing with PBS, a live bacterial suspension (approximately 10 μL) was added. 8(CFU / mL), incubate for 1 hour, then wash repeatedly with sterile PBS to remove unadhered bacteria, lyse epithelial cells and count the number of adhered colonies, and calculate the adhesion rate of the strain to Caco-2 cells (the percentage of adhered bacteria to the initial number of bacteria added).

[0060] III. Results and Analysis The three strains, aFMT-202, aFMT-104, and aFMT-501, all exhibited good survival rates under strong acid and high bile salt conditions and possessed a certain degree of intestinal epithelial cell adhesion ability. The results are summarized in Table 1.

[0061] Table 1 Results of acid resistance, bile salt resistance, and in vitro adhesion tests of the three strains.

[0062] Note: Data are mean ± standard error (n=3).

[0063] Table 1 shows that the survival rate of all three strains was above 75% after 2 hours in a strongly acidic environment (pH 2.0), with *Lactobacillus mucosa* aFMT-501 exhibiting the best acid resistance, reaching a survival rate of 92%. After 4 hours in the presence of 0.3% bile salts, the survival rates of all strains were close to 90% or higher, indicating good tolerance to small intestinal bile salts. Adhesion assays showed that all three strains had a certain adhesion ability to human intestinal epithelial cells, with aFMT-501 showing an adhesion rate of approximately 12.5%, slightly higher than the two *Bifidobacterium* strains. This may be related to surface proteins of *Lactobacillus* (such as adhesins). In summary, these results demonstrate that the strains selected in this invention can survive well through the adverse environment of the gastrointestinal tract and adhere to the surface of intestinal epithelial cells, laying the foundation for colonization and functional performance.

[0064] Experimental Example 2: Animal Experiment on the Antidepressant Effect of Formula-D Formulation: I. Experimental Objective To verify the ameliorative effect of Formula-D formulation, composed of aFMT-202, aFMT-104, and aFMT-501 in a 1:2:1 ratio, on a mouse model of chronic stress-induced depressive-like behavior.

[0065] II. Experimental Methods Thirty male ICR mice (6 weeks old, weighing 20-25g) were randomly divided into three groups (n=10 per group): a normal control group, a depression model group, and a depression model + Formula-D group. The depression model was established using the Chronic Unpredictable Mild Stress (CUMS) method: for 6 consecutive weeks, mice in the model group were subjected to randomized mild stressors (such as day-night reversal, wet nesting, and light stimulation). Specific treatments for each group are as follows: Depression model + Formula-D group: A depression model was established using the Chronic Unpredictable Mild Stress (CUMS) method. Starting one week after the onset of stress, the mice in the depression model were administered Formula-D bacterial solution daily by gavage (Example 2: the mixed bacterial sludge was resuspended in physiological saline to obtain the corresponding bacterial solution, with a concentration of 1.0 × 10⁻⁶). 10 (CFU / mL), administered continuously for 2–4 weeks; the dosage volume is calculated at 0.1 mL / 10g body weight, and the daily dose is set at 1.0 × 10⁻⁶ CFU / mL. 9 CFU / mouse / day (total viable bacteria count).

[0066] Depression model group: A depression model was established using the chronic unpredictable mild stress (CUMS) method. Starting one week after the onset of stress, the depression model mice were given physiological saline by gavage daily (the same volume of Formula-D bacterial solution as the depression model + Formula-D group).

[0067] Normal control group: Mice were not subjected to stress, were fed normally, and were given physiological saline by gavage daily.

[0068] Throughout the experiment, changes in mouse body weight and behavioral alterations were recorded regularly. Behavioral assessments were conducted at the end of week 6, including a sucrose preference test (SPT, used to assess anhedonia) and a forced swimming test (FST, used to assess behavioral despair). In the sucrose preference test, the percentage of total water intake consumed by mice with 2% sucrose was measured; in the forced swimming test, the time mice remained stationary in the swimming tank for 6 minutes was recorded.

[0069] III. Experimental Results Mice in the "normal control group" showed normal indicators. After CUMS stress, mice in the "depression model group" exhibited typical depressive-like behavioral changes: slow weight gain, significantly reduced interest in sucrose water, and significantly prolonged periods of immobility. Mice in the "depression model + Formula-D group" treated with Formula-D showed significant improvement in behavioral indicators compared to the model group. Key data are shown in Table 2.

[0070] Table 2. Effects of Formula-D on behavioral parameters in a mouse model of chronic stress-induced depression.

[0071] *Note: Data are mean ± standard error (n=10). Compared with the normal control group: **P<0.01; compared with the model group: P<0.05.

[0072] As shown in Table 2, the sucrose preference rate in the chronic stress-induced "depression model group" mice was only 55.4%, significantly lower than the 88.3% in the "normal control group," indicating a significant loss of pleasure. The forced swimming immobility time increased from approximately 100 seconds in the "normal control group" to over 180 seconds, reflecting increased behavioral despair. After 6 weeks of Formula-D intervention, the sucrose preference rate in mice rebounded to 79.6%, approaching normal levels (an increase of approximately 44%), and the forced swimming immobility time decreased to 121.3 seconds, significantly lower than the untreated "depression model group" (a decrease of approximately 33%). This demonstrates that Formula-D can effectively alleviate depressive-like behaviors induced by chronic stress. Observation of the general condition of the mice showed that the "depression model + Formula-D group" mice had glossy fur and higher activity levels, significantly better than the model group.

[0073] In addition, serum was collected at the end of the experiment for the detection of stress hormones (such as corticosterone). The results also showed that the "depression model + Formula-D group" had a lower level than the "depression model group", suggesting that the overactivation of the stress axis was suppressed.

[0074] In summary, the Formula-D formulation demonstrated good antidepressant effects in animal models, confirming the synergistic effect of multi-strain combination on mood regulation.

[0075] Experiment Example 3: Animal experiment on the anti-inflammatory and intestinal-soothing effects of Formula-I formulation: I. Experimental Objective The anti-inflammatory and soothing effects of Formula-I, composed of aFMT-202, aFMT-104, and aFMT-501 in a 1:1:2 ratio, on a mouse colitis model were verified.

[0076] II. Experimental Methods Twenty-four 8-week-old male C57BL / 6 mice were randomly divided into three groups (n=8 per group): a normal control group, a colitis model group, and a colitis model + Formula-I group. The acute colitis model was induced using sodium dextran sulfate (DSS). Specific treatments are as follows: Colitis model + Formula-I group: Mice were allowed free access to 2.5% DSS solution for 7 days to induce colonic mucosal inflammation. Simultaneously, Formula-I bacterial solution was administered by gavage daily while receiving DSS (Example 3: the mixed bacterial sludge was resuspended in physiological saline to prepare the corresponding bacterial solution, with a concentration of 1.0 × 10⁻⁶). 10 (CFU / mL); the gavage volume is calculated at 0.1 mL / 10g body weight, and the daily dose is 1.0 × 10⁻⁶. 9 CFU / mouse / day (total viable bacteria count).

[0077] Colitis model group: Mice were allowed to drink 2.5% DSS (sodium dextran sulfate) solution for 7 days to induce colonic mucosal inflammation. At the same time, PBS (with the same volume of Formula-I bacterial solution as the colitis model + Formula-I group) was administered by gavage daily while DSS was being administered.

[0078] Normal control group: Mice were fed normally and given pure water.

[0079] During the observation period, changes in mouse body weight, fecal characteristics, and fecal hemorrhage were recorded to calculate the Disease Activity Index (DAI). Mice were sacrificed at the end of day 7, colon length was measured, and distal colon tissue was collected for inflammatory marker detection, including myeloperoxidase (MPO) activity assay, ELISA quantification of pro-inflammatory cytokines such as TNF-α, and colonic histological inflammation scoring.

[0080] III. Experimental Results Following DSS administration, mice in the "colitis model group" exhibited typical colitis symptoms: weight loss, bloody diarrhea, and elevated DAI scores. Compared to the "normal control group," the colon in the "colitis model group" mice was significantly shortened, and histological examination revealed extensive mucosal ulceration and neutrophil infiltration, with significantly elevated levels of inflammatory factors. Mice in the "colitis model + Formula-I group" receiving Formula-I intervention showed significantly reduced symptoms: less weight loss, gradually formed stools with reduced bloody stools, and improved histological mucosal ulceration and neutrophil infiltration. Figure 4 The main objective indicators are listed in Table 3.

[0081] Table 3 Effects of Formula-I on inflammatory markers in DSS-induced colitis mice

[0082] *Note: Data are presented as mean ± standard error (n=8). Compared with the normal control group: **P<0.01; Compared with the model group: P<0.05 。

[0083] As shown in Table 3, the colon length of mice in the "colitis model group" was only about 5.5 cm, shortened by about 32% compared to the "normal control group," indicating severe inflammation and tissue damage in the colon. The level of the pro-inflammatory factor TNF-α in the colon tissue increased to more than three times that of the normal group, showing a strong inflammatory response. After intervention with Formula-I, the colon length of mice recovered to 6.9 cm, significantly longer than the untreated model group, indicating improved mucosal damage. Simultaneously, the TNF-α content in the colon tissue decreased to 15.8 pg / mg, nearly 1.5 times the normal level, significantly lower than the model group. This indicates that Formula-I effectively reduced the production of pro-inflammatory mediators in the colon. Combined with histopathological observation (… Figure 4The intestinal mucosal structure of the "colitis model + Formula-I group" was relatively intact, the ulcer area was small, and the infiltration of inflammatory cells was greatly reduced; conversely, the "colitis model group" had large-area ulcers accompanied by severe inflammation.

[0084] In addition, regarding the clinical symptom scores in mice, the DAI score of the "colitis model + Formula-I group" decreased from 3.5 in the model group to 1.2 (close to mild), which also supports its alleviating effect on enteritis.

[0085] It is evident that Formula-I significantly reduces DSS-induced acute colitis response through gut microbiota regulation, demonstrating good efficacy in protecting the intestinal mucosa and inhibiting inflammation.

[0086] Experiment Example 4: Animal experiment on the antioxidant and anti-aging effects of Formula-A formulation: I. Experimental Objective The antioxidant and anti-aging effects of Formula-A, composed of aFMT-202, aFMT-104, and aFMT-501 in a 2:1:1 ratio, were verified in a mouse model of premature aging.

[0087] II. Experimental Methods Eighteen healthy male Kunming mice aged 6 months were randomly divided into three groups (n=6 per group): a normal control group, a premature aging model group, and a model + Formula-A group. Specific treatments were as follows: Premature aging model group: Mice were injected intraperitoneally with D-galactose (120 mg / kg body weight / day) for 8 weeks to induce accelerated aging and oxidative stress accumulation in mice.

[0088] Model + Formula-A group: Mice were intraperitoneally injected with D-galactose (120 mg / kg body weight / day) for 8 weeks to induce accelerated aging and oxidative stress accumulation in mice; during the modeling period, Formula-A bacterial solution (prepared by resuspending the mixed bacterial sludge from Example 4 in sterile physiological saline, concentration 1×10⁻⁶) was administered by gavage daily. 10 CFU / mL); administer at a dose of 0.1 mL / 10 g body weight, ensuring a daily dose of approximately 1 × 10⁻⁶. 9 CFU / Rat.

[0089] Normal control group: Mice were injected intraperitoneally with an equal volume of physiological saline.

[0090] During the 8-week experiment, changes in the general condition of the mice (coat luster, activity level, etc.) were recorded weekly. After the last administration, serum and major organs of the mice were collected to assess in vivo oxidative damage and antioxidant capacity indicators, including malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) activity; at the same time, peripheral blood routine and histological changes of major organs of mice in each group were observed.

[0091] III. Experimental Results Mice in the "premature aging model group" began to show signs of aging in the middle of the experiment: dull fur, reduced activity, and some mice exhibited loose skin and kyphosis on their backs. Compared with normal age-matched mice, the "premature aging model group" had significantly higher serum MDA levels and lower activity of antioxidant enzymes such as SOD, indicating significant oxidative damage. Mice in the "model + Formula-A group" treated with Formula-A showed reduced signs of aging: relatively shiny fur, near-normal activity levels, and more sensitive responses to external stimuli. The main biochemical indicators are shown in Table 4.

[0092] Table 4. Effects of Formula-A on oxidative stress indices in a D-galactose-induced premature aging model mouse.

[0093] *Note: Data are mean ± standard error (n=6). Compared with the normal group: **P<0.01; compared with the model group: P<0.05.

[0094] As shown in Table 4, the serum MDA level in the "premature aging model group" mice increased to 12.1 μmol / L, approximately 2.3 times that of the normal control group (indicating increased lipid peroxidation damage); simultaneously, SOD activity decreased from 102 U / mL in the normal group to approximately 61 U / mL, a decrease of nearly 40% (indicating damage to the antioxidant enzyme system). However, in the "model + Formula-A group" treated with Formula-A, the MDA level significantly decreased to 6.3 μmol / L, only slightly higher than the "normal control group," reducing peroxide accumulation by nearly 50%; SOD activity recovered to 91.4 U / mL, restoring 89% of the normal level, indicating a significant improvement in endogenous antioxidant capacity.

[0095] Regarding other antioxidant indicators, the Formula-A group mice showed significantly higher GSH-Px activity and total antioxidant capacity (T-AOC) compared to the model group. Histological examination also revealed varying degrees of lipid accumulation and cell degeneration in the liver and kidneys of the "premature aging model group" mice, while the tissue morphology of the "model + Formula-A group" was close to normal. Figure 5These results demonstrate that Formula-A effectively reduces D-galactose-induced oxidative stress damage and has the effect of delaying the aging process.

[0096] Based on the above experimental results, it is clear that the effects of the three-strain combination described in this invention in different functional directions are supported by experimental evidence: In the antidepressant direction, the Formula-D formulation can significantly improve anhedonia and behavioral hopelessness in animals with chronic stress-induced depression, confirming its alleviating effect on depressive-like behaviors; in the intestinal anti-inflammatory direction, the Formula-I formulation can reduce colonic damage in animals with DSS-induced colitis and reduce the level of the pro-inflammatory factor TNF-α in colonic tissue, demonstrating a clear intestinal anti-inflammatory effect; in the antioxidant and anti-aging direction, the Formula-A formulation can reduce oxidative damage in animals with D-galactose premature aging and simultaneously increase the activity of endogenous antioxidant enzymes, effectively delaying aging-related physiological changes. Furthermore, no significant adverse reactions were observed in any of the experiments, further verifying the safety and functional reliability of this three-strain combination, providing sufficient experimental evidence for its application in the treatment of corresponding diseases.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A structural microbial community-based drug combination, characterized in that, It consisted of three strains: *Bifidobacterium pseudobulbarum* aFMT-202, *Bifidobacterium longum* subsp. *longum* aFMT-104, and *Lactobacillus mucosa* aFMT-501; among them, *Bifidobacterium pseudobulbarum* aFMT-202 was classified as... Bifidobacterium pseudocatenulatum aFMT-202, deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No. 65183; the taxonomic name of *Bifidobacterium longum* subsp. *longum* aFMT-104 is... Bifidobacterium longum subsp. Longum aFMT-104, deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No. 66087; the taxonomic name of Lactobacillus mucosa aFMT-501 is... Limosilactobacillus mucosae aFMT-501, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No. 66090; all three strains were present in the assemblage in live form.

2. The structural microbial community drug combination according to claim 1, characterized in that, The ratio of viable bacteria of the three strains is 1:2:1, that is, Bifidobacterium pseudosporidis aFMT-202: Bifidobacterium longum subsp. longum aFMT-104: Lactobacillus mucosa aFMT-501 = 1:2:

1. This ratio is used to prepare antidepressant drugs.

3. The structural microbial community drug combination according to claim 1, characterized in that, The ratio of viable bacteria of the three strains is 1:1:2, that is, Bifidobacterium pseudosporidis aFMT-202: Bifidobacterium longum subsp. longum aFMT-104: Lactobacillus mucosa aFMT-501 = 1:1:

2. This ratio is used to prepare intestinal anti-inflammatory drugs.

4. The structural microbial community drug combination according to claim 1, characterized in that, The ratio of viable bacteria of the three strains is 2:1:1, that is, Bifidobacterium pseudosporidis aFMT-202: Bifidobacterium longum subsp. longum aFMT-104: Lactobacillus mucosa aFMT-501 = 2:1:

1. This ratio is used to prepare antioxidant and anti-aging drugs.

5. A microbial pharmaceutical preparation, characterized in that, The drug combination comprises the structural microbial community of any one of claims 1 to 4, and pharmaceutically acceptable protectants and excipients.

6. The microbial pharmaceutical preparation according to claim 5, characterized in that, The protective agent is selected from one or more of 5-10% skim milk powder, 5% trehalose, and 1% monosodium glutamate; the excipients are selected from one or more of enteric coating materials, fillers, and binders; the dosage form of the pharmaceutical preparation is freeze-dried powder, enteric capsules, enteric tablets, or granules; the total viable count of the three strains in each gram of the pharmaceutical preparation is 10. 9 ~10 11 CFU.

7. A method for preparing a microbial pharmaceutical preparation as described in claim 5 or 6, characterized in that, Includes the following steps: (1) Culture expansion: Three strains of Bifidobacterium pseudosmata aFMT-202, Bifidobacterium longum subsp. longum aFMT-104 and Lactobacillus mucosa aFMT-501 were inoculated into the culture medium and cultured to the logarithmic growth phase. (2) Large-scale fermentation: The seed liquids of each strain are separately inoculated into fermentation tanks and fermented anaerobically with stirring until the viable cell count in the liquid reaches 10. 9 CFU / mL or higher; (3) Collection and mixing of bacterial cells: Collect the bacterial cell precipitates of each strain by centrifugation and mix them; (4) Addition of protective agent and freeze drying: Add a pharmaceutically acceptable protective agent to the mixed bacteria, pre-freeze to below -40°C and then freeze-dry under vacuum to obtain freeze-dried powder; (5) Formulation: The freeze-dried powder is mixed with pharmaceutically acceptable excipients to form enteric capsules, enteric tablets or granules.

8. The application of a structural microbial community drug combination as described in any one of claims 1 to 4 in the preparation of an antidepressant, characterized in that, The antidepressant is used to improve depressive-like behaviors caused by chronic stress.

9. The application of a structural microbial community drug combination as described in any one of claims 1 to 4 in the preparation of an intestinal anti-inflammatory drug, characterized in that, The intestinal anti-inflammatory drugs are used to relieve inflammation associated with inflammatory bowel disease or intestinal mucosal damage.

10. The application of a structural microbial community drug combination as described in any one of claims 1 to 4 in the preparation of antioxidant and anti-aging drugs, characterized in that, The antioxidant and anti-aging drugs are used to reduce oxidative stress damage in the body and improve age-related functional decline.

Images