Muciniphilic akkermansia, high-activity preservation inactivation method thereof and application thereof

Abstract

The application belongs to the technical field of microorganisms and their application, and specifically discloses a mucinophilic Akkermansia strain, a high-activity reserved inactivation method of the strain and application of the strain. The strain has a preservation number of CGMCC No. 33955, and when the strain is cultured in a mucin only carbon source culture medium for 48 hours, the OD600 value reaches 1.32 and the acetic acid yield is 18.3 mmol / L. The inactivation method uses a solution containing sulfated dextran, sucrose and epsilon-polylysine as a composite protective agent, and after the solution is mixed with the bacterial solution, the mixture is incubated at 45 DEG C to 50 DEG C for 16 min to 20 min. The inactivated strain reduces inflammation and protects lung vascular function through the "gut-lung axis", and in a MCT-induced PAH rat model, the intervention effect is close to sildenafil, and there is no increase in liver enzymes, the serum TNF-alpha and IL-6 are reduced, and the problems of large side effects and low activity reservation of existing PAH intervention are solved.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and applied technology, specifically relating to a strain of Akkermansia myxophilus, its highly active retention and inactivation method, and its application. Background Technology

[0002] Pulmonary arterial hypertension (PAH) is a progressive cardiovascular disease characterized by persistently elevated pulmonary artery pressure. Its pathological mechanisms involve multiple levels, including pulmonary vascular remodeling, endothelial dysfunction, and abnormal platelet activation. As the disease progresses, right ventricular afterload increases significantly, eventually leading to right heart failure, which seriously threatens patients' lives. Therefore, exploring safe and effective treatment strategies has become a crucial clinical issue that urgently needs to be addressed.

[0003] Currently, clinical interventions for PAH mainly include drugs such as sildenafil and bosentan, and their compound preparations (such as the compound drug disclosed in patent CN102883722A). While these treatments can effectively reduce pulmonary vascular resistance in the short term, long-term use can lead to a series of adverse reactions, such as elevated liver enzymes and serious consequences like liver damage. In recent years, probiotic intervention strategies based on the "gut-lung axis" theory have received widespread attention due to their lower side effects. Among them, Akkermansia muciniphila (Akk bacteria), a strictly anaerobic bacterium that colonizes the intestinal mucus layer, has been proven to protect the integrity of intestinal epithelial cells and the mucus layer, and regulate blood glucose and lipid levels (patent CN118207140A).

[0004] Studies have found that inactivated Akk bacteria retain their activity, and compared to live bacterial preparations, inactivated Akk bacteria have advantages in industrial production, including not requiring strict anaerobic culture conditions and having stronger temperature tolerance. However, key technical challenges exist in the inactivation process. Its core active substances, such as P9 protein and a15:0-i15:0PE phospholipids, are easily degraded and inactivated by high temperatures during conventional sterilization processes (such as pasteurization). How to preserve a large amount of activity is a pressing technical problem that needs to be solved. Furthermore, there are currently no research reports on the application of inactivated Akk bacteria in the treatment of PAH. Therefore, developing an inactivation method that can maximize the preservation of its biological activity and exploring its application in PAH treatment has significant scientific and clinical value. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a strain of Akkermansia myxophilus, a highly active inactivation method, and the application of the inactivated strain in improving pulmonary hypertension.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A strain of Akkermansia myxophilus ( Akkermansia muciniphila This bacterium was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 24, 2025, with accession number CGMCC No. 33955 and strain number AKKLF. This strain exhibits excellent physiological activity; after 48 hours of culture in a medium with mucin as the sole carbon source, its OD600 value reaches 1.32, and its acetic acid production is 18.3 mmol / L. Gram staining and optical microscopy revealed no spores, consistent with the classification characteristics of Akkermansia myxophilus.

[0008] The highly active retention and inactivation method for Akkermansia myxophilus as described above includes: mixing a bacterial suspension containing Akkermansia myxophilus with a composite protective agent solution containing sulfated dextran, saccharides and cationic polypeptides, and then incubating at 40°C to 55°C for 12 min to 25 min.

[0009] In the highly active retention and inactivation method described above, preferably, the mass concentration of sulfated dextran in the composite protective agent solution is 1.5%~3.0%, the mass concentration of carbohydrate compounds is 1.0%~2.0%, and the mass concentration of cationic polypeptides is 0.05%~0.12%.

[0010] In the highly active retention and inactivation method described above, preferably, the sulfated dextran has a molecular weight of 25kDa to 50kDa and a degree of sulfation of 1.0 to 1.8; the saccharide compound is at least one of sucrose, glucose, trehalose, and lactose; and the cationic polypeptide is at least one of ε-polylysine, polylysine, and protamine.

[0011] The highly active retention inactivation method described above preferably involves a bacterial count of 1 × 10⁻⁶ in the Akkermansia myxophilus culture. 9 CFU / mL ~1×10 11 CFU / mL; the bacterial culture and the compound protective agent solution were mixed at a volume ratio of 0.6~1.4:1.

[0012] The highly active retention and inactivation method described above, preferably, involves preparing the bacterial suspension of Akkermansia myxophilus by inoculating Akkermansia myxophilus into a modified culture medium, culturing it in an anaerobic environment at a temperature of 35°C to 39°C and a CO2 concentration of 4% to 6% for 40 to 56 hours, collecting the bacterial cells by centrifugation, and resuspending it in sterile physiological saline to obtain the bacterial suspension.

[0013] Furthermore, the modified culture medium is formulated as follows: per 1L of distilled water, there are 8.0g~12.0g of casein peptone, 8.0g~12.0g of beef extract, 4.0g~6.0g of yeast powder, 4.0g~6.0g of glucose, 4.0g~6.0g of sodium acetate, 1.5g~2.5g of diammonium citrate, 0.8g~1.2g of Tween-80, 1.5g~2.5g of K2HPO4, 0.15g~0.25g of MgSO4·7H2O, 0.04g~0.06g of MnSO4·H2O, and 18.0g~22.0g of CaCO3, with the pH value adjusted to 6.5~7.2.

[0014] In the highly active retention and inactivation method described above, preferably, the composite protectant may also contain 0.5%~1.5% glycerol or 0.3%~0.8% mannitol as an auxiliary protectant.

[0015] In the high-activity retention inactivation method described above, preferably, the holding temperature is 42℃~52℃, and the corresponding holding time is 14min~22min; more preferably, the holding temperature is 45℃ for 18min-22min, or the holding temperature is 50℃ for 14min-18min.

[0016] The use of Akkermansia myxophilus obtained as described above or by the above-described high-activity retention inactivation method in the preparation of products for the prevention and / or treatment of pulmonary hypertension.

[0017] Furthermore, the *Ackermania* species is used to prepare products with the following applications:

[0018] 1) Used to downregulate serum inflammatory factor levels;

[0019] 2) Used to improve right ventricular systolic pressure, mean pulmonary artery pressure, and right ventricular hypertrophy index in pulmonary hypertension model animals.

[0020] The dosage forms of the products mentioned above are injections and oral preparations.

[0021] Furthermore, the product contains 5 × 10⁻⁶ inactivated Akkermansia muciniphila. 8 CFU / unit dose ~5×10 9 CFU / unit dose, which can be mixed with pharmaceutically acceptable excipients to prepare a product; said excipients include at least one of fillers, stabilizers, pH adjusters, and suspending agents.

[0022] Furthermore, the injectable preparation is an intravenous injection or an intraperitoneal injection; the oral preparation is a tablet, capsule, powder, or suspension; the enteric-coated preparation is an enteric-coated tablet or an enteric-coated capsule; wherein the pH value of the injectable preparation is adjusted to 6.8-7.4, and 0.1%-0.5% of a gastric-soluble coating material may be added to the oral preparation.

[0023] The application of inactivated Akkermansia myxophilus in improving pulmonary hypertension, as described in this invention, relies on highly active substances (P9 protein, a15:0-i15:0PE phospholipids) retained through a highly active retention inactivation method, which work synergistically via a dual gut-lung axis pathway.

[0024] 1. Immune regulatory pathway: P9 protein can bind to Toll-like receptor 4 (TLR4) on the surface of intestinal mucosa, activate intestinal macrophages to differentiate into the M2 anti-inflammatory phenotype, reduce the release of pro-inflammatory factors TNF-α and IL-6, and reduce the damage of systemic inflammatory response to pulmonary vessels;

[0025] 2. Pulmonary vascular protection pathway: a15:0-i15:0PE Phospholipids can be absorbed through the intestines and enter the bloodstream, targeting and binding to phospholipid receptors on the surface of pulmonary vascular endothelial cells, inhibiting endothelial-mesenchymal transition (EndMT), reducing the proliferation of pulmonary vascular smooth muscle cells, and delaying pulmonary vascular remodeling.

[0026] The beneficial effects of this invention are as follows:

[0027] The present invention provides a strain of Akkermansia myxophilus, numbered CGMCC No. 33955, which has stable activity. When cultured for 48 hours with myxin as the sole carbon source, the OD600 value and acetic acid production show better carbon source utilization efficiency and metabolic activity than ordinary Akkermansia myxophilus.

[0028] The inactivation method provided by this invention uses a composite protectant for gentle treatment, which not only achieves the required bacterial inactivation but also retains key components such as P9 protein. It is more resistant to degradation than pasteurization and superior to sterilization methods without protectants. The protectant residue is compliant with regulations, which is conducive to industrialization.

[0029] The inactivated Akkermansia strain provided by this invention has great potential for treating pulmonary hypertension. Animal experiments have shown that it can regulate inflammatory factors, improve hemodynamics, and has no risk of liver damage. It is safe and has clinical translational value. Attached Figure Description

[0030] Figure 1 The colony morphology of Akkermansia myxophilus strain AKKLF in a modified petri dish;

[0031] Figure 2An optical microscope image of Akkermansia myxophilus strain AKKLF;

[0032] Figure 3 This is a growth curve of Akkermansia myxophilus strain AKKLF in a medium with mucin as the sole carbon source. Detailed Implementation

[0033] This invention provides a strain of Akkermansia myxophilus with accession number CGMCC No. 33955; when cultured in a medium with myxophilus as the sole carbon source at 37℃±2℃ for 48h, the strain has an OD600 value of 1.32±0.05 and an acetic acid yield of 18.3±0.5mmol / L.

[0034] This invention also provides a highly active retention and inactivation method for *Akkermansia muciniphila*, the core of which is the synergistic effect of a composite protective agent and a mild temperature to achieve a balance between efficient sterilization and high retention of active ingredients, especially in the retention of P9 protein, which is significantly superior to conventional pasteurization methods. The method specifically includes the following steps:

[0035] (1) Preparation of bacterial culture: Akkermansia myxophilus strain was inoculated into a modified medium and cultured in an anaerobic environment at a temperature range of 35℃~39℃ and a CO2 concentration of 4%~6% for 40h~56h. After centrifugation, the bacterial cells were collected and resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL ~1×10¹¹ CFU / mL;

[0036] (2) Preparation of composite protective agent: The composite protective agent contains 1.5%~3.0% 25kDa~50kDa sulfated dextran (sulfation degree 1.0~1.8), 1.0%~2.0% carbohydrate compounds, 0.05%~0.12% cationic polypeptides, and the remainder is sterile physiological saline; after mixing the components, stir for 20min~40min until completely dissolved, filter through a 0.22μm filter membrane for sterilization, and set aside for use;

[0037] (3) Mixed inactivation treatment: The bacterial solution prepared in step (1) is mixed with the composite protective agent in step (2) at a volume ratio of (0.6~1.4):1, and placed in a constant temperature water bath or a programmable temperature control device. The mixture is kept at 40℃~55℃ for 12min~25min, and the temperature fluctuation during the holding process is controlled within ±1.0℃.

[0038] (4) Post-inactivation treatment: After the heat preservation is completed, the mixture is rapidly cooled to 2℃~8℃ and can be used directly or freeze-dried (pre-frozen at -40℃ to -80℃ for 2h~6h, freeze-dried at a vacuum of 0.1mbar-0.5mbar for 12h~24h) to make dry powder; after freeze-drying, the activity test results are as follows: the P9 protein retention rate is 85.2%±2.3%, the a15:0-i15:0PE phospholipid retention rate is 80.1%±2.5%, and the viable bacteria residue rate is ≤1×10⁻ 8 .

[0039] After treatment by the inactivation method, the viable bacterial survival rate is ≤1×10⁻ 8 P9 protein retention rate ≥85%, a15:0-i15:0PE phospholipid retention rate ≥80%.

[0040] In the inactivation method described above, preferably, the sulfated dextran in step (2) is 30kDa to 40kDa, with a sulfate degree of 1.2 to 1.6, more preferably 35kDa and a sulfate degree of 1.4; the sugar compound is at least one of sucrose, glucose, trehalose, and lactose, preferably sucrose; the cationic polypeptide is at least one of ε-polylysine, polylysine, and protamine, preferably ε-polylysine with a purity ≥95%.

[0041] In the inactivation method described above, preferably, the modified culture medium in step (1) has the following formula: per 1L of distilled water, there are 8.0g~12.0g of casein peptone, 8.0g~12.0g of beef extract, 4.0g~6.0g of yeast powder, 4.0g~6.0g of glucose, 4.0g~6.0g of sodium acetate, 1.5g~2.5g of diammonium citrate, 0.8g~1.2g of Tween-80, 1.5g~2.5g of K2HPO4, 0.15g~0.25g of MgSO4·7H2O, 0.04g~0.06g of MnSO4·H2O, and 18.0g~22.0g of CaCO3, with the pH value adjusted to 6.5~7.2.

[0042] In the inactivation method described above, preferably, the heat preservation temperature in step (3) is 42℃~52℃, and the corresponding heat preservation time is 14min~22min; more preferably, the heat preservation temperature is 45℃ for 18min~22min, or the heat preservation temperature is 50℃ for 14min~18min.

[0043] In the inactivation method described above, preferably, the composite protectant in step (2) may also contain 0.5%~1.5% glycerol or 0.3%~0.8% mannitol as an auxiliary protectant to further improve the retention rate of P9 protein and a15:0-i15:0PE phospholipids, so that the retention rate of P9 protein is ≥87% and the retention rate of a15:0-i15:0PE phospholipids is ≥81%.

[0044] The present invention preferably employs sulfated dextran, sucrose, and ε-polylysine in the composite protective agent. This combination can minimize P9 protein denaturation while protecting a15:0-i15:0PE phospholipids from oxidative degradation; wherein sulfated dextran can insulate the thermal environment through dense encapsulation, ε-polylysine can synergistically improve sterilization efficiency and inhibit phospholipid aggregation, and sucrose further stabilizes the protein conformation by regulating osmotic pressure.

[0045] Furthermore, in a preferred embodiment, the specific conditions of the inactivation method are as follows:

[0046] Method A: Mix the bacterial culture with the composite protectant at a volume ratio of 1:1 (the composite protectant contains 2.0% 35kDa sulfated dextran, 1.5% sucrose, and 0.08% ε-polylysine), and incubate in a 45℃ constant temperature water bath for 20 minutes, with a temperature control accuracy of ±0.5℃;

[0047] Method B: Mix the bacterial culture with the composite protectant at a volume ratio of 1:1 (the composite protectant contains 2.5% 35kDa sulfated dextran, 1.5% sucrose, and 0.08% ε-polylysine), and incubate in a 50℃ constant temperature water bath for 16 minutes, with a temperature control accuracy of ±0.5℃;

[0048] Both methods require strict control of temperature fluctuations to no more than ±0.5℃ to avoid sudden temperature changes that could lead to degradation of active ingredients and ensure the stability of sterilization effect and activity retention rate.

[0049] The effects of the two methods described above: After treatment with methods A and B, the viable bacterial survival rate was ≤1×10⁻ 8 The sterilization effect is comparable to that of pasteurization. The retention rates of the key active ingredient P9 protein are 87.8%±2.7% (Method A) and 87.3%±2.9% (Method B), respectively, which are much higher than the 42.3%±3.5% of pasteurization, representing an increase of more than 100%. The retention rates of a15:0-i15:0PE phospholipids are 83.2%±3.0% (Method A) and 81.5%±3.1% (Method B), respectively, effectively solving the technical problem of low retention rate of core active ingredients in conventional inactivation methods.

[0050] In a preferred embodiment, in an MCT (limonene)-induced pulmonary hypertension rat model, after intervention with inactivated strains prepared by methods A and B:

[0051] Method A: The right ventricular systolic pressure (RVSP) of rats reached 28.92±2.71 mmHg, and the mean pulmonary artery pressure (mPAP) reached 0.32±0.04 kPa;

[0052] Method B: The rats' RVSP reached 28.26±2.76 mmHg, and mPAP reached 0.30±0.04 kPa;

[0053] In the model group, the RVSP of rats was 40.30±4.33 mmHg and the mPAP was 0.55±0.10 kPa. The improvement in hemodynamic parameters in both groups A and B was similar to that of sildenafil (RVSP 27.20±1.34 mmHg, mPAP 0.29±0.06 kPa), a commonly used clinical drug. During the intervention, the serum ALT and AST levels of rats remained within the normal range (25.2~25.6 U / L, 42.7~43.0 U / L), with no adverse reactions of liver damage, and the safety was better than that of traditional drugs.

[0054] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0055] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and unless otherwise specified, all reagents used in the embodiments are analytical grade or higher.

[0056] Example 1: Isolation, identification, and preservation of Akkermansia myxophilus CGMCC No. 33955 (strain AKKLF)

[0057] 1. Separation steps:

[0058] Take 0.5g of stool sample from a healthy volunteer (approved through ethical review, with informed consent signed by the volunteer), and serially dilute it 10-fold with sterile saline to a concentration of 10⁻⁻⁶. 6 ~10⁻ 8 (That is, each 1 mL of diluted solution contains 0.5 g × 10⁻ of the original fecal sample) 6 ~0.5g×10⁻ 8Within the specified concentration range, 100 μL of the diluted solution was spread onto modified PYG medium and placed in an anaerobic incubator (85% N2, 10% H2, 5% CO2) for static incubation at 37°C for 48 h. After incubation, single colonies with a diameter of 1-2 mm, exhibiting a milky white color, round shape, and well-defined edges were selected (colony morphology as shown in the image). Figure 1 As shown in the figure, pure strains were obtained by streaking three times on LB solid medium. A total of 5 candidate strains of Akkermansia myxophilus (numbered AKK1~AKK5) were isolated. AKK3 (i.e. AKKLF) was selected by mucin utilization capacity test (OD600 value) and acetic acid production determination. Its OD600 value (1.35) and acetic acid production (18.8 mmol / L) were significantly higher than the other 4 strains (AKK1: OD600=0.91, acetic acid=11.2 mmol / L; AKK2: OD600=0.87, acetic acid=10.5 mmol / L; AKK4: OD600=0.93, acetic acid=11.8 mmol / L; AKK5: OD600=0.89, acetic acid=10.9 mmol / L).

[0059] The modified PYG medium formula is as follows (per 1L of distilled water): 10.0g casein peptone, 10.0g beef extract, 5.0g yeast powder, 5.0g glucose, 5.0g sodium acetate, 2.0g diammonium citrate, 1.0g Tween-80, 2.0g K2HPO4, 0.2g MgSO4·7H2O, 0.05g MnSO4·H2O, 20.0g CaCO3, and 15.0g agar; adjust the pH to 6.8±0.2, sterilize by moist heat at 121℃ for 15min, cool to 50℃, and then pour into plates for later use.

[0060] 2. Identification steps:

[0061] (1) Molecular identification:

[0062]

[0063] (2) Physiological and biochemical identification:

[0064] Growth curve determination: Strain strain AKKLF was inoculated into a medium containing only mucin as the carbon source and cultured anaerobically at 37°C with shaking (150 rpm). Samples were taken at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h, and the OD600 values ​​were measured using a Shimadzu UV-2600 microscope to plot the growth curve. Figure 3 As shown, the OD600 value reached 1.32 at 48h and entered a stable period after 72h (OD600 value 1.42±0.04).

[0065] The formulation of the culture medium containing the sole carbon source of mucin is as follows: per 1L of distilled water, there are 20.0g of mucin, 1.0g of NH4Cl, 1.5g of K2HPO4, 0.2g of MgSO4・7H2O, and 0.05g of CaCl2, with the pH value adjusted to 7.0±0.2.

[0066] Acetic acid yield determination: The bacterial culture after 48 h was centrifuged at 4000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter and analyzed using an Agilent 7890A gas chromatograph. Chromatographic conditions: HP-FFAP capillary column (30 m × 0.32 mm × 0.25 μm), initial column temperature 60 °C (2 min), increased to 180 °C (5 min) at 10 °C / min; injection port 200 °C, FID detector 250 °C; N2 carrier gas (1.0 mL / min), injection volume 1 μL, split ratio 10:1. The acetic acid yield of the strain was calculated using the external standard method (acetic acid standard 0.1–10 mmol / L) to be 18.3 ± 0.5 mmol / L.

[0067] Morphological observation: Logarithmic-phase bacterial culture was Gram-stained and observed under an Olympus BX53 optical microscope. The strains were as follows: Figure 2 As shown, it is non-spore-forming, Gram-negative, and conforms to the morphological characteristics of Akkermansia myxophilus.

[0068] 3. Preservation steps:

[0069] The strain AKKLF was inoculated into modified PYG medium and anaerobically cultured at 37℃ for 48 h. 1 mL of the bacterial culture was then mixed with 50% sterile glycerol at a 1:1 volume ratio and stored at -80℃. Simultaneously, freshly cultured bacterial colonies were inoculated onto modified solid culture slant agar and sent to the China General Microbiological Culture Collection Center (CGMCC, Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) for patent preservation. The strain was classified and named *Ackermania myxophila*. Akkermansia muciniphilaThe date of deposit is March 24, 2025, and the accession number is CGMCC No. 33955.

[0070] Example 2 Method A: Preparation of inactivated myxotrophic Akkermansia

[0071] 1. Preparation of bacterial culture:

[0072] The AKKLF strain isolated in Example 1 was inoculated into a modified PYG liquid medium (the formulation differs from Example 1 only in that it does not contain agar) and cultured anaerobically at 37°C with shaking (150 rpm) for 48 h. After the culture, the bacterial cells were collected by centrifugation at 4000 rpm for 10 min (centrifugation radius 10 cm), resuspended in 0.9% sterile physiological saline, and counted using a hemocytometer (trypan blue staining to exclude dead bacteria). The bacterial concentration was adjusted to 1 × 10¹. 0 CFU / mL, for later use.

[0073] 2. Formulation of composite protective agent:

[0074] Weigh 2.0g of 35kDa sulfated dextran (sulfation degree 1.4, Shanghai Yuanye Biotechnology Co., Ltd.), 1.5g of sucrose (analytical grade, Sinopharm Group), and 0.08g of ε-polylysine (purity ≥95%, Shandong Freda Biotechnology Co., Ltd.). Add sterile physiological saline to a final volume of 100mL. Stir magnetically for 30min (25℃) until completely dissolved. Filter through a 0.22μm filter membrane for sterilization.

[0075] 3. Mixed inactivation treatment:

[0076] Mix the bacterial suspension prepared in step 1 with the composite protectant prepared in step 2 at a volume ratio of 1:1 (total system 100mL), place in a constant temperature water bath (Shanghai Jinghong DK-S24), set the temperature to 45℃, and control the temperature fluctuation within ±0.5℃ (monitored in real time by a thermometer), and keep warm for 20min; after the incubation is completed, quickly place the mixture in an ice bath to cool to room temperature to obtain the inactivated bacterial suspension.

[0077] 4. Performance Testing:

[0078] (1) Determination of viable bacterial survival rate: The plate count method was used. 100 μL of the inactivated bacterial solution was taken and serially diluted 10-fold to 10⁻ using sterile physiological saline as the dilution medium. 8 The samples were spread on modified PYG solid medium and anaerobic incubated for 72 hours before being counted. The results were calculated using the following formula:

[0079] Viable colony survival rate = (Number of colonies after inactivation × Dilution factor / Inoculation volume) / (Number of colonies before inactivation × Dilution factor / Inoculation volume) × 100%

[0080] (2) Determination of P9 protein retention rate: ELISA was used to coat the outer membrane proteins of the bacteria before and after inactivation (specifically, 10 mL of bacterial solution before and after inactivation was collected by centrifugation at 4000 rpm for 10 min, resuspended in RIPA lysis buffer (containing protease inhibitor), lysed on ice for 30 min, centrifuged at 12000 rpm for 15 min, and the supernatant was collected as the outer membrane protein extract) onto a 96-well plate. Rabbit anti-human P9 polyclonal antibody (Abcam ab28456) was used as the primary antibody, and HRP-labeled goat anti-rabbit IgG was used as the secondary antibody for incubation. After TMB color development and sulfuric acid termination, the absorbance at 450 nm was measured using a microplate reader, and the P9 protein retention rate was calculated using the following formula:

[0081] P9 protein retention rate = (absorbance value of outer membrane protein after inactivation / absorbance value of outer membrane protein before inactivation) × 100%.

[0082] (3) Determination of the retention rate of a15:0-i15:0PE phospholipids: High performance liquid chromatography-mass spectrometry (HPLC-MS, Agilent 1290-6460) was used. The chromatographic column was ZORBAX SB-C18 (2.1×150mm, 1.8μm), the mobile phase was acetonitrile-water (90:10, containing 0.1% formic acid by volume), the flow rate was 0.3mL / min, and the mass spectrometer used ESI source (positive ion mode). The retention rate was calculated by external standard method (a15:0-i15:0PE standard, Avanti Polar Lipids). Specifically, the test sample is the phospholipid extract of the bacterial suspension before and after inactivation: Take 10 mL of bacterial suspension, add 20 mL of chloroform-methanol (2:1, v / v), vortex for 20 min, centrifuge at 4000 rpm for 10 min, collect the lower organic phase, dry it under nitrogen, redissolve it with 1 mL of mobile phase, filter through a 0.22 μm filter membrane, and then detect it. Calculate the phospholipid retention rate using the following formula: 15:0-i15:0PE Phospholipid retention rate = (phospholipid concentration after inactivation × extraction volume / total bacterial count) / (phospholipid concentration before inactivation × extraction volume / total bacterial count) × 100%, where the phospholipid concentration is calculated using a standard curve: Concentration = (sample peak area / standard peak area) × standard concentration.

[0083] (4) Determination of ε-polylysine residue: High performance liquid chromatography (Shimadzu LC-20A) was used. The chromatographic column was a Shim-pack VP-ODS (4.6×250mm, 5μm), the mobile phase was 0.1% trifluoroacetic acid-acetonitrile (95:5), the detection wavelength was 210nm, and the residue was calculated using the external standard method. The formula is as follows:

[0084] ε-polylysine residue = (sample peak area / standard peak area) × standard concentration × dilution factor.

[0085] The performance indicators of the inactivated Akkermansia myxophilus prepared by method A are shown in Table 1.

[0086] Table 1. Indicators for the preparation of inactivated Akkermansia myxophilia by Method A

[0087]

[0088] As shown in Table 1, the inactivated bacteria prepared by method A showed excellent performance in all indicators: the viable bacteria retention rate met the strict sterilization safety standards; the core active ingredients were sufficiently retained; the residual amount of ε-polylysine was far below the limit of GB2760-2024 Food Additives Use Standard, with no safety risks. The overall method has both high sterilization efficiency and activity retention capability.

[0089] Example 3 Method B Preparation of inactivated myxotrophic Akkermansia

[0090] 1. Preparation of bacterial culture:

[0091] Consistent with step 1 of Example 2, strain CGMCC No. 33955 was cultured anaerobically in modified liquid medium for 48 hours, centrifuged at 4000 rpm to collect the bacterial cells, and resuspended in physiological saline to a concentration of 1×10¹. 0 CFU / mL.

[0092] 2. Formulation of composite protective agent:

[0093] Weigh 2.5g of 35kDa sulfated dextran (sulfation degree 1.4), 1.5g of sucrose, and 0.08g of ε-polylysine. Add sterile physiological saline to a final volume of 100mL, stir to dissolve, and then filter to sterilize.

[0094] 3. Mixed inactivation treatment:

[0095] The bacterial suspension and the composite protectant were mixed at a volume ratio of 1:1, placed in a constant temperature water bath, set at 50℃, and kept at that temperature for 16 minutes. After the mixture was removed, it was cooled to room temperature in an ice bath to obtain an inactivated bacterial suspension.

[0096] 4. Performance testing: The testing method is the same as in Example 2. The indicators for preparing inactivated Akkermansia bacillus by method B are shown in Table 2.

[0097] Table 2. Indicators for the preparation of inactivated Akkermansia myxophilus by Method B

[0098] index Survival rate (%) P9 protein retention (%) a15:0-i15:0PE Phospholipid Retention Rate (%) ε-polylysine residue (mg / mL) numerical values <![CDATA[1×10⁻ 8 ]]> 85.5±2.5 80.8±2.8 0.007±0.001

[0099] As shown in Table 2, Method B achieved inactivation with a temperature of 50℃ for 16 min. Although the retention rates of P9 protein and a15:0-i15:0PE phospholipids decreased slightly compared to Method A due to the slightly higher temperature, they were still within the high retention range, and the viable cell retention rate also met the high-efficiency sterilization standard. The residual amount of ε-polylysine was far below the safety limit, ensuring both efficiency and safety.

[0100] Comparative Example 1: Preparation of inactivated Akkermansia myxophilia by pasteurization

[0101] 1. Preparation of bacterial culture:

[0102] Consistent with step 1 of Example 2, the bacterial concentration was adjusted to 1×10¹. 0 CFU / mL.

[0103] 2. Inactivation treatment:

[0104] Without adding any preservatives, take 100 mL of bacterial culture directly and place it in a 65℃ constant temperature water bath for 30 min (standard pasteurization parameters), and then cool it in an ice bath after the process.

[0105] 3. Performance testing: The testing method is the same as in Example 2. The indicators for preparing inactivated Akkermansia myxophilus by this method are shown in Table 3.

[0106] Table 3. Indicators for preparing inactivated Akkermansia bacilli using pasteurization method

[0107]

[0108] As shown in Table 3, this method involves maintaining the temperature in a 65℃ constant water bath for 30 minutes without adding any preservatives. While the viable bacteria retention rate is comparable to methods A and B, meeting basic sterilization requirements, the retention rate of core active ingredients is significantly lower. This is mainly due to the high temperature directly damaging the protein's spatial structure and triggering thermal oxidation and degradation of phospholipids. Furthermore, despite the absence of preservative residues, the large-scale inactivation of active ingredients fails to support the improvement effect on pulmonary hypertension.

[0109] Comparative Example 2: Preparation of inactivated Akkermansia myxophilus by a method without protective agent at 45°C

[0110] 1. Preparation of bacterial culture:

[0111] Consistent with step 1 of Example 2, the bacterial concentration is 1×10¹ 0 CFU / mL.

[0112] 2. Inactivation treatment:

[0113] Dilute the bacterial solution by 1:1 with 0.9% sterile saline (without compound protective agent), place it in a 45℃ constant temperature water bath, keep it warm for 20 minutes (same temperature and time as in Method A), and then cool it in an ice bath.

[0114] 3. Performance testing: The testing method is the same as in Example 2. The indicators for obtaining inactivated Akkermansia myxophilus are shown in Table 4.

[0115] Table 4. Indicators for preparing inactivated Akkermansia myxophilus by the 45℃ method without protective agent

[0116] index Survival rate (%) P9 protein retention (%) a15:0-i15:0PE Phospholipid Retention Rate (%) ε-Polylysine residue (mg / mL) numerical values <![CDATA[1×10⁻ 4 ]]> 68.5±3.2 65.2±3.8 0

[0117] As shown in Table 4, this method uses the same temperature (45℃) and incubation time (20 min) as Method A, the difference being the absence of a composite preservative. Tests showed that the viable cell survival rate was 10% of that of Method A. 4 The sterilization efficiency is severely insufficient due to the lack of synergistic sterilization effect of ε-polylysine; although the retention rate of P9 protein and a15:0-i15:0PE phospholipids is higher than that of pasteurization, it is lower than that of method A. This is because there is no encapsulation stabilizing effect of sulfated dextran. The protein and phospholipids are easily denatured and degraded by temperature, and there is no protective agent residue. Due to the dual defects of sterilization and preservation, it is still difficult to use in practice.

[0118] Comparative Example 3: Preparation of inactivated Akkermansia muciniphilis using a method without protective agent at 50°C

[0119] 1. Preparation of bacterial culture:

[0120] Consistent with step 1 of Example 2, the bacterial concentration is 1×10¹ 0 CFU / mL.

[0121] 2. Inactivation treatment:

[0122] Dilute the bacterial solution 1-fold with 0.9% sterile saline (without composite protectant), place it in a 50℃ constant temperature water bath, and keep it at that temperature for 16 minutes (same temperature and time as in Method B). After that, cool it in an ice bath.

[0123] 3. Performance Testing:

[0124] The testing method was the same as in Example 2, and the indicators for obtaining inactivated Akkermansia myxophilus are shown in Table 5.

[0125] Table 5. Indicators for preparing inactivated Akkermansia myxophilus by the 50℃ method without protective agent

[0126] index Survival rate (%) P9 protein retention (%) a15:0-i15:0PE Phospholipid Retention Rate (%) ε-Polylysine residue (mg / mL) numerical values <![CDATA[1×10⁻ 5 ]]> 61.8±3.4 58.7±4.0 0

[0127] As shown in Table 5, this method follows the temperature of 50 °C and the heat preservation time of 16 min of Method B, and no composite protective agent is added. The results show that the sterilization efficiency is slightly improved, but the effect of retaining activity deteriorates. Affected by the increase in temperature, the viable bacteria survival rate is lower than that of Comparative Example 2, but the comprehensive performance is still inferior to Method B.

[0128] Example 4 Verification of animal experiments on the improvement of pulmonary hypertension by inactivated strains prepared by Methods A and B

[0129] 1. Experimental animals and grouping:

[0130] Sixty SPF-grade male SD rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number SCXK (Beijing) 2024-0001), weighing 200-220 g, were adaptively fed for 1 week (temperature 22±2 °C, humidity 50±5%, 12 h light-dark cycle, free access to food and water). The rats were randomly divided into 8 groups, with 8 rats in each group, and were respectively designated as the normal group, model group, sildenafil group, Method A group, Method B group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group.

[0131] 2. Establishment of pulmonary hypertension model:

[0132] Except for the normal group, the rats in the remaining groups were subcutaneously injected with MCT (Sigma-Aldrich, purity ≥98%) once in the neck, with a dose of 60 mg / kg (MCT was dissolved in sterile saline, concentration 20 mg / mL); the rats in the normal group were injected with an equal volume of sterile saline.

[0133] 3. Intervention plan:

[0134] Normal group, model group: From the day of model establishment, ddH2O was administered by gavage every day, with a dose of 10 mL / kg, for 28 consecutive days;

[0135] Sildenafil group: From the 7th day of model establishment, sildenafil (Pfizer Pharmaceuticals, dissolved in saline, concentration 1 mg / mL) was intraperitoneally injected every day, with a dose of 10 mg / kg, for 21 consecutive days;

[0136] Method A group, Method B group: From the 7th day of model establishment, the inactivated strain suspension prepared by the corresponding method was injected into the tail vein every day (bacterial concentration 1×10¹ 0 CFU / mL), with a dose of 0.2 mL / rat (i.e., 2×10 9 CFU / rat), for 21 consecutive days;

[0137] Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group: From the 7th day of model establishment, the inactivated strain suspension prepared by the corresponding method was injected into the tail vein every day, with the same dose as the Method A group, for 21 consecutive days.

[0138] 4. Detection indicators and methods:

[0139] (1) Measurement of hemodynamic parameters: On day 28 of modeling, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL / kg), the right external jugular vein was separated, a catheter was inserted into the right ventricle, and a pressure sensor (PowerLab 8 / 35, ADInstruments) was connected to record the right ventricular systolic pressure (RVSP); the left common carotid artery was separated, a catheter was inserted into the pulmonary artery, and the mean pulmonary artery pressure (mPAP) was recorded.

[0140] (2) Right ventricular hypertrophy index (RVHI) determination: After hemodynamic testing, the rats were sacrificed, the heart was separated, the atria and great vessels were cut off, the weight of the right ventricle (RV) and the weight of the left ventricle + interventricular septum (LV+S) were weighed, and RVHI=RV / (LV+S) was calculated.

[0141] (3) Serum inflammatory factor determination: Blood was collected from the abdominal aorta and centrifuged at 3000 rpm for 15 min to separate the serum. The levels of TNF-α and IL-6 were detected using an ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.). The operation was strictly performed in accordance with the kit instructions.

[0142] (4) Measurement of liver injury indicators: Serum ALT and AST levels were measured using a fully automated biochemical analyzer.

[0143] 5. Experimental Results:

[0144] (1) Hemodynamic and right ventricular hypertrophy indices (see Table 6):

[0145] Table 6 Comparison of hemodynamic and right ventricular hypertrophy indices among rats in each group (x̄±s, n=8)

[0146] Group Right ventricular systolic pressure (RVSP, mmHg) Mean pulmonary artery pressure (mPAP, kPa) Right ventricular hypertrophy index (RVHI) normal group 23.44±1.45 0.24±0.02 0.24±0.02 Model group 40.30±4.33*** 0.55±0.10*** 0.55±0.06*** Sildenafil group 27.20±1.34**# 0.29±0.06**# 0.29±0.05**# Method A 28.92±2.71**# 0.30±0.04**# 0.30±0.03**# Method B group 28.26±2.76**# 0.32±0.04**# 0.31±0.04**# Comparative Example 1 38.85±3.12* 0.50±0.08* 0.51±0.05* Comparative Example 2 36.52±3.47* 0.47±0.09* 0.48±0.06* Comparative Example 3 37.64±3.29* 0.48±0.08* 0.49±0.05*

[0147] Note: In the table, *** indicates a highly significant difference compared to the normal group (P<0.001); ** indicates a highly significant difference compared to the model group (P<0.01); * indicates a significant difference compared to the model group (P<0.05); and # indicates no statistically significant difference compared to the sildenafil group (P>0.05).

[0148] As shown in Table 6, the RVSP, mPAP, and RVHI of groups A and B were significantly lower than those of the model group (P<0.01), and there was no significant difference compared with the sildenafil group (P>0.05). Although the indicators of comparative groups 1 and 2 were lower than those of the model group, they were significantly higher than those of groups A and B (P<0.05), indicating that the inactivated strains prepared by methods A and B had a better effect on improving pulmonary hypertension.

[0149] (2) Serum inflammatory factor levels (see Table 7):

[0150] Table 7 Comparison of serum TNF-α and IL-6 levels in rats of different groups (x̄±s, n=8, pg / mL)

[0151] Group TNF-α (pg / mL) IL-6 (pg / mL) normal group 15.2±2.3 12.5±1.8 Model group 68.7±5.9*** 56.3±4.7*** Sildenafil group 32.4±3.5**# 28.6±3.1**# Method A 33.6±4.1**# 292±3.4**# Method B group 35.8±3.8**# 30.1±3.2**# Comparative Example 1 56.3±4.8* 49.7±4.2* Comparative Example 2 51.6±5.2* 45.9±4.5* Comparative Example 3 53.8±5.0* 46.9±4.3*

[0152] Note: In the table, ***: compared with the normal group, P<0.001; **: compared with the model group, P<0.01; *: compared with the model group, P<0.05; #: compared with the sildenafil group, P>0.05.

[0153] As shown in Table 7, the serum TNF-α and IL-6 levels in groups A and B were significantly lower than those in the model group (P<0.01) and similar to those in the sildenafil group (P>0.05). Although the levels of inflammatory factors in comparative groups 1, 2, and 3 decreased, they were significantly higher than those in groups A and B (P<0.05), indicating that the inactivated strains prepared by methods A and B had more significant anti-inflammatory effects.

[0154] (3) Liver injury indicators (see Table 8):

[0155] Table 8 Comparison of serum ALT and AST levels in rats of different groups (x̄±s, n=8, U / L)

[0156] Group ALT (U / L) AST (U / L) normal group 25.3±3.1 42.5±4.2 Model group 26.1±3.5# 43.8±4.5# Sildenafil group 31.8±3.7* 44.2±2.3# Method A 25.2±3.3# 42.7±4.4# Method B group 25.6±3.4# 43.0±3.2# Comparative Example 1 26.0±3.6# 43.2±4.6# Comparative Example 2 25.8±3.2# 43.1±4.3# Comparative Example 3 25.9±2.7# 43.7±3.8#

[0157] Note: In the table, * indicates P < 0.05 compared with the normal group; # indicates P > 0.05 compared with the normal group.

[0158] As shown in Table 8, the serum ALT level in the sildenafil group was significantly higher than that in the normal group (P<0.05), indicating a potential risk of liver damage. The ALT and AST levels in methods A and B were not significantly different from those in the normal group (P>0.05), indicating that the inactivated Akkermansia myxophilus prepared by methods A and B of this invention is safer than sildenafil.

[0159] This invention provides Akkermansia myxophilus (CGMCC No. 33955), which has an OD600 value of 1.32 and an acetic acid production of 18.3 mmol / L when cultured in a medium with mucin as the sole carbon source for 48 h, exhibiting excellent physiological activity.

[0160] The two methods developed in this invention, Method A (45℃×20min, 2.0% 35kDa sulfated dextran composite protectant) and Method B (50℃×16min, 2.5% 35kDa sulfated dextran composite protectant), can achieve a viable bacteria residue rate of ≤1×10⁻. 8 The P9 protein retention rate was 87.3%-87.8%, and the a15:0-i15:0PE phospholipid retention rate was 81.5%-83.2%, which were significantly better than pasteurization and non-protective inactivation methods.

[0161] In an MCT-induced PAH rat model, this inactivated strain showed RVSP and mPAP levels close to those of sildenafil after intervention, with no liver damage, and significantly reduced serum TNF-α and IL-6. This invention addresses the problems of significant side effects and low retention of activity of conventionally inactivated proteins in existing PAH intervention drugs, achieving stable efficacy, high safety, and ease of industrialization, thus possessing significant clinical and industrial value.

Claims

1. A high activity retention inactivation method of Akkermansia muciniphila, characterized in that, It comprises: Mixing the Akkermansia muciniphila bacterial solution with the complex protective agent solution containing sulfated dextran, saccharide compound and cationic polypeptide, and then incubating at 40-55℃ for 12-25 min, and then rapidly cooling the mixture to room temperature in ice bath; The preservation number of the Akkermansia muciniphila is CGMCC No. 33955; The mass concentration of the sulfated dextran in the complex protective agent solution is 1.5-3.0%, the mass concentration of the saccharide compound is 1.0-2.0%, and the mass concentration of the cationic polypeptide is 0.05-0.12%; The molecular weight of the sulfated dextran is 35 kDa, and the sulfation degree is 1.4; the saccharide compound is sucrose, and the cationic polypeptide is ε-polylysine; The bacteria content of the Akkermansia muciniphila bacterial solution was 1 x 10 9 CFU / mL ~1x10 11 CFU / mL; the bacterial solution was mixed with the composite protective agent solution at a volume ratio of 1:

1.

2. The high activity retention inactivation method of claim 1, wherein, The preparation method of the Akkermansia muciniphila bacterial solution comprises inoculating Akkermansia muciniphila into a modified culture medium, and culturing at 35-39℃ in an anaerobic environment with a CO2 concentration of 4-6% for 40-56 h, then centrifuging to collect the bacterial cells, resuspending with sterile normal saline to obtain the bacterial solution.

3. The high activity retention inactivation method of claim 1, wherein, The complex protective agent further adds 0.5-1.5% glycerol or 0.3-0.8% mannitol as an auxiliary protective agent.

4. The application of the Akkermansia muciniphila obtained by the high-activity-retained inactivation method of any one of claims 1-3 in the preparation of a product for preventing or treating pulmonary arterial hypertension.

5. The use according to claim 4, wherein the compound is ###0002### The application comprises: 1) preparing a product for down-regulating the level of inflammatory factors in serum; 2) preparing a product for improving the right ventricular systolic pressure, mean pulmonary arterial pressure and right ventricular hypertrophy index of a pulmonary arterial hypertension model animal.

6. The use according to claim 4, wherein the compound is ###0002### The dosage form of the product is injection, oral preparation.

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