Luciphil Ackermann strain and application thereof in preparation of anti-weakness or healthy life prolonging product

Abstract

The invention discloses an ackermania muciniphila strain and application of the ackermania muciniphila strain in preparation of a product for resisting weakness or prolonging healthy life, and particularly relates to the technical field of microorganisms, the ackermania muciniphila strain is preserved in China General Microbiological Culture Collection Center (CCTCC), the preservation number is CCTCC NO: 46390, the preservation date is April 15, 2025, and the preservation number is CCTCC NO: 46390. The preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing. The application refers to the application in preparation of anti-weakening products and health life prolonging products. The ackermansiella muciniphila has the effects of resisting weakness and prolonging the healthy life, has the application potential in preparation of products for relieving weakness and prolonging the healthy life, is low in preparation cost, high in preparation efficiency and small in side effect, and has a good industrial application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a strain of Akkermansia muciniphila and its application in preparing products for resisting frailty and prolonging healthy lifespan. Background Art

[0002] Authoritative research indicates that the main risk factors for disability in the elderly include geriatric syndromes such as frailty and various chronic diseases. This frail state, characterized by a decline in physiological functional reserve, has attracted considerable attention due to its intervention potential. Because frailty can be prevented and controlled through targeted measures, timely identification of high-risk groups and implementation of effective interventions can not only slow the progression of disease and reduce the incidence of disability, but also become a key focus for extending the healthy lifespan of the population. Effective frailty intervention programs have important public health value for improving the quality of life of the elderly and extending the healthy lifespan of the population.

[0003] Different from simply extending lifespan, extending healthy lifespan specifically refers to the time spent in perfect health, rather than simply increasing total lifespan. Its core goal is to maintain physical function, cognitive ability, and the ability to care for oneself in old age, minimizing the decline in quality of life caused by chronic disease or disability.

[0004] Akkermansia belongs to the phylum Verrucomicrobia. Akkermansia muciniphila, a member of this genus, is a core commensal in the human gut. It stimulates mucin synthesis by degrading mucins, playing a crucial role in maintaining intestinal barrier function and improving lipid metabolism. Numerous studies have revealed that Akkermansia muciniphila is closely linked to human metabolic health and possesses anti-anxiety and anti-metabolic properties, such as diabetes and hyperlipidemia.

[0005] Currently, there are no reports showing that Akkermansia muciniphila has direct anti-frailty and healthy lifespan-extending effects. Summary of the Invention

[0006] In order to overcome the above-mentioned defects of the prior art, the present invention provides a strain of Akkermansia muciniphila and its use in preparing anti-frailty and healthy life-extending products, so as to solve the problems raised in the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions: On the one hand, the present invention discloses a strain of Akkermansia muciniphila, which is deposited in the General Microbiology Center of the China Culture Collection Administration Committee, with a deposit number of CCTCC NO: 46390, a deposit date of April 15, 2025, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Akkermansia muciniphila CCTCC NO: 46390 comprises a 16S sequence consisting of the nucleotide sequence of SEQ ID NO: 1.

[0008] The above-mentioned Akkermansia muciniphila is obtained as follows: By preparing a fecal suspension from fresh fecal samples of 14-year-old adolescents, isolating the bacteria in the fecal suspension, and finally selecting and identifying the isolated Akkermansia muciniphila, the obtained strain was identified as a new human Akkermansia muciniphila, which was named Akkermansia muciniphila P02H01.

[0009] In another aspect, the present invention provides a pharmaceutical preparation comprising a fermentation mixture, fermentation broth precipitate, fermentation supernatant, live bacteria, dead bacteria, lyophilized powder or lysate of Akkermansia muciniphila.

[0010] Specifically, the preparation method of the above-mentioned live bacteria includes the following steps: S1: Inoculate Akkermansia muciniphila CCTCC NO: 46390 and culture anaerobically in liquid culture medium to obtain viable bacteria; S2: After centrifugation, the precipitate is washed with physiological saline, centrifuged again, and precipitated again to obtain the final precipitate; S3: resuspend the final precipitate in physiological saline or liquid culture medium containing 25% glycerol by volume to prepare a bacterial liquid preservation solution, which is stored in a deep cryogenic environment at -80 degrees Celsius; S4: Anaerobically culturing the prepared bacterial liquid preservation solution in a solid culture medium, and counting the number of viable bacteria in the preservation solution.

[0011] Preferably, the components of the solid culture medium are 3.7 g / L brain heart infusion broth powder, 0.05 g / L L-cysteine, 1 g / L agar powder, and 0.05 g / L sterile mucin; the components of the liquid culture medium are 37 g / L brain heart infusion broth powder, 0.5 g / L L-cysteine, and 0.5 g / L sterile mucin.

[0012] In another aspect, the present invention further discloses the use of the above-mentioned Akkermansia muciniphila CCTCC NO: 46390 in the preparation of foods, medicines, health products, bacterial agents, food additives, and health product additives.

[0013] The present invention also discloses the use of the Akkermansia muciniphila CCTCC NO: 46390 in the preparation of anti-asthenia products.

[0014] The present invention also discloses the use of the Akkermansia muciniphila CCTCC NO: 46390 in preparing a product for prolonging healthy lifespan.

[0015] The Akkermansia muciniphila P02H01 of the present invention has the effects of resisting frailty and prolonging healthy life span, and has the potential for application in preparing products for alleviating frailty and prolonging healthy life span.

[0016] Microbial Deposit Information Strain name: Akkermansia muciniphila P02H01 Storage time: April 15, 2025 Depository: General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Deposit number: CCTCC NO:46390 BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a growth curve diagram of three strains of Akkermansia muciniphila isolated from three real stool samples of the present invention; Figure 2 This is a graph showing the anti-frailty effect of Akkermansia muciniphila P02H01 of the present invention on an elderly frail mouse model; Figure 3 This is a graph showing the experimental results of the effect of Akkermansia muciniphila P02H01 of the present invention on the age-related frailty in elderly mice; Figure 4 This is a graph showing the experimental results of the present invention on the effect of age on the occurrence of renal function decline in elderly mice using Akkermansia muciniphila P02H01; Figure 5 This is a graph showing the experimental results of the present invention on the effect of age on liver function decline in elderly mice using Akkermansia muciniphila P02H01; Figure 6 This is a graph showing the experimental results of the effect of Akkermansia muciniphila P02H01 on the healthy lifespan of mice; Figure 7This is a graph showing the experimental results of the effect of Akkermansia muciniphila P02H01 on the survival time of elderly mice. DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0019] In the following examples, the experimental methods without specific conditions were carried out according to conventional methods and conditions, or according to the product specifications. All reagents used in the present invention are commercially available and their sources are not specifically limited. The sources of some reagents are shown in Table 1 below.

[0020] Table 1: Reagent sources Reagents Manufacturer / Company Item No. Brain Heart Infusion Broth Powder Huankai Microbiology Technology Co., Ltd. 024053 L-Cysteine Sangon Bioengineering (Shanghai) Co., Ltd. A422882-0001 L-Arginine hydrochloride Sangon Bioengineering (Shanghai) Co., Ltd. A100877-0100 agar powder Sangon Bioengineering (Shanghai) Co., Ltd. A505255 mucin MedChemExpress HY-B2196 Polymyxin B MedChemExpress HY-K1044 Pepsin MedChemExpress HY-P1635 pancreatic enzymes Sangon Bioengineering (Shanghai) Co., Ltd. A430529-0010 Anaerobic gas production bags Shanghai Baimeng / Shibo Biotechnology C-11 Anaerobic culture bags Shanghai Baimeng / Shibo Biotechnology C-41 Creatinine test kit Sangon Bioengineering (Shanghai) Co., Ltd. D799853-0096 Mouse Albumin ELISA Kit Sangon Bioengineering (Shanghai) Co., Ltd. D721206-0096 Example 1: Isolation and cultivation of strains 1.1 Preparation of solid culture medium for separation Mucin-brain heart infusion broth (BHI) Gram-negative solid isolation medium: Add 3.7 g brain heart infusion broth powder, 0.05 g L-cysteine, 1 g L-arginine hydrochloride, and 1 g agar powder to 90 mL of deionized water and heat to boiling in a microwave oven. After boiling, cool the solution to 70°C, dilute to 100 mL, and sterilize by autoclaving. After cooling, add 0.05 g sterile mucin and 2 mg / L polymyxin B. Sonicate at 60°C to dissolve the solution. After dissolving the mucin, pour 10 mL into sterile plates. After cooling and solidifying, place the solution upside down in a sterile plastic bag. Before use or storage, deoxygenate the solid isolation medium overnight (>16 hours) in an anaerobic workstation. Store the solid isolation medium in an airtight anaerobic container at 4°C.

[0021] 1.2 Preparation of culture and subculture medium: Mucophilic Ackermann Broth: Add 37 g of brain heart infusion broth powder and 0.5 g of L-cysteine ​​to 900 mL of deionized water and bring to a boil in a microwave oven. Cool the boiled solution, dilute to 1 L, and sterilize by autoclaving. After the solution cools, add 0.5 g of sterile mucin per 100 mL of brain heart infusion broth and sonicate to dissolve the solution.

[0022] Mucophilic Ackermann solid medium: Add 3.7 g brain heart infusion broth powder, 0.05 g L-cysteine, and 1 g agar powder to 90 mL of deionized water and heat to boiling in a microwave oven. After boiling, cool the solution to 70°C, dilute to 100 mL, and sterilize by autoclaving. After cooling, add 0.05 g sterile mucin and sonicate at 60°C. Once the mucin is dissolved, pour 10 mL into sterile petri dishes. Once cooled and solidified, place the solution upside down in a sterile plastic bag and store sealed at 4°C.

[0023] Deoxygenation of the culture system: The prepared Ackermann liquid medium or corresponding solid culture medium is placed in an anaerobic workstation with a gas atmosphere of 80% nitrogen, 10% hydrogen and 10% carbon dioxide for overnight deoxygenation (more than 24 hours and no more than 48 hours). The deoxygenated culture medium or solid culture medium is placed in a sealed anaerobic culture bag containing an anaerobic gas-producing bag and stored in a sealed environment (4-8 degrees Celsius, away from light).

[0024] 1.3 Preparation of fecal suspension for bacterial isolation Fresh stool samples from 14-year-old adolescents were quickly transferred to an anaerobic workstation (Don WhitleyScientific, DG250; anaerobic gas environment of 80% nitrogen, 10% carbon dioxide, and 10% hydrogen). A sterile scoop was used to collect a mung bean-sized stool sample and homogenized in 1 mL of sterile Akkerman's liquid medium containing 25% glycerol. The homogenized suspension was filtered through 100 μm and 40 μm filters to remove impurities. The filtered suspension was placed in a cryovial and stored at -80°C.

[0025] 1.4 Bacterial Isolation from Fecal Suspensions Take out 20µL of the suspension filtered in 1.2 and add 180µL of BHI solution to dilute it 10 times, which is recorded as 10 -1 Dilute the fraction; remove 20µL of 10 -1 Dilute the components and add 180µL of new BHI solution, which is recorded as 10 -2 In this way, the original fecal suspension was diluted downwards in 8 steps, which were recorded as 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8180 µL of eight dilution gradients of fecal fractions were spread on mucin-brain heart infusion broth (BHI) Gram-negative solid isolation medium. After anaerobic culture for 48 h, dilutions with appropriate clonal density were selected for plating. Pinhole-sized, off-white, translucent monoclonal colonies were picked and transferred to mucophilic Akkerman liquid medium containing 0.05% L-cysteine, 1% arginine hydrochloride, 0.05% mucin, and 2 mg / L polymyxin B for anaerobically cultured for 24 h.

[0026] 1.5 Selection and identification of Akkermansia muciniphila To identify the species of the selected monoclonal clones, we amplified and sequenced the V3V4 regions of the isolated strain 16s using strain V3V4 region identification PCR combined with Sanger sequencing (BGI). The specific primer forward sequences and amplification procedures are shown in Tables 2 and 3. Amplified sequences with a single peak (monoclonal) in the Sanger sequencing results were compared with the NCBI (monoclonal cultures were labeled with the species ranked first in similarity).

[0027] Specifically, we selected 96 single colonies, of which 3 were identified as Akkermansia muciniphila (see Table 4). Culture fluid obtained from amplified single colonies identified as Akkermansia muciniphila in the V3V4 region was added to an equal volume of 50% glycerol, transferred to cryovials, and stored at -80°C.

[0028] Table 2: 16S PCR primer sequences for identification of bacterial monoclonal cultures Among them: H, V, W, Y are degenerate bases; H represents A or T or C; V represents G or A or C; W represents A or T; Y represents C or T; Table 3: PCR amplification program for identification of bacterial monoclonal cultures The enzyme used in the identification PCR was a high-fidelity enzyme (Thermo-Fisher Phusion Green Hot Start II High-Fidelity PCR Master Mix, F565S). Table 4: Species taxonomy of the 96 isolated bacterial monoclonal strains 1.6 Strain selection 1.6.1 Strain growth evaluation: Three strains of Akkermansia muciniphila were inoculated into Akkermansia muciniphila liquid culture medium at 1% inoculum and cultured anaerobically in a 37°C incubator for 9 hours. The number of bacteria was detected every hour (by measuring the 600 nm absorbance OD of the bacterial culture). 600 accomplish).

[0029] Specifically, such as Figure 1 As shown, among the three strains of Akkermansia muciniphila, the growth ability of Akkermansia muciniphilaP02H01 was significantly higher than that of the other two strains.

[0030] 1.6.2 Evaluation of strain tolerance to gastrointestinal fluid: Preparation of gastric juice containing pepsin: Take 16.4 ml of dilute hydrochloric acid (equivalent to 3.84 ml of concentrated hydrochloric acid), add about 800 ml of water and 10 g of pepsin, shake well, and dilute with water to 1000 ml.

[0031] Preparation of intestinal juice containing pancreatic enzymes: Take 6.8g of potassium dihydrogen phosphate, add 500ml of water to dissolve it, and adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution; take another 10g of pancreatic enzymes, add appropriate amount of water to dissolve it, mix the two liquids, and dilute with water to 1000ml.

[0032] Three strains of Akkermansia muciniphila were inoculated with 2% inoculum into Akkermansia muciniphila liquid culture medium. After anaerobic culture in a 37°C incubator for 9 hours, 2 mL of culture solution was taken for each strain and centrifuged at 8000g speed and 4°C for 10 minutes. The centrifuged bacterial pellet was resuspended in 1 mL of gastric juice or intestinal fluid, where the gastric juice was cultured for 1.5 hours and the intestinal juice was cultured for 4 hours. After the culture was completed, the cultured Akkermansia muciniphila strains were washed and centrifuged, and then spread on Akkermansia muciniphila solid culture medium. The number of bacterial colonies was counted after 48 hours. The survival rate was calculated as follows: Here, C(treated) is the number of clones after treatment with gastric juice or intestinal juice, and C(control) is the number of clones before treatment with gastric juice or intestinal juice.

[0033] Specifically, among the three strains of Akkermansia muciniphila, as shown in Table 5, Akkermansia muciniphila P02H01 had higher tolerance to gastric juice and intestinal juice than the other two strains.

[0034] Table 5: Evaluation of the gastrointestinal fluid resistance of the three strains 1.6.3 Species Classification of Akkermansia muciniphila P02H01 To confirm the strain identity of Akkermansia muciniphila P02H01, we amplified the full-length 16S sequence from a pure bacterial suspension. We sequenced the 16S sequence of Akkermansia muciniphila P02H01 in both forward and reverse directions using Sanger sequencing. We assembled the sequencing results into contiguous sequences using SnapGene software to obtain a full-length 16S sequence. The resulting full-length amplified 16S sequence was then aligned with known Akkermansia muciniphila sequences using NCBI.

[0035] The 16S sequence of Akkermansia muciniphila P02H01 is: SEQ ID NO: 1, and the full-length nucleic acid sequence of the gene is as follows: The full-length 16S rRNA gene sequence of the strain obtained above was compared and analyzed using the NCBI database. Akkermansia muciniphila P02H01 was assigned to the species classification of Akkermansia muciniphila. The comparison results with some existing reference strains are shown in Table 6.

[0036] Table 6: Comparison of the full-length amplified sequence of Akkermansia muciniphila P02H01 16s with the reference strains Example 2: Anti-frailty intervention experiment of Akkermansia muciniphila P02H01 Preferably, we will use the Akkermansia muciniphila P02H01 strain screened in Specific Embodiment 1, which has strong growth ability and strong resistance to gastric juice and intestinal juice, for specific implementation.

[0037] Preparation of live Akkermansia muciniphila P02H01 cells: 50 mL of a 9-hour culture of Akkermansia muciniphila P02H01 (starting inoculum size: 0.1% by volume) was centrifuged at 8000 g for 10 minutes at 4°C. The bacterial pellet was washed with saline and centrifuged again under the same conditions. After washing, the pellet was dissolved in 10 mL of saline containing 50% glycerol by volume, aliquoted, and stored at -80°C. A portion of the aliquot was plated onto solid culture medium and incubated anaerobically for 48 hours. Colonies were counted and the number of colony-forming units (CFU) per milliliter of the aliquot was calculated.

[0038] Establishment of the frailty model: Specific pathogen-free (SPF) C57 / BL6N mice were purchased from Weitonglihua. Twenty-month-old mice were used for the frailty model. Mice were gavaged with Akkermansia muciniphila P02H01 once daily for one week at a dose of 2 × 10 8CFU (suspended in saline containing 25% glycerol). A control group of mice was gavaged with an equal volume of saline containing 25% glycerol. To assess frailty in mice, we calculated a frailty index score monthly based on four parameters: body weight, muscle strength (grip test), gait speed (rotarod test), and decreased exercise tolerance (rotarod test). See Table 7 for details.

[0039] Among them, FI is the comprehensive frailty index score, FIBW is the frailty index of weight loss, FIMS is the frailty index of decreased muscle strength, FIWS is the frailty index of slowed gait speed, and FIEE is the frailty index of decreased exercise endurance.

[0040] Table 7: Specific assessment methods for each frailty index The experimental results are as follows Figure 2 As shown in the results, intervention with Akkermansia muciniphila P02H01 significantly reduced the frailty index of aged mice (P<0.05).

[0041] Example 3: Experiment on the extension of healthy lifespan of mice by intervention with Akkermansia muciniphila P02H01 Establishment of a frailty model in aged mice: Specific pathogen-free (SPF) C57 / BL6N male mice were purchased from Vital River. Based on the literature, the frailty model was established using 22-23 month-old mice. Mice were gavaged with Akkermansia muciniphila P02H01 once daily for one week at a dose of 2 × 10 8 CFU (suspended in saline containing 25% glycerol). The control group of mice was gavaged with an equal volume of saline containing 25% glycerol.

[0042] To evaluate the healthy lifespan of mice (the time they survive in a fully healthy state; i.e., the age at which any one of frailty, renal function decline, or liver function decline occurs), we detected and compared the age at which the frailty index of the control mice was greater than 0.45, the age at which renal function declined (defined as blood urea nitrogen or blood creatinine above the upper limit of normal as renal aging, i.e., blood urea nitrogen > 11.5 mmol / L or blood creatinine > 13.2 µmol / L), the age at which liver function declined (albumin < 22 g / L), and the extension of total lifespan between the control and Akkermansia muciniphila P02H01-treated aged mice.

[0043] Specifically, before and during the Akkermansia muciniphila P02H01 intervention, mice were tail-clipped and blood was collected monthly. The collected blood was diluted and centrifuged, and the supernatant was assayed for serum creatinine and mouse albumin according to the instructions of the creatinine test kit and mouse albumin ELISA kit, respectively. At the end of the intervention, the age at which each mouse reached a frailty index greater than 0.45, as well as decreased renal and liver function, was calculated and compared. The healthy lifespan of the mice was also compared.

[0044] The experimental results are as follows Figure 3 As shown, intervention with Akkermansia muciniphila P02H01 significantly prolonged the age at which the frailty index reached greater than 0.45.

[0045] like Figure 4 As shown, intervention with Akkermansia muciniphila P02H01 significantly prolonged the decline in renal function in mice.

[0046] like Figure 5 As shown, intervention with Akkermansia muciniphila P02H01 delayed the age at which liver function decline occurred in aged mice.

[0047] like Figure 6 and Figure 7 As shown, intervention with Akkermansia muciniphila P02H01 significantly extended the healthy lifespan and total lifespan of mice.

[0048] 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, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A strain of Akkermansia muciniphila, characterized in that: It is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CCTCC NO: 46390 and the deposit date of April 15, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. The strain of Akkermansia muciniphila according to claim 1, characterized in that The invention also comprises a 16S sequence consisting of the nucleotide sequence of SEQ ID NO:

1.

3. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the fermentation mixture, fermentation broth precipitate, fermentation supernatant, live bacteria, dead bacteria, lyophilized powder or lysate of Akkermansia muciniphila according to claim 1.

4. The pharmaceutical preparation according to claim 3, characterized in that The preparation method of the viable bacteria comprises the following steps: S1: Inoculate Akkermansia muciniphila CCTCC NO: 46390 and culture anaerobically in liquid culture medium to obtain viable bacteria; S2: After centrifugation, the precipitate is washed with physiological saline, centrifuged again, and precipitated again to obtain the final precipitate; S3: resuspending the final precipitate in physiological saline or liquid culture medium containing 25% glycerol by volume to prepare a bacterial liquid preservation solution, which is stored in a deep cryogenic environment at -80 degrees Celsius; S4: Anaerobically culturing the prepared bacterial liquid preservation solution in a solid culture medium, and counting the number of viable bacteria in the preservation solution.

5. The pharmaceutical preparation according to claim 4, characterized in that The components of the solid culture medium are 3.7 g / L brain heart infusion broth powder, 0.05 g / L L-cysteine, 1 g / L agar powder, and 0.05 g / L sterile mucin. The components of the liquid culture medium are 37 g / L brain heart infusion broth powder, 0.5 g / L L-cysteine, and 0.5 g / L sterile mucin.

6. Use of the Akkermansia muciniphila according to claim 1 in the preparation of foods, medicines, health products, microbial agents, food additives, and health product additives.

7. Use of Akkermansia muciniphila according to claim 1 in the preparation of anti-asthenia products.

8. Use of Akkermansia muciniphila according to claim 1 in the preparation of a product for prolonging healthy lifespan.

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