Application of Akkermansia myxophilus YG2645 or compositions thereof in the preparation of life-extending products

Abstract

This invention provides the application of Akkermansia myxophilus YG2645 or compositions thereof in the preparation of life-extending products, particularly relating to the fields of C12N1 / 20 bacteria and fermentation products or A61P biopharmaceuticals. It provides a compound microbial agent comprising Akkermansia myxophilus YG2645 (AKK2645 for short) and / or its derivatives (e.g., inactivated AKK2645 cells or fermentation products containing inactivated cells), which can be used to extend lifespan (e.g., extend the lifespan of Alzheimer's patients or insulin-resistant patients), delay neurodegeneration (optionally delaying the progression of Alzheimer's disease), and slow aging. This invention is the first to discover that Akkermansia myxophilus can play a role in different dimensions of aging scenarios and can have a synergistic effect with compositions of Bifidobacterium lactis, providing a novel microbial source solution for a wider range of anti-aging populations.

Description

Technical Field

[0001] This invention relates to the field of microbial and fermentation product technology, particularly to the field of C12N1 / 20 bacteria and fermentation products or A61P biopharmaceuticals, specifically to the application of Akkermansia myxophilus YG2645 or compositions thereof in the preparation of life-extending products. Background Technology

[0002] Anti-aging is an eternal topic of human pursuit. As we age, the body gradually experiences functional decline, accompanied by increased oxidative stress, metabolic disorders, and neurodegenerative diseases, among other physiological changes. These processes collectively drive the aging process. Most anti-aging products currently on the market target only a single mechanism, such as anti-oxidation, gut health regulation, or sleep enhancement, making it difficult to comprehensively address the complex aging process mediated by multiple factors including metabolic disorders and neurodegenerative diseases. Furthermore, existing drug-based anti-aging methods (such as metformin) are not primarily indicated for anti-aging purposes, and long-term or unauthorized use may pose potential risks.

[0003] Current technologies lack the means to comprehensively address the complex aging process mediated by multiple factors such as metabolic disorders and neurodegenerative diseases. Summary of the Invention

[0004] Purpose of the invention

[0005] The purpose of this invention is to provide the application of Akkermansia myxophilus YG2645 or compositions thereof in the preparation of life-extending products. This invention is the first to discover that Akkermansia myxophilus can play a role in various aspects of aging and can have a synergistic effect with compositions containing Bifidobacterium lactis, providing a novel microbial source solution for a wider range of anti-aging populations. The probiotics of this invention have a synergistic effect, comprehensively intervening in the aging process on multiple levels, including extending lifespan, enhancing stress resistance, improving metabolic function, and delaying neurodegeneration, providing a more comprehensive, efficient, and safe anti-aging solution for a broad population.

[0006] Solution To achieve the objectives of this invention, the technical solution adopted is as follows: In a first aspect, a compound microbial agent comprising Akkermansia myxophilus YG2645 and / or its derivatives is provided, including Akkermansia myxophilus YG2645 and / or its derivatives (the derivatives may include inactivated AKK2645 cells or fermentation products containing inactivated cells), and Bifidobacterium lactis YG2013 and / or its derivatives. The accession number of the myxotrophic Akkermansia YG2645 is CGMCC No.30598; The preservation number of the Bifidobacterium lactis YG2013 is CGMCC No.27579.

[0007] As a feasible embodiment, the Akkermansia xylophilus YG2645 derivative is its inactivated cells, extract, culture, culture supernatant, fermentation product, fermentation supernatant, or a combination thereof.

[0008] As a feasible embodiment, Bifidobacterium lactis YG2013 is a live bacterium, and optionally, Bifidobacterium lactis YG2013 derivatives are its inactivated cells, extracts, cultures, culture supernatants, fermentation products, fermentation supernatants, or combinations thereof.

[0009] As a feasible implementation example, Bifidobacterium lactis ( Bifidobacterium lactis The cell ratio of YG2013 and / or its derivatives to Akkermansia xylophilus YG2645 and / or its derivatives is 1:(0.1~10), optionally 1:(0.25~4), optionally 1:1.

[0010] As one feasible embodiment, it includes Bifidobacterium lactis (Bifidobacterium lactis) with a bacterial count ratio of 1:(0.1~10). Bifidobacterium lactis The ratio of live YG2013 bacteria to inactivated YG2645 Akkermansia myxophilus cells can be optionally 1:(0.25~4), or optionally 1:1.

[0011] Optionally, the concentration of inactivated Akkermansia myxophilus YG2645 (AKK2645) cells is 1×10⁻⁶. 7 -1×10 13 TFU / mL, optionally ≥1×10 9 TFU / mL, optionally 1×10 9 -1×10 13 TFU / mL.

[0012] Optionally, the live bacteria concentration of YG2013 is 1×10⁻⁶. 7 -1×10 13 CFU / mL, optionally ≥1×10 9 CFU / mL, optionally 1×10 9 -1×10 13 CFU / mL.

[0013] In a second aspect, a composition is provided comprising an effective amount of the compound microbial agent described in the first aspect.

[0014] As a possible embodiment, the composition is a pharmaceutical composition, a food composition, or a feed composition.

[0015] As a possible embodiment, the composition further includes other probiotics, postbiotics, prebiotics, antibacterial agents, immunomodulators, anticancer agents, anti-inflammatory agents, or combinations thereof; Optionally, the concentration of inactivated Akkermansia myxophilus YG2645 (AKK2645) cells is 1×10⁻⁶. 7 -1×10 13 TFU / mL, optionally ≥1×10 9 TFU / mL, optionally 1×10 9 -1×10 13 TFU / mL.

[0016] Optionally, the live bacteria concentration of YG2013 is 1×10⁻⁶. 7 -1×10 13 CFU / mL, optionally ≥1×10 9 CFU / mL, optionally 1×10 9 -1×10 13 CFU / mL.

[0017] Thirdly, the use of the compound microbial agent described in the first aspect, or the composition described in the second aspect, in the preparation of products, preferably pharmaceutical products, for the following purposes: 1) Improve heat stress resistance; 2) Extend lifespan; optionally extend the lifespan of Alzheimer's patients; optionally extend the lifespan of insulin-resistant patients; 3) It can delay neurodegeneration, optionally slow the progression of Alzheimer's disease, and optionally prevent or treat Alzheimer's disease; 4) Improve reproductive capacity; 5) Improves metabolic function, and optionally improves insulin resistance; 6) Antioxidant stress; 7) Lowering lipids; 8) Delay aging.

[0018] Fourthly, a method for treating or alleviating Alzheimer's disease is provided, comprising administering an effective amount of the compound bacterial agent described in the second aspect, or the composition described in the third aspect, to a subject in need.

[0019] Fifthly, a method for treating or alleviating insulin resistance is provided, comprising administering an effective amount of the compound bacterial agent described in the first aspect or the composition described in the second aspect to a subject in need.

[0020] Optionally, the concentration of inactivated Akkermansia myxophilus YG2645 (AKK2645) cells is 1×10⁻⁶. 7 -1×10 13 TFU / mL, optionally ≥1×109 TFU / mL, optionally 1×10 9 -1×10 13 TFU / mL.

[0021] Optionally, the live bacteria concentration of YG2013 is 1×10⁻⁶. 7 -1×10 13 CFU / mL, optionally ≥1×10 9 CFU / mL, optionally 1×10 9 -1×10 13 CFU / mL.

[0022] Sixthly, the present invention provides the use of Akkermansia myxophilus YG2645 and / or its derivatives in the preparation of products for any or at least two of the following uses: 1) Improve heat stress resistance; 2) Extend lifespan; optionally extend the lifespan of Alzheimer's patients; optionally extend the lifespan of insulin-resistant patients; 3) It can delay neurodegeneration, optionally slow the progression of Alzheimer's disease, and optionally prevent or treat Alzheimer's disease; 4) Improve reproductive capacity; 5) Improves metabolic function, and optionally improves insulin resistance; 6) Antioxidant stress; 7) Lowering lipids; 8) Delay aging.

[0023] In particular, this invention provides the use of Akkermansia mucinosa or compositions containing it in the preparation of products for delaying aging and / or prolonging life (optionally prolonging the life of patients with Alzheimer's disease; optionally prolonging the life of patients with insulin resistance).

[0024] As a feasible embodiment, the Akkermansia myxophilus YG2645 derivative is its inactivated cells, extract, culture, culture supernatant, fermentation product, fermentation supernatant or a combination thereof; And / or, the product is a medicine; Optionally, the concentration of inactivated Akkermansia myxophilus YG2645 (AKK2645) cells is 1×10⁻⁶. 7 -1×10 13 TFU / mL, optionally ≥1×10 9 TFU / mL, optionally 1×10 9 -1×10 13 TFU / mL.

[0025] Beneficial effects (1) This invention is the first to discover that Akkermansia 2645 can play a role in different dimensions of aging scenarios and can have a synergistic effect with Bifidobacterium lactis 2013, thereby comprehensively intervening in the aging process on multiple levels such as prolonging life, enhancing stress resistance, improving metabolic function, and delaying neurodegeneration, providing a new microbial source solution for a wider range of anti-aging populations.

[0026] (2) This invention is the first to discover that the combination of Akkermansia muciniphila and Bifidobacterium lactis has stronger antioxidant stress and lipid-lowering effects than a single strain, thereby more effectively reducing aging-related indicators and prolonging life.

[0027] (3) This invention develops a food or health product with a multi-mechanism synergistic anti-aging function. By selecting a combination of strains with complementary functions (AKK2645 and YG2013) and verifying their synergistic effect through rigorous modeling, it comprehensively intervenes in the aging process at multiple levels, such as prolonging life, enhancing stress resistance, improving metabolic function, and delaying neurodegeneration, providing a more comprehensive, efficient, and safe anti-aging solution for a wide range of people.

[0028] (4) The Acetobacter xanthophyte and Bifidobacterium lactis of the present invention are microorganisms that exist in the human intestine. They are a combination of novel second-generation and traditional first-generation bacteria, and have the characteristics of being natural, safe and without toxic side effects. Attached Figure Description

[0029] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0030] Figure 1 The effect of different single bacterial agents on the lifespan of *C. elegans* N2 in Test Example 1 of this invention is shown. Figure 2 The effects of different bacterial agents in Test Example 1 of this invention on the lifespan (A), lipofuscin (B), total number of eggs laid (C), and heat stress resistance (D) of Caenorhabditis elegans N2.

[0031] Figure 3 This is the effect of different bacterial agents in Test Example 2 of this invention on the insulin-resistant nematode model; where A represents the lifespan of N2 nematodes, B represents relative lipid content, and C represents lipofuscin accumulation. The differences between groups marked with different letters in the figure are significant, p < 0.05. Among them, " "###" indicates that compared with group 2013 (and group 2645), p < 0.001; "####" indicates that compared with group 2013 (and group 2645), p < 0.0001.

[0032] Figure 4 This is the effect of different bacterial agents on the lifespan of AD model nematodes in Test Example 3 of this invention; wherein, (A) is the lifespan of CL2006 nematodes, and (B) is the non-paralysis rate of CL4176 nematodes. "This indicates that compared to the model group (CL2006 or CL4176), P < 0.001." "##" indicates that compared with the model group (CL2006 or CL4176), p < 0.0001. Among them, "##" indicates that compared with the 2013 group (and the 2645 group), p < 0.01; "###" indicates that compared with the 2013 group (and the 2645 group), p < 0.001; and "####" indicates that compared with the 2013 group (and the 2645 group), p < 0.0001. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0035] All raw materials used in the following examples are commercially available. For example, Escherichia coli OP50 was purchased from Fujian Shangyuan Biotechnology Co., Ltd., China. The present invention uses the Caenorhabditis elegans N2 strain, purchased from the website of CGC (Caenorhabditis Genetics Center), USA.

[0036] The *Bifidobacterium lactis* YG2013, YG4617, YG8091, *Ackermania xylophilus* YG2645, YG1033, and YG3611 of this invention were obtained from healthy individuals through screening.

[0037] Among them, Akkermansia muciniphila ( Akkermansia muciniphila YGK2645 has the accession number CGMCC No.30598. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing. The deposit date is May 10, 2024 (see CN 119242495 A for details).

[0038] Bifidobacterium lactis ( Bifidobacterium lactis YG2013 has the accession number CGMCC No.27579. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing (Postcode 100101). The deposit date is June 7, 2023 (see CN117417863A for details).

[0039] Example 1: Isolation and identification of Akkermansia muciniphila YG1033 and YG3611 The methods for isolating and identifying Akkermansia muciniphila strains YG1033 and YG3611 are similar to those in Example 1 of CN 119242495 A.

[0040] Example 2: Bifidobacterium lactis lactis Separation and identification of YG4617 and YG8091 Bifidobacterium lactis ( Bifidobacterium lactis The methods for isolating and identifying strains YG4617 and YG8091 are similar to those in Example 1 of CN117417863A.

[0041] Example 3: Bacterial culture dishes for culturing Caenorhabditis elegans (1) Preparation of live bacteria of Bifidobacterium lactis YG2013, YG8091 and YG4617 Frozen tubes of Bifidobacterium lactis strains YG2013, YG8091, and YG4617, stored at -80°C, were thawed in a 37°C water bath and then activated by inoculation onto MRS solid medium in a biosafety cabinet. The activated strains were then transferred to MRS liquid medium and cultured under suitable conditions to obtain sufficient bacterial cells.

[0042] (2) Preparation of inactivated cells of Akkermansia myxophilus YG1033, AKK2645 and YG3611 Frozen tubes of Akkermansia myxophilus strains YG1033, AKK2645, and YG3611, stored at -80℃, were thawed in a 37℃ water bath and then activated in a biosafety cabinet by inoculation into modified BHI solid medium (composition: brain heart extract 12.5 g / L, bovine heart extract 5.0 g / L, peptone 10.0 g / L, glucose 2.0 g / L, sodium chloride 5.0 g / L, disodium hydrogen phosphate 2.5 g / L, L-cysteine ​​hydrochloride 0.5 g / L, L-threonine 5 g / L, N-acetyl-D-glucosamine 5 g / L, and agar 16 g / L). The activated strains were further inoculated into modified BHI liquid medium for expansion culture. After cell collection, the cells were heat-treated at 70℃ for 30 minutes to obtain pasteurized inactivated bacterial cultures.

[0043] (3) Escherichia coli OP50 ( E. coli Cultivation of OP5 E. coli OP50 (Sunybiotech, Fujian) After thawing, the cells were inoculated onto LB solid medium for activation, then transferred to LB liquid medium and cultured at 37°C in a shaker at 150 rpm / min for 16 hours. The cells were collected, washed three times with M9 buffer, and finally concentrated to 1×10¹. 0 CFU / mL, stored at 4℃ for later use as a regular feed for nematodes.

[0044] (4) Preparation of bacterial culture dishes for culturing Caenorhabditis elegans The live Bifidobacterium lactis (YG2013, YG4617, and YG8091) and the pasteurized Akkermansia myxophilus (YG1033, AKK2645, and YG3611) from steps (1) and (2) above were washed three times with M9 buffer. Single or mixed cultures (with a 1:1 ratio of live Bifidobacterium lactis (CFU) to inactivated Akkermansia myxophilus (TFU)) were then washed with 1×10¹⁰ buffer. 0 Resuspend E. coli culture at CFU / mL to achieve a single cell concentration of 1×10¹ 0 TFU / mL or 1×10¹ 0 CFU / mL, so that the concentration of each single cell in the mixed bacterial cell reaches 5×10. 9 TFU / mL or 5×10 9 CFU / mL. Place 100 μL of the intervention bacterial suspension or mixed bacterial suspension in the center of NGM solid medium, dry in a biosafety cabinet, and incubate overnight at 20°C to obtain the plates for each intervention group. (Direct addition...) E. coliThe OP50 suspension is a Con group plate.

[0045] In the case of grouping single or mixed bacterial cells: Blank control group (Con group); YG2013 live bacteria group (2013 group), YG8091 live bacteria group (8091 group), YG4617 live bacteria group (4617 group), YG1033 inactivated bacteria group (1033 group), AKK2645 inactivated bacteria group (2645 group), YG3611 inactivated bacteria group (3611 group), YG2013 live bacteria + YG1033 inactivated bacteria group (2013 + 1017 group). Group 33, Mix 1 (mixed bacterial culture), YG2013 live bacteria + AKK2645 inactivated bacteria group (2013+2645 group, Mix 2 group, mixed bacterial culture), YG8091 live bacteria + YG1033 inactivated bacteria group (8091+1033 group, Mix 3 group, mixed bacterial culture), and YG8091+AKK2645 inactivated bacteria group (8091+2645 group, Mix 4 group, mixed bacterial culture).

[0046] Test Example 1 (1) Synchronization of Caenorhabditis elegans N2 After washing the culture dish containing a large number of oviposition-stage adults with M9 buffer, the nematodes were collected into 15 mL centrifuge tubes and allowed to settle naturally for 5 min. The tubes were then centrifuged at 1,500 rpm for 1 min to remove the supernatant. Lysis buffer (a mixture of 5M sodium hydroxide solution, 5% sodium hypochlorite solution, and M9 buffer at a volume ratio of 1:2:7) was added for 5 min of lysis. The tubes were then centrifuged at 5000 rpm for 2 min, and the supernatant and adult fragments were discarded. The eggs were collected, washed five times with an equal volume of M9 buffer, and then transferred to NGM solid medium containing OP50. After incubation at 20°C for 56 h, the eggs matured into L4 stage larvae, which were used for subsequent experiments.

[0047] (2) Determination of nematode lifespan The L4 stage larvae synchronized in step (1) were transferred to NGM solid culture medium inoculated with OP50 and corresponding intervention substances as in Example 3, and simultaneously inoculated in equal amounts until only the medium was coated with... E. coli OP50 NGM medium served as a blank control (Con group). All lifespan experiments were conducted at 20°C. The lifespan determination began at day T0, with nematodes synchronized to stage L4. They were then transferred to fresh culture dishes every two days until all individuals died. Three plates were used for each lifespan experiment, totaling 90 nematodes (30 per plate). The number of dead nematodes was recorded daily; individuals unresponsive to mechanical stimulation were considered dead. The lifespans of nematodes under different treatment conditions were statistically analyzed, and survival curves were plotted.

[0048] This experiment first investigated the effects of different single-agent bacterial agents: YG2013 live bacteria group (2013 group), YG8091 live bacteria group (8091 group), YG4617 live bacteria group (4617 group), YG1033 inactivated bacteria group (1033 group), AKK2645 inactivated bacteria group (2645 group), and YG3611 inactivated bacteria group (3611 group) (with a live bacterial cell concentration of 1×10⁻⁶). 10 CFU / mL, inactivated bacterial cell concentration is 1×10⁻⁶ 10 The effect of TFU / mL on nematode lifespan, the results are as follows: Figure 1 The results showed that, compared with the control group (Con group) fed only with *E. coli*, the live *Bifidobacterium lactis* YG2013 (mean lifespan increased by 14.2% compared to the control group) and YG8091 (mean lifespan increased by 10.9% compared to the control group), and the inactivated *Ackermannii myxotroph* YG1033 (mean lifespan increased by 12.2% compared to the control group) and AKK2645 (mean lifespan increased by 12.9% compared to the control group) groups significantly prolonged the lifespan of *N. 2* nematodes. However, *Bifidobacterium lactis* YG4617 and *Ackermannii myxotroph* YG3611 did not significantly prolong the lifespan of nematodes. In other words, the ability of *Bifidobacterium lactis* and *Ackermannii myxotroph* to delay natural aging in nematodes is strain-specific.

[0049] To further investigate the effects of pairwise combinations of *Bifidobacterium lactis* YG2013 and YG8091 with *Ackermania pseudomallei* YG1033 and AKK2645, *Ackermania pseudomallei* and *Bifidobacterium lactis* were mixed at a 1:1 ratio and divided into four groups (Mix1, Mix2, Mix3, and Mix4). Survival curves were plotted, and the results are as follows: Figure 2 The results showed that, compared with the single-strain intervention groups (2013, 8091, 1033 and 2645 groups), only the Mix2 group showed a further statistically significant improvement in the mean lifespan of nematodes (p<0.05).

[0050] Simultaneously, the reproductive capacity, heat stress resistance, and lipofuscin accumulation of single and mixed bacterial agents of Bifidobacterium lactis YG2013, YG8091, and Akkermansia myxophilus YG1033 and AKK2645 were determined. (3) Determination of nematode reproductive capacity The L4 stage larvae synchronized in step (1) were transferred to NGM culture media inoculated with OP50+ intervention. Simultaneously, an equal amount was inoculated onto NGM culture media coated only with OP50 as a blank control (Con group). Five plates were used per group, with two nematodes per plate. The nematodes on each plate were transferred to new plates daily, and the old plates were incubated at 20 ℃ for 48 h. Oviposition was then recorded daily until the nematodes ceased oviposition, and the total number of ovipositions was tallied. Results are as follows: Figure 2 C in the middle.

[0051] (4) Determination of nematode heat stress resistance The L4 stage larvae synchronized in step (1) were transferred to NGM culture media inoculated with OP50+ intervention, and inoculated in equal amounts until only the surface was coated with the intervention material. E. coli OP50 NGM medium was used as a blank control (Con group) and cultured in a constant temperature incubator at 20 ℃. The plates were changed daily. On the 5th day of sample intervention, nematodes were picked and transferred to new corresponding NGM plates, 20 nematodes / plate, with three replicates per group. The plates were then placed in a 34 ℃ incubator, and the number of surviving nematodes was recorded after 48 hours of incubation at 34 ℃. The results are as follows: Figure 2 D in the middle.

[0052] (5) Assay for lipofuscin accumulation in nematodes The L4 stage larvae synchronized in step (1) were transferred to NGM culture media inoculated with OP50+ intervention. Simultaneously, an equal amount was inoculated onto NGM culture media coated only with OP50 as a blank control (Con group). The media were incubated at 20 ℃, with the plates changed daily. On the 8th day of the intervention, 30 nematodes from each group were picked, fixed with 4% paraformaldehyde solution, transferred to 3% agarose slides, and observed and imaged using a fluorescence microscope. The fluorescence intensity was statistically analyzed using ImageJ software. The results are as follows: Figure 2 B in the middle.

[0053] Figure 2Results A, B, C, and D indicate that, compared to the control group (Con group) fed only with *E. coli*, all four single-strain intervention groups significantly prolonged the average lifespan of nematodes while improving several key lifespan-related indicators, including reducing lipofuscin accumulation, increasing egg production, and enhancing heat stress resistance. These results demonstrate that live bacteria YG2013 and YG8091, along with inactivated bacteria YG1033 and AKK2645, can extend lifespan and delay natural aging by enhancing antioxidant and heat stress resistance without negatively impacting reproductive capacity. Compared to the single-strain intervention groups (2013, 8091, 1033, and 2645 groups), only the Mix2 group showed further statistically significant improvements in nematode average lifespan, lipofuscin accumulation, egg production, and heat stress resistance. p <0.05). This indicates that live bacteria YG2013 and inactivated bacteria AKK2645 have a synergistic effect in delaying natural aging and prolonging lifespan, and this effect is strain-specific.

[0054] Test Example 2: AKK2645 and its combined bacterial agents prolonged the lifespan of *C. elegans* N2 in an insulin resistance model. This experiment used the Caenorhabditis elegans N2 strain, which was purchased from the official website of CGC (Caenorhabditis Genetics Center) in the United States.

[0055] (1) Determination of nematode lifespan Nematode larvae synchronized to the L4 stage were transferred to NGM solid medium fed with OP50 (Con group), NGM solid medium containing 2% glucose fed with OP50 (Glu group), and NGM solid medium containing 2% glucose fed with OP50 and corresponding interventions (2013 group, 2645 group, and Mix AE group) respectively (the amount of food and intervention added to each group is shown in Table 1), and cultured in a constant temperature incubator at 20℃. During the experiment, the medium was replaced with fresh medium every two days, and the number of nematode deaths was recorded daily until all individuals died. Finally, the lifespan data of nematodes under different treatment conditions were statistically analyzed, and their survival curves were plotted, as shown in Table 1. Figure 3 As shown in A in the diagram.

[0056] Table 1. Amounts of food and intervention added to each group in the insulin resistance model ; (2) Oil Red O staining of nematodes (lipid content) L4 stage larvae were transferred to NGM medium containing the interventions shown in Table 1 and cultured for 3 days. Nematodes from each group were collected, washed three times with M9-T buffer containing 0.1% Triton X-100, and then fixed with 60% isopropanol solution for 5 minutes. After removing the supernatant, Oil Red O staining solution was added, and staining was performed at room temperature in the dark for 1 hour. After staining, the nematodes were washed five times with PBS buffer, transferred to a glass slide, and imaged using an inverted microscope in bright field. The staining results were then quantitatively and statistically analyzed using ImageJ software. Figure 3 As shown in B in the diagram.

[0058] (3) Determination of lipofuscin in nematodes The L4-stage larvae synchronized in step (1) of Test Example 1 were transferred to NGM culture media containing the interventions shown in Table 1 for each group, with the culture medium changed every two days. On day 8 of the intervention, 30 nematodes were picked from each group, washed with M9 buffer, and fixed with 4% paraformaldehyde solution. The fixed nematodes were then transferred to slides coated with 3% agarose, and autofluorescence images were observed and acquired using a fluorescence microscope. The fluorescence intensity of lipofuscin in the images was quantitatively statistically analyzed using ImageJ software, and the results are as follows: Figure 3 C in the middle.

[0059] Figure 3 Figures A, B, and C indicate that, compared to the control group (Con group), the glucose-treated (Glu) nematodes exhibited significantly shorter lifespans, increased lipid content, and increased lipofuscin accumulation, indicating the successful establishment of the insulin resistance model. Under various probiotic intervention conditions, the shortened lifespan, increased lipid content, and lipofuscin accumulation in the model nematodes were all alleviated to varying degrees, demonstrating that both live bacteria YG2013 and inactivated bacteria AKK2645 can prolong the lifespan of nematodes under insulin resistance. Furthermore, compared to the single-strain treatment groups (2013 group or 2645 group), Mix B, Mix C, and Mix D in the mixed groups showed stronger improving effects in prolonging lifespan, reducing lipofuscin accumulation, and decreasing lipid accumulation, with Mix C showing the most significant effect. These results indicate that within the range of 1:4 to 4:1 ratio of live bacteria YG2013 to inactivated bacteria AKK2645, the two have a synergistic effect in prolonging the lifespan of nematodes under insulin resistance, with the optimal effect observed at a 1:1 ratio.

[0060] Example 3: AKK2645 and its composition prolong the lifespan of *Caenorhabditis elegans* in an AD model (Alzheimer's disease model). Experimental animals: The transgenic Caenorhabditis elegans strains CL4176 and CL2006 (two different AD models) were used in this experiment and were purchased from the official website of CGC (Caenorhabditis Genetics Center) in the United States.

[0061] (1) Synchronization treatment of Caenorhabditis elegans CL4176 and CL2006 Take a culture dish with a high concentration of adult nematodes in the oviposition stage, rinse with M9 buffer, collect the nematodes into a 15 mL centrifuge tube, let it stand for 5 min to allow it to settle naturally, then centrifuge at 1,500 rpm for 1 min, discard the supernatant, and retain about 1 mL of liquid. Add lysis buffer (prepared by mixing 5 M sodium hydroxide solution, 5% sodium hypochlorite solution, and M9 buffer at a volume ratio of 1:2:7), lyse for 5 min, then centrifuge at 5,000 rpm for 2 min, discard the supernatant and adult fragments, and collect the eggs. Wash the eggs five times with an equal volume of M9 buffer, and finally transfer the eggs to NGM medium and incubate them in a 16℃ biochemical incubator for 36 h and 72 h, respectively, to obtain L1 and L4 stage larvae for subsequent experiments.

[0062] (2) Lifespan determination of nematode CL2006 CL2006 nematodes synchronized to stage L4 were transferred to NGM medium containing or without live YG2013 bacteria, inactivated AKK2645 bacteria, or their combinations (food and intervention amounts for each group are shown in Table 2). Lifespan experiments were conducted at 25°C. Starting with L4 nematodes as day 0, the medium was replaced with fresh medium every two days until all nematodes died. A total of 90 nematodes (30 per dish) were used in three culture dishes. The number of dead nematodes was recorded daily; those unresponsive to mechanical stimulation were considered dead. Finally, the lifespan data of nematodes under each treatment condition were statistically analyzed, and survival curves were plotted. The results are shown in Table 2. Figure 4 A in the middle.

[0063] Table 2. Food and intervention amounts in each group of the CL2006 AD model of Nematode ; (3) Detection of paralysis rate of nematode CL4176 CL4176 nematodes synchronized to the L1 stage were inoculated onto solid NGM medium with the combinations shown in Table 3. After incubation at 16°C for 36 h, the temperature was raised to 25°C to induce Aβ protein expression in muscle cells. Abnormal Aβ aggregation led to motor dysfunction in CL4176 nematodes, manifesting as progressive paralysis, thus establishing a nematode model of Alzheimer's disease. The experiment was conducted in triplicate, with approximately 30 nematodes per replicate. Starting 36 h after raising the temperature to 25°C, the number of paralyzed individuals in the entire nematode population was observed and recorded every 2 h until all nematodes exhibited the paralyzed phenotype. The results are shown in Table 3. Figure 4 B in the middle.

[0065] Table 3. Food and intervention amounts in each group of the CL4176 AD model of Nematode ; Figure 4 Results A and B in the study showed that, compared with the model group, both groups 2013 and 2645 prolonged the lifespan of nematode CL2006 and increased the non-paralysis rate of nematode CL4176 to varying degrees, indicating that both live bacteria YG2013 and inactivated bacteria AKK2645 have the effect of increasing the lifespan of patients with Alzheimer's disease. Furthermore, compared with single strains, the Mix BD group further prolonged the lifespan of nematodes and reduced the paralysis rate, suggesting that the ratio of live bacteria YG2013 to inactivated bacteria AKK2645 within the range of 1:4 to 4:1 has a synergistic effect in prolonging lifespan in Alzheimer's disease, with the best effect observed when the bacterial count of live YG2013 and inactivated AKK2645 is 1:1.

[0067] This invention utilizes the complementary mechanism of Akkermansia muciniphila (AKK) and Bifidobacterium lactis, especially Bifidobacterium lactis 2013 and Akkermansia muciniphila 2645, which exhibit synergistic anti-aging effects in multiple scenarios. It can delay natural aging and extend lifespan by enhancing antioxidant and heat stress capabilities without negatively impacting reproductive capacity. It can also extend lifespan in insulin resistance and Alzheimer's disease states, demonstrating the advantages of multi-pathway and systemic intervention.

[0068] The bacterial strains selected in this invention (Ackermania myxophila and Bifidobacterium lactis) are both native to the human intestinal flora, with good biocompatibility and high safety; among them, AKK bacteria are inactivated, which further avoids the potential risks of live bacteria and is more suitable for long-term use.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound bacterial agent comprising Akkermansia myxophilus YG2645 and / or its derivatives, characterized in that, Including the aforementioned Akkermansia xylophilus YG2645 and / or its derivatives, and Bifidobacterium lactis YG2013 and / or its derivatives; The accession number of the myxotrophic Akkermansia YG2645 is CGMCC No.30598; The preservation number of the Bifidobacterium lactis YG2013 is CGMCC No.27579.

2. The compound microbial agent according to claim 1, characterized in that, The derivatives of Akkermansia myxophilus YG2645 are its inactivated cells, extracts, cultures, culture supernatants, fermentation products, fermentation supernatants, or combinations thereof; And / or, Bifidobacterium lactis YG2013 is a live bacterium, and Bifidobacterium lactis YG2013 derivatives are its inactivated cells, extracts, cultures, culture supernatants, fermentation products, fermentation supernatants, or combinations thereof.

3. The compound microbial agent according to claim 1 or 2, characterized in that, The cell ratio of Bifidobacterium lactis YG2013 and / or its derivatives to Akkermansia xylophilus YG2645 and / or its derivatives is 1:(0.1~10) or 1:(0.25~4). And / or, including live Bifidobacterium lactis YG2013 and inactivated Akkermansia xylophilus YG2645 in a cell count ratio of 1:(0.1~10), wherein the cell count ratio is 1:(0.25~4) or 1:1; And / or, the concentration of inactivated Akkermansia myxophilus YG2645 cells is 1×10⁻⁶. 7 -1×10 13 TFU / mL or 1×10 9 -1×10 13 TFU / mL; And / or, the viable concentration of Bifidobacterium lactis YG2013 is 1×10⁻⁶. 7 -1×10 13 CFU / mL or 1×10 9 -1×10 13 CFU / mL.

4. A composition, characterized in that, Includes an effective amount of the compound microbial agent according to any one of claims 1 to 3.

5. The composition according to claim 4, characterized in that, The composition is a pharmaceutical composition, a food composition, or a feed composition; And / or, the concentration of inactivated Akkermansia myxophilus YG2645 cells is 1×10⁻⁶. 7 -1×10 13 TFU / mL or 1×10 9 -1×10 13 TFU / mL; And / or, the viable bacterial concentration of YG2013 is 1×10⁻⁶. 7 -1×10 13 CFU / mL or 1×10 9 -1×10 13 CFU / mL; And / or, the composition may further include other probiotics, postbiotics, prebiotics, antibacterial agents, immunomodulators, anticancer agents, anti-inflammatory agents, or combinations thereof.

6. The use of the compound microbial agent according to any one of claims 1 to 3, or the composition according to claim 4 or 5, in the preparation of products for any one or at least two of the following uses: 1) Improve heat stress resistance; 2) Extend lifespan; 3) Delays nerve degeneration; 4) Improve reproductive capacity; 5) Improves metabolic function; 6) Antioxidant stress; 7) Lowering lipids; 8) Delay aging.

7. The use of the compound microbial agent according to any one of claims 1 to 3, or the composition according to claim 4 or 5, in the preparation of products for any one or at least two of the following uses: I) Extend the lifespan of Alzheimer's patients; II) Prolonging the lifespan of patients with insulin resistance; III) Slowing the progression of Alzheimer's disease; IV) Prevention or treatment of Alzheimer's disease; V) Improves insulin resistance.

8. Use of Akkermansia myxophilus YG2645 and / or its derivatives in the preparation of products intended for any one or at least two of the following uses: 1) Improve heat stress resistance; 2) Extend lifespan; 3) Delays nerve degeneration; 4) Improve reproductive capacity; 5) Improves metabolic function; 6) Antioxidant stress; 7) Lowering lipids; 8) Delay aging.

9. Use of Akkermansia myxophilus YG2645 and / or its derivatives in the preparation of products intended for any one or at least two of the following uses: I) Extend the lifespan of Alzheimer's patients; II) Prolonging the lifespan of patients with insulin resistance; III) Slowing the progression of Alzheimer's disease; IV) Prevention or treatment of Alzheimer's disease; V) Improves insulin resistance.

10. The application according to claim 8 or 9, characterized in that, The derivatives of Akkermansia myxophilus YG2645 are its inactivated cells, extracts, cultures, culture supernatants, fermentation products, fermentation supernatants, or combinations thereof; And / or, the product is a medicine; And / or, the concentration of AKK2645 inactivated bacteria is 1×10⁻⁶. 7 -1×10 13 TFU / mL or 1×10 9 -1×10 13 TFU / mL.

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