Isolated ackermansiella muciniphila, composition comprising same and anti-aging and / or anti-oxidation use thereof

Abstract

The invention provides isolated Ackermansiella muciniphila, a composition containing the same and an anti-aging and / or anti-oxidation application of the Ackermansiella muciniphila. The Akkermansia muciniphila has the advantages that the average life and the longest life of the Akkermansia muciniphila in a nematode model can be prolonged, the movement activity, the swallowing function and the reproductive capacity of the Akkermansia muciniphila can be improved, the expression of insulin / IGF-1 pathway genes (daf-16, daf-2 and age-1), the expression of antioxidant genes (gst-4 and sod-3) and the expression of longevity related genes (sir-2.1) of the Akkermansia muciniphila can be up-regulated, and the antioxidant capacity and the heat stress tolerance of the Akkermansia muciniphila can be enhanced. It is shown that the Akkermansia muciniphila provided by the invention can be used for preparing or developing the anti-oxidation and anti-aging products, and the anti-oxidation and anti-aging products can be prepared from the Akkermansia muciniphila. The invention provides an efficient and safe solution for anti-aging and / or anti-oxidation.

Description

Technical Field

[0001] The present disclosure relates to the field of microorganisms, and more particularly to a novel strain of Akkermansia muciniphila obtained by separation, a composition containing the strain, and uses thereof. Background Art

[0002] Akkermansia muciniphila ( Akkermansia muciniphila ) is a mucin-degrading bacterium that normally colonizes the intestines of humans and many animals.

[0003] The screening of new strains of Akkermansia muciniphila faces great difficulties because it mainly exists in the intestines of humans or animals, is a strictly anaerobic microorganism, has extremely high requirements for nutrition and culture environment, and has a long growth cycle. In addition, when verifying the efficacy of the drug, the bacteria of this genus are difficult to verify through in vitro cell experiments because they are anaerobic; during in vivo animal experiments, the bacteria of this genus are difficult to maintain a stable number of viable bacteria due to the difficulty of culture and high degree of anaerobiosis, and there is a problem of insufficient repeatability. These in vivo and in vitro related technical difficulties have limited the discovery and application of new strains of Akkermansia muciniphila.

[0004] In addition, Akkermansia muciniphila grows slowly and has difficulty competing with other microbial strains, so there is a problem of difficulty in accessibility during the screening process.

[0005] The hydrophobicity of the AKK PROBIO strain disclosed in CN116925975B reached 31% at 60 min; the self-aggregation tended to stabilize at 52% at 20 h. Some literature suggests that AKK PROBIO has a weak ability to form biofilms, and in vitro test results show that AKK PROBIO has a low ability to colonize in the intestine (https: / / doi.org / 10.3390 / foods13030442, Section 3.1). If the AKK strain has a weak ability to colonize and adhere, it will limit its commercial product development.

[0006] Aging is a natural phenomenon, and the process is often accompanied by changes in antioxidant levels, organ tissues, and immune factors. Cytokines undergo complex changes, such as pro-inflammatory cytokines IL-6, IL-4, and TNF-α, which show an increasing trend. IL-6 and TNF-a are both considered to play an important role in the occurrence of senile diseases. With the development of genetics and molecular biology, research on the mechanism of biological aging has made gratifying progress. Through single gene mutation experiments on some model organisms, such as mice, fruit flies, and Caenorhabditis elegans, researchers have found that some genes can significantly increase the lifespan of these organisms by as much as 6 times.

[0007] More than a century ago, Nobel Prize winner Metchnikoff first emphasized the importance of intestinal microorganisms to human health and aging, and that the intestinal flora also changes with the aging process. With the in-depth study of the intestinal flora, Akkermansia muciniphila has attracted a lot of attention and research resources due to its beneficial effects found in many metabolic diseases, obesity, etc.

[0008] The AKK-PL001 strain disclosed in CN118909825A can extend the lifespan of nematodes by improving their antioxidant capacity. However, oxidative damage is only one of the factors of aging. Studies have shown that the aging process is also affected by a combination of factors such as mitochondrial function, telomeres, inflammation, genes, metabolic pathways, epigenetics, biological clocks, malfunction of nutritional perception, and dysbiosis of bacterial flora (Campisi, J., Kapahi, P., Lithgow, GJ et al. From discoveries inaging research to therapeutics for healthy ageing. Nature 571, 183–192(2019). https: / / doi.org / 10.1038 / s41586-019-1365-2). Among them, mitochondrial function is closely related to cell aging and brain aging. The aging of nerve cells and the brain is closely related to motor ability, forgetfulness, Parkinson's syndrome (PD), Alzheimer's disease and other diseases. Mitochondrial dysfunction is an important cause of PD. For example, the expression of Sir2 can induce the mitochondrial unfolded protein response (UPRmt), improve mitochondrial function and significantly extend the lifespan of nematodes. Studies have found that Sir2 overexpression significantly inhibited the degeneration of motor neurons in transgenic fruit flies with Parkinson's disease (PD) and significantly improved the motor ability of fruit flies. After UPRmt was inhibited, the protective effect of Sir2 was significantly weakened. Conclusion Sir2 has a neuroprotective effect on transgenic fruit flies with PD, and this neuroprotective effect is related to UPRmt (Qi Xinxin et al., The role of mitochondrial unfolded protein response in Sir2 inhibiting Parkinson's transgenic fruit flies, Journal of Anhui Medical University, 2016).

[0009] No studies have shown that Akkermansia muciniphila can regulate the expression of genes related to lifespan. Therefore, screening for new species or strains that can be effectively used to delay aging is a huge challenge, but also represents a huge unmet need. Summary of the invention

[0010] The present invention is based on the isolation of a novel strain of Akkermansia muciniphila ( Akkermansia muciniphila ).

[0011] The muciniphilic Akkermansia of the present invention can effectively delay aging and achieve healthy longevity, including extending lifespan, improving brain aging, and improving athletic ability and reproductive ability, by anti-inflammatory and anti-oxidation effects and regulating the expression of multiple genes such as insulin / IGF-1 signaling pathway genes, antioxidant genes, and mitochondrial unfolded protein response regulatory genes.

[0012] Akkermansia muciniphila of the present invention ( Akkermansia muciniphila ) can be used in the preparation or development of anti-aging, anti-oxidation and anti-inflammatory products. The present invention provides an efficient and safe solution for anti-aging, anti-oxidation and anti-inflammatory.

[0013] Compared with the existing muciniphilic Akkermansia strains, the new strain disclosed in the present invention has a stronger epithelial cell adhesion ability and can be well colonized in the human or animal intestines, thereby being able to prolong the residence time in the intestines and better exert its efficacy in the intestines; at the same time, due to the excellent colonization and adhesion ability of the strain of the present invention, it can effectively prevent the adhesion of pathogens, which is beneficial to maintaining intestinal homeostasis and health.

[0014] In a first aspect, the present disclosure provides a composition for anti-aging and / or anti-oxidation, the composition comprising an effective amount of Akkermansia muciniphila or a culture of the Akkermansia muciniphila, the Akkermansia muciniphila deposit number is GDMCC No: 63782, wherein the Akkermansia muciniphila includes any one of live bacteria, lyophilized bacteria and inactivated bacteria of the Akkermansia muciniphila, and the culture includes any one of the following A) to D): A) a fermentation broth of the Akkermansia muciniphila; B) the supernatant of the fermentation broth of the Akkermansia muciniphila; C) a killed product of fermentation broth of the Akkermansia muciniphila; D) The concentrated or dried product of any one of A) to C) above.

[0015] In a second aspect, the present disclosure provides use of the Akkermansia muciniphila, a culture of the Akkermansia muciniphila, or the composition of the first aspect in the preparation of anti-aging or anti-oxidation drugs, health products, or foods.

[0016] In a third aspect, the present disclosure provides use of the Akkermansia muciniphila, the culture of the Akkermansia muciniphila, or the composition of the first aspect in anti-aging or anti-oxidation for non-disease treatment purposes.

[0017] In a fourth aspect, the present disclosure provides a non-therapeutic method for anti-aging or anti-oxidation, wherein a subject in need thereof ingests an effective amount of the Akkermansia muciniphila, the culture of the Akkermansia muciniphila, or the composition of the first aspect. The non-therapeutic method for anti-aging or anti-oxidation can be a method for health care, regimen, beauty, etc.

[0018] In some embodiments, the Akkermansia muciniphila ( Akkermansia muciniphila ) and Akkermansia muciniphila ( Akkermansia muciniphila ) (GCF_000020225.1) strain has an alignment score (AF) of at least 90%, at least 90.5%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100%.

[0019] In some embodiments, the Akkermansia muciniphila ( Akkermansia muciniphila ) having a 16S rRNA sequence that is at least 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to the sequence shown in SEQ ID NO. 1.

[0020] In some embodiments, the Akkermansia muciniphila is an Akkermansia muciniphila species ( Akkermansia muciniphila )) and the new strain is different from Akkermansia muciniphila ( Akkermansia muciniphila) (GCF_000020225.1) has an average nucleotide identity (ANI) value of at least 95%, such as at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, 96.8%, at least 96.9%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, 97.8%, at least 97.9%, at least 97.1%, at least 97.2 %, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100%.

[0021] In some embodiments, the Akkermansia muciniphila ( Akkermansia muciniphila ) and Akkermansia muciniphila ( Akkermansia muciniphila ) strain MNH19250 has an average nucleotide identity (ANI) value of at least 95.1%, such as at least 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9 ... %, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

[0022] In some embodiments, the metabolites of the muciniphilic Akkermansia include short-chain fatty acids, and the short-chain fatty acids include at least one of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid and decanoic acid; preferably, the muciniphilic Akkermansia can produce high acetic acid and / or propionic acid. In some embodiments, the acetic acid content secreted by the muciniphilic Akkermansia is not less than 1000 μg / mL, 1100 μg / mL, 1200 μg / mL, 1300 μg / mL, 1400 μg / mL or 1500 μg / mL. In some embodiments, the acetic acid content of the muciniphilic Akkermansia is not less than 50 μg / mg, 60 μg / mg, 70 μg / mg, 80 μg / mg, 90 μg / mg, 95 μg / mg or 100 μg / mg.

[0023] In some embodiments, the propionic acid content of the Akkermansia muciniphila is not less than 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL or 230 μg / mL. In some embodiments, the propionic acid content of the Akkermansia muciniphila is not less than 5 μg / mg, 6 μg / mg, 7 μg / mg, 8 μg / mg, 9 μg / mg, 10 μg / mg, 11 μg / mg, 12 μg / mg, 13 μg / mg, 14 μg / mg or 15 μg / mg.

[0024] In some embodiments, the Akkermansia muciniphila ( Akkermansia muciniphila )MNH19250, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCCNo: 63782, and the deposit date is June 21, 2024.

[0025] In some embodiments, the culture of Akkermansia muciniphila includes a solid culture of Akkermansia muciniphila, a fermentation culture, a supernatant of a fermentation culture, an inactivated fermentation broth, or a concentrated or dried product of any one of the foregoing.

[0026] In some embodiments, the fermentation culture or fermentation culture supernatant is a fermentation culture or fermentation culture supernatant obtained under anaerobic culture conditions using a liquid culture medium.

[0027] In some embodiments, the composition is provided in liquid form or in solid form.

[0028] In some embodiments, the composition comprises 1×10 4 Up to 1×10 12 cfu / mL or 1×10 4Up to 1×10 12 cfu / mg of live Akkermansia muciniphila.

[0029] In some embodiments, the composition comprises 1×10 5 Up to 1×10 11 cfu / mL or 1×10 5 Up to 1×10 11 cfu / mg of live Akkermansia muciniphila.

[0030] In some embodiments, the composition comprises 1×10 6 Up to 1×10 10 cfu / mL or 1×10 6 Up to 1×10 10 cfu / mg of live Akkermansia muciniphila.

[0031] In some embodiments, the composition comprises 1×10 7 Up to 1×10 9 cfu / mL or 1×10 7 Up to 1×10 9 cfu / mg of live Akkermansia muciniphila.

[0032] In some embodiments, each mg of the composition comprises 1×10 3 Up to 1×10 17 colony forming units (CFU); for example, 1×10 4 Up to 1×10 12 pcs, 1×10 5 Up to 1×10 11 or 1×10 6 Up to 1×10 10 Colony forming units (CFU), for example, 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 ,7×10 3 , 8×10 3 ,9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 ,7×10 4 , 8×10 4 ,9×104 、1×10 5 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11, 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 ,7×10 11 , 8×10 11 ,9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 ,7×10 12 , 8×10 12 ,9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 ,7×10 13 , 8×10 13 ,9×10 13 or any value in between of colony forming units (CFU).

[0033] In some embodiments, the Akkermansia muciniphila in the composition is an attenuated bacterium, a killed bacterium, a freeze-dried bacterium, or an irradiated bacterium, for example, a heat-inactivated bacterium, preferably a pasteurized bacterium.

[0034] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0035] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral solution, suspension, emulsion, liquid preparation, sustained release preparation, nanoformulation, or microencapsulated capsule.

[0036] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0037] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers or excipients or auxiliary materials. The pharmaceutically acceptable auxiliary materials are well known to those skilled in the art.

[0038] In some embodiments, the auxiliary material may be at least one selected from a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor.

[0039] In some embodiments, the composition comprises one or more of a buffer (e.g., sodium bicarbonate, infant formula or sterilized human milk or other agents that allow bacteria to survive and grow (e.g., survive in the acidic environment of the stomach and grow in the intestinal environment)), a lyoprotectant, a preservative, a stabilizer, a binder, a compacting agent, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0040] In some embodiments, the composition further comprises one or more other active agents for anti-aging.

[0041] In some embodiments, the other active agent may be one or more of a probiotic, a prebiotic, or a combination thereof; Preferably, the probiotics are selected from one or more of lactic acid bacteria, lactobacillus, lactococci, butyrate-producing bacteria, bifidobacteria, thermophilic streptococci, faecal streptococci, and mesenteric leuconostoc.

[0042] Preferably, the probiotic is selected from at least one of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus krum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis, and Lactococcus creamer.

[0043] Preferably, the prebiotic is selected from inulin, mulberry leaf extract, berberine, ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast β-glucan, ginseng or its extract, nutritional compounds, biotin, polydextrose, fructooligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharide, manno-oligosaccharide (MOS), inulin rich in fructooligosaccharide, oligo-glucose, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS) and any combination thereof. In some embodiments, the other active agent can be an antioxidant, for example, vitamin C, vitamin E, vitamin A and its precursors (such as β-carotene), tea polyphenols, anthocyanins, resveratrol, coenzyme Q10, ergothioneine, lipoic acid, selenium, zinc, and any combination thereof.

[0044] In some embodiments, the other active agents can be anti-aging drugs, such as anti-inflammatory drugs (such as NSAIDs, glucocorticoids); metabolic regulators (such as metformin, rapamycin); growth hormone, DHEA; drugs for treating or preventing aging (such as Senolytics, NAD+ enhancers); immunomodulators (such as immunoenhancers, immunosuppressants); etc.

[0045] In some embodiments, the composition can be formulated as a frozen composition, such as a frozen composition prepared by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0046] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0047] In some embodiments, the strain in the composition is freeze-dried or spray-dried. In some embodiments, the strain in the composition is electrostatically spray-dried. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is alive. In some embodiments, the strain in the composition is freeze-dried or spray-dried and can partially or completely colonize the intestine. In some embodiments, the strain is reconstituted before administration. In some cases, the reconstitution is carried out by using a diluent as described herein.

[0048] In some embodiments, the composition can be administered alone or in combination with a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.

[0049] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric preparation. In some embodiments, the enteric preparation is a dosage form with an enteric coating. For example, the enteric preparation can be an enteric granule, an enteric tablet, or an enteric capsule. In some embodiments, the composition is a capsule. In some embodiments, the capsule is a hard capsule or a soft capsule; or the capsule is a sustained release capsule, a controlled release capsule, or an enteric capsule, or the capsule can be a microencapsulated capsule, or a microcapsule.

[0050] In some embodiments, the composition is a medicine, or a health product or a food.

[0051] In some embodiments, the composition is an infant-suitable dosage form, a child-suitable dosage form, or an adult-suitable dosage form. In some embodiments, the composition is an enteral dosage form or a parenteral dosage form.

[0052] As used herein, microbial population generally refers to the microbial population consisting of a single strain, species or genus, which may be the case when the subgroup culture group is separated and purified from such strains, species or genus. Therefore, for a given microbial population, if cultivated from a separated microbial species or strain, such a population will be referred to as purified or substantially pure in this article. The resulting population can be at least 80% purity for the microbial species or strain, at least 90% purity, at least 95% purity, at least 98% purity, at least 99% purity, at least 99.5% purity, or at least 99.9% purity relative to other microbial species or strains in this particular population. On the contrary, the level of non-desired strains in any specific desired microbial population will be less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5% or less than 0.1%. For example, the impurity level in the purified population aggregate, such as other undesirable microbial strains or species, can be the above-mentioned level of each desired population in proportion or less than the above-mentioned level. Where a composition comprises a consortium of multiple populations of microorganisms, each population may have the above-described purity, either prior to its incorporation into the composition, or when the consortium is measured in aggregate.

[0053] The pharmaceutical compositions of the present disclosure may also include cellular components, metabolites, secreted molecules and compounds metabolized by Akkermansia muciniphila, and the like. Cellular components or cell fractions, metabolites or secreted compounds may be obtained, for example, by recovering the supernatant of Akkermansia muciniphila culture or by extracting from Akkermansia muciniphila culture; may correspond to components in isolated form from Akkermansia muciniphila, or any mixture of one or more components from Akkermansia muciniphila.

[0054] The Akkermansia muciniphila isolated and obtained in the present invention ( Akkermansia muciniphila ) MNH19250 has strong self-aggregation and hydrophobicity, easily adheres to epithelial cells, and has good pH, NaCl and bile salt tolerance, so it can quickly and stably colonize in the human or animal intestines.

[0055] The Akkermansia muciniphila isolated and obtained in the present invention ( Akkermansia muciniphila ) MNH19250 can also synthesize a large amount of short-chain fatty acids, so it can effectively resist aging.

[0056] In some embodiments, anti-aging includes at least one of the following effects: extending the lifespan of the subject; improving the subject's motor activity, swallowing function or reproductive ability, such as the decline in motor activity, swallowing function and / or reproductive ability caused by aging; regulating the expression of insulin / IGF-1 signaling pathway genes, antioxidant genes or longevity-related genes; enhancing the subject's tolerance to heat stress or oxidative stress.

[0057] In some embodiments, the Akkermansia muciniphila of the present invention ( Akkermansia muciniphila ), or the composition can extend the maximum lifespan of a subject and / or the average lifespan of a subject population.

[0058] In some embodiments, the Akkermansia muciniphila of the present invention ( Akkermansia muciniphila ), or the composition can significantly improve the subject's motor activity, movement rate and physiological function.

[0059] In some embodiments, the Akkermansia muciniphila of the present invention ( Akkermansia muciniphila ), or the composition can enhance the reproductive capacity of the subject.

[0060] In some embodiments, the Akkermansia muciniphila of the present invention ( Akkermansia muciniphila ), or the composition can regulate the expression of insulin / IGF-1 pathway related genes or their homologous genes in mammals, antioxidant genes or their homologous genes in mammals, mitochondrial unfolded protein response regulatory genes or their homologous genes in mammals. For example, the insulin / IGF-1 signaling pathway genes include daf-16, daf-2, age-1 or their homologous genes in mammals, the antioxidant genes include sod-3, gst-4 or their homologous genes in mammals, and the mitochondrial unfolded protein response regulatory genes include sir-2.1.

[0061] In some embodiments, the Akkermansia muciniphila described in the present invention regulates the expression of daf-16, daf-2, age-1, gst-4, sod-3, and sir-2.1.

[0062] In some embodiments, the Akkermansia muciniphila of the present invention ( Akkermansia muciniphila ), or the composition can significantly improve antioxidant capacity. In some embodiments, aging also includes aging-related diseases, such as Parkinson's syndrome, Alzheimer's disease, etc. In some embodiments, the muciniphilic Akkermansia ( Akkermansia muciniphila ), or the composition can delay the development of aging-related diseases such as Parkinson's syndrome and Alzheimer's disease.

[0063] Beneficial effects of the present invention Akkermansia muciniphila of the present invention ( Akkermansia muciniphila) has high auto-aggregation ability and strong surface hydrophobicity, can effectively adhere to intestinal epithelial cells, prolong intestinal residence time, and improve treatment effect. The strain adapts to the intestinal environment and has high stability. The muciniphilic Akkermansia ( Akkermansia muciniphila ) in the nematode model to extend the average lifespan and the maximum lifespan, improve motor activity, swallowing function and reproductive ability, regulate the expression of insulin / IGF-1 pathway-related genes (such as daf-16, daf-2, age-1), antioxidant genes (such as gst-4, sod-3), mitochondrial unfolded protein response regulatory genes (such as sir-2.1), and enhance antioxidant capacity and heat stress tolerance. This shows that the muciniphilic Akkermansia ( Akkermansia muciniphila ) can be used in the preparation or development of anti-oxidation and anti-aging products. The present invention provides an efficient and safe solution for anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The embodiments will be described below in conjunction with the accompanying drawings so that the above and other aspects and advantages of the present invention will become apparent and easily understood.

[0065] Figure 1 : Shows the colony morphology photos of strains MNH19250, MNH20651, and MNH26240.

[0066] Figure 2 : Gram staining photographs of strains MNH19250, MNH20651, and MNH26240 are shown.

[0067] Figure 3 : Electron micrographs of strains MNH19250, MNH20651, and MNH26240 are shown.

[0068] Figure 4 : Shows the results of the tolerance of strain MNH19250 to different pH values. The horizontal axis is the pH value and the vertical axis is the survival percentage.

[0069] Figure 5 : Shows the tolerance results of strain MNH19250 to different concentrations of NaCl.

[0070] Figure 6 : Shows the tolerance results of strain MNH19250 to different concentrations of bile salts.

[0071] Figure 7 : Shows the 6h autoaggregation ability of strain MNH19250.

[0072] Figure 8 : The phylogenetic tree of strain MNH19250 is shown.

[0073] Figure 9: A bar graph showing the relative fluorescence values ​​of IFNβ expression in the presence of strain MNH19250.

[0074] Figure 10A : Shows the survival curves of nematodes under different treatment conditions. Compared with the control group fed with Escherichia coli OP50, supplementation with pasteurized or live bacteria MNH19250 significantly extended the maximum lifespan of nematodes.

[0075] Figure 10B : Shows the average lifespan of nematodes under different treatment conditions. Compared with the control group fed with Escherichia coli OP50, pasteurized MNH19250 significantly increased the average lifespan of nematodes.

[0076] Figure 11A : The effects of different treatments on the movement activity of nematodes are shown. The activity score shows that supplementation with MNH19250 significantly reduced the proportion of dead (category D) and immobile (category C) nematodes, and increased the proportion of nematodes with normal movement (category A).

[0077] Figure 11B : The effects of different treatments on the movement activity of nematodes are shown. The activity score shows that supplementation with MNH19250 significantly reduced the proportion of dead (category D) and immobile (category C) nematodes, and increased the proportion of nematodes with normal movement (category A).

[0078] Figure 11C :Shows the effects of different treatments on the movement rate of nematodes. The significant increase in movement rate indicates improved motor activity.

[0079] Figure 11D : Shows the effects of different treatments on the physiological functions of nematodes. The increase in swallowing pump rate shows that MNH19250 improves the feeding function of nematodes.

[0080] Figure 12A : Shows the effect of MNH19250 supplementation on the life cycle of nematodes, and supplementation with MNH19250 significantly shortens the life cycle of nematodes.

[0081] Figure 12B : Shows the effect of MNH19250 supplementation on nematode body size. Nematode body size measurements showed that MNH19250 significantly increased the projected area of ​​nematodes, indicating that it promoted growth.

[0082] Figure 13A : The effect of MNH19250 on the reproductive capacity of nematodes was shown. Supplementation of MNH19250 significantly increased the daily egg laying of nematodes, indicating that it enhances reproductive capacity.

[0083] Figure 13B: The effect of MNH19250 on the reproductive capacity of nematodes was shown. Supplementation of MNH19250 significantly increased the total number of offspring in nematodes, indicating that it enhances reproductive capacity.

[0084] Figure 14 : The effects of MNH19250 on anti-aging-related gene pathways in nematodes were shown. Pasteurized inactivated MNH19250 significantly upregulated the expression of insulin / IGF-1 pathway genes (daf-16), antioxidant genes (gst-4, sod-3) and mitochondrial unfolded protein response regulatory genes (sir-2.1).

[0085] Figure 15A : The effect of MNH19250 on nematode resistance to heat stress was shown. Supplementation of MNH19250 (pasteurized) significantly prolonged the survival time of nematodes under heat stress (35°C).

[0086] Figure 15B : The results show the effect of MNH19250 on nematodes' resistance to heat stress. Supplementation with MNH19250 (live bacteria) significantly prolonged the survival time of nematodes under heat stress (35°C).

[0087] Figure 15C : The effects of MNH19250 on the antioxidant capacity of nematodes are shown. MNH19250 (pasteurized or live) can significantly prolong the lifespan of nematodes under ultraviolet radiation stress and improve the survival rate of nematodes against ultraviolet aging.

[0088] Figure 16A : Shows different Akkermansia The influence of strains on the lifespan of nematodes: MNH19250, MNH39567, MNH19631, MNH26240, and MNH20651 strains all significantly extended the maximum lifespan of nematodes, among which MNH19250 had the best effect.

[0089] Figure 16B : Shows different Akkermansia The influence of strains on the lifespan of nematodes: MNH19250, MNH39567, MNH19631, MNH26240, and MNH20651 strains all significantly extended the average lifespan of nematodes, among which MNH19250 had the best effect.

[0090] Figure 17 : Shows different Akkermansia The influence of strains on the expression of anti-aging genes in nematodes, MNH19250, MNH39567, MNH19631, MNH26240, and MNH20651 strains all significantly upregulated the expression of insulin / IGF-1 pathway genes (daf-16, daf-2, age-1), antioxidant genes (gst-4, sod-3), and longevity-related genes (sir-2.1).

[0091] Figure 18A : Shows different Akkermansia The effect of strains on the antioxidant capacity of nematodes. Pasteurized MNH19250 and MNH19631 significantly prolonged the survival time of nematodes under oxidative stress.

[0092] Figure 18B : Shows different Akkermansia The effect of strains on the antioxidant capacity of nematodes. Pasteurized MNH19250, MNH19631, MNH39567, and MNH26240 significantly prolonged the lifespan of nematodes under ultraviolet irradiation stress and increased the survival rate of nematodes against ultraviolet aging.

[0093] Deposit of strains The strain Akkermansia muciniphila MNH19250 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit name of Akkermansia muciniphila MNH19250 and the deposit number of the strain being GDMCC No: 63782. The deposit date is June 21, 2024.

[0094] The strain Akkermansia muciniphila MNH26240 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit name of Akkermansia muciniphila MNH26240 and the deposit number of the strain being GDMCC No: 65547. The deposit date is November 27, 2024.

[0095] The strain Akkermansia muciniphila MNH20651 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit name of Akkermansia muciniphila MNH20651 and the deposit number of the strain being GDMCC No: 65542. The deposit date is November 27, 2024.

[0096] The strain Akkermansia muciniphila MNH19631 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit name of Akkermansia muciniphila MNH19631 and the deposit number of the strain being GDMCC No: 65809. The deposit date is January 16, 2025.

[0097] The strain Akkermansia muciniphila MNH39567 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit name of Akkermansia muciniphila MNH39567 and the deposit number of the strain being GDMCC No: 65810. The deposit date is January 16, 2025. DETAILED DESCRIPTION

[0098] The present invention isolates a new strain of Akkermansia muciniphila, with a deposit number of GDMCC No: 63782, and identifies it using traditional classification methods and molecular biology methods. The identification results show that the strain is a new strain of Akkermansia muciniphila. Further, the present invention studies the biochemical properties and therapeutic uses of the strain.

[0099] It is known in the art that bacterial species can be classified and identified by traditional classification methods and molecular biological methods. Traditional classification methods include, but are not limited to, cell morphology observation, Gram staining, flagella staining, various metabolic experiments, etc. Molecular biological methods include, but are not limited to, ribosomal RNA sequence determination methods, determination methods based on whole genome sequencing, etc.

[0100] As used herein, the term "prebiotic" may be a general term referring to chemicals and / or ingredients that can affect the growth and / or activity of microorganisms in a host (e.g., can allow for specific changes in the composition and / or activity of a microbiome).

[0101] The terms "subject," "subject," "individual," "host," and "patient" are used interchangeably herein to refer to any animal subject, including: humans, mammals, laboratory animals, livestock, and domestic pets.

[0102] Compositions or preparations disclosed herein can be used as pharmaceutical preparations, therapeutic compositions, dietary supplements, nutritional supplements, medical probiotics or medical foods. In some cases, the composition is applied in the form of a pharmaceutical preparation. In some cases, the composition is applied in the form of a nutritional supplement. In some cases, the composition is applied in the form of a dietary supplement. In some cases, the composition is applied in the form of a medical food. In some cases, the composition is applied in the form of a medical probiotic. In some cases, the composition (e.g., dietary supplements, nutritional supplements, medical probiotics or medical foods) can be orally administered, for example as a capsule, pill or tablet.

[0103] In the context of the present invention, the term "polypeptide" is equivalent to "protein". A polypeptide has a specific amino acid sequence. A "variant" of a polypeptide of the present invention preferably has an amino acid sequence that has at least 50% sequence identity with a polypeptide of the present invention.

[0104] The term "homeostasis" refers to the mechanisms of processes that contribute to maintaining a balanced internal state in an organism, eg, glucose regulation homeostasis, triglyceride homeostasis, cholesterol homeostasis.

[0105] 16S rRNA is a ribosomal RNA of prokaryotes. The 16S rRNA gene consists of a variable region and a conserved region. The conserved region is common to all bacteria, while the variable region has different degrees of difference between different bacteria. By comparing the 16S rRNA gene sequences of bacteria, according to the base number of sequence differences and their evolutionary distance, an evolutionary tree can be drawn. When the identity between the 16S rRNA gene sequences of two strains is less than 98.65%, they can be judged to belong to different species (see Kim, M., Oh, H.-S., Park, S.-C.,&Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology, 64(Pt 2), 346–351, and Liu, C., Du, M.-X., Abuduaini,R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J.(2021). Enlightening the taxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), P23).

[0106] The "identity" between two nucleic acid molecule sequences can be determined using known computer algorithms, such as the "FASTA" program, the GCG program package, BLASTN or FASTA. Commercial or publicly available programs may also be, for example, the DNAStar "MegAlign" program.

[0107] The second generation sequencing technology can also be used to identify bacterial species based on whole genome sequencing, making the identification results more accurate. The average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two bacterial genomes. The alignment fraction (AF) refers to the fraction of orthologous genes shared between two genomes. The ANI value can be calculated using methods such as BLAST. In the field of bacterial taxonomy, it is generally believed that the ANI value needs to reach more than 95% to be identified as belonging to the same species (Jain C, Rodriguez-R LM, Phillippy AM, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[J]. Nature Communications, 2018, 9 (1): 5114.).

[0108] Various existing mature ANI and AF value calculation tools can be used, such as local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0109] Using the above method, a person skilled in the art can determine whether an isolated strain belongs to the species of Akkermansia muciniphila identified by the inventors. Akkermansia muciniphila) (GCF_000020225.1) has an average nucleotide identity ANI value of at least 95%, such as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.9 When the content of the bacterial strains is 7.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, it can be determined that they belong to the same species.

[0110] For another example, when its 16S rRNA sequence has at least 98.65% identity with the sequence shown in SEQ ID NO. 1, for example, at least 98.7%, at least 98.8%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100%, it can be determined to belong to the same bacterial species.

[0111] The alignment fraction (AF) refers to the fraction of orthologous genes shared between two genomes. Based on ANI>95%, the researchers proposed further using the alignment fraction (AF, i.e. the fraction of orthologous genes shared between two genomes) as an additional threshold to restrict species to ensure that the ANI value is not based on a small set of conserved genes. There is no unified standard for the AF threshold. Some literature uses 65% (Parks, DH, Chuvochina, M., Chaumeil, PA. et al. A complete domain-to-species taxonomy for Bacteria and Archaea. Nat Biotechnol 38, 1079–1086(2020). ), and some literature uses 30% (Zeng, S., Patangia, D., Almeida, A. et al. Acompendium of 32,277 metagenome-assembled genomes and over 80 million genesfrom the early-life human gut microbiome. Nat Commun 13, 5139 (2022). ). In short, the AF value can be used as an auxiliary additional threshold.

[0112] A "strain" refers to a member of a bacterial species that has a genetic characteristic that allows it to be distinguished from closely related members of the same bacterial species. The genetic characteristic can be the total or partial absence of at least one gene, the total or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosomal binding site), the absence ("cure" of at least one native plasmid), the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosomal binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In the case of a strain that gains or loses antibiotic resistance (compared to another strain of the same species) or gains or loses a biosynthetic ability (e.g., an auxotrophic strain), the strains can be distinguished by selection or counter-selection using antibiotics or nutrients / metabolites.

[0113] "Supernatant" or "supernatant" within the meaning herein refers to the culture supernatant of the bacterial strain according to the present disclosure, optionally comprising compounds and / or cell debris of said strain, and / or metabolites and / or molecules secreted by said strain.

[0114] Compositions can be prepared using the Akkermansia muciniphila described herein, for example, by using a pharmaceutically acceptable excipient. The pharmaceutical composition comprises a pharmaceutically effective amount of the Akkermansia muciniphila, for example, the Akkermansia muciniphila with a deposit number of GDMCC NO: 63782. Similarly, the Akkermansia muciniphila with a deposit number of GDMCC NO: 63782 can also be prepared into a pharmaceutical composition, for example, by using a pharmaceutically acceptable excipient, which comprises a pharmaceutically effective amount of the Akkermansia muciniphila.

[0115] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavors.

[0116] The compositions herein can be formulated into any form suitable for enhancing the abundance of Akkermansia muciniphila in a subject. The compositions can be administered orally (e.g., by oral gavage), intramuscularly, by inhalation, intracranially, intralymphatically, intraocularly, intraperitoneally, intrapleurally, intrathecally, intratracheally, intrauterinely, intravascularly, intravenously, intravesically, intranasally, intragastrointestinally, by bile infusion, by cardiac infusion, anteriorly, rectally, subcutaneously, sublingually, topically, intravaginally, transdermally, by ureteral or urethral routes.

[0117] Examples of dosage forms suitable for the composition herein include, but are not limited to, tablets, aerosols, chewable sticks, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, and concentrates.

[0118] In some embodiments, the composition is a sugar-coated tablet, a gel capsule, a gel, an emulsion, a tablet, a sheet capsule, a hydrogel, a nanofiber gel, an electrospun fiber, a food bar, a candy, a fermented milk, a fermented cheese, a chewing gum, a powder or a toothpaste, etc.

[0119] In some embodiments, administration can also be by inclusion in a subject's diet, such as inclusion in a functional food for humans or companion animals.

[0120] The compositions provided herein may include a pharmaceutically acceptable excipient, diluent or carrier. The pharmaceutically acceptable excipient, diluent or carrier is well known in the art.

[0121] In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is lyophilized. In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is spray-dried. In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is lyophilized or spray-dried and is alive. In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is lyophilized or spray-dried and can partially or completely colonize in the intestine. In some embodiments, the lyophilized Akkermansia muciniphila is reconstituted before administration. In some embodiments, the reconstitution is performed using a diluent described herein.

[0122] In some embodiments, the compositions of the present disclosure are administered orally. Oral administration may involve swallowing, thereby allowing the composition to enter the gastrointestinal tract, and / or administration through the mouth, tongue, or sublingually.

[0123] In some embodiments, the composition is prepared by freeze drying or spray drying. The composition disclosed herein includes a pharmaceutical composition, a health product or a food.

[0124] The subject of the present disclosure may be a human or an animal, and the animal includes but is not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0125] The pharmaceutical composition disclosed herein can be used for anti-aging.

[0126] Amuc-1100 and Amuc_1631 (P9) proteins are derived from Akkermansia muciniphila ( Akkermansia muciniphila The outer membrane proteinAmuc_1100 of . Amuc-1100 protein can interact with Toll-like receptor 2 (TLR2) of host cells, affecting intestinal health and immune regulation (Wang J, Xu W, Wang R, et al., The outer membrane proteinAmuc_1100 of Akkermansia muciniphila promotes intestinal 5-HT biosynthesisand extracellular availability through TLR2 signaling. [J]. Food & function,2021, 12(8): 3597-3610. DOI:10.1039 / d1fo00115a.). Amuc-1100 has the effect of promoting GLP-1 secretion, which helps to improve blood sugar control and metabolic disorders; "YOON HS, CHO CH, YUN MS, et al., Akkermansia muciniphilasecretes a glucagon-like peptide-1-inducing proteinthat improves glucose homeostasis and ameliorates metabolic disease in mice[J]. Nature Microbio-logy, 2021, 6: 563-573";Amuc-1100 remains stable during pasteurization, and can inhibit the expression of intestinal CB1 receptors and enhance the expression of tight junction proteins; "Ding, G.,Yang, X., Li, Y.et al., Gut microbiota regulates gut homeostasis, mucosal immunity andinfluences immune-related diseases. Mol Cell Biochem (2024). https: / / doi.org / 10.1007 / s11010-024-05077-y".

[0127] Amuc_1631 (P9) protein can promote the secretion of GLP-1 in intestinal L cells and has the potential to treat diabetes, obesity and other metabolic diseases (Wenxuan D, Yuchen Z, Xinyuan Z, et al., Heterologous expression of P9 from Akkermansia muciniphila increases the GLP-1 secretion of intestinal L cells[J]. World Journal of Microbiology&Biotechnology, 2024(7):40. DOI:10.1007 / s11274-024-04012-z.). P9 protein also exhibits a strong anti-inflammatory effect and can regulate intestinal barrier function, thus playing a role in inflammatory bowel disease. P9 protein enhances intestinal barrier function by increasing the number of goblet cells, mucus production, and differentiation of stem cells into secretory cells."Patrice D. Cani, Clara Depommier, Muriel Derrien, et al., Akkermansia muciniphila: paradigm for next-generation beneficial microorganisms. Nature Reviews Gastroenterology&Hepatology volume 19, pages 625-637 (2022)".

[0128] Therefore, the Akkermansia muciniphila ( Akkermansia muciniphila ) can also affect or modulate immune signaling and / or affect intestinal barrier function and / or affect glucose homeostasis and / or cholesterol homeostasis and / or triglyceride homeostasis.

[0129] The anti-aging mentioned in the present invention includes "treating, preventing or slowing down aging", which can also be understood as "delaying aging", "preventing or slowing down signs of aging", "extending life" and other similar meanings. In this article, the above terms can be used interchangeably. "Treatment, prevention or slowing down of diseases related to aging" is also included in the scope of the present invention. "Diseases related to aging" include, for example, Parkinson's disease, etc.

[0130] The application of the strain, bacterial agent or its fermentation product or fermentation extract in the above aspects also falls within the protection scope of the present invention.

[0131] The aging research model animal Caenorhabditis elegans N2 used in the present invention is a lifespan research model animal and is a simple invertebrate model with the advantages of short lifespan, simple living environment, large-scale synchronization, clear genetic background, and many genes being highly conserved / homological with mammals (including humans) in evolution. The aging mechanism can be studied from the behavioral, molecular and genetic levels, and several major theories of the aging mechanism have been confirmed in nematodes. Therefore, Caenorhabditis elegans is a common model organism for lifespan and aging research, and products that have lifespan extension and anti-aging effects on nematodes are generally believed to have the same effects on humans.

[0132] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature of the art or in accordance with the product or instrument instructions are used. All reagents or instruments are indicated by the manufacturer and can be purchased commercially. Example

[0133] The liquid MM01 culture medium involved in the embodiment is composed of: peptone 5g / L, tryptic casein 5g / L, yeast powder 10g / L, beef extract 5g / L, glucose 5g / L, K2HPO4 2g / L, sodium acetate 2g / L, Tween 80 1mL / L, heme 5mg / L, L-cysteine ​​hydrochloride 0.5g / L, vitamin K1 1μL / L, inorganic salt solution 8ml / L (each 1L includes calcium chloride 0.25g, K2HPO4 1g, KH2PO4 1g, magnesium sulfate 0.5g, sodium bicarbonate 10g, sodium chloride 2g).

[0134] The solid MM01 medium involved in the embodiment is composed of: peptone 5g / L, tryptic casein 5g / L, yeast powder 10g / L, beef extract 5g / L, glucose 5g / L, K2HPO4 2g / L, sodium acetate 2g / L, Tween 80 1mL / L, heme 5mg / L, L-cysteine ​​hydrochloride 0.5g / L, vitamin K1 1μL / L, inorganic salt solution 8ml / L (each 1L includes calcium chloride 0.25g, K2HPO4 1g, KH2PO4 1g, magnesium sulfate 0.5g, sodium bicarbonate 10g, sodium chloride 2g), agar 15g / L.

[0135] Anaerobic blood agar plates were purchased from Huankai Microorganisms, with the following formula: 10 g / L casein pancreatic digest, 3 g / L heart pancreatic digest, 1 g / L corn starch, 5 g / L meat peptic digest, 5 g / L yeast extract powder, 5 g / L sodium chloride, 15 g / L agar, 50-100 mL / L sterile defibrinated sheep blood, pH 7.3±0.2.

[0136] The above culture medium can be prepared by conventional preparation methods and sterilization methods.

[0137] The experimental data were plotted and statistically analyzed using GraphPad Prism 9.3.1 software. Unpaired t test was used for pairwise comparisons; One-Way ANOVA was used for comparisons between more than three groups; significance was indicated by *, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****p < 0.0001, and no significance was not marked.

[0138] Example 1: Isolation and identification of strains

[0139] 1.1 Isolation and purification of strain MNH19250 An intestinal strain numbered MNH19250 was isolated from a sample of a healthy male volunteer in Guangzhou, Guangdong, China. Intestinal strains numbered MNH26240, MNH20651, MNH19631, and MNH39567 were isolated from samples of healthy volunteers in different regions of China. The isolation method adopted the conventional strain isolation method, using gradient dilution, and then picking a single colony for isolation and culture, and then purifying the strain and anaerobic culture at 37°C. The pure culture strain was prepared into a 20% glycerol / water-bacteria solution and stored at -80°C.

[0140] Specifically, the strain isolation method is as follows: The donor takes 5 g of sample and puts it into a sample collection and storage tube. After shaking and homogenization, the processed sample is placed in an ice box and sent to the laboratory for strain isolation within 24 hours.

[0141] Dispense physiological saline in a biosafety cabinet, 9 mL / tube; prepare anaerobic blood agar plates for strain isolation culture medium, and transfer them to the anaerobic workstation 24 hours in advance, marking the sample information, culture medium type, isolation date, etc.

[0142] Take a new sample and place it in the anaerobic workstation and shake it on a vortex shaker for 1 min to mix it. Pipette 1 mL of the sample into 9 mL of normal saline and mix it for 10 -1 dilution, and then serially diluted to 10 -6 Dilution solution for later use.

[0143] Take 10 -6 Drop the diluted solution onto the anaerobic blood agar plate at a rate of 100 μL / plate. Spread evenly. After the surface of the plate is dry, invert the plate and culture at 37°C for 3 to 5 days.

[0144] Observe the growth of the strain on the isolation medium (anaerobic blood agar plate) and pick a single colony with a sterilized toothpick for strain purification. The purified strain is placed at 37°C for anaerobically cultured. The pure culture strain is prepared into a 20% glycerol / water-bacteria solution and stored at -80°C.

[0145] 1.2 Morphological characteristics of strain MNH19250 Culture and morphological characteristics The strain MNH19250 was inoculated into MM01 medium and cultured anaerobically at 37℃ for 72h. Visible colonies were formed on the MM01 plate medium. The colonies were round, with regular and smooth edges, about 0.5mm in diameter, light yellow, and translucent. The strain was Gram-negative. Microscopic morphology showed that it had no flagella, was non-motile, rod-shaped, and about 0.5~1µm×1.5~3µm in size. For photos of the colony morphology of strain MNH19250 after 72h of culture on MM01, MNH20651, and MNH26240 on anaerobic blood plates, see Figure 1Gram staining photos of strains MNH19250, MNH20651 and MNH26240 are available at Figure 2 , electron microscope photos see Figure 3 .

[0146] The strains MNH20651 and MNH26240 were inoculated on anaerobic blood agar plates. After anaerobic culture at 37°C for 72 hours, visible colonies were formed on the blood agar plates. The colonies were round, with regular and smooth edges, white and translucent. The strains were Gram-negative. Microscopic morphological observation revealed that the strains had no flagella, were non-motile, and were rod-shaped.

[0147] 1.3 Physiological characteristics of strain MNH19250 The strain MNH19250 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow in the pH range of 6.0 to 9.0, with the optimal growth pH being around 8.0 (see the results of strain tolerance to different pH values ​​for details). Figure 4 ); growth was significantly inhibited on medium containing more than 2% (w / v) NaCl (for the results of strain tolerance to different concentrations of NaCl, see Figure 5 ); strain MNH19250 can survive and grow in the bile salt concentration range of 0%~0.25% (w / v), and its growth is significantly inhibited when the bile salt concentration is greater than or equal to 0.3% (for the results of strain tolerance to different concentrations of bile salt, see Figure 6 ).

[0148] 1.4 Results of biochemical identification of strain MNH19250 by API 20A API 20A (purchased from BioMérieux, CN2030025) was used to perform biochemical identification of strain MNH19250. For specific experimental operations, please refer to the conventional API reagent operation guide. The culture conditions of strain MNH19250 were: 37°C, anaerobic. The experimental results are shown in Table 1.

[0149]

[0150] MNH19250 can utilize glucose, lactose, and mannose to ferment and produce acid. Therefore, during the fermentation or cultivation of strain MNH19250, glucose, lactose, mannose, and their derivatives can be used as carbon sources.

[0151] MNH19250 can hydrolyze esculin (ESC), that is, it can synthesize β-glucosidase. β-glucosidase belongs to the cellulase class and can hydrolyze cellobiose and short-chain cellooligosaccharides to produce glucose.

[0152] MNH19250 can hydrolyze gelatin (GEL), that is, it can synthesize protease. The function of protease is to convert the protein that cannot be absorbed into polypeptides or amino acids that can penetrate the bacteria. This enzyme is mainly an extracellular enzyme, which can hydrolyze gelatin into polypeptides first, and then further hydrolyze it into amino acids, losing the gel property and liquefying, allowing the black substance in the reaction well to diffuse, and a positive reaction is shown.

[0153] 1.5 Minimum inhibitory concentration test of antibiotics for strain MNH19250 The minimum inhibitory concentration of antibiotics for strain MNH19250 was determined using E-test (purchased from Liofilchem) paper. The test results are shown in Table 2.

[0154]

[0155] The results showed that MNH19250 was sensitive to ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampicin, imipenem, penicillin, and cefquinome. It can be seen that MNH19250 is sensitive to most types of antibiotics, and the risk of long-term use of MNH19250 leading to antibiotic resistance in subjects is low.

[0156] 1.6 Determination of the autoaggregation ability of strain MNH19250 The strain MNH19250 was inoculated in MM01 liquid culture medium and cultured anaerobically at 37°C for 48 hours. 20 mL of the fermentation broth was collected by centrifugation (4500 rpm, 4°C, 10 min), washed twice with sterile PBS (pH 7.2), resuspended in PBS buffer, and the OD600 value was adjusted to 0.5, recorded as A0.

[0157] The bacterial solution was placed at 37°C (directly in a cuvette), and its OD600 value was measured every 30 minutes, recorded as At, for 6 hours, and repeated three times.

[0158] The auto-aggregation ability of the strain was calculated according to the following formula: auto-aggregation ability of the strain (%) = [1-(At / A0)] × 100%.

[0159] The 6h autoaggregation capacity of MNH19250 was 54.21% (see Figure 7 ), compared with other strains in the literature, the ability of automatic aggregation is more than 20% stronger (Xin Ma 1, Meng Tian2, Xueping Yu, et al., Foods. 2024, Jan30; 13(3):442. Characterization and Preliminary Safety Evaluation of Akkermansia muciniphilaPROBIO (https: / / doi.org / 10.3390 / foods13030442); Figure 1d, A. muciniphila PROBIO has an auto-aggregation ability of about 30% (8h). Strains with strong auto-aggregation ability have stronger epithelial cell adhesion ability and can better colonize in the human intestine, thereby exerting a probiotic effect.

[0160] 1.7 Determination of surface hydrophobicity of strain MNH19250 The strain MNH19250 was inoculated into MM01 liquid culture medium and cultured anaerobically at 37°C for 48 hours. 20 mL of the fermentation broth was collected by centrifugation (4500 rpm, 4°C, 10 min), washed twice with sterile PBS (pH 7.2), resuspended in PBS buffer, and the OD600 value was adjusted to 0.5, recorded as A0.

[0161] 4 mL of xylene was added to 4 mL of the resuspension, the two-phase system was vortexed for 5 min, incubated at room temperature for 1 h, and then the xylene phase was carefully removed. The absorbance value A of the aqueous phase was measured at 600 nm, and repeated three times. The surface hydrophobicity of the test strain was calculated according to the following formula: H%=[(A0-A) / A0]×100%, where A0 and A represent the absorbance values ​​before and after organic solvent extraction, respectively.

[0162] The surface hydrophobicity of strain MNH19250 was 57.06%, which was more than 25% higher than that of other strains in the literature (Xin Ma 1, Meng Tian 2, Xueping Yu, et al., Foods. 2024, Jan 30;13(3):442. Akkermansia muciniphila PROBIO (https: / / doi.org / 10.3390 / foods13030442); Figure 1c, A.muciniphila PROBIO surface hydrophobicity is 31% (1h)). Strains with strong surface hydrophobicity have stronger epithelial cell adhesion ability and can better colonize in the human intestine to play a probiotic role. At the same time, they can inhibit the adhesion of other pathogens and play an intestinal probiotic function.

[0163] 1.8 Amplification of 16S rRNA gene of strains Take fresh culture of the strain to extract genomic DNA of the strain, and use the extracted genomic DNA of the strain as a template to amplify the 16S rRNA gene.

[0164] The primer pairs used for PCR of 16S rRNA gene are: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 6) 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO. 7). The PCR reaction procedure is as follows: Pre-denaturation: 94℃, 4 min; denaturation: 94℃, 50 sec; annealing: 52℃, 40 sec; extension: 72℃, 70sec; final extension: 72℃, 10 min (36 cycles).

[0165] 1.916S rRNA gene sequencing The PCR product was purified and sequenced by Bioengineering Corporation to obtain the 16S rRNA gene sequence (1355 bp), as shown in SEQ ID NO. 1:

[0166] The 16S rRNA gene sequence of MNH20651 is shown in SEQ ID NO. 2:

[0167] The 16S rRNA gene sequence of MNH26240 is shown in SEQ ID NO. 3:

[0168] The 16S rRNA gene sequence of MNH19631 is shown in SEQ ID NO. 4:

[0169] The 16S rRNA gene sequence of MNH39567 is shown in SEQ ID NO. 5:

[0170] 1.10 Identification of strain MNH19250 The 16S rRNA gene sequence as shown in SEQ ID NO. 1 was analyzed using the NCBI Basic Local Alignment Search Tool to confirm the strain classification information.

[0171] The measured sequences were compared with the data in GenBank by BLAST analysis. The results showed that the strain with the highest similarity to MNH19250 was Akkermansia muciniphila ( Akkermansia muciniphila ), the similarity is 100%, so the strain MNH19250 is judged to be Akkermansia muciniphila ( Akkermansia muciniphila ) strains planted.

[0172] The strain MNH19250 was compared with Akkermansia genus obtained from GenBank and other databases ( Akkermansia sp. ) were compared with the 16S rRNA gene sequences of related strains to construct a phylogenetic tree.

[0173] The 16S rRNA gene sequence of strain MNH19250 was compared with the sequences of model strains with high 16S rRNA gene sequence similarity in the NCBI database, and then a phylogenetic tree was constructed using the software MEGA 5 (the phylogenetic tree was constructed using the maximum likelihood method) (see Figure 8 ), Figure 8 Only the Bootstrap values ​​greater than 50% are displayed in the developmental tree nodes.

[0174] From the phylogenetic tree, it can be seen that strain MNH19250 is closely related to Akkermansia spp. Akkermansia sp. )and Akkermansia muciniphila Muc AY271254, therefore, strain MNH19250 was identified as Akkermansia muciniphila ( Akkermansiamuciniphila ) new strains planted.

[0175] 1.11 Genome analysis of strain MNH19250 Genome analysis of strain MNH19250 The genome of the MNH19250 strain was fragmented by ultrasonic method, and then the Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was sequenced with NovaSeq (Illumina) for double-end 150bp. The sequencing obtained 3.01Gbp data, of which Q20 accounted for 97.42%.

[0176] The original genome sequencing data was filtered using fastp (version: 0.20.0). The filtered raw data was assembled using SPAdes (version: v3.14.0). The total gene length of the genome assembly was 2.82 Mbp, the N50 length was 378.4 kbp, and the GC content was 55.19%.

[0177] The genomic genes were predicted and analyzed using the prokaryotic analysis software genome annotation pipeline prokka (version: 1.14.5). A total of 2376 CDS sequences were predicted, with an average CDS sequence length of 1039 bp.

[0178] Potential antibiotic resistance genes in the genome were analyzed using RGI (version: 4.2.2), where the antibiotic resistance gene database was CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). Detailed comparison information is shown in Table 3.

[0179]

[0180] The analysis of potential virulence factors and related genes in the genome was performed by comparing the virulence factor database VFDB (virulence factor database, http: / / www.mgc.ac.cn / cgibin / VFs / v5 / main.cgi, updated on September 19, 2019) using NCBI blastp (version: 2.7.1+). The detailed comparison results are shown in Table 4.

[0181]

[0182] The analysis of potential secondary metabolism gene clusters in the genome was performed using antiSMASH6 (version: 6.0.1). The detailed alignment results are shown in Table 5.

[0183]

[0184] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version: 1.0.0). The detailed alignment results are shown in Table 6.

[0185]

[0186] Analysis of the Amuc_1100 protein and P9 protein coding genes of strain MNH19250. The Amuc_1100 protein sequence (WP_197738471) and the Amuc_1631 (P9) protein sequence (ACD05451) were compared using BLAST.

[0187]

[0188] Sequence: >MNH19250_01469 hypothetical protein MSNWITDNKPAAMVAGVGLLLFLGLSATGYIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSKFIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEATAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED (SEQ ID NO. 8)

[0189] Sequence: >MNH19250_00667 Tail-specific protease (SEQ ID NO. 9) Example 2: Fatty acid composition analysis of strain MNH19250 2.1 Analysis of fatty acid composition of strain MNH19250 The strain MNH19250 was inoculated on MM01 plates and cultured anaerobically at 37°C for 72 hours. The bacteria were then collected and their fatty acids were extracted and methylated. The fatty acid composition of the strain MNH19250 was analyzed using the fully automated bacterial identification system from MIDI (Microbial ID, Inc., Newark, Del.).

[0190] The main fatty acid (>10%) of the experimental strain MNH19250 was C15:0 anteiso 48.73%.

[0191] 2.2 Analysis of short-chain fatty acids (SCFA) of strain MNH19250 Bacteria preparation The strain MNH19250 was inoculated into MM01 liquid culture medium and cultured anaerobically at 37°C for 48 hours. The bacterial cells and the supernatant were collected by centrifugation and stored at -80°C for later use.

[0192] (1) After thawing, vortex the sample for 3 min to mix; (2) Pipette 50 μL of sample into a 1.5 mL centrifuge tube, add 100 μL of phosphoric acid solution (0.5%, v / v), and vortex at 2500 rpm for 3 min; (3) Add 750 n of MTBE extractant containing internal standard, vortex at 2500 r / min for 3 min, sonicate at 4 m for 5 min, and centrifuge at 4 m and 12000 r / min for 10 min; (4) Pipette 200 ml of supernatant into the liner of the injection bottle and store it in a -20 °C refrigerator until GC-MS / MS analysis.

[0193] Pretreatment of bacterial samples (1) After the sample is thawed, add 100% ultrapure water extract to resuspend it evenly; (2) Pipette 50 μL of bacterial suspension sample into a 1.5 mL centrifuge tube, add 100 μL of phosphoric acid solution (0.5%, v / v), and vortex for 3 min to mix; (3) Soak in liquid nitrogen for 2 min, take out and thaw completely on ice, vortex at 2500 rpm for 3 min, and repeat 3 times; (4) Add 150 nr of MTBE extractant containing internal standard, vortex at 2500 r / min for 3 min, sonicate at 4 m for 5 min, and centrifuge at 4 m and 12000 r / min for 10 min; (5) aspirating 90% of the supernatant into the liner tube of a sampling bottle for GC-MS / MS analysis;

[0194] (6) The remaining 50 mL of bacterial suspension was repeatedly frozen and thawed in liquid nitrogen three times. After centrifugation at 12,000 rpm for 10 min, the supernatant was collected and the protein concentration was determined by the BCA method.

[0195] Standard preparation: Prepare 0.005 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, 20 μg / mL of different concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid as standard solution. Use the conditions shown in Table 9 for chromatography-mass spectrometry detection.

[0196] Obtain the chromatographic peak intensity data of the corresponding quantitative signal of each concentration standard. Use the external standard to internal standard concentration ratio (Concentration Ratio) as the horizontal axis and the external standard to internal standard peak area ratio (Area Ratio) as the vertical axis to draw standard curves for different substances.

[0197]

[0198] The integrated peak area ratios of all detected samples were substituted into the linear equation of the standard curve for calculation. After the sample calculation formula was entered, the content data of the substance in the actual sample was finally obtained. The results showed that both the bacterial supernatant and the bacteria contained short-chain fatty acids such as acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. The contents of acetic acid and propionic acid are shown in Table 10.

[0199]

[0200] The test results show that strain MNH19250 can synthesize short-chain fatty acids during its growth, including a large amount of acetic acid and propionic acid.

[0201] Example 3. Effect of strain MNH19250 on IFNβ expression Inflammation is now considered a biomarker of accelerated aging and one of the characteristics of aging biology. Extended lifespan and healthspan may be the result of a good balance between pro-inflammatory and anti-inflammatory processes. Studies have shown that although centenarians have increased levels of inflammatory molecules (e.g., interleukin-6), the adverse consequences of these pro-inflammatory molecules are offset by high levels of anti-inflammatory molecules. (Campisi, J., Kapahi, P., Lithgow, GJ et al. From discoveries inageing research to therapeutics for healthy ageing. Nature 571, 183–192(2019). https: / / doi.org / 10.1038 / s41586-019-1365-2).

[0202] Type I interferon β (IFNβ) has been shown to have anti-inflammatory effects and can promote macrophage reprogramming to promote anti-inflammatory phenotypes, thereby helping to terminate inflammation (Kumaran Satyanarayanan, S., El Kebir, D., Soboh, S.et al. IFN-β is a macrophage-derived effector cytokine facilitating the resolution of bacterial inflammation. Nat Commun 10, 3471 (2019). https: / / doi.org / 10.1038 / s41467-019-10903-9). IFNβ can also regulate immunity and achieve antiviral and anti-tumor effects through immune regulation.

[0203] To verify whether MNH19250 can promote the expression of IFNβ, this study used THP-1 cells carrying the IFNβ gene promoter reporter gene (THP-1-IFNβ-promoter reporter cells, a cell line built by Muen) to evaluate the effect of MNH19250 on the transcriptional activity of IFNβ.

[0204] The construction of THP-1-IFNβ-promoter reporter cells includes the following steps: inserting the reporter gene into a vector, infecting cells with the vector, and screening to obtain cell lines expressing the reporter gene (see the following literature for details: Huashan Du, Tianmin Xu, Manhua Cui, "cGAS-STING signaling in cancer immunity and immunotherapy" Biomedicine & Pharmacotherapy 133 (2021) 110972; Jiang et al., "cGAS-STING, an important pathway in cancer immunotherapy" Journal of Hematology & Oncology (2020) 13:81; Khiem C. Lam et al., "Microbiota triggers STING-type I IFN-dependent monocyte reprogramming of the tumor microenvironment" Cell 184, 5338-5356).

[0205] Preparation of culture supernatant of strain MNH19250: inoculate strain MNH19250 in MM01 liquid culture medium, culture anaerobically at 37°C for 48 hours, remove bacteria by centrifugation, filter the culture supernatant with a 0.22 μm filter, aliquot, and store the collected material at -80°C for later use.

[0206] Control group (Control): DMEM complete medium (Gibco, containing 10% FBS) containing 10% volume of MM01 liquid medium; MSA-2 group (positive control group): DMEM complete medium containing 10 μM MSA-2 (purchased from Taoshu Biological); Strain MNH19250 group: DMEM complete medium containing 10% volume of the culture supernatant of strain MNH19250.

[0207] THP-1-IFNβ-promoter reporter cells were seeded in 96-well plates at 1 × 10 5 cells. Treat the cells according to the set groups. After culturing for 24 hours, the cells were centrifuged at 300g for 5 minutes, the culture supernatant was removed, and 50 μL 1×Luminescence (Promega) was added to normalize the control group (Control) to evaluate the effect of strain MNH19250 on IFNβ transcription activity.

[0208] The experimental results are as follows Figure 9 As shown, the metabolites of strain MNH19250 can significantly promote the transcriptional activity of IFNβ. Therefore, these results indicate that MNH19250 and its metabolites have anti-inflammatory and immune-regulating functions, thereby delaying the aging of the body.

[0209] Example 4: Lifespan test of wild-type Caenorhabditis elegans N2 (hereinafter referred to as nematodes) supplemented with MNH19250 1. Cultivation of strain MNH19250 After thawing the glycerol cryopreservation tube of MNH19250 strain at 37°C, inoculate it into MM01 medium in a biological safety cabinet for activation, and then inoculate the activated strain into MM01 liquid medium for culture to obtain a sufficient amount of culture.

[0210] 2. Cultivation of Escherichia coli OP50 Take out the OP50 strain frozen in a -80℃ refrigerator (the Escherichia coli OP50 strain used in this experiment was donated by the Institute of Vegetable and Floriculture, Chinese Academy of Agricultural Sciences), inoculate it into LB medium, and culture it at 37℃ with shaking at 225 rpm for about 16 hours. Aliquot the bacterial solution and store it in a 2~8℃ refrigerator for later use.

[0211] 3. Preparation of bacterial culture dishes for feeding nematodes The Caenorhabditis elegans N2 (hereinafter referred to as nematodes) used in this experiment was donated by the Institute of Vegetable and Floriculture, Chinese Academy of Agricultural Sciences.

[0212] 100 μL of the overnight cultured OP50 bacterial solution was spread on NGM plate medium (Shandong Top Bioengineering Co., Ltd., M2328B), which is the OP50 nematode feeding plate (labeled as OP50); Use M9 buffer (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl and 1 mL 1 M MgSO4, add water to 1 L, and sterilize with high temperature and high pressure before use) Akkermansia muciniphila MNH19250 was washed three times and then resuspended in E. coli OP50 to form different concentration gradients, and 100 μL of each gradient was spread on NGM plate medium, i.e., plates for feeding OP50 and live MNH19250 nematodes (labeled as OP50+AKK); Before resuspending in E. coli OP50, different concentrations of Akkermansia muciniphila MNH19250 was heated at 70°C for 30 min and then resuspended in E. coli OP50 to form different concentration gradients. 100 μL of each gradient was spread on NGM medium, i.e., OP50 and pasteurized MNH19250 nematode feeding plates (labeled as OP50+Pas-AKK).

[0213] 4. Synchronization of nematodes The nematode culture dish with more eggs was washed several times with sterile water, and the washing liquid was collected into a 15 mL centrifuge tube. After the nematodes and eggs were naturally precipitated, the supernatant was removed and 3.5 mL of sterile water for eggs was retained. Then 1.5 mL of lysis solution (5 mol / L sodium hydroxide solution and 5% sodium hypochlorite solution were mixed in a volume ratio of 1:2 to prepare the lysis solution) was added, and the centrifuge tube was shaken at 200 rpm with a vortex oscillator, and repeated every 2 minutes for a total of 10 minutes, and then centrifuged at 1300 g for 30 seconds to precipitate the eggs, remove the supernatant, add sterile water to wash once, and transfer the eggs to a new NGM plate, incubate in a 20°C biochemical incubator, and grow into L4 larvae after about 48 hours for subsequent experiments.

[0214] 5. Determination of nematode lifespan L4 larvae were transferred to bacterial NGM culture dishes inoculated with OP50 or OP50+Pas-AKK or OP50+AKK for feeding nematodes. All lifespan experiments were performed at 20°C. The synchronized L4 worms were used as t = 0 for lifespan determination and transferred to fresh culture dishes every two days until all worms died. 90 worms were tested on 3 plates for each experiment (30 worms per plate). The survival rate of nematodes was determined every 1-2 days. Nematodes that did not respond to mechanical stimulation were judged to be dead. The maximum number of days and average lifespan of nematodes under different treatment conditions were counted and survival curves were drawn.

[0215] 6. Test results GraphPad Prism 9.3.1 software was used for drawing and statistical analysis. The survival curve was analyzed by simple survival analysis (Kaplan-Meier); for pairwise comparison, t test (Unpaired t test) was used for analysis; for comparison between three or more groups, one-way analysis of variance (One-Way ANOVA) was used for analysis; significance was indicated by *, *: p < 0.05, **: p < 0.01, ***: p < 0.001, **** p < 0.0001, and no significance was not marked.

[0216] The results are as follows Figure 10A-10B As shown, ( Fig. 10A ) Compared with the average lifespan and maximum lifespan of nematodes fed with E. coli OP50, pasteurized MNH19250 and live MNH19250 significantly extended the maximum lifespan of nematodes. ( Fig. 10B ) Pasteurized MNH19250 significantly prolonged the average lifespan of nematodes, while live MNH19250 prolonged the average lifespan of nematodes, indicating that MNH19250 can delay the aging of nematodes.

[0217] Example 5: Effect of MNH19250 supplementation on locomotor activity of nematodes 1. Activity Ability Scoring Activity is one of the key indicators for evaluating the healthy lifespan of nematodes. The movement status and movement ability will gradually deteriorate and weaken with the aging of the body. During the lifespan test, the activity of nematodes was scored to evaluate the activity of nematodes and then evaluate the effect of MNH19250 on the healthy lifespan of nematodes.

[0218] The synchronized L4 larvae were placed on bacterial NGM plates inoculated with OP50, OP50+Pas-AKK or OP50+AKK for feeding nematodes. The activity scores were measured on day 4, day 6, day 8, day 10, day 12, day 14, day 16 and day 17. During observation, the nematodes were picked up and placed on NGM plates without food. At least 90 nematodes were randomly scored for each experiment.

[0219] Activity Scoring Rules:

[0220] 2. Movement speed The movement rate is one of the key indicators for evaluating the movement activity of nematodes. Nematodes move forward by bending their bodies, so the body bending frequency is used to represent the movement rate. Thirty synchronized L4 larvae were randomly selected and placed on bacterial NGM culture plates inoculated with OP50, OP50+Pas-AKK, ​​or OP50+AKK for feeding nematodes. Their movement rates were measured on day 4, day 6, day 8, day 10, day 12, day 14, day 16, and day 17. When observing, the nematodes were selected to the NGM plates without food. The nematodes were allowed to move freely for 2 minutes. The number of body bends of the nematodes within 1 minute was observed and recorded under a microscope as an indicator of body bending frequency. One bend is defined as the movement of the nematode relative to one wavelength in the direction of the long axis of the body.

[0221] 3. Swallowing pump rate measurement Nematodes mainly ingest food through the swallowing pump. Thirty synchronized L4 larvae were randomly selected and placed on bacterial NGM culture dishes inoculated with OP50, OP50+Pas-AKK, ​​or OP50+AKK for feeding nematodes. Their swallowing frequency was measured on day 4, day 6, day 8, day 10, day 12, day 14, day 16, and day 17. The swallowing pump rate was measured by observation under a stereomicroscope. The number of pharyngeal contractions of the terminal pharyngeal bulb at intervals of 30 s was calculated.

[0222] 4. Test results The results are as follows Figure 11A-11B As shown, with the aging of the nematodes, the activity of nematodes fed with Escherichia coli OP50 gradually deteriorated and weakened, and the number of dead (category D) and immobile (category C) nematodes in the activity score gradually increased; compared with the activity score of nematodes fed with Escherichia coli OP50, supplementary feeding with pasteurized MNH19250 and live bacteria MNH19250 significantly improved the healthy survival and activity of nematodes, and the number of dead (category D) and immobile (category C) nematodes was significantly less than that of nematodes fed with OP50, and there were more nematodes in normal sinusoidal movement (category A).

[0223] ( Fig. 11C )Supplementary feeding of pasteurized MNH19250 and live MNH19250 significantly increased the sinusoidal movement frequency of nematodes and improved the healthy movement vitality of the body. Fig.11D ) Supplementation of pasteurized MNH19250 and live MNH19250 significantly increased the swallowing frequency of the nematode's swallowing pump.

[0224] It can be seen that MNH19250 can significantly enhance the motor activity and physiological function of nematodes, and improve the decline in motor activity and physiological function caused by aging.

[0225] Example 6: Effects of MNH19250 supplementation on the life cycle and body size of nematodes 1. Life cycle time To determine the life cycle of nematodes, 20 eggs were placed on 20 bacterial NGM dishes seeded with OP50 or OP50+Pas-AKK or OP50+AKK for nematode support, 1 egg per culture medium, and life cycle analysis was performed. The cells were observed under a stereomicroscope every 1 hour starting from the 60th hour until the first egg was laid. When the nematode laid the first egg, this was the time when the life cycle was completed.

[0226] 2. Body measurement Randomly pick 20 synchronized L4 larvae and place them on NGM culture dishes inoculated with OP50, OP50+Pas-AKK or OP50+AKK for feeding nematodes. Use a stereomicroscope to take images on Day 1, Day 3 and Day 5 of culture, and use ImageJ software to measure the body projection area of ​​the nematodes as body size data.

[0227] 3. Test results The results are shown in the figure Figures 12A-12B As shown, ( Fig. 12A ) Compared with the life cycle of nematodes fed with Escherichia coli OP50, supplementary feeding with pasteurized MNH19250 and live MNH19250 significantly shortened the life cycle of nematodes and promoted the maturation of nematodes.

[0228] ( Fig. 12B ) Compared with nematodes fed with Escherichia coli OP50, supplementary feeding with pasteurized MNH19250 and live MNH19250 significantly increased the body size of nematodes and promoted the maturation of nematodes.

[0229] Example 7: Effect of MNH19250 supplementation on nematode reproductive capacity Randomly pick 20 synchronized L4 larvae and place them on NGM culture plates inoculated with OP50, OP50+Pas-AKK or OP50+AKK for feeding nematodes. Every 24 hours, transfer the nematodes to new NGM culture plates inoculated with OP50, OP50+Pas-AKK or OP50+AKK for feeding nematodes. The replaced old plates are inverted and cultured at 20°C for 24 hours. The number of larvae is recorded until the nematodes lose their reproductive capacity completely. The sum of the number of offspring produced each day is the lifetime egg production of the nematodes.

[0230] The results are as follows Figures 13A-13B As shown, compared with the number of progeny of nematodes fed with E. coli OP50, supplementation with pasteurized MNH19250 and live MNH19250 significantly increased the number of progeny per day of nematodes ( Fig.13A ) and the total number of offspring ( Fig. 13B ). It can be seen that MNH19250 promotes the reproductive ability of nematodes.

[0231] Example 8: Effects of MNH19250 supplementation on anti-aging-related gene pathways in nematodes The insulin / insulin-like growth factor 1 (IGF-1) signaling pathway is one of the most important signaling pathways regulating the aging of biological individuals. The daf-16 gene is a key gene in the insulin signaling pathway in nematodes, and the homologous gene of its signaling pathway can be found in humans. At present, a large number of literature reports have shown that the daf-16 gene can be regulated by providing specific nutrients to delay lifespan. The higher the activity of daf-16, the longer the lifespan of nematodes and the better the immunity against infection. Inhibition of DAF-2 and age-1 genes can activate DAF-16 and promote DAF-16 to enter the nucleus from the cytoplasm, thereby widely regulating the expression levels of downstream genes related to stress resistance, detoxification and metabolism.

[0232] Sir-2.1 is a member of the sirtuin protein family and plays an important role in DNA damage repair, cell cycle control, inhibition of apoptosis, resistance to oxidative stress, energy metabolism, and protection of mitochondrial function. Recent studies have shown that Sir2 can increase the ratio between mitochondrial-encoded MTCO1 (mitochondrial oxidative respiratory chain complex IV subunit) and nuclear-encoded ATP5A (mitochondrial oxidative respiratory chain complex IV subunit), destroy the imbalance of mitochondrial nuclear proteins, induce mitochondrial unfolded protein response (UPRmt), improve mitochondrial function and significantly extend the lifespan of nematodes (Qi Xinxin et al., The role of mitochondrial unfolded protein response in Sir2 inhibition of Parkinson's transgenic fruit flies, Journal of Anhui Medical University, 2016). This study confirmed that Sir2 overexpression significantly inhibited the degeneration of motor neurons in transgenic fruit flies with Parkinson's disease (PD) and significantly improved the movement ability of fruit flies. After UPRmt was inhibited, the protective effect of Sir2 was significantly weakened. Conclusion Sir2 has a neuroprotective effect on PD transgenic flies, and this neuroprotective effect is related to UPRmt.

[0233] At the same time, the results showed that exosomes derived from mesenchymal stem cells can significantly increase the expression of SIRT1 gene (homologous gene of Sir2.1) in aged mice, resist cell apoptosis, and reduce the effects of oxidative stress in the brains of aged mice. SIRT1 is a viable therapeutic target and plays an important role in the anti-aging process, especially in anti-brain aging (Zhang, Xiaowen et al. "Exosomes secreted by mesenchymal stem cells delaybrain aging by upregulating SIRT1 expression." Scientific reports vol. 13,113213. 14 Aug. 2023, doi:10.1038 / s41598-023-40543-5).

[0234] Antioxidant genes include glutathione S-transferase 4 (gst-4) and superoxide dismutase 3 (sod-3).

[0235] The synchronized L4 larvae were randomly picked and placed on bacterial NGM culture plates inoculated with OP50 or OP50+Pas-AKK or OP50+AKK for feeding nematodes. To ensure sufficient bacteria for feeding nematodes, the number of nematodes in each culture plate was no more than 150. After culturing on the culture plates at 20°C for 4 days, no less than 500 nematodes were collected from each treatment group and washed 5 times with M9 buffer to remove the remaining bacteria. Total RNA of nematodes was extracted using MiniBEST Universal RNA Extraction Kit (Takara, cat.no9767), and 1ug of total RNA was reverse transcribed into cDNA according to the method described in PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, catalog number RR047A). Real-time fluorescence quantitative PCR was performed on AppliedBiosystems QuantStudio 3 real-time fluorescence quantitative PCR system using PrimeScript RT Enzyme Mix reagent (Takara, cat.no RR037Q) reaction system. 2 -ΔΔCt The relative gene expression was calculated by the primer sequence as shown in the following table.

[0236]

[0237] like Fig.14 As shown, compared with the number of nematode offspring fed with Escherichia coli OP50, supplementation with pasteurized MNH19250 significantly increased the relative expression of genes related to the insulin / IGF-1 growth factor lifespan signaling pathway (daf-16) in Caenorhabditis elegans, significantly increased the relative expression of key genes in the antioxidant pathway (gst-4, sod-3), and significantly increased the relative expression of Sir-2.1 protein, a regulator of mitochondrial unfolded protein response in glial cell signaling.

[0238] Therefore, MNH19250 can slow down the aging rate by upregulating the expression of genes related to the insulin / IGF-1 growth factor lifespan signaling pathway in Caenorhabditis elegans (daf-16), upregulating the expression of key genes in the antioxidant pathway (gst-4, sod-3), and upregulating the expression of Sir-2.1 protein, a regulator of mitochondrial unfolded protein response in glial cell signaling.

[0239] Example 9: Effect of MNH19250 supplementation on the resistance of nematodes to heat stress and ultraviolet stress Heat stress test: Randomly select 30 synchronized L4 larvae and place them on bacterial NGM culture dishes inoculated with OP50 or OP50+Pas-AKK or OP50+AKK for feeding nematodes. Culture at 20°C for 4 days, then transfer the above treatment groups to a 35°C constant temperature incubator, and record the number of nematode deaths every hour or every 30 minutes. Worms that did not respond to mechanical stimulation were judged to be dead. The maximum number of days and average lifespan of nematodes under different treatment conditions were counted and survival curves were drawn.

[0240] Anti-ultraviolet aging test: L4 larvae were washed from NGM plates with sterile water, the larvae suspension was centrifuged at 156 g for 1 minute, the supernatant was removed, M9 buffer was added to suspend the suspension to about 30 larvae in 50 μl, and OP50 or OP50 and different treatments were inoculated into each well. Akkermansia muciniphila MNH19250 was adapted overnight in 6-well plates with 3 replicates for each sample. The next day, the plates were irradiated with UV at 254 nm at a dose of 1000 J / m2 for 8 min 40 s and immediately transferred to a 20°C incubator. Animals were counted every 24 h until all worms died in the experiment.

[0241] like Fig.15A and Fig. 15B As shown, compared with the lifespan of nematodes fed with E. coli OP50, 10 10 CFU / mL, 10 9 CFU / mL, 10 8 CFU / mL Pas-AKK and AKK, pasteurized and live Akkermansia muciniphila MNH19250 can significantly prolong the lifespan of nematodes under heat stress and improve the survival rate of nematodes against heat stress.

[0242] like Fig. 15C As shown, compared with the lifespan of nematodes fed with E. coli OP50, 10 11 CFU / mL, 10 10 CFU / mLPas-AKK pasteurized Akkermansia muciniphila MNH19250 and 10 11 CFU / mL live bacteria Akkermansiamuciniphila MNH19250 can significantly prolong the lifespan of nematodes under UV stress and improve the survival rate of nematodes against UV aging.

[0243] Embodiment 10: Akkermansia muciniphila Lifespan test of nematodes supplemented with different strains Strains MNH26240, MNH20651, MNH39567, MNH19631, and MNH19250 were isolated from the intestines of healthy volunteers in different regions. Akkermansia muciniphila strains, for testing Akkermansia muciniphila The anti-aging effects of different strains were tested in the following experiments. The present invention only involves the anti-aging and antioxidant activities of the MNH19250 strain, and the MNH26240, MNH20651, MNH39567, and MNH19631 strains are only used for control experiments. The implementation of the present invention does not involve the MNH26240, MNH20651, MNH39567, and MNH19631 strains.

[0244] 1. Cultivation of strains Strains MNH26240, MNH20651, MNH39567, MNH19631 and MNH19250 were inoculated into MM01 liquid culture medium and cultured anaerobically at 37°C for 48-72 hours, then the cells were collected.

[0245] 2. Preparation of bacterial culture dishes for feeding nematodes MNH26240, MNH20651, MNH39567, MNH19631 and MNH19250 were washed three times with M9 buffer, heated at 70°C for 30 min, and then resuspended in Escherichia coli OP50. 100 μL of each gradient was spread on NGM medium to prepare nematode feeding plates (labeled as OP50+MNH26240, OP50+MNH20651, OP50+MNH39567, OP50+MNH19631, OP50+MNH19250).

[0246] 3. Determination of nematode lifespan L4 larvae were transferred to bacterial NGM culture dishes inoculated with OP50+MNH26240, OP50+MNH20651, OP50+MNH39567, OP50+MNH19631, and OP50+MNH19250 for feeding nematodes. All lifespan experiments were performed at 20°C. The synchronized L4 worms were used as t = 0 for lifespan determination and transferred to fresh culture dishes every two days until all worms died. 90 worms were tested on 3 plates for each experiment (30 worms per plate). The survival rate of nematodes was determined every 1 to 2 days. Worms that did not respond to mechanical stimulation were judged to be dead. The maximum number of days and average lifespan of nematodes under different treatment conditions were counted and survival curves were plotted.

[0247] 4. Test results The results are as follows Figure 16A-16B As shown, compared with the average lifespan and the maximum lifespan of E. coli OP50, the pasteurized Akkermansia muciniphila The strains all extended the maximum lifespan of nematodes to varying degrees ( Fig.16A), MNH19250, MNH39567, MNH19631, MNH26240, and MNH19250 can significantly extend the average lifespan of nematodes ( Fig. 16B Among them, MNH19250 has the best effect.

[0248] Embodiment 11: Akkermansia muciniphila Effects of supplementary feeding of different strains on anti-aging-related gene pathways in nematodes The insulin / insulin-like growth factor 1 (IGF-1) signaling pathway is one of the most important signaling pathways regulating the aging of biological individuals. The daf-16 gene is a key gene in the insulin signaling pathway in nematodes, and the homologous gene of its signaling pathway can be found in humans. At present, a large number of literature reports have shown that the daf-16 gene can be regulated by providing specific nutrients to prolong life. The higher the activity of daf-16, the longer the life of the nematode and the better the immunity against infection. Inhibition of DAF-2 and age-1 genes can activate DAF-16 and promote DAF-16 to enter the nucleus from the cytoplasm, thereby widely regulating the expression levels of downstream genes related to stress resistance, detoxification and metabolism. Sir-2.1 is another evolutionarily conserved longevity regulator and a cofactor of daf-16. It can extend the life of Caenorhabditis elegans by regulating the insulin / insulin growth factor signaling pathway and binding to daf-16 to activate the expression of daf-16.

[0249] Randomly pick the synchronized L4 larvae to the bacterial NGM culture dishes inoculated with OP50+MNH26240, OP50+MNH20651, OP50+MNH39567, OP50+MNH19631, and OP50+MNH19250 for feeding nematodes. To ensure sufficient bacteria for feeding nematodes, the number of nematodes in each culture dish is no more than 150. Culture on a 20°C culture dish for 4 days, collect no less than 500 worms from each treatment group, and wash 5 times with M9 buffer to remove the remaining bacteria. Perform total RNA extraction, reverse transcription, and real-time quantitative PCR according to the method of Example 6. The experimental method is the same as Example 8.

[0250] like Fig.17 As shown, compared with nematodes fed with E. coli OP50, nematodes supplemented with pasteurized Akkermansia muciniphilaMNH19250, MNH39567, MNH19631, MNH26240 and MNH20651 significantly increased the relative expression levels of genes related to the insulin / IGF-1 growth factor lifespan signaling pathway of Caenorhabditis elegans (Daf-16, Sir-2.1), and significantly increased the relative expression levels of key genes in the antioxidant pathway (gst-4, sod-3), thereby achieving the effect of delaying the aging rate.

[0251] Embodiment 12: Akkermansia muciniphila Effects of supplementary feeding of different bacterial strains on antioxidant capacity and resistance to UV aging in C. elegans Antioxidant test: L4 larvae were washed from NGM plates with sterile water, the worm suspension was centrifuged at 156 g for 1 minute, the supernatant was removed, and M9 buffer was added to suspend about 30 larvae in 50 μl. Each well was inoculated into 6-well plates containing OP50 or OP50+MNH26240, OP50+MNH20651, OP50+MNH39567, OP50+MNH19631, and OP50+MNH19250 for overnight adaptation, and each sample was repeated in 3 wells. The next day, 25 mM H2O2 was added to each well for antioxidant test. The live worms in the wells were counted every 30 minutes until all worms died in the test.

[0252] Anti-UV aging test: L4 larvae were washed from NGM plates with sterile water, the worm suspension was centrifuged at 156 g for 1 minute, the supernatant was removed, and M9 buffer was added to suspend about 30 larvae in 50 μL. Each well was inoculated into 6-well plates containing OP50 or OP50+MNH26240, OP50+MNH20651, OP50+MNH39567, OP50+MNH19631, and OP50+MNH19250 for overnight adaptation, and each sample was repeated 3 wells. The next day, the wells were exposed to UV irradiation at a dose of 1000 J / m2 at 254 nm for 8 min 40 s and immediately transferred to a 20°C incubator. Animals were counted every 24 hours until all worms died in the experiment.

[0253] like Fig.18A As shown, compared with feeding with Escherichia coli OP50, pasteurized MNH19250 and MNH19631 (OP50+MNH19250 and OP50+MNH19631) significantly extended the lifespan of nematodes under oxidant stress and improved the survival rate of nematodes against oxidative stress.

[0254] like Fig.18BAs shown, compared with feeding Escherichia coli OP50, pasteurized MNH19250, MNH19631, MNH39567, MNH26240 (OP50+MNH19250, OP50+MNH19631, OP50+MNH39567 and OP50+MNH26240) significantly prolonged the lifespan of nematodes under UV irradiation stress and improved the survival rate of nematodes against UV aging.

[0255] According to Examples 10-12, this application Akkermansia muciniphila Different strains of this herb have anti-aging and antioxidant capabilities.

[0256] Although the present invention has been disclosed with reference to certain embodiments, it is apparent that modifications and variations may be made without departing from the spirit and scope of the present invention as disclosed herein and provided in the appended claims. In addition, it should be understood that although all examples disclosed herein illustrate embodiments of the present invention, they are provided only as non-limiting examples and therefore should not be considered as limiting the various aspects of the present invention thus described. The present invention is intended to have the full scope defined by the language of the present disclosure, the following claims, and any equivalents thereof. Therefore, the drawings and detailed description should be considered illustrative rather than restrictive.

Claims

1. A composition for anti-aging and / or anti-oxidation, the composition comprising an effective amount of Akkermansia muciniphila or a culture of the Akkermansia muciniphila, the Akkermansia muciniphila comprising at least one strain of Akkermansia muciniphila deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 63782, GDMCC No: 65547, GDMCC No: 65542, GDMCC No: 65809 or GDMCC No: 65810, wherein: The Akkermansia muciniphila includes any one of live bacteria, lyophilized bacteria and inactivated bacteria of the Akkermansia muciniphila, and the culture includes any one of the following A) to D): A) a fermentation broth of the Akkermansia muciniphila; B) the supernatant of the fermentation broth of the Akkermansia muciniphila; C) a fermentation broth of the Akkermansia muciniphila that has been inactivated; D) The concentrated or dried product of any one of A) to C) above.

2. The composition according to claim 1, characterized in that The inactivated bacteria are bacteria obtained by pasteurization or lysis; the inactivated fermentation broth is a product obtained by pasteurization or lysis of the fermentation broth of Akkermansia muciniphila.

3. The composition according to claim 1 or 2, characterized in that The composition further comprises one or more food or pharmaceutically acceptable carriers, excipients and / or adjuvants.

4. The composition according to claim 1 or 2, characterized in that The composition may also contain one or more other active agents for anti-aging and / or anti-oxidation.

5. The composition according to claim 4, characterized in that The other active agents are one or more of probiotics, prebiotics, antioxidants and anti-aging drugs.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of anti-aging or anti-oxidation drugs, health products or foods in subjects in need thereof.

7. The use according to claim 6, characterized in that: The anti-aging treatment includes at least one of the following: (1) Extend the life of the object; (2) Improve the decline in motor ability, swallowing function and / or reproductive ability caused by aging; (3) Regulate the expression of insulin / IGF-1 signaling pathway genes, antioxidant genes, and mitochondrial unfolded protein response regulatory genes; (4) Alleviate damage or aging caused by heat stress, oxidative stress or ultraviolet stress.

8. The use according to claim 7, characterized in that: The insulin / IGF-1 signaling pathway genes include daf-16, daf-2, age-1 or their homologous genes in mammals; the antioxidant genes include sod-3, gst-4 or their homologous genes in mammals; the mitochondrial unfolded protein response regulatory genes include sir-2.1 or their homologous genes in mammals.

9. The use according to claim 6, characterized in that: The anti-aging includes treating, preventing or alleviating diseases associated with aging.

10. The use according to claim 9, characterized in that: The diseases associated with aging are Parkinson's syndrome and / or Alzheimer's disease.

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