Ackermansia guanciensis, which has antioxidant, fat-reducing, and tumor growth-inhibiting effects, and its products and uses.

Abstract

This invention discloses Akkermansia, its products and use, wherein the Akkermansia are Akkermansia guanciensis N21116 and N21169, with deposit numbers GDMCC No: 62888 and GDMCC No: 62889, respectively. Akkermansia guanciensis N21116 and N21169 in this invention are new species of Akkermansia that have the effect of tolerating artificial intestinal fluid and have antioxidant, tumor cell proliferation inhibitory, and weight loss / lipid reduction effects.

Description

[Technical Field]

[0001] This invention relates to the art of microorganisms, and more particularly to Ackermansia guanciensis, which has antioxidant, fat-reducing, and tumor growth-inhibiting effects, as well as its products and uses. [Background technology]

[0002] Population aging is one of the most talked-about and important social issues of the 21st century. According to relevant studies, the proportion of the elderly population (65 years and older) is projected to reach 16% by 2050. Countries around the world, including China, are all facing the problem of aging populations. Currently, China's elderly population aged 65 and over exceeds 200 million, accounting for 14.2% of the total population. It is projected that by around 2035, China's elderly population aged 60 and over will surpass 400 million.

[0003] The emergence of an aging population has increased global concern about health and aging. Furthermore, with the rise of global economic standards, there is a widespread consensus among people to seek longer healthy lifespans. Currently, the academic community universally recognizes that while the mechanisms of longevity are complex, they are inextricably linked to mechanisms such as antioxidant mechanisms, chronic inflammation mechanisms, and metabolic disorders. Therefore, developing drugs or supplements with antioxidant, anti-inflammatory, and metabolic disorder-improving effects is becoming an important means of achieving healthy longevity.

[0004] Guangxi is one of the provinces in China with the highest concentration of longevity counties, and research on its elderly population and the phenomenon of longevity is currently a hot spot. In particular, studying the characteristics of the gut microbiota of elderly people and its relationship to chronic diseases in the elderly is a current research hotspot. The gut microbiota is broadly associated with various chronic diseases, and the evidence that is emerging shows that the gut microbiota plays an important and essential role in antioxidant, anti-inflammatory, and metabolic improvement. However, there are relatively few probiotics with the above anti-aging functions currently available, so developing new probiotics with relevant functions derived from elderly people is of great significance for the development of anti-aging products for humans. [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention provides Akkermansia guanciensis, which has antioxidant, fat-reducing, and tumor-suppressing effects, as well as products containing the bacterium and their use. The two strains of Akkermansia in the present invention were isolated from fecal samples of centenarians in Guangxi and have been found to have stronger antioxidant capacity, fat-reducing capacity, and stronger tumor cell proliferation inhibitory capacity compared to other Akkermansia strains, thus possessing extremely high development and practical value and providing new concepts for the development and use of products such as antioxidant, tumor-suppressing, and fat-reducing products. [Means for solving the problem]

[0006] In a first aspect of the present invention, a single strain of Akkermansia is provided, which is Akkermansia guangxiensis N21116, with the taxonomic name Akkermansia guangxiensis sp., deposited with the Guangdong Provincial Microbial Strain Preservation Center on December 2, 2022, with deposit number GDMCC No: 62888.

[0007] In a second aspect of the present invention, a single strain of Akkermansia is provided, which is Akkermansia guangxiensis N21169, with the taxonomic name Akkermansia guangxiensis sp., deposited with the Guangdong Provincial Microbial Strain Preservation Center on December 2, 2022, with deposit number GDMCC No: 62889.

[0008] In some embodiments of the present invention, Ackermansia N21116 and N21169 were both screened and isolated from fecal samples of centenarians in Guangxi.

[0009] In some embodiments of the present invention, the colony morphology of Ackermansia guanciensis N21116 and N21169 is that of a light gray colony that is circular, convex, has a smooth surface, and has orderly edges.

[0010] In some embodiments of the present invention, the 16S sequence of Ackermansia guanciensis N21116 is shown as SEQ ID NO: 5.

[0011] In some embodiments of the present invention, the 16S sequence of Ackermansia guanciensis N21169 is shown as SEQ ID NO:6.

[0012] In some embodiments of the present invention, the screening and isolation method for Ackermansia guanxiensis N21116 and N21169 involves adding a fecal sample from a centenarian in Guangxi to a mucin solid medium, culturing it under anaerobic conditions for 3 to 6 days, then collecting light gray colonies from the medium that have a circular and convex morphology, a smooth surface, and orderly edges, and identifying them by PCR amplification to obtain Ackermansia guanxiensis N21116 and N21169.

[0013] In this invention, through testing of antioxidant levels (hydroxyl radical scavenging rate, superoxide anion scavenging rate, and superoxide dismutase (SOD) activity), it was confirmed that Ackermansia guanciensis N21116 and N21169 obtained by the above manual screening isolation have stronger antioxidant capacity than strains of the same genus, and thus products prepared therefrom exhibit superior antioxidant effects.

[0014] In some embodiments of the present invention, the culture time for Ackermansia guanciensis N21116 and N21169 is 40 to 72 hours.

[0015] In some embodiments of the present invention, Ackermansia guanciensis N21116 and N21169 are cultured at a temperature of 30°C to 37°C and a rotation speed of 150 rpm to 220 rpm.

[0016] In some embodiments of the present invention, the identification by PCR amplification uses an Ackermansia-specific amplification primer.

[0017] In some embodiments of the present invention, the Ackermansia-specific amplification primers are shown in SEQ ID NO: 1 and 2.

[0018] In some embodiments of the present invention, the amplification system for identification by PCR amplification is shown in Table 1.

[0019] In some embodiments of the present invention, the thermal cycling parameters for identification by PCR amplification are set to include pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 40 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, repeated 35 times, and final extension at 72°C for 10 minutes.

[0020] Of course, those skilled in the art can also reasonably adjust the content, concentration, and amplification program in the PCR amplification system according to actual usage requirements, thereby achieving the identification of Akkermansia.

[0021] In a third aspect of the present invention, there is provided a product containing Akkermansia described in the first and / or second aspect of the present invention, and the product includes food, food additives, feed, feed additives, drugs, and cosmetics.

[0022] In some embodiments of the present invention, the product further includes a product containing the bacterial agent, bacterial liquid, or culture of Akkermansia described in the first and / or second aspect of the present invention.

[0023] In some embodiments of the present invention, the mass fraction of Akkermansia muciniphila in the product is 5% or more. <000009​​​​​​​​​​​​​​​​​​​​​​In some embodiments of the present invention, the pharmaceutically acceptable adjuvants include, but are not limited to, diluents (e.g., starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (e.g., calcium sulfate, calcium hydrogen phosphate, etc.), wetting agents (e.g., ethanol), binders (e.g., hypromellose, povidone, etc.), disintegrants (e.g., sodium carboxymethyl starch, crospovidone, etc.), lubricants (e.g., talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (e.g., titanium dioxide, methylene blue, etc.), coating substrates, solvents, pH adjusters, antimicrobial agents (e.g., sodium sulfite, sodium thiosulfate, etc.), isotonic modifiers (e.g., glucose, sodium chloride, etc.), and chelating agents (e.g., disodium EDTA).

[0030] In some embodiments of the present invention, the food additive includes, but is not limited to, colorants, enzymes, thickeners, leavening agents, and sweeteners.

[0031] In some embodiments of the present invention, the cosmetic auxiliary substances include, but are not limited to, fragrances, colorants, preservatives, and antioxidants.

[0032] In some embodiments of the present invention, the form of Akkermansia guanciensis in the product includes freeze-dried powder, bacterial suspension, and granular inoculant. Of course, those skilled in the art may rationally select and use an appropriate Akkermansia form depending on the actual usage requirements and existing processing processes, and such Akkermansia form includes, but is not limited to, the above-mentioned freeze-dried powder, bacterial suspension, and granular inoculant.

[0033] In this invention, the term "freeze-dried powder" refers to a freeze-dried product obtained by pre-freezing the water in a drug solution or bacterial solution using a freeze-drying machine, and then sublimating the frozen water in the drug solution or bacterial solution under a vacuum and sterile environment.

[0034] In this invention, the term "granular inoculant" refers to a fungal agent used to prevent powdered fungal agents from coming into direct contact with fungicides or chemical fertilizers during use, thereby reducing their effectiveness. Generally, it refers to a type of fungal agent obtained by mixing a fungal solution with a granular carrier (e.g., biochar, vermiculite, etc.).

[0035] In some embodiments of the present invention, the number of viable Ackermansia guanciensis cells in the product is 10 7 cfu / mL ~ 10 9 The concentration is cfu / mL.

[0036] Of course, those skilled in the art can rationally adjust the activity of Ackermansia guanciensis in the product according to its actual form, thereby achieving stable technical effects.

[0037] A fourth aspect of the present invention provides a food additive containing akkermansia as described in the first and / or second aspect of the present invention, wherein the food additive includes freeze-dried akkermansia powder, plant extract, maltodextrin, and dietary fiber as described in the first and / or second aspect of the present invention.

[0038] In some embodiments of the present invention, the plant extract includes, but is not limited to, plant essential oils, saponins, alkaloids, polysaccharides, polyphenols, and flavonoids.

[0039] In some embodiments of the present invention, the plant extract is tea polyphenol and bayberry anthocyanin extract.

[0040] In some embodiments of the present invention, the dietary fiber includes water-soluble dietary fiber and insoluble dietary fiber.

[0041] In some embodiments of the present invention, the dietary fiber is water-soluble dietary fiber.

[0042] In some embodiments of the present invention, the food additive comprises, by mass percent, 5-15% of Akkermansia freeze-dried powder according to the first and / or second aspect of the present invention, 15-40% of plant extract, 40-60% of hydrolyzed starch polysaccharides, and 5-15% of dietary fiber.

[0043] In some embodiments of the present invention, the food additive comprises, by mass percent, 10% freeze-dried Ackermansia guanciensis N21116 or N21169 powder, 15% tea polyphenols, 15% bayberry anthocyanin extract, 50% maltodextrin, and 10% water-soluble dietary fiber.

[0044] In some embodiments of the present invention, the method for preparing the food additive is to thoroughly mix the Akkermansia freeze-dried powder, plant extract, maltodextrin, and dietary fiber described in the first and / or second aspects of the present invention.

[0045] A fifth aspect of the present invention provides the use of akkermansia according to the first and / or second aspect of the present invention in the manufacture of food, food additives, feed, feed additives, pharmaceuticals and cosmetics.

[0046] In this invention, safety evaluations have shown that the two strains of Akkermansia do not contain pathogenic or toxic genes, and resistance tests in a simulated gastrointestinal environment have shown that both strains can tolerate the gastrointestinal environment. Based on these characteristics, the two strains are shown to be probiotics with antioxidant, fat-reducing, and tumor growth-inhibiting effects.

[0047] In some embodiments of the present invention, the food, food additive, feed, feed additive, pharmaceutical and cosmetic is (1) Antioxidant, (2) Fat reduction, and (3) Inhibition of tumor growth, and has at least one of these functions.

[0048] In this invention, the fat-reducing effect is verified using a nematode (Caenorhabditis elegans) screening platform. Caenorhabditis elegans has a simple structure, a transparent body, is easy to observe, has a short developmental cycle, is easy to culture artificially, and its signaling pathways are highly conserved. Therefore, the Caenorhabditis elegans model has relatively high accuracy in verifying the effect and low experimental costs. In this invention, through fat-reducing tests using Caenorhabditis elegans, it was effectively demonstrated that Ackermansia guanciensis N21116 and N21169 have the effect of reducing body weight and lipid levels.

[0049] In this invention, through testing of antioxidant levels (hydroxyl radical scavenging rate, superoxide anion scavenging rate, and superoxide dismutase (SOD) activity), it was confirmed that both strains of Ackermansia guanciensis N21116 and N21169 in this invention have antioxidant capabilities exceeding those of other strains of the same genus, demonstrating excellent antioxidant capacity.

[0050] In some embodiments of the present invention, the tumor is a gastrointestinal tumor.

[0051] In some embodiments of the present invention, the gastrointestinal tumor includes colon cancer.

[0052] In this invention, by testing the effects of two strains of Ackermansia guanciensis, N21116 and N21169, on the proliferation of the human colon cancer cell line HCT116, it was confirmed that both strains could significantly suppress the proliferation of human colon cancer cells and have the potential to inhibit tumor growth.

[0053] A sixth aspect of the present invention provides a method for the prevention and / or treatment of a tumor, the method comprising administering a therapeutically effective amount of Ackermansia to a tumor patient, wherein the Ackermansia is Ackermansia guanciensis N21116 and / or Ackermansia guanciensis N21169. In some embodiments of the present invention, the tumor is a gastrointestinal tumor.

[0054] In some embodiments of the present invention, the gastrointestinal tumor includes colon cancer. [Effects of the Invention]

[0055] The beneficial effects of this invention are as follows:

[0056] 1. This invention is the first to discover a new strain of Akkermansia that possesses extremely high resistance to artificial intestinal fluid and excellent antioxidant, tumor cell proliferation inhibitory, and weight loss / lipid reduction effects. As a result, related products can be effectively developed based on this strain, giving it extremely high practical value.

[0057] 2. The new species of Akkermansia in this invention has stronger attachment ability and antioxidant, tumor cell proliferation inhibitory, and weight loss / lipid reduction effects compared to existing Akkermansia species, and therefore has extremely high utility and commercial development value. [Brief explanation of the drawing]

[0058] [Figure 1] This figure shows the phylogenetic trees of Ackermansia guanciensis N21116 and N21169 according to embodiments of the present invention. [Figure 2]This figure shows the ANI analysis of Akkermansia guangxiensis according to an embodiment of the present invention, where Agl[T] represents Akkermansia glycaniphila Pyt[T], Amu[T] represents Akkermansia muciniphila ATCC BAA-835[T], AmuYL44 represents Akkermansia muciniphila YL44, Amu22959 represents Akkermansia muciniphila DSM 22959[T], AguN21116 represents Akkermansia guangxiensis N21116, and AguN21169 represents Akkermansia guangxiensis N21169. [Figure 3] This figure shows the results of the resistance of Ackermansia guanciensis N21116 and N21169 to artificial intestinal fluid according to the embodiment of the present invention, where AmucT represents the Ackermansia muciniphila standard strain ATCC BAA-835. [Figure 4] This figure shows the results of antioxidant level tests for Ackermansia guanciensis N21116 and N21169 according to examples of the present invention, where a is the test result for hydroxyl radical scavenging activity, b is the test result for superoxide anion scavenging activity, c is the test result for superoxide dismutase (SOD) activity, AmucT represents Ackermansia muciniphila standard strain ATCC BAA-835, and A21028 represents Ackermansia muciniphila A21028. [Figure 5] This figure shows the inhibitory effect of Ackermansia guanciensis N21116 and N21169 on nematode fat accumulation according to embodiments of the present invention, where AmucT represents the Ackermansia muciniphila standard strain ATCC BAA-835 and A21028 represents Ackermansia muciniphila A21028. [Figure 6] This figure shows the results of fat staining of nematodes given different Akkermansia strains, where AmucT represents the Akkermansia muciniphila standard strain ATCC BAA-835 and A21028 represents Akkermansia muciniphila A21028. [Figure 7] This figure shows the inhibitory effect of Ackermansia guanciensis N21116 and N21169 according to the embodiment of the present invention on the human colon cancer cell line HCT116, where AmucT represents the Ackermansia muciniphila standard strain ATCC BAA-835. [Modes for carrying out the invention]

[0059] The present invention will be described in more detail below with reference to specific examples. Unless otherwise specified, the raw materials, reagents, or apparatus used in the examples and comparative examples are all available through ordinary commercial channels or obtained by prior art methods. Unless otherwise specified, the tests or test methods are all conventional methods in the art.

[0060] In the following example, the mucin solid medium used contained, at its final concentration, 38 g / L of BHI (Brain and Heart Extract Medium), 4 g / L of mucin, and 15 g / L of agar. The above components were uniformly mixed in the specified ratio and then autoclaved at 115°C for 20 minutes.

[0061] In the following examples, the synthetic liquid culture medium used contained, at its final concentration, 38 g / L of BHI (Brain and Heart Extract Medium), 16 g / L of soy peptone, 4 g / L of threonine, 25 mM of glucose, and 25 mM of N-acetylglucosamine. The above components were uniformly mixed in the specified ratio and then autoclaved at 115°C for 20 minutes.

[0062] Isolation and identification of Ackermansia guanciensis N21116 and N21169 The isolation and purification steps for Ackermansia guanciensis in this example are specifically as follows:

[0063] Appropriate amounts of fecal samples were taken from two centenarians in Guangxi, diluted with PBS, and uniformly spread onto mucin solid medium. The medium was then placed on an anaerobic workstation. The samples were incubated at 37°C for 2 days under gas conditions of 90% (v / v)N2 and 10%CO2. After incubation, pale gray colonies with a circular, convex morphology, smooth surface, and orderly edges were collected from the plate and identified using PCR amplification.

[0064] The PCR identification method for Ackermansia guanciensis in this example is as follows:

[0065] Using a sterilized toothpick, colonies satisfying the above-described Akkermansia morphology characteristics were collected and transferred to a 2× PCR premix solution, with upstream and downstream specific primers and sterile water added in sequence (the specific PCR amplification system is shown in Table 1). The thermal cycling parameters were set to pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 40 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, repeated 35 times, and final extension at 72°C for 10 minutes.

[0066] [Table 1]

[0067] Ackermansia-specific amplification primers for identification are: Upstream primer F:5'-CAGCACGTGAAGGTGGGGAC-3' (SEQ ID NO:1), and The downstream primer R was 5'-CCTTGCGGTTGGCTTCAGAT-3' (SEQ ID NO: 2).

[0068] The amplified product was detected by agarose gel electrophoresis, and if the length of the amplified fragment matched the expected length, the isolated strain could be tentatively identified as a strain suspected to belong to the genus Akkermansia.

[0069] For bacterial strains identified as suspected to belong to the genus Akkermansia by PCR amplification, further 16S rRNA molecule identification was performed.

[0070] The 16S rRNA molecular identification system is the same as in Table 1, and the thermal cycling parameters are as follows: pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 40 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 90 seconds, repeated 30 times, and final extension at 72°C for 10 minutes.

[0071] The specific amplification primers used for identifying 16S rRNA molecules are: Upstream primer 16S-F:5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:3), and The downstream primer was 16S-R:5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO:4).

[0072] The amplified products were sent to Shanghai Biotechnology for sequencing.

[0073] The 16S sequence of suspected Akkermansia strain 1 (number N21169), obtained by sequencing, is shown in SEQ ID NO: 5. The 16S sequence of suspected Akkermansia strain 2 (number N21116) is shown in SEQ ID NO: 6. Sequence searches using the NCBI BLAST tool revealed that the 16S rRNA sequences of both strains were 95.45% and 95.53% similar to those of the Akkermansia muciniphila ATCC BAA-835 standard strain, respectively. As can be seen from this, these two strains may be new species of Akkermansia that have not been previously discovered.

[0074]

[0075]

[0076] To further determine the evolutionary relationships of the above strains, the whole genomes of two suspected strains were sequenced, and ANI analysis was performed on them and the two current members of the genus Akkermansia.

[0077] The results are shown in Figures 1 and 2.

[0078] As a result, N21116 and Akkermansia muciniphila ATCC BAA-835 of the genus Akkermansia T , and Akkermansia saccharophila Py isolated from pythons T The ANI values ​​for N21169 and Akkermansia muciniphila ATCC BAA-835 were 68.95% and 68.34%, respectively. T , and Akkermansia saccharophila Py isolated from pythons T The ANI values ​​for N21116 and N21169 were 68.83% and 68.49%, respectively, meeting the ANI criteria (95%-96%) for new species classification. Furthermore, the sequence identity between N21116 and N21169 was 98.59%. Therefore, both belonged to the same bacterial species.

[0079] Considering that two strains of microorganism were isolated from Guangxi, the inventors tentatively named them Akkermansia guangxiensis and tentatively determined its Latin name to be Akkermansia guangxiensis sp. nov.

[0080] The phylogenetic tree of the 16S rRNA sequences of the two isolated Ackermansia guanciensis strains in this example is shown in Figure 1, indicating that the phylogenetic tree belongs to the phylum Verrucomicrobium, class Verrucomicrobium, order Verrucomicrobiales, family Ackermansia, and genus Ackermansia.

[0081] The two Akkermansia strains (N21116 and N21169) obtained in the above example were deposited and preserved on December 2, 2022, at the Guangdong Provincial Microbial Strain Preservation Center (GDMCC, located at 5th Floor, Building 59, Dayuan, 100 Xianlie Middle Road, Guangzhou). Both strains were taxonomically named Akkermansia sp., and their deposit numbers were GDMCC No: 62888 and 62889, respectively.

[0082] Tolerance levels of Ackermansia guanciensis N21116 and N21169 to artificial intestinal fluid The specific experimental steps are as follows: The Ackermansia guanciensis N21116 and N21169 obtained in the above example were cultured and activated overnight (30°C to 37°C, rotation speed 150 rpm to 220 rpm), and the bacterial suspension concentration was adjusted to OD with PBS. 600 The solution was diluted to 1. The bacterial suspension was inoculated into artificial intestinal fluid at pH=8 at a 10% volume ratio. After 2h, 4h, 6h, and 8h, 100 μL of the solution was spread onto mucin solid medium plates and incubated at 37°C for 72h on an anaerobic workstation. The number of single colonies was then counted. The viability of Ackermansia guanciensis N21116 and N21169 in artificial intestinal fluid was calculated based on the number of single colonies.

[0083] The method for preparing the artificial intestinal fluid is as follows: 6.8 g of potassium dihydrogen phosphate is taken, dissolved in 500 mL of water, the pH is adjusted to 8.0 with a 0.1 mol / L sodium hydroxide solution, 10 g of pancreatin is added, and then water is added to dilute it to 1000 mL.

[0084] At the same time, the standard strain of Ackermansia muciniphila, ATCC BAA-835, was used as a control group.

[0085] The results are shown in Figure 3.

[0086] Within a short time, the resistance of Akkermansia muciniphila standard strain ATCC BAA-835 to artificial intestinal fluid was superior to that of Akkermansia guanciensis N21116 and N21169. However, after a treatment time of 8 hours, it was found that Akkermansia guanciensis N21116 and N21169 had survival rates of 120% and 129% respectively after 8 hours of treatment with artificial intestinal fluid at pH=8.0, which was significantly higher than that of Akkermansia muciniphila standard strain ATCC BAA-835 (91%). This indicates that Akkermansia guanciensis N21116 and N21169 according to the examples of the present invention have relatively high resistance to intestinal fluid, have the potential to colonize the human digestive tract over a long period, and can meet the conditions for being considered a probiotic.

[0087] Safety evaluation of Ackermansia guanciensis N21116 and N21169 To ensure the safety of Ackermansia guanciensis N21116 and N21169 obtained in the above examples for various uses, a safety evaluation of the strains was performed on both in this example.

[0088] The specific detection items include the following:

[0089] The entire genomes of Ackermansia guanciensis N21116 and N21169 obtained in the above examples were sequenced to detect the presence of pathogenic factors and pathogenic genes. Sequence similarity ≥ 85% and e-value < 10 -5 Under these conditions, a search was conducted using the existing VFDB (Virulence Factor Database) pathogenicity factor database. As a result, no pathogenic factors or pathogenic genes were detected (see Table 2 for details). Therefore, it was determined from a genetic level that Ackermansia guanciensis N21116 and N21169 pose no pathogenicity risk.

[0090] [Table 2]

[0091] Antioxidant levels of Akkermansia guanschensis N21116 and N21169 After anaerobic culture of Akkermansia guanschensis N21116 and N21169 obtained in the above examples in a synthetic liquid medium at 37 °C for 72 hours, they were centrifuged at 8000 rpm for 10 minutes under 4 °C conditions, thereby separating the supernatant and cell precipitate. The cell precipitate was resuspended in PBS, and the cell concentration was adjusted to 1×10 10 CFU / mL. After sonication, it was centrifuged at 8000 rpm for 10 minutes under 4 °C conditions, and the supernatant was collected to obtain a cell-free extract (CFE).

[0092] The hydroxyl radical, superoxide anion radical scavenging ability and SOD activity of the cell-free extract were detected (using the hydroxyl radical measurement kit, glutathione peroxidase (GSH-PX) measurement kit and total superoxide dismutase (SOD) measurement kit from Nanjing Jiancheng Bioengineering Institute), and the measurement method was referred to the instruction manual.

[0093] At the same time, Akkermansia muciniphila standard strain ATCC BAA-835 and Akkermansia muciniphila A21028 were used as the control group.

[0094] The results are shown in Figure 4.

[0095] Akkermansia guanciensis N21116 demonstrated significantly superior performance (P<0.001) compared to the standard Akkermansia muciniphila strain ATCC BAA-835 and Akkermansia muciniphila A21028 in three indicators: hydroxyl radical scavenging rate, superoxide anion scavenging rate, and SOD activity. In particular, in terms of hydroxyl radical scavenging rate, both Akkermansia guanciensis N21116 and N21169 strains reached 90%, far higher than the standard strain's 65.56% and A21028's 73.38%, indicating extremely superior antioxidant capacity. Furthermore, Akkermansia guanciensis N21169 also performed similarly better than the two Akkermansia muciniphila strains, leading to the conclusion that both Akkermansia N21116 and N21169 possess relatively high antioxidant capacity.

[0096] Fat reduction experiment of nematodes using Ackermansia guanciensis N21116 and N21169 In the above example, Ackermansia guanciensis N21116 and N21169 were cultured in synthetic liquid medium, and Escherichia coli OP50 was cultured in LB liquid medium. The culture conditions for both were a temperature of 37°C, a time of 20 hours, and a rotation speed of 200 rpm. The bacterial suspensions of Ackermansia guanciensis N21116 and N21169 and Escherichia coli OP50 were respectively (the concentration of the bacterial suspensions was 10%). 7 The sample (which was CFU / mL) was spread onto nematode growth medium (NGM) and incubated overnight.

[0097] The NGM medium consisted of 3 g of sodium chloride, 17 g of agar powder, 2.5 g of peptone, and 975 mL of deionized water. After thoroughly mixing the above components, the medium was sealed with tin foil. It was sterilized by high-pressure steam for 20 minutes and then cooled to 55°C in a constant-temperature water bath. Under sterile conditions, 1 mL of sterile 1M CaCl2, 1 mL of 1M MgSO4, 25 mL of 1M KPO4 buffer, and 1 mL of 5 mg / mL cholesterol (dissolved in 95% ethanol) were added.

[0098] The thawed nematodes (Caenorhabditis elegans) were centrifuged, and then each was added to NGM medium containing OP50 and incubated in a 20°C incubator for 3 days.

[0099] Nematodes for the experiment were synchronized, i.e., suspended and cultured in M9 buffer. The nematodes were then dispensed into culture tubes, and cell lysate (5N NaOH by mass and 5% sodium hypochlorite by volume) was added to each tube. Dissolution was performed for 6 minutes, followed by centrifugation at 3500 r / min for 1 minute. The supernatant was discarded, and the tubes were washed four times with M9 buffer. Centrifugation was performed again, and the supernatant was discarded. The precipitate was transferred to NGM medium and incubated overnight at 20°C to obtain L1 nematode larvae. After washing with M9 buffer and centrifuging once, the nematode larvae were transferred to an NGM plate containing OP50 and cultured at 20°C for 28-30 hours to obtain synchronized nematodes, i.e., L4 nematodes.

[0100] The obtained synchronized nematodes were randomly divided into three groups: a blank group (OP50), an N21116 group, and an N21169 group, with 150 nematodes in each group. The day of L4 nematode collection was recorded as day 0. During the experiment, the nematodes were replaced every two days with NGM medium containing OP50 and Ackermansia guanciensis N21116 and N21169. On day 7, after washing three times with cooled M9 buffer, the nematodes were resuspended in 4% paraformaldehyde. They were gently shaken at room temperature for 1 hour, centrifuged at 3000-4000 rpm for 1 minute, the supernatant was removed, and then washed twice with M9 buffer. Subsequently, the nematodes were resuspended in a mixed PBS solution containing 60% (v / v) isopropanol and 0.01% (v / v) Triton X-100 and incubated for 15 minutes. After the nematodes settled, the isopropanol was removed, 1 mL of 40% Oil Red O staining solution was added, and the mixture was incubated on a vibrating table at 25°C for 1-2 hours to allow for thorough staining. The dye was removed, and the mixture was washed twice with M9 buffer. 200 μL of M9 buffer was added, and each nematode was imaged individually using an inverted fluorescence microscope. Light intensity data of the stained regions of the images were statistically analyzed using ImageJ.

[0101] Simultaneously, the standard strain of Akkermansia muciniphila ATCC BAA-835 and Akkermansia muciniphila A21028 were used as control groups.

[0102] The results are shown in Figures 5 and 6.

[0103] The inhibition rate of lipid granule formation in nematodes given the standard strain Akkermansia muciniphila ATCC BAA-835 was found to be 33.9%. The inhibition rates of lipid granule formation in nematodes given Akkermansia muciniphila A21028 and Akkermansia guanciensis N21169 were 42.1% and 44.8%, respectively, with no significant difference from the standard strain group (P>0.05). However, the inhibition rate of lipogenesis in the group given Akkermansia guanciensis N21116 reached 89.0%, which was 55.1% (P<0.01), 46.9% (P<0.0001), and 44.2% (P<0.001), respectively, compared to the above three groups. Furthermore, as can be seen from the lipid-stained microscopic images of the nematodes (Figure 4), the nematodes in the group given Ackermansia guanciensis N21116 have sparsely distributed lipid granules, smaller particle size, and more elongated bodies. As can be seen from this, Ackermansia guanciensis N21116 has a remarkable lipid-reducing effect.

[0104] Suppression of colon cancer cell proliferation by Ackermansia guanciensis N21116 and N21169 Human colon cancer cell line HCT116 was cultured in RPMI1640 (TransGen) medium containing 10% fetal bovine serum. When the cells proliferated in the logarithmic phase, they were digested with pancreatin, and then the cells were counted using a hemocytometer. The cells were counted in 5 × 10⁶ units. 3 The cells were uniformly inoculated into 96-well plates at a rate of 1 / well and incubated overnight in a cell incubator containing 5% CO2 at 37°C. After centrifugation of activated Ackermansia N21116 or N21169, a bacterial suspension was prepared with a bacterial concentration of 1 × 10⁶. 8The concentration was CFU / mL. A portion of the bacterial suspension was centrifuged to obtain cells, which were then washed twice with PBS and inactivated in a 70°C constant temperature water bath for 30 minutes to obtain inactivated bacteria. Six repetitions were performed for each group, depending on whether it was a blank group (with the same amount of PBS added), a N21116 viable cell group, a N21169 viable cell group, a N21116 inactivated cell group, or a N21169 inactivated cell group. After incubating the cells for a total of 72 hours, the growth status of HCT116 cells was detected using the CCK-8 reagent (Solarbio) (procedure followed the instructions in the manual).

[0105] At the same time, the standard strain of Ackermansia muciniphila, ATCC BAA-835, was used as a control group.

[0106] The results are shown in Figure 7.

[0107] The growth inhibition rate of Ackermansia guanciensis N21116 and N21169 against the human colon cancer cell line HCT116 was 85%, with the live and inactive bacterial groups being nearly similar (P>0.05), and not lower than that of the Ackermansia muciniphila standard strain ATCC BAA-835 (P>0.05). As can be seen from this, the morphology of both the live and inactive Ackermansia guanciensis N21116 and N21169 exhibited growth inhibition rates against the human colon cancer cell line HCT116 that were not lower than those of the standard strain, indicating that they have the ability to significantly inhibit the proliferation of human colon cancer cells.

[0108] Products based on Ackermansia guanciensis N21116 or N21169 that have antioxidant, tumor-suppressing, and fat-reducing effects. Akkermansia guanciensis strains N21116 or N21169 were fermented and grown, and then the resulting bacterial solution was centrifuged and freeze-dried to obtain freeze-dried powder. Next, a food additive or supplement containing Akkermansia guanciensis N21116 or N21169 was obtained by formulating it according to the following ingredients.

[0109] Based on human usage amounts and calculated by weight percentage, the ingredients of a food additive or supplement containing Akkermansia guanciensis N21116 or N21169 are: 10% freeze-dried Akkermansia guanciensis N21116 or N21169 powder, 15% tea polyphenols (purchased from Thankcome Biological Science and Technology Co., Ltd.), 15% bayberry anthocyanin extract (purchased from Thankcome Biological Science and Technology Co., Ltd.), 50% maltodextrin, and 10% water-soluble dietary fiber (purchased from Thankcome Biological Science and Technology Co., Ltd.). The preparation method is to thoroughly mix the above raw materials.

[0110] In short, as can be seen from the above test results, among the existing Akkermansia species, only Akkermansia guanciensis N21116 and N21169 simultaneously possess excellent antioxidant, weight loss / lipid reduction, and tumor growth inhibition effects, and are able to survive even in an artificial intestinal fluid environment. Therefore, it has been found that they can colonize the human digestive tract and stably exert antioxidant, weight loss / lipid reduction, and tumor growth inhibition effects over a long period of time.

[0111] While the above embodiments are preferred embodiments of the present invention, the embodiments of the present invention are not limited thereto. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principles of the present invention should be equivalent substitutions and fall within the scope of protection of the present invention.

Claims

1. Products containing Akkermansia, The aforementioned products include food, food additives, animal feed, animal feed additives, pharmaceuticals, and cosmetics. The aforementioned Ackermansia is Ackermansia guanciensis N21116 and / or Ackermansia guanciensis N21169. The aforementioned Akkermansia guangxiensis N21116 has the taxonomic name Akkermansia guangxiensis sp., and was deposited with the Guangdong Provincial Microbial Strain Preservation Center on December 2, 2022, with deposit number GDMCC No: 62888. The aforementioned Akkermansia guangxiensis N21169 is characterized by its taxonomic name being Akkermansia guangxiensis sp., its deposit with the Guangdong Provincial Microbial Strain Preservation Center on December 2, 2022, and its deposit number GDMCC No: 62889.

2. The product according to claim 1, characterized in that the mass fraction of akkermancia in the product is 1% or more, preferably 8% or more, and more preferably 10% or more.

3. The product according to claim 1, characterized in that the product further comprises other auxiliary substances, the auxiliary substances include pharmaceutically acceptable auxiliaries, food additives, and cosmetic auxiliary substances.

4. A food additive containing Akkermansia, A food additive characterized in that the food additive contains freeze-dried powder of Ackermansia guanciensis N21116 and / or Ackermansia guanciensis N21169, plant extract, maltodextrin, and dietary fiber.

5. The food additive according to claim 4, characterized in that the food additive contains, by mass percentage, 5 to 15% freeze-dried powder of Ackermansia guanciensis N21116 and / or Ackermansia guanciensis N21169, 15 to 40% plant extract, 40 to 60% maltodextrin, and 5 to 15% dietary fiber.

6. The food additive according to claim 4 or 5, characterized in that the plant extract contains tea polyphenols and bayberry anthocyanin extract.

7. Use of Ackermansia in the manufacture of food, food additives, feed, feed additives, pharmaceuticals and cosmetics, wherein Ackermansia is Ackermansia guanciensis N21116 and / or Ackermansia guanciensis N21169.

8. The aforementioned foods, food additives, animal feed, animal feed additives, pharmaceuticals, and cosmetics are (1) Antioxidant, (2) Fat reduction, and The use according to claim 7, characterized in that it has at least one of the functions of (3) tumor growth inhibition.

9. The use according to claim 8, characterized in that the tumor is a gastrointestinal tumor.

10. A method for the prevention and / or treatment of tumors, The method comprises administering a therapeutically effective dose of Ackermansia to a tumor patient, wherein the Ackermansia is Ackermansia guanciensis N21116 and / or Ackermansia guanciensis N21169. A method for preventing and / or treating a tumor, wherein the tumor is preferably a gastrointestinal tumor.

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