A postbiotic immunomodulatory agent based on Bifidobacterium longum CD11 and its preparation method

Abstract

This invention provides a postbiotic immunomodulatory agent based on *Bifidobacterium longum* CD11 and its preparation method. The postbiotic is prepared using *Bifidobacterium longum* CD11. Heat-inactivated metabolites of *Bifidobacterium longum* CD11, cell wall, cell membrane, and cell contents postbiotic components all promote the immune activity of RAW264.7 macrophages. Specifically, the cell wall component dose-dependently promotes RAW264.7 macrophage proliferation and inhibits the relative expression of inflammatory cytokines TNF-α and IL-1β mRNA genes. Furthermore, the peptidoglycan (PGN) postbiotic derived from the cell wall component significantly inhibits the relative expression of TNF-α and IL-1β mRNA genes in RAW264.7 macrophages, restoring inflammation to a healthy level (NC group level). Therefore, *Bifidobacterium longum* CD11 can serve as a potential immunomodulatory postbiotic strain.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to the application and preparation method of Bifidobacterium longum CD11 postbiotic in immune regulation. Background Technology

[0002] The immune system is crucial in protecting the host from external viruses, pathogens, parasites, fungi, yeasts, and internal abnormal cells (such as tumor cells), serving as a vital line of defense against disease. The immune system maintains homeostasis by sustaining immune tolerance and regulating the immune system. Insufficient or excessive immune activity, as well as dysregulation of immune organs, cells, and molecules, can lead to immune disorders.

[0003] There is bidirectional communication between the host immune system and the gut microbiota. Both innate and reactive immune systems participate in shaping the microbial composition. The body can recognize microorganisms and their derivatives (e.g., short-chain fatty acids), activating immune signaling molecules and generating an immune defense response.

[0004] Recent research on probiotics has shifted its focus from live microbial cells to non-living components that are beneficial to human health, often referred to as "metapiogenics," "paraprobiotics," or "heat-inactivated probiotics" components. In 2021, the International Scientific Association for Probiotics and Prebiotics (ISAPP) defined metabiotics as "non-living microorganisms or components thereof that produce beneficial properties for the health of the host."

[0005] Postbiotics have been reported to have immunomodulatory effects, enhancing immune regulation and maintaining bodily health by protecting the intestinal barrier. Heat-inactivated *Lactobacillus plantarum* nF1 promoted intestinal health in rats with lobelanamide-induced constipation. Heat-inactivated *Lactobacillus rhamnosus* LGG alleviated oxidative stress and inflammation in diet-induced NAFLD rats. Heat-inactivated fermented milk CCFM1139 and its supernatant had a allergic effect on rats with periodontitis.

[0006] Heat treatment temperature and duration are related to the immune-related properties of probiotic surfaces. Studies have found that heat treatment of *Lactobacillus rhamnosus* at 80°C for 20 min can reduce the levels of LPS-induced pro-inflammatory mediators IL-10 and IL-1β, modulating inflammation levels in rats. Metabiotic components derived from *Bifidobacterium longum* CECT-7347 after heat treatment at 121°C for 20 min alleviate inflammation and protect intestinal barrier function by activating immune pathways. TEM scanning of the cell walls of spray-dried LGG cells revealed reduced cell surface adhesion after heat treatment. Metabiotics exhibit immunomodulatory activity, providing health benefits to the host. For immunocompromised or immunodeficient populations such as the elderly, patients with chronic diseases, premature newborns, and patients undergoing chemotherapy, metabiotics, possessing the health benefits and safety of "live" probiotics, may be a better alternative.

[0007] Therefore, this invention proposes a postbiotic immunomodulatory agent based on Bifidobacterium longum CD11 and its preparation method. Summary of the Invention

[0008] In view of the above-mentioned prior art, this invention provides a technical solution to address the problems of insufficient efficacy and questionable safety of existing immune modulation products.

[0009] The purpose of this invention is to provide a metabiotic, its products, compositions, and their applications in immunomodulation, as well as a method for their preparation. This invention not only screens the heat treatment temperature and duration of the metabiotic but also further explores its bioactive components. The functional components of this invention's metabiotic not only promote the proliferation of RAW264.7 macrophages but also possess certain immunomodulatory capabilities.

[0010] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0011] On one hand, the present invention provides a Bifidobacterium longum, wherein the Bifidobacterium longum is Bifidobacterium longum CD11; the preservation number of the Bifidobacterium longum CD11 is CGMCC No. 6273, and the preservation information of this strain has been recorded in the prior art document CN104046573B.

[0012] On the other hand, the present invention provides a metabiotic component prepared from the aforementioned *Bifidobacterium longum* CD11. The metabiotic component includes heat-inactivated *Bifidobacterium longum* CD11 cells and their metabolites, *Bifidobacterium longum* CD11 cell wall components, *Bifidobacterium longum* CD11 cell membrane components, and *Bifidobacterium longum* CD11 cell contents components. Furthermore, it includes metabiotic components derived from the *Bifidobacterium longum* CD11 cell wall, including *Bifidobacterium longum* CD11 cell wall peptidoglycan (PGN), *Bifidobacterium longum* CD11 cell wall capsular polysaccharide (CPS), *Bifidobacterium longum* CD11 cell wall teichoic acid (WTA), *Bifidobacterium longum* CD11 cell wall lipoteichoic acid (LTA), and *Bifidobacterium longum* CD11 surface protein (SLP), etc.

[0013] Furthermore, the postbiotic component can be dose-dependent, and the concentration of the heat-inactivated Bifidobacterium longum CD11 and the postbiotic components of the above-mentioned products is 1.0 × 10⁻⁶. 5 CFU / mL~1.0×10 11 CFU / mL, preferably, the concentration of the post-genetic component is 10. 6 10 7 10 8 10 9 10 10 More preferably, it is 1.0 × 10 8 CFU / mL, 5.0×10 8 CFU / mL and 1.0×10 9 CFU / mL.

[0014] This invention provides a method for preparing postgenetic agents, comprising one or more of the following steps:

[0015] a. Culture Bifidobacterium longum CD11;

[0016] b. The absorbance (OD) of the Bifidobacterium longum CD11 cultured in step a was measured at 600 nm using a UV spectrophotometer. 600 The data was measured every 1 hour for 24 hours, and a growth curve was plotted to determine the late logarithmic growth phase.

[0017] c. The Bifidobacterium longum CD11 cultured in step a during the late logarithmic growth stage was subjected to heat treatment at different temperatures and durations. The effect of temperature on bacterial morphology was observed by transmission electron microscopy. The survival of Bifidobacterium longum CD11 at different temperatures and durations was analyzed by inverted plate analysis.

[0018] d. Heat inactivation step a: Obtain heat-inactivated Bifidobacterium longum CD11 cells and its metabolites from the cultured Bifidobacterium longum CD11 cells in the late logarithmic growth stage.

[0019] e. Wash the heat-inactivated Bifidobacterium longum CD11 cells from the late logarithmic growth stage in step b with phosphate-buffered saline (PBS) to remove the supernatant, washing three times. The precipitate is then suspended in sterile distilled water and disrupted using a non-contact ultrasonic disruptor. The disrupted liquid is then centrifuged using differential centrifugation to obtain postbiotic components such as the Bifidobacterium longum CD11 cell wall, cell membrane, and cell contents.

[0020] f. Extracting the cell wall components of Bifidobacterium longum CD11 from step e, obtaining postbiotic components such as peptidoglycan (PGN), capsular polysaccharide (CPS), wall teichoic acid (WTA), lipid teichoic acid (LTA), and surface protein (SLP) of the cell wall of Bifidobacterium longum CD11.

[0021] Furthermore, step a includes the following one or more steps:

[0022] a1. Inoculate Bifidobacterium longum CD11 into MRS medium and incubate in a 37℃ constant temperature incubator to obtain the first generation strain;

[0023] a2. The first generation strain obtained in step a1 is inoculated into fresh MRS anaerobic test tubes at an inoculation rate of 2% (v / v%) and cultured under the same culture conditions as in step a1 to obtain the second strain.

[0024] a3. The second-generation strain obtained in step a2 was inoculated into a new MRS anaerobic test tube at an inoculation rate of 2% (v / v%) and cultured under the same conditions as a1 to obtain the activated Bifidobacterium longum CD11 bacterial culture in the late logarithmic growth stage.

[0025] Step b includes:

[0026] The cultured Bifidobacterium longum CD11 was inoculated into new MRS anaerobic test tubes at an inoculation rate of 2% (v / v%). Starting from 0 h, the OD of the Bifidobacterium longum CD11 bacterial culture was measured every 1 h. 600 The value was continuously monitored for 24 hours, and a growth curve was plotted to determine the late logarithmic phase.

[0027] Step c includes:

[0028] c1. The Bifidobacterium longum CD11 cultured in step a, which has reached the late logarithmic growth stage in step b, is heat-treated under five different temperature gradients and two time gradients: 60℃ for 30 min; 75℃ for 10 min; 80℃ for 10 min; 100℃ for 10 min; and 121℃ for 10 min.

[0029] The effects of heat-killing temperature and duration on Bifidobacterium longum CD11 cells were observed using transmission electron microscopy (C2).

[0030] c3 uses the inverted plate method to culture colonies at each temperature to verify the presence of viable bacteria under the given temperature and duration conditions.

[0031] Step d includes

[0032] d1. Centrifuge the cultured Bifidobacterium longum CD11 bacterial solution that meets the logarithmic growth stage of b to obtain heat-inactivated Bifidobacterium longum CD11 cells and its metabolites.

[0033] d2. Wash the heat-inactivated Bifidobacterium longum CD11 cells obtained in step d1 with sterile PBS, repeating the washing 3 times;

[0034] d3. Prepare a bacterial suspension by resuspending the Bifidobacterium longum CD11 bacterial cell pellet washed with PBS buffer in sterile distilled water to achieve a final bacterial concentration of 10. 10 CFU / mL.

[0035] d4. The bacterial suspension was heated in a water bath to inactivate Bifidobacterium longum CD11 in order to obtain the post-thermal biotic components of Bifidobacterium longum CD11 containing Bifidobacterium longum CD11.

[0036] Step e includes:

[0037] e1. The heat-inactivated Bifidobacterium longum CD11 cells from step d5 were disrupted using a non-contact ultrasonic disruptor. The disruption conditions were: Time: 1 hour; Temperature: 4°C; Power: 35%; Energy: 900,000 Joules. Frequency: 9 seconds on, 9 seconds off, 200 cycles.

[0038] e2. Centrifuge the bacterial cell lysate from step d5 at 10000×g for 10 min at 4℃, and collect the supernatant and precipitate separately. Resuspend the precipitate in distilled water to obtain the cell wall components of Bifidobacterium longum CD11. Centrifuge the supernatant again at 25000×g for 90 min at 4℃, and collect the precipitate to obtain the cell membrane components of Bifidobacterium longum CD11. The supernatant contains the cell contents and other postbiotic components of Bifidobacterium longum CD11.

[0039] Furthermore, the concentrations of the heat-inactivated Bifidobacterium longum CD11 cells and their metabolites, the Bifidobacterium longum CD11 cell wall components, the Bifidobacterium longum CD11 cell membrane components, and the supernatant containing the Bifidobacterium longum CD11 cell contents postbiotic components, as described in the postbiotic, were adjusted to 1.0 × 10⁻⁶. 8 CFU / mL, 5.0×10 8 CFU / mL and 1.0×10 9 CFU / mL.

[0040] On the other hand, the present invention provides a method for preparing the post-biotic functional components of the *Bifidobacterium longum* CD11 cell wall, and the resulting post-biotic functional component formulation includes post-biotic components such as *Bifidobacterium longum* CD11 cell wall peptidoglycan (PGN), *Bifidobacterium longum* CD11 cell wall capsular polysaccharide (CPS), *Bifidobacterium longum* CD11 cell wall teichoic acid (WTA), *Bifidobacterium longum* CD11 cell wall lipoteichoic acid (LTA), and *Bifidobacterium longum* CD11 cell wall surface protein (SLP). All formulations are in liquid or powder form.

[0041] On the other hand, the postbiotic provided by the present invention or the postbiotic prepared by the above-mentioned method can be used in the preparation of immune-enhancing dietary supplements, health products, pharmaceuticals, fermented milk, food and other functional foods.

[0042] On the other hand, the metabiotics provided by the present invention can be used in combination with the following microorganisms. The microorganisms can be bacteria, fungi, or any combination thereof; preferably, the microorganisms are probiotics; preferably, the microorganisms are yeasts; preferably, the yeasts are selected from *Saccharomyces cerevisiae*, *Saccharomyces boulardii*, *Kluyveromyces marxianus*, or any combination thereof;

[0043] Preferably, the bacteria are selected from the genera *Lactobacillus*, *Bifidobacterium*, *Bacillus*, *Propionibacterium*, *Streptococcus*, *Lactococcus*, *Pediococcus*, *Enterococcus*, *Staphylococcus*, or any combination thereof. The Lactobacillus species mentioned are selected from: *Lactobacillus paracasei*, *Lactobacillus acidophilus*, *Lactobacillus brevis*, *Lactobacillus jensenii*, *Lactobacillus iners*, *Lactobacillus casei*, *Lactobacillus crispatus*, *Lactobacillus curvatus*, *Lactobacillus delbrueckii*, *Lactobacillus fermentum*, *Lactobacillus gasseri*, *Lactobacillus helveticus*, *Lactobacillus johnsonii*, *Lactobacillus plantarum*, and *Lactobacillus reuteri*. Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, or any combination thereof;

[0044] The bacteria of the genus Bifidobacterium are selected from: Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium adolescentis, or any combination thereof;

[0045] The bacteria of the genus Bacillus are selected from: Bacillus subtilis, Bacillus coagulans, or any combination thereof;

[0046] The bacteria of the genus Propionibacterium are selected from: Propionibacterium shermanii, Propionibacterium freudenreichii, Propionibacterium acidipropionici, or any combination thereof.

[0047] The bacteria of the genus Streptococcus are selected from: Streptococcus thermophilus, Streptococcus salivarius, or any combination thereof;

[0048] The bacteria in the genus Lactococcus mentioned are Lactococcus lactis;

[0049] The bacteria of the genus Enterococcus are selected from: Enterococcus faecalis, Enterococcus faecium, Enterococcus mundtii, or any combination thereof.

[0050] Preferably, the effective amount of postgenetic agent is 0.001g-0.1g; for example, 0.001-0.005g, 0.005-0.01g, 0.01-0.05g, or 0.05-0.1g.

[0051] Preferably, the subject is a mammal;

[0052] Preferably, the mammal is selected from rats, pigs, rabbits, monkeys, sheep, and humans.

[0053] On the other hand, the present invention provides a Bifidobacterium longum CD11 postbiotic and its composition, preferably obtained by culturing Bifidobacterium longum CD11 and then heat-treating it at 100°C for 10 min. The postbiotic components are one or more combinations of inactivated bacterial cells, metabolites, cell walls, cell membranes and / or intracellular substances.

[0054] Preferably, apart from the inactivated bacteria, the other metagenic components are non-toxic to RAW264.7 macrophages and can promote macrophage proliferation. Preferably, the macrophages are RAW264.7.

[0055] On the other hand, the present invention provides a Bifidobacterium longum CD11 postbiotic and its composition, wherein heat-inactivated Bifidobacterium longum CD11 cells and their metabolites, Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane, and Bifidobacterium longum CD11 cell contents postbiotic components can reduce the expression levels of inflammatory cytokines IL-1β and TNF-α mRNA genes in RAW264.7 macrophages. Preferably, the postbiotic component is preferably a Bifidobacterium longum CD11 cell wall postbiotic component.

[0056] Preferably, the metagenic component is heat-inactivated bacterial cells, cell wall metagenic components, cell membrane components, and / or Bifidobacterium longum CD11 cell contents.

[0057] Preferably, the postbiotic component may also be Bifidobacterium longum CD11 cell wall peptidoglycan (PGN), Bifidobacterium longum CD11 cell wall capsular polysaccharide (CPS), Bifidobacterium longum CD11 cell wall teichoic acid (WTA), Bifidobacterium longum CD11 cell wall lipid teichoic acid (LTA), and Bifidobacterium longum CD11 cell wall surface protein (SLP), which are prepared by heat-inactivated Bifidobacterium longum CD11.

[0058] This invention provides a Bifidobacterium longum CD11 postbiotic component, wherein the Bifidobacterium longum CD11 cell wall postbiotic component inhibits the relative expression levels of TNF-α and IL-1β mRNA genes secreted by RAW264.7 macrophages in a dose-dependent manner.

[0059] Preferably, at concentrations of Bifidobacterium longum CD11 cell wall peptidoglycan (PGN), Bifidobacterium longum CD11 cell wall capsular polysaccharide (CPS), Bifidobacterium longum CD11 cell wall teichoic acid (WTA), Bifidobacterium longum CD11 cell wall lipid teichoic acid (LTA), and Bifidobacterium longum CD11 cell wall surface protein (SLP) postbiotic components of 1×10⁸ CFU / mL, 5×10⁸ CFU / mL, and 1×10⁹ CFU / mL, each postbiotic significantly inhibited the relative expression levels of TNF-α and IL-1β mRNA genes secreted by RAW264.7 macrophages; more preferably, the postbiotic is Bifidobacterium longum CD11 cell wall peptidoglycan (PGN).

[0060] The *Bifidobacterium longum* strain of this invention was deposited on June 25, 2012, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (CGMCC), with accession number CGMCCNo. 6273. This strain is named CD11 in this application.

[0061] Compared with the prior art, the beneficial effects of the post-biotic formulations described in this invention include, but are not limited to:

[0062] (1) This invention selects Bifidobacterium longum CD11, which has the strongest microbial metabolic activity and biological activity in the late logarithmic stage, through growth curve detection, to clarify the growth cycle and suitable growth conditions of Bifidobacterium longum CD11, and to explore the way to obtain highly active postbiotic products.

[0063] (2) By subjecting Bifidobacterium longum CD11 to heat treatment with different heat treatment temperature gradients and duration gradients, the effects of heat treatment temperature and duration on the immune-related properties of probiotic cells and probiotic cell surfaces were investigated.

[0064] (3) The metabolites, cell wall components, cell membrane components and cell contents of Bifidobacterium longum CD11 of the present invention are not only non-toxic to RAW264.7 macrophages, but also promote cell proliferation at a certain concentration. The cell wall components of Bifidobacterium longum CD11 have the best effect on the proliferation and immune regulation of RAW264.7 macrophages.

[0065] (4) This invention extracts components from the cell wall of Bifidobacterium longum CD11, which has the best immunomodulatory effect. The extracted components include Bifidobacterium longum CD11 cell wall peptidoglycan (PGN), Bifidobacterium longum CD11 cell wall capsular polysaccharide (CPS), Bifidobacterium longum CD11 cell wall teichoic acid (WTA), Bifidobacterium longum CD11 cell wall lipid teichoic acid (LTA), and Bifidobacterium longum CD11 cell wall surface protein (SLP), which are post-genetic components that are non-toxic to RAW264.7 macrophages and promote RAW264.7 macrophage cell proliferation at a certain concentration. The Bifidobacterium longum CD11 cell wall peptidoglycan post-genetic component has the best cell proliferation effect.

[0066] (5) The Bifidobacterium longum CD11 postbiotic of the present invention has a certain immunomodulatory effect on RAW264.7 macrophages. The postbiotic can significantly inhibit the expression of LPS-induced RAW264.7 macrophage secreted cytokine genes, effectively enhance the body's immune defense capabilities and maintain immune balance.

[0067] (6) The heat-inactivated Bifidobacterium longum CD11 cell metabolites and their metabolites of the present invention, including Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, Bifidobacterium longum CD11 cell contents components, and Bifidobacterium longum CD11 cell wall components, including Bifidobacterium longum CD11 cell wall peptidoglycan (PGN), Bifidobacterium longum CD11 cell wall capsular polysaccharide (CPS), Bifidobacterium longum CD11 cell wall wall teichoic acid (WTA), Bifidobacterium longum CD11 cell wall lipid teichoic acid (LTA), and Bifidobacterium longum CD11 cell wall surface protein (SLP), can all promote the immune activity of RAW264.7 macrophages. Therefore, Bifidobacterium longum CD11 can be a potential candidate strain for immunomodulatory metabolites.

[0068] (7) The method for preparing Bifidobacterium longum CD11 postbiotic of the present invention is simple, low in cost and can be mass-produced. Since the postbiotic has strain specificity and Bifidobacterium longum is the main beneficial bacteria in the body's gastrointestinal microbiota, Bifidobacterium longum CD11 postbiotic has high safety and no side effects and is a potential immunomodulator. Attached Figure Description

[0069] Figure 1 This is a graph showing the growth curve of Bifidobacterium longum CD11 in one embodiment of the present invention.

[0070] Figure 2 This is a transmission electron microscope (TEM) image showing the effect of different heat treatment temperatures and durations on Bifidobacterium longum CD11 cells in one embodiment of the present invention. Red circles: intracellular material leakage; blue circles: cell breakage / depression.

[0071] Figure 3 This is a bar graph illustrating the effect of Bifidobacterium longum CD11 postbiotic on the cell viability of RAW264.7 macrophages in one embodiment of the present invention.

[0072] Figure 4 This is a bar graph illustrating the effect of Bifidobacterium longum CD11 postbiotic on the expression of TNF-α inflammatory factor mRNA in RAW264.7 macrophages in an embodiment of the present invention.

[0073] Figure 5This is a bar graph illustrating the effect of Bifidobacterium longum CD11 postbiotic on the expression of IL-1β inflammatory factor mRNA in RAW264.7 macrophages in an embodiment of the present invention.

[0074] Figure 6 This is a bar graph illustrating the effect of post-biotic components of the Bifidobacterium longum CD11 cell wall on the cell viability of RAW264.7 macrophages in an embodiment of the present invention.

[0075] Figure 7 This is a bar graph illustrating the effect of post-biotic components of the Bifidobacterium longum CD11 cell wall on the expression of TNF-α inflammatory factor mRNA in RAW264.7 macrophages in an embodiment of the present invention.

[0076] Figure 8 This is a bar graph illustrating the effect of post-biotic components of the Bifidobacterium longum CD11 cell wall on the expression of IL-1β inflammatory factor mRNA in RAW264.7 macrophages in an embodiment of the present invention. Detailed Implementation

[0077] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0078] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0079] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0080] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0081] The strains, reagents, and basal culture media involved in this invention include:

[0082] Bifidobacterium longum CD11 was isolated from the feces of a healthy centenarian and is deposited at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 6273 and deposit date of June 25, 2012.

[0083] The mouse mononuclear macrophage RAW264.7 macrophage cell line was purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0084] De Man, Rogosa and Sharpe (MRS) broth culture medium:

[0085] 110.0g peptone, 5.0g anhydrous sodium acetate, 5.0g yeast powder, 20.0g glucose, 1.0g Tween 80, 0.19g manganese sulfate tetrahydrate, 2.0g diammonium hydrogen citrate, 0.58g magnesium sulfate, 2.0g dipotassium hydrogen phosphate, 5.0g beef extract, 0.5g cysteine ​​salt, diluted to 1L with distilled water, adjusted pH to 6.5, and sterilized at 121℃ for 15min.

[0086] De Man, Rogosa and Sharpe (MRS) solid culture medium:

[0087] 110.0g peptone, 5.0g anhydrous sodium acetate, 5.0g yeast powder, 20.0g glucose, 1.0g Tween 80, 0.19g manganese sulfate tetrahydrate, 2.0g diammonium hydrogen citrate, 0.58g magnesium sulfate, 2.0g dipotassium hydrogen phosphate, 5.0g beef extract, 0.5g cysteine, 15g agar powder, diluted to 1L with distilled water, adjusted pH to 6.5, and sterilized at 121℃ for 15min.

[0088] Preparation of lipopolysaccharide (LPS) solution:

[0089] Accurately weigh 0.001 g of LPS powder into 10 mL of sterile PBS buffer, vortex to mix, and prepare a 1 mg / mL LPS stock solution. Store at -20°C for later use. When using, dilute the 1 mg / mL LPS stock solution to a 1 μg / mL LPS solution using DMEM high-glucose medium.

[0090] RAW264.7 macrophage culture:

[0091] Take out 1 mL of the cell line stored at low temperature, thaw it quickly, place it in a 15 mL sterile centrifuge tube, add 5 mL of 10% fetal bovine serum culture medium (10% FBS), mix well, centrifuge at 1500 rpm / min for 3 min, remove the supernatant, transfer the cell pellet to a 100 mm cell culture dish, and culture at 37°C under saturated humidity.

[0092] When the cells reach about 80% confluence, wash them twice with PBS, digest them with 0.25% trypsin-EDTA digestion solution for 3-4 minutes, and then detach the cells by pipetting them off the bottom of the container with serum-free culture medium.

[0093] Centrifuge the cell suspension at 1500 rpm for 3 min, discard the supernatant, and add fresh culture medium to suspend the cell pellet at the bottom. Passage the cell suspension at a 1:3 ratio into three culture dishes, add culture medium to each dish, and continue culturing at 37°C under saturated humidity.

[0094] Other reagents can be purchased commercially or prepared according to a biology lab manual.

[0095] Example 1: Bacterial Culture

[0096] The Bifidobacterium longum CD11 of this application was isolated from the feces of healthy centenarians.

[0097] Pure colonies were picked and inoculated into MRS broth medium. The culture was incubated at 37°C for 24 hours to activate the strain. After subculturing three times using the same method at a 2% (v / v) inoculation rate, the culture was stored at 4°C for later use.

[0098] Example 2: Preparation of post-genetic agents and post-genetic components

[0099] The metabiotics of this application include heat-inactivated Bifidobacterium longum CD11 cells and their metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, Bifidobacterium longum CD11 cell contents components, and Bifidobacterium longum CD11 cell wall peptidoglycan (PGN), Bifidobacterium longum CD11 cell wall capsular polysaccharide (CPS), Bifidobacterium longum CD11 cell wall teichoic acid (WTA), Bifidobacterium longum CD11 cell wall lipoteichoic acid (LTA), and Bifidobacterium longum CD11 cell wall surface protein (SLP), etc.

[0100] The concentration of the biotic components after heat inactivation of Bifidobacterium longum CD11 was 1.0 × 10⁻⁶. 8 CFU / mL, 5.0×10 8 CFU / mL and 1.0×10 9 CFU / mL.

[0101] The specific method for preparing postgenes in this application includes the following steps:

[0102] a. Culture Bifidobacterium longum CD11.

[0103] The third-generation activation technology was used to activate Bifidobacterium longum CD11.

[0104] The activated strains were stored in a 4°C refrigerator for later use.

[0105] The activation steps are as follows:

[0106] a1. Activated Bifidobacterium longum CD11 was inoculated at a rate of 2% (v / v%) into an anaerobic test tube containing MRS broth medium. The mixture was vortexed and incubated at 37°C for 24 hours. The first generation strain obtained was then stored at 4°C for later use.

[0107] a2. The first generation strain was inoculated at an inoculation rate of 2% (v / v%) into an anaerobic test tube containing fresh MRS broth medium, vortexed to mix, and incubated in a constant temperature incubator at 37℃ for 24h to obtain the second generation strain, which was then stored in a refrigerator at 4℃ for later use.

[0108] a3. The second-generation strain was inoculated at an inoculation rate of 2% (v / v%) into an anaerobic test tube containing fresh MRS broth medium, vortexed and mixed, and then incubated in a constant temperature incubator at 37℃ for 10h to obtain the logarithmic late stage bacterial culture of Bifidobacterium longum CD11, which was then stored in a refrigerator at 4℃ for later use.

[0109] OD of Bifidobacterium longum CD11 bacterial culture 600 The value is 1.774.

[0110] b. Heat inactivation of Bifidobacterium longum CD11 cultured in step a, to obtain Bifidobacterium longum CD11 post-biotic.

[0111] b1. Centrifuge the cultured Bifidobacterium longum CD11 solution at 6000×g for 15 min. Obtain Bifidobacterium longum CD11 cell precipitate and Bifidobacterium longum CD11 metabolites.

[0112] b2. Wash the precipitate of Bifidobacterium longum CD11 strain with sterile PBS, washing more than 3 times to completely remove the culture medium.

[0113] b3. Prepare a Bifidobacterium longum CD11 bacterial suspension by resuspending the PBS-washed Bifidobacterium longum CD11 bacterial cell pellet in sterile distilled water. The final concentration of the bacterial suspension should be 10. 10 CFU / mL.

[0114] b4. Heat the Bifidobacterium longum CD11 bacterial suspension in a water bath to inactivate the bacterial strains in each suspension, thereby obtaining Bifidobacterium longum CD11 bacterial post-biotics, which can be stored in a refrigerator at 4℃ for later use.

[0115] Heat treatment was performed at five different temperature gradients and two time gradients, namely 65℃ for 30 min, 75℃ for 10 min, 80℃ for 10 min, 100℃ for 10 min, and 121℃ for 10 min, with water bath heating temperatures and durations of 65℃ for 30 min, 75℃ for 10 min, 80℃ for 10 min, 100℃ for 10 min, and 121℃ for 10 min.

[0116] The morphology of Bifidobacterium CD11 cells was observed using a transmission electron microscope after heat inactivation at different temperatures and durations. If the cells were concave and intracellular fluid was released, it indicated that the cells had ruptured.

[0117] The heat-inactivated Bifidobacterium longum CD11 bacterial suspensions were mixed at each temperature and cultured in MRS solid medium for 48 hours using the inverted plate method. If no colonies appeared in the medium, it indicated that the bacterial cells were completely inactivated.

[0118] c. The heat-inactivated Bifidobacterium longum CD11 cells, which have reached the heat-killing temperature, are placed in a non-contact ultrasonic disruptor for disruption.

[0119] c1. The crushing conditions for the non-contact ultrasonic crusher are as follows: Time: 1 hour; Temperature: 4°C; Amplitude: 35%; Energy: 900,000 Joules; Frequency: 9 seconds on, 9 seconds off, 200 cycles.

[0120] c2. Differential centrifugation was used to separate the cell wall components, cell membrane components, and cell contents of Bifidobacterium longum CD11.

[0121] c3. The lysate of *Bifidobacterium longum* CD11 cells was centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatant and precipitate were collected separately. The precipitate was resuspended in distilled water to obtain the cell wall components of *Bifidobacterium longum* CD11; the supernatant was further centrifuged at 25,000 × g for 90 min at 4 °C, and the precipitate was collected to obtain the cell membrane components of *Bifidobacterium longum* CD11, while the supernatant contained the cell contents and other postbiotic components of *Bifidobacterium longum* CD11.

[0122] d. Extracting the components from the cell wall of Bifidobacterium longum CD11.

[0123] d1. Extraction method of Bifidobacterium longum CD11 peptidoglycan (PGN): The bacterial cells cultured in step a were centrifuged at 2,000×g for 10 min to collect Bifidobacterium longum CD11 cells and boiled in 4% sodium dodecyl sulfate (SDS) for 30 min.

[0124] The suspension was cooled at 4°C, centrifuged at 6000×g at room temperature for 5 min, and washed with distilled water until no SDS was found, at least 3 times.

[0125] The precipitate was treated with an ultrasonic processor of 700W for 10 minutes, centrifuged at 6000×g for 5 minutes at room temperature, and washed three times with distilled water.

[0126] The precipitate was resuspended in 10 mL of 100 mM Tris-HCl (pH 7.5) and incubated with DNase I (5 μL, 1 mg / mL), RNase A (5 μL, 5 mg / mL) and MgSO4 (20 mM) in a shaker at 37 °C for 140 rpm for 2 h.

[0127] Add trypsin (10 μL, 10 mg / mL) and CaCl2 (10 mM) and incubate at 37°C on a shaker at 140 rpm for 12 h.

[0128] Centrifuge at 6000×g for 5 min, boil the precipitate in 1% SDS for 10 min, wash the SDS-insoluble cell wall components until SDS-free, and resuspend in trichloroacetic acid (10%) at 4 °C for 24 h to remove cell wall lipid teichoic acid (LTA).

[0129] Wash the water-insoluble components with pre-cooled distilled water until the pH is neutral. Collect the PGN, freeze-dry it, and store it at -20°C for later use.

[0130] d2. Extraction method of Bifidobacterium longum CD11 capsular polysaccharide (CPS): The bacterial cells cultured in step a were collected by centrifugation, resuspended in digestion buffer (PBS with 5mM MgCl2 and 1mM CaCl2 added), and lysozyme and lysozyme (concentration of 100μg / mL) were added and digested at 37℃ for 18h.

[0131] Centrifuge at 6000×g for 5 min at 4℃ to remove insoluble substances. Incubate the supernatant with nucleases (DNase I and RNase A, 100 μg / mL) at 37℃ for 4 h.

[0132] Incubate with proteinase K (100 μg / mL) at 56 °C for 18 h.

[0133] Add 4 times the volume of anhydrous ethanol to treat the solution, and collect the precipitate by centrifugation.

[0134] The precipitate was resuspended in distilled water and transferred to a prepared dialysis bag (8000-14000 Da). Dialysis was performed at 4°C for 4 days. The dialysis water was changed every 8 hours during the process to thoroughly remove solvent molecules and small molecule impurities.

[0135] d3. Extraction method of CD11 surface protein (SLP) of Bifidobacterium longum: The bacterial cells cultured in step a were harvested by centrifugation at 6000×g for 10 min and washed three times with sterile PBS (pH 7.4).

[0136] The washed cell resuspension was placed in 4 mol / L LiCl (pH 2.0), and a protease inhibitor was added. The mixture was incubated at 37°C for 1 h. The sample was then centrifuged at 12800×g for 30 min at 4°C, and the supernatant containing SLP was collected.

[0137] The SLP was placed in a prepared dialysis bag (8000-14000 Da) and dialyzed at 4°C for 4 days. The dialysis water was changed every 8 hours to thoroughly remove solvent molecules and small molecule impurities.

[0138] d4. Extraction method of Bifidobacterium longum CD11 wall teichoic acid (WTA): Collect bacterial cells by centrifugation at 2,000×g for 10 min from the bacterial cells cultured in step a.

[0139] The precipitate was washed once with 30 mL of buffer 1 [50 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5].

[0140] Centrifuge at 6000×g for 5 min at 4℃ to collect the precipitate, and resuspend it in 30 mL of buffer 2 (4% [wt / vol] sodium dodecyl sulfate [SDS], 50 mM MES, pH 6.5). Place the sample in a boiling water bath for 1 h.

[0141] Cells were collected by centrifugation at 4°C, 10,000×g for 10 min. The precipitate was resuspended in buffer 2 and transferred to a 2 mL microcentrifuge tube. The precipitate was collected by centrifugation at 4°C, 14,000×g for 10 min.

[0142] The precipitate was washed once with buffer 2, once with buffer 3 (2% NaCl, 50mM MES, pH 6.5), and finally washed with buffer 1. After the last wash, the sample was treated with proteinase K digestion solution (1 mL of 20mM Tris-HCl [pH 8.0], 0.5% [wt / vol] SDS and 20 μg proteinase K) and incubated at 50°C for 4 h.

[0143] After digestion, wash the sample once with buffer 3, and then wash with distilled water for at least 3 times to remove SDS.

[0144] The sample was resuspended in 1 mL of 0.1 M NaOH and hydrolyzed at room temperature for 16 h.

[0145] Insoluble cell wall fragments were removed by centrifugation at 4°C, 14,000×g, for 10 min, and the supernatant was collected.

[0146] The supernatant sample was neutralized to neutral with 0.1M acetic acid, dialyzed with distilled water, the molecular weight cutoff was 1 kDa, and the sample was freeze-dried to obtain a white powder, which was stored at -20℃ for later use.

[0147] d4. Extraction of Bifidobacterium longum CD11 lipoteichoic acid (LTA): Collect Bifidobacterium longum CD11 bacterial cells by centrifugation at room temperature for 10 min at 2,000×g.

[0148] Bifidobacterium longum CD11 bacterial cells were resuspended in distilled water.

[0149] The bacterial culture was treated with an ultrasonic processor of 700W for 10 minutes, and then centrifuged at 4℃ for 6000×g for 5 minutes.

[0150] At room temperature, the sample containing the broken cell walls was mixed with an equal volume of water-saturated n-butanol and stirred continuously for 60 minutes.

[0151] Dispense into centrifuge tubes and centrifuge at 4°C, 13000×g for 30 min in a high-speed centrifuge.

[0152] Collect the aqueous phase layer and centrifuge again under the same conditions to completely remove the precipitate, obtaining crude LTA extract.

[0153] The crude LTA extract was transferred to a prepared dialysis bag (molecular weight cutoff 3500 Da) and dialyzed at 4°C for 4 days. The dialysis water was changed every 8 hours to thoroughly remove solvent molecules and small molecule impurities.

[0154] Example 3: Functional Verification of Postbiotics and Postbiotic Components

[0155] Experimental Example 1: Determination of CD11 growth curve of Bifidobacterium longum

[0156] The time of the late logarithmic growth phase was determined by analyzing the growth curve of Bifidobacterium longum CD11.

[0157] S11 was inoculated at a rate of 2% (v / v%) into a culture medium containing fresh MRS broth and cultured in a constant temperature incubator at 37°C.

[0158] S12 was measured using an ultraviolet spectrophotometer at 600 nm. nm OD of absorbance at the point of detection 600 Values, plot growth curves;

[0159] S13 was counted using the inverted plate method to count the number of Bifidobacterium longum CD11 colonies in the late logarithmic phase of S12.

[0160] Experiment Example 2: Effects of heat inactivation temperature and duration on the cell viability of Bifidobacterium longum CD11

[0161] S11 was set to perform heat treatment under five different temperature gradients and two time gradients: 65℃ for 30 min; 75℃ for 10 min; 80℃ for 10 min; 100℃ for 10 min; and 121℃ for 10 min.

[0162] S12 was inoculated into anaerobic test tubes of fresh MRS broth medium at an inoculation rate of 2% (v / v%). After being cultured to the late logarithmic phase, the culture was subjected to heat inactivation treatment under the temperature and duration conditions described in S11. Then, the bacterial suspensions treated at each temperature were cultured in MRS solid medium for 48 hours using the inverted plate method. If no colonies appeared in the medium, it indicated that the bacterial cells were completely inactivated. The temperature and duration at which no viable cells were found were determined as the heat-killing temperature and time.

[0163] Experiment Example 3: Observation of the morphology of heat-inactivated Bifidobacterium longum CD11 cells using transmission electron microscopy.

[0164] The steps are as follows:

[0165] Take the cultured Bifidobacterium longum CD11 in the late logarithmic growth stage, vortex mix well, and aliquot into 2mL sterile enzyme-free centrifuge tubes. Heat treat at different temperatures and durations. Centrifuge at 4℃, 12000rpm for 5min. Discard the supernatant and collect the precipitate. Wash the precipitate three times with sterile water and PBS. Add 200μL of fresh 25% glutaraldehyde and fix overnight at 4℃. Wash three times with sterile PBS for 10min each time.

[0166] Freeze-cut: The sample is rapidly frozen with liquid nitrogen and then gently tapped with a hammer to allow it to break naturally.

[0167] Dehydration was performed using gradients of 30%, 50%, 70%, 90%, and 100% ethanol, with each gradient dehydrating for 10 minutes.

[0168] Replacement: Replace ethanol with ethyl pentyl acetate for 10 min;

[0169] Supercritical drying: CO2 supercritical instrument was used for drying for 1.5 h at 32 °C and 73 atmospheres (7.373 MPa);

[0170] Coating: Platinum film was deposited using an ion sputtering apparatus;

[0171] The sample was photographed using a transmission electron microscope.

[0172] The results are from Experiments 1-3. Figure 1 , 2It was found that by plotting the growth curve of *Bifidobacterium longum* CD11, the late logarithmic growth phase obtained according to the above culture method was 10 hours. After heat treatment at 100℃ and 121℃ for 10 minutes, no viable bacteria appeared in the inverted plate culture, indicating that *Bifidobacterium longum* CD11 cells died after heat treatment at 100℃ for 10 minutes. Transmission electron microscopy showed that the bacterial cells ruptured and became concave during this period, with the cell contents flowing out. The degree of bacterial cell rupture increased and the number of cell concavities increased after heat treatment at 121℃ for 10 minutes. Therefore, heat treatment of *Bifidobacterium longum* CD11 at 100℃ for 10 minutes can produce a postbiotic product for subsequent experiments.

[0173] Experiment Example 4: Effect of Bifidobacterium longum CD11 postbiotic on the proliferation capacity of RAW264.7 macrophages

[0174] Take 1×10⁻⁶ RAW264.7 macrophages in the late logarithmic growth phase. 5 Cells were seeded at a density of 200 μL / well in 96-well plates and incubated at 37°C in a humidified incubator for 24 h.

[0175] Remove the culture medium from the 96-well plate, wash the non-adherent cells with sterile PBS, add Bifidobacterium longum CD11 post-generic in the 96-well plate, and incubate at 37°C in a saturated humidity incubator for 24 hours.

[0176] Different concentration groups were set up: heat-inactivated Bifidobacterium longum CD11 cells, Bifidobacterium longum CD11 metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, and Bifidobacterium longum CD11 cell contents components, with a concentration of 1×10⁻⁶ for each functional component. 8 CFU / mL, 5×10 8 CFU / mL, 1×10 9 CFU / mL, 200 μL per well, and continue culturing for 24 h.

[0177] Remove the post-biotic liquid from the 96-well plate, wash twice with sterile PBS, add 10% CCK8 solution to each well, incubate at 37°C in the dark for 1-4 hours for color development, measure the absorbance at 450 nm using a multi-functional microplate reader, and prepare a blank control group.

[0178] The formula for calculating cell viability is as follows:

[0179]

[0180] In the formula: OD0 is the zeroing hole; OD1 is the blank hole; OD2 is the experimental hole.

[0181] Experiment 5: Lipopolysaccharides (LPS) induced an inflammation model in RAW264.7 macrophages.

[0182] The groups were set up as blank control group (NC), LPS model group (LPS), LPS + positive drug dexamethasone group (Positive), and LPS + different functional component treatment groups.

[0183] Take a suspension of RAW264.7 macrophages and adjust the density to 1.2 × 10⁻⁶. 6 Cells were seeded at a density of 1 μg / mL in 12-well cell culture plates. After 12 hours of cell adhesion culture, cells in all groups except the NC group were stimulated with 1 μg / mL LPS for 24 hours to induce cell polarization.

[0184] Cells that successfully modeled LPS polarization were treated with heat-inactivated Bifidobacterium longum CD11 cells, Bifidobacterium longum CD11 metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, and Bifidobacterium longum CD11 cell contents components, respectively, at a concentration of 1×10⁻⁶ mL / well. 8 CFU / mL, 5×10 8 CFU / mL, 1×10 9 Different postbiotic functional components at CFU / mL.

[0185] The results are as follows Figure 3 As shown, apart from the heat-inactivated Bifidobacterium longum CD11 bacterial cell component, the other metagenic components of this invention are not only non-toxic to RAW264.7 macrophages, but can also promote the proliferation of RAW264.7 macrophages at a certain concentration.

[0186] like Figure 4 , 5 As shown, compared with the blank control group (NC group), lipopolysaccharide (LPS) induced the secretion of inflammatory cytokines IL-1β and TNF-α by RAW264.7 macrophages. Heat-inactivated Bifidobacterium longum CD11 cells and their metabolites, Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane, and Bifidobacterium longum CD11 cell contents, as well as other postbiotic components, significantly reduced the mRNA gene expression levels of inflammatory cytokines IL-1β and TNF-α in RAW264.7 macrophages (P<0.05).

[0187] Compared with the positive drug dexamethasone group, heat-inactivated Bifidobacterium longum CD11 cells, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, and Bifidobacterium longum CD11 cell contents, as well as other postbiotic components, inhibited the secretion of TNF-α and IL-1β by RAW264.7 macrophages.

[0188] Compared with heat-inactivated Bifidobacterium longum CD11 cell fraction, the Bifidobacterium longum CD11 cell wall post-biotic fraction showed better inhibitory effects on the secretion of IL-1β and TNF-α by RAW264.7 macrophages.

[0189] Compared with Bifidobacterium longum CD11 metabolites, heat-inactivated Bifidobacterium longum CD11 cell fractions, Bifidobacterium longum CD11 cell wall postgenetic fractions, Bifidobacterium longum CD11 cell membrane fractions, and Bifidobacterium longum CD11 cell contents postgenetic fractions showed the best inhibitory effect on the secretion of TNF-α and IL-1β by RAW264.7 macrophages.

[0190] In summary, combining experimental examples 4 and 5, Figure 3-5 As shown, the heat-inactivated Bifidobacterium longum CD11 cells, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, and Bifidobacterium longum CD11 cell contents of the present invention, etc., have certain immunomodulatory capabilities. Furthermore, the Bifidobacterium longum CD11 cell wall components exhibit the strongest inhibitory effect on TNF-α and IL-1β secreted by RAW264.7 macrophages, at a concentration of 1×10⁻⁶. 8 CFU / mL, 5×10 8 CFU / mL and 1×10 9 A CFU / mL concentration gradient showed dose-dependent inhibition of the mRNA gene expression of inflammatory cytokines TNF-α and IL-1β.

[0191] Experimental Example 6: Effects of Bifidobacterium longum CD11 cell wall post-biotic components on the proliferation capacity of RAW264.7 macrophages

[0192] Unlike Experiment 4, the postbiotics of *Bifidobacterium longum* CD11 were replaced with postbiotic components of the *Bifidobacterium longum* CD11 cell wall, including postbiotic components such as *Peptidoglycan* (PGN), *CPS* (CPS), *WTA* (Wasteteichoic acid), *LTA* (Lipoteichoic acid), and *SLP* (Symplocanediol). All other experimental procedures remained unchanged.

[0193] Experiment Example 7: Effects of Bifidobacterium longum CD11 postbiotic on the mRNA expression of IL-1β and TNF-α inflammatory cytokines in RAW264.7 macrophages

[0194] RAW264.7 macrophages in the logarithmic growth phase were harvested and their density adjusted to 1.2 × 10⁻⁶ cells. 6 Cells were seeded per well in 12-well cell culture plates and incubated at 37°C in a saturated humidity incubator for 24 hours.

[0195] Aspirate the culture medium from the 12-well plate with a pipette, wash twice with sterile PBS buffer, add Bifidobacterium longum CD11 postbiotic at a volume of 1 mL / well, and incubate at 37℃ for 24 h.

[0196] Different concentration groups were set up: the concentrations of heat-inactivated Bifidobacterium longum CD11 and its metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, and Bifidobacterium longum CD11 cell contents components were 1×10⁻⁶. 8 CFU / mL, 5×10 8 CFU / mL, 1×10 9 CFU / mL.

[0197] Take the cells cultured in the previous steps and extract total RNA according to the RNA extraction kit operation procedure.

[0198] The qRT-PCR kit 5×All In One RT-MasterMix kit and PowerUp were used. TM SYBR TM The Green premix kit was used for reverse transcription and real-time quantitative PCR, and a blank control group was also used.

[0199] The experiment consisted of four parallel groups, with a reaction volume of 20 μL. The reverse transcription amplification conditions are shown in Table 1.

[0200] Table 1. qRT-PCR amplification conditions

[0201]

[0202] The primers for real-time quantitative qRT-PCR are shown in Table 2, and the internal control is GAPDH.

[0203] Use 2 ^(-△△Ct) The method is used to analyze data and calculate the relative fold change in gene expression.

[0204] Table 2 Primer Sequence List for Real-Time Quantitative PCR

[0205]

[0206] Experiment Example 8: Effects of various components of the Bifidobacterium longum CD11 cell wall on the mRNA expression of IL-1β and TNF-α inflammatory cytokines in RAW264.7 macrophages

[0207] Unlike Experiment 7, the heat-inactivated Bifidobacterium longum CD11 and its metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, and Bifidobacterium longum CD11 cell contents components were replaced with Bifidobacterium longum CD11 cell wall peptidoglycan (PGN), Bifidobacterium longum CD11 cell wall capsular polysaccharide (CPS), Bifidobacterium longum CD11 cell wall teichoic acid (WTA), Bifidobacterium longum CD11 cell wall lipid teichoic acid (LTA), and Bifidobacterium longum CD11 cell wall surface protein (SLP), etc., as postbiotic components.

[0208] like Figure 6 As shown, compared with the blank control group (NC group), the peptidoglycan (PGN) component, capsular polysaccharide (CPS) component, wall teichoic acid (WTA) component, lipid teichoic acid (LTA) component, and surface protein (SLP) component of Bifidobacterium longum CD11 cell wall not only had no toxicity to RAW264.7 macrophages, but also promoted the proliferation of RAW264.7 macrophages at certain concentrations.

[0209] Compared with the positive control group treated with dexamethasone, the peptidoglycan (PGN) component, capsular polysaccharide (CPS) component, wheyteichoic acid (WTA) component, lipoteichoic acid (LTA) component, and surface protein (SLP) component of *Bifidobacterium longum* CD11 cell wall significantly promoted the proliferation of RAW264.7 macrophages (P<0.05). Furthermore, the peptidoglycan (PGN) and SLP components of *Bifidobacterium longum* CD11 cell wall promoted RAW264.7 macrophage proliferation in a dose-dependent manner. Specifically, 1 × 10-1 8 5×10 8 and 1×10 9 At a CFU / mL concentration, the inhibitory effect on the proliferation of RAW264.7 macrophages and the inhibition of inflammatory cytokines increased with the increase of PGN post-biotic concentration; the higher the PGN post-biotic concentration, the better the effect.

[0210] like Figure 7-8 It was found that, compared with the LPS model group (NC group), the post-biotic components of the Bifidobacterium longum CD11 cell wall significantly reduced the relative mRNA expression levels of TNF-α and IL-1β genes in RAW264.7 macrophages, and the effect was significant (P<0.05).

[0211] Compared with the positive drug dexamethasone group, the metagenic components of Bifidobacterium longum CD11 cell wall peptidoglycan (PGN), Bifidobacterium longum CD11 cell wall capsular polysaccharide (CPS), Bifidobacterium longum CD11 cell wall teichoic acid (WTA), Bifidobacterium longum CD11 cell wall lipid teichoic acid (LTA), and Bifidobacterium longum CD11 cell wall surface protein (SLP) inhibited the relative mRNA expression levels of TNF-α and IL-1β genes in RAW264.7 macrophages.

[0212] Compared with other metagenic components such as the capsule polysaccharide (CPS) component, wall teichoic acid (WTA) component, lipid teichoic acid (LTA) component, and surface protein (SLP) component of the cell wall of Bifidobacterium longum CD11, the peptidoglycan (PGN) component of the cell wall of Bifidobacterium longum CD11 showed a better inhibitory effect on the relative expression of TNF-α and IL-1β genes in RAW264.7 macrophages.

[0213] In summary, based on Experiments 3-5, the heat-inactivated Bifidobacterium longum CD11 and its metabiotic components of this application, including the heat-inactivated Bifidobacterium longum cells, metabolites, cell wall, cell membrane, and cell contents, can significantly inhibit the relative mRNA expression levels of TNF-α and IL-1β genes in RAW264.7 macrophages. Therefore, the heat-inactivated Bifidobacterium longum CD11 metabiotic of this invention possesses a certain immunomodulatory capacity, and the Bifidobacterium longum CD11 cell wall component dose-dependently inhibits the relative mRNA expression levels of TNF-α and IL-1β genes in RAW264.7 macrophages.

[0214] Combined with Experiments 6-8, the post-biotic components of the Bifidobacterium longum CD11 cell wall, including peptidoglycan (PGN), capsular polysaccharide (CPS), wheyteichoic acid (WTA), lipoteichoic acid (LTA), and surface protein (SLP), can inhibit the relative mRNA expression levels of TNF-α and IL-1β genes in RAW264.7 macrophages. Therefore, the post-biotic components of the Bifidobacterium longum CD11 cell wall of this invention possess certain immunomodulatory capabilities, and the post-biotic Bifidobacterium longum CD11 cell wall peptidoglycan (PGN) component significantly inhibits the relative mRNA expression levels of TNF-α and IL-1β genes in RAW264.7 macrophages, restoring inflammation to healthy (NC group) levels.

[0215] Conclusion: Analysis of the in vitro proliferation capacity of RAW264.7 macrophages showed that the concentration of biotic components after heat inactivation of Bifidobacterium CD11 cells was 1×10⁻⁶. 8 CFU / mL, 5×108 CFU / mL and 1×10 9 At CFU / mL, it showed some toxicity to RAW264.7 macrophages, reducing cell viability. However, the effects of Bifidobacterium longum CD11 metabolites, Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane, and Bifidobacterium longum CD11 intracellular substances were superior to those of heat-inactivated Bifidobacterium longum CD11 post-biotic components.

[0216] When the concentration of postbiotic components of Bifidobacterium longum CD11 metabolites, Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane, and Bifidobacterium longum CD11 intracellular substances is 1×10⁻⁶ 8 CFU / mL, 5×10 8 CFU / mL and 1×10 9 At CFU / mL, all postbiotics significantly promoted the activity of RAW264.7 macrophages, but the postbiotic components of Bifidobacterium longum CD11 cells showed a significant increase at 1×10⁻⁶ CFU / mL. 9 At high concentrations of CFU / mL, CFU / mL showed some toxicity to RAW264.7 macrophages, reducing cell viability. The post-biotic components of the Bifidobacterium longum CD11 cell wall were not toxic to RAW264.7 macrophages and promoted their proliferation in a dose-dependent manner.

[0217] Furthermore, regarding the cell wall components of the metagenic bifidobacteria CD11 cell wall components, peptidoglycan (PGN), capsular polysaccharide (CPS), wheyteichoic acid (WTA), lipoteichoic acid (LTA), and surface protein (SLP) components showed no toxicity to RAW264.7 macrophages, and these components were effective at a concentration of 1×10⁻⁶. 8 CFU / mL ~ 1×10 9 CFU / mL significantly promoted the proliferation of RAW264.7 macrophages (P<0.05). However, the cell wall peptidoglycan (PGN) and surface protein (SLP) post-biotic components of Bifidobacterium longum CD11 showed better promoting effects than the capsular polysaccharide (CPS), wall teichoic acid (WTA), and lipoteichoic acid (LTA) post-biotic components of Bifidobacterium longum CD11.

[0218] LPS-induced conversion of RAW264.7 macrophages from anti-inflammatory M2 macrophages to pro-inflammatory M1 macrophages leads to the secretion of inflammatory cytokines such as TNF-α and IL-1β, resulting in immune dysregulation. Therefore, the in vitro immune activity of each metagener was evaluated by detecting the relative expression levels of TNF-α and IL-1β mRNA secreted by RAW264.7 macrophages.

[0219] When heat-inactivated Bifidobacterium longum CD11 cells and its metabolites, Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane, and Bifidobacterium longum CD11 intracellular substances are subjected to a concentration of 1×10⁻⁶, the postbiotic components are... 8 CFU / mL ~ 1×10 9 At CFU / mL, each metagener significantly inhibited the relative expression of TNF-α and IL-1β mRNA genes secreted by RAW264.7 macrophages, and the metagenerer component of the Bifidobacterium longum CD11 cell wall inhibited the relative expression of TNF-α and IL-1β mRNA genes secreted by RAW264.7 macrophages in a dose-dependent manner.

[0220] Functional components were further extracted from the postbiotic components of the Bifidobacterium longum CD11 cell wall, including Bifidobacterium longum CD11 cell wall peptidoglycan (PGN), Bifidobacterium longum CD11 cell wall capsular polysaccharide (CPS), Bifidobacterium longum CD11 cell wall teichoic acid (WTA), Bifidobacterium longum CD11 cell wall lipoteichoic acid (LTA), and Bifidobacterium longum CD11 cell wall surface protein (SLP) postbiotic components at a concentration of 1×10⁻⁶. 8 CFU / mL, 5×10 8 CFU / mL and 1×10 9 At CFU / mL, all metagenes significantly inhibited the relative expression of TNF-α and IL-1β mRNA genes secreted by RAW264.7 macrophages, and the peptidoglycan (PGN) metagenetic component of Bifidobacterium longum CD11 cell wall significantly inhibited the relative expression of TNF-α and IL-1β mRNA genes secreted by RAW264.7 macrophages, restoring inflammation to a healthy level (NC level).

[0221] In summary, *Bifidobacterium longum* CD11 reached the late logarithmic phase after 10 hours of incubation at 37℃, with a colony count of 2.0 × 10⁻⁶ at this stage. 10CFU / mL. At 100℃ for 10 min, bacterial cell rupture occurred, intracellular fluid was released, and no viable bacteria remained. At 121℃ for 10 min, bacterial cell indentation increased, indicating greater cell rupture. Furthermore, in addition to heat-inactivated *Bifidobacterium longum* CD11, heat-inactivated *Bifidobacterium longum* CD11 metabolites, cell wall, cell membrane, and post-biotic components of cell contents all promoted the immune activity of RAW264.7 macrophages. Specifically, the cell wall component dose-dependently promoted RAW264.7 macrophage proliferation and inhibited the relative expression of inflammatory cytokines TNF-α and IL-1β mRNA genes. Moreover, the peptidoglycan (PGN) post-biotic from the cell wall component significantly inhibited the relative expression of TNF-α and IL-1β mRNA genes in RAW264.7 macrophages, restoring inflammation to healthy levels (NC group level). Therefore, *Bifidobacterium longum* CD11 can serve as a potential immunomodulatory post-biotic strain.

[0222] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that any modifications, equivalent substitutions, or improvements made by those skilled in the art without departing from the technical principles of the present invention should be included within the protection scope of the present invention.

[0223] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An epigenetic agent, characterized in that, The metagener was obtained by culturing and then inactivating Bifidobacterium longum CD11, wherein the metagener is peptidoglycan (PGN), a component of the cell wall of Bifidobacterium longum CD11, and the accession number of Bifidobacterium longum CD11 is CGMCC No. 6273.

2. The epigenetic agent as described in claim 1, characterized in that, Peptidoglycan (PGN), a cell wall component of Bifidobacterium longum CD11, was prepared using the following method: Collect Bifidobacterium longum CD11 cells by centrifugation and boil them in 4% sodium dodecyl sulfate (SDS); cool and centrifuge, and wash with distilled water until no SDS is present; The sediment is treated with ultrasound, followed by centrifugation and washing with distilled water. The precipitate was resuspended and incubated with DNase I, RNase A and MgSO4 in a shaker at 37°C; trypsin and CaCl2 were added and incubated in a shaker at 37°C. Centrifuge, boil the precipitate in 1% SDS, and resuspend it in trichloroacetic acid; wash with pre-cooled distilled water until the pH is neutral, collect the PGN and freeze-dry.

3. The method for preparing postgenetic agents as described in claim 1 or 2, characterized in that, Includes the following steps: Culture Bifidobacterium longum CD11, collect the bacterial cells by centrifugation and boil them in 4% sodium dodecyl sulfate; The suspension was cooled, centrifuged, and washed with distilled water. The precipitate was treated with ultrasound, centrifuged at room temperature, and washed with distilled water. The precipitate was resuspended in Tris-HCl and incubated with DNase I, RNase A and MgSO4; Add trypsin and CaCl2 and incubate; Centrifuge, boil the precipitate in SDS, wash until free of SDS, and resuspend in trichloroacetic acid; Wash the water-insoluble components with pre-cooled distilled water until the pH is neutral; collect the PGN and freeze-dry it.

4. The method for preparing postgenetic agents as described in claim 3, characterized in that, Includes the following steps: Culture Bifidobacterium longum CD11, centrifuge at 2,000×g for 10 min to collect the bacterial cells of Bifidobacterium longum CD11 and boil in 4% sodium dodecyl sulfate for 30 min; The suspension was cooled at 4°C, centrifuged at 6000×g at room temperature for 5 min, and washed with distilled water until no SDS was found, at least 3 times. The precipitate was treated with an ultrasonic processor of 700W for 10 minutes, centrifuged at 6000×g for 5 minutes at room temperature, and washed three times with distilled water. The precipitate was resuspended in 10 mL of 100 mM Tris-HCl and incubated with 5 μL of 1 mg / mL DNase I, 5 μL of 5 mg / mL RNase A and 20 mM MgSO4 in a shaker at 37 °C and 140 rpm for 2 h. Add 10 μL of 10 mg / mL trypsin and 10 mM CaCl2 and incubate at 37°C and 140 rpm for 12 h on a shaker. Centrifuge at 6000×g for 5 min, boil the precipitate in 1% SDS for 10 min, wash the SDS-insoluble cell wall components until SDS-free, and resuspend in 10% trichloroacetic acid at 4°C for 24 h to remove cell wall lipoteichoic acid (LTA). Wash the water-insoluble components with pre-cooled distilled water until the pH is neutral; collect the PGN, freeze-dry it, and store it at -20°C for later use.

5. A post-genetic composition, characterized in that, The preparation includes the postgenetic agent as described in any one of claims 1 or 2, or the postgenetic agent prepared by the preparation method as described in claims 3 or 4.

6. The use of the post-biotic as described in claim 1 or 2, or the post-biotic prepared by the method described in claim 3 or 4, or the post-biotic composition as described in claim 5, in the preparation of immunomodulatory drugs, characterized in that, The drug can promote macrophage proliferation and is used for immune regulation, which involves inhibiting the expression of cytokines TNF-α and IL-1β mRNA genes and alleviating inflammation.

7. The use as described in claim 6, characterized in that, The macrophages were RAW264.7 macrophages.

8. The use as described in claim 6 or 7, characterized in that, The medicine also contains a pharmaceutically acceptable carrier; The medicine is in the form of pills, powders, capsules, tablets, orally soluble granules, liquids, or suppositories; The amount of metabolites added to the drug is 0.001g-0.1g.

Images