Bifidobacterium longum CD11-based postbiotic immunomodulatory preparation and preparation method thereof

Abstract

The invention provides a postbiotic immunomodulatory preparation based on bifidobacterium longum CD11 and a preparation method of the postbiotic immunomodulatory preparation. Bifidobacterium longum CD11 is used to prepare a metagen. The immunocompetence of RAW264.7 macrophages can be promoted by thermal inactivation of bifidobacterium longum CD11 metabolites, cell walls, cell membranes and cell content metabolite components, and the cell wall component dose-dependently promotes the proliferation of the RAW264.7 macrophages and inhibits the relative expression of inflammatory factor TNF-alpha and IL-1 beta mRNA (messenger Ribonucleic Acid) genes. In addition, a peptidoglycan (PGN) metagen from a cell wall component can remarkably inhibit relative expression of TNF-alpha and IL-1 beta mRNA genes of RAW264.7 macrophages, so that inflammation is recovered to a health level (NC group level). Therefore, the bifidobacterium longum CD11 can be used as a potential immunomodulatory metabiotic strain.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to an application of Bifidobacterium longum CD11 postbiotics in immunoregulation and a preparation method thereof. Background Art

[0002] The immune system plays a vital role in protecting the host from external viruses, pathogens, parasites, fungi, yeasts and internal abnormal cells (such as tumor cells), and is an important line of defense for the human body against diseases. The immune system maintains the stability of the internal environment by maintaining autoimmune tolerance and immune regulation. Insufficient or excessive immune function, dysregulation of immune organs, cells and molecules can lead to immune disorders.

[0003] There is a two-way communication between host immunity and intestinal microbiota. The innate and adaptive immune systems are involved in shaping the composition of microorganisms. The body can recognize microorganisms and microbial derivatives (such as short-chain fatty acids), activate immune signaling molecules, and produce immune defense responses.

[0004] Recent research on probiotics has shifted from living microbial cells to non-living substances that are beneficial to human health, which are often referred to as "postbiotics", "parabiotics", or "heat-killed probiotics". In 2021, the International Scientific Association for Probiotics and Prebiotics (ISAPP) defined postbiotics as "inanimate microorganisms and / or their components that have beneficial properties for host health".

[0005] Postbiotics have been reported to have immunomodulatory effects and can maintain the health of the body by protecting the intestinal barrier and enhancing the ability of immunomodulation. Heat-killed Lactobacillus plantarum nF1 promoted the intestinal health of rats with loberamide-induced constipation. Heat-killed Lactobacillus rhamnosus LGG can alleviate oxidative stress and inflammation in diet-induced NAFLD rats. Heat-killed fermented milk CCFM1139 and its supernatant have a relieving effect on rats with periodontitis.

[0006] Heat treatment temperature and duration are related to the immune-related properties of the probiotic surface. Studies have found that heat treatment of Lactobacillus rhamnosus at 80°C for 20 minutes can reduce the levels of LPS-induced proinflammatory mediators IL-10 and IL-1β and regulate the level of inflammation in rats. The postbiotic components derived from Bifidobacterium longum CECT-7347 after heat treatment at 121°C for 20 minutes relieve inflammation and protect intestinal barrier function by activating immune pathways. TEM scanning of the spray-dried LGG cell wall found that the cell surface adhesion decreased after heat treatment. Postbiotics show immunomodulatory activity, which provides health benefits to the host. This benefit is for immunocompromised or immunosuppressed groups such as the elderly, patients with chronic diseases, premature newborns, and clinical patients undergoing chemotherapy. Postbiotics have the health properties and safety of "live" probiotics and may be a better alternative.

[0007] Therefore, the present invention proposes a postbiotic immunomodulatory preparation based on Bifidobacterium longum CD11 and a preparation method thereof. Summary of the invention

[0008] In view of the above-mentioned prior art, the technical solution of the present invention is provided to solve the problems that the existing immunomodulatory products are insufficiently effective and have questionable safety.

[0009] The object of the present invention is to provide a postbiotic, and its products, compositions, applications in immunomodulation, and preparation methods thereof. The present invention not only screens the heat treatment temperature and duration of postbiotics, but also further explores the bioactive components of postbiotics. The postbiotic functional components of the present invention can not only promote the proliferation of RAW264.7 macrophages, but also have certain immunomodulatory capabilities.

[0010] To achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0011] In one aspect, 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 the strain has been recorded in the prior art document CN104046573B.

[0012] On the other hand, the present invention provides a postbiotic component, prepared by the above-mentioned Bifidobacterium longum CD11. The postbiotic component includes heat-inactivated Bifidobacterium longum CD11 bacteria and metabolites thereof, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, Bifidobacterium longum CD11 cell content components, and in addition includes postbiotic components from Bifidobacterium longum CD11 cell walls, 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) and other postbiotic components.

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

[0014] The present invention provides a method for preparing postbiotics, comprising one or more of the following steps:

[0015] a. Cultivate Bifidobacterium longum CD11;

[0016] b. Detect the absorbance OD of the Bifidobacterium longum CD11 cultured in step a at 600 nm using an ultraviolet spectrophotometer. 600 , test once every 1 hour for 24 hours, and draw a growth curve to determine the late logarithmic growth period.

[0017] c. The Bifidobacterium longum CD11 in the late logarithmic growth stage cultured in step a was heat-treated at different temperatures and durations, and the effect of temperature on bacterial morphology was observed using a transmission electron microscope. The survival of Bifidobacterium longum CD11 at different temperatures and durations was analyzed by pouring plates.

[0018] d. Heat inactivation of the Bifidobacterium longum CD11 in the late logarithmic growth stage cultured in step a to obtain heat-inactivated Bifidobacterium longum CD11 cells and metabolites thereof.

[0019] e. The inactivated cells of Bifidobacterium longum CD11 in the late logarithmic growth stage heat-inactivated in step b were washed with phosphate buffered saline (PBS) to remove the supernatant and washed three times. The precipitate was suspended in sterile distilled water and crushed in a non-contact ultrasonic crusher, and the crushed liquid was centrifuged by differential centrifugation to obtain postbiotic components such as Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane and Bifidobacterium longum CD11 cell contents.

[0020] f. Extract the cell wall components of Bifidobacterium longum CD11 in step e to obtain postbiotic components such as 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 lipoteichoic acid (LTA) and Bifidobacterium longum CD11 cell wall surface protein (SLP).

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

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

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

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

[0025] The step b comprises:

[0026] The cultured Bifidobacterium longum CD11 was inoculated into a new MRS anaerobic test tube at a 2% (v / v%) inoculum volume. Starting from 0 h, the OD of the Bifidobacterium longum CD11 bacterial solution was tested every 1 h. 600 The growth curve was drawn to determine the late logarithmic phase after continuous monitoring for 24 h.

[0027] The step c comprises:

[0028] c1. Heat treatment of the Bifidobacterium longum CD11 cultured in step a and reaching the late logarithmic growth stage in step b at 60°C, 30 min; 75°C, 10 min; 80°C, 10 min; 100°C, 10 min and 121°C, 10 min under 5 different temperature gradients and 2 time gradients

[0029] c2 Transmission electron microscopy was used to observe the effects of thermal lethal temperature and duration on Bifidobacterium longum CD11 cells.

[0030] c3 uses the pour plate method to culture colonies at each temperature to verify whether there are live bacteria under the conditions of temperature and duration.

[0031] The step d comprises

[0032] d1. Centrifuge the bacterial solution of Bifidobacterium longum CD11 cultured in step a and reaching the late logarithmic growth stage in step b to obtain heat-killed Bifidobacterium longum CD11 cells and their metabolites;

[0033] d2. Wash the heat-killed Bifidobacterium longum CD11 cells obtained in step d1 with sterile PBS, and repeat the washing three times;

[0034] d3. Resuspend the Bifidobacterium longum CD11 bacterial precipitate washed with PBS buffer in sterile distilled water to prepare a bacterial suspension, so that the final concentration of the bacterial cell is 10 10 CFU / mL.

[0035] d4. The bacterial suspension is heated in a water bath to inactivate Bifidobacterium longum CD11, so as to obtain a Bifidobacterium longum CD11 heat-killed bacterial postbiotic component including Bifidobacterium longum CD11.

[0036] The step e comprises:

[0037] e1. The heat-killed Bifidobacterium longum CD11 cells in step d5 were crushed in a non-contact ultrasonic crusher. The crushing conditions were: time: 1h; temperature: 4°C; power: 35%; energy: 900000 Joules. Frequency: crushing for 9 seconds, pause for 9 seconds, 200× cycles (Time: 1h, Temperature: 4°C, Amplitude: 35%, Energy: 9000000 Joules, Pulse on: 9sec; Pulse off: 9sec, 200xcycles).

[0038] e2. Centrifuge the cell disruption solution in step d5 at 4°C 10000×g for 10 min, and collect the supernatant and precipitate respectively. Resuspend the precipitate in distilled water to obtain the cell wall component of Bifidobacterium longum CD11, and continue to centrifuge the supernatant at 4°C 25000×g for 90 min. Collect the precipitate as the cell membrane component of Bifidobacterium longum CD11, and the supernatant as the postbiotic components such as the cell contents of Bifidobacterium longum CD11.

[0039] Further, the concentration of the heat-killed Bifidobacterium longum CD11 bacteria and its metabolites, the cell wall components, the cell membrane components and the supernatant of Bifidobacterium longum CD11 cell contents of Bifidobacterium longum CD11 described in the postbiotics was adjusted to 1.0×10 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 a postbiotic functional component of the cell wall of Bifidobacterium longum CD11, and a postbiotic functional component preparation prepared, wherein the postbiotic functional component preparation includes 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 lipoteichoic acid (LTA) and Bifidobacterium longum CD11 cell wall surface protein (SLP) and other postbiotic components. The preparation types are all liquid or powder.

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

[0042] On the other hand, the above-mentioned postbiotics provided by the present invention can be used in combination with the following microorganisms. The microorganism can be bacteria, fungi, or any combination thereof; preferably, the microorganism is a probiotic; preferably, the microorganism is a yeast; preferably, the yeast is selected from Saccharomyces cerevisiae, Saccharomyces boulardii, Kluyveromyces marxianus, or any combination thereof;

[0043] Preferably, the bacteria is selected from Lactobacillus spp., Bifidobacterium spp., Bacillus spp., Propionibacterium spp., Streptococcus spp., Lactococcus spp., Pediococcus spp., Enterococcus spp., Staphylococcus spp., or any combination thereof. The bacteria of the genus Lactobacillus are selected from the group consisting of 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, Lactobacillus reuteri, Lactobacillus spp. reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, or any combination thereof;

[0044] The bacterium of the genus Bifidobacterium is selected from the group consisting of Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium adolescentis, or any combination thereof;

[0045] The bacterium of the genus Bacillus is 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 of the genus Lactococcus is Lactococcus lactis;

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

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

[0051] Preferably, the subject is a mammal;

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

[0053] On the other hand, the present invention provides a Bifidobacterium longum CD11 postbiotic and a composition thereof, preferably obtained by culturing Bifidobacterium longum CD11 and heat treating it at 100°C for 10 minutes. The postbiotic component is one or more combinations of inactivated bacteria, metabolites, cell walls, cell membranes and / or intracellular substances.

[0054] Preferably, except for the inactivated bacteria, other postbiotic 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 a composition thereof, heat-killed Bifidobacterium longum CD11 bacteria and metabolites thereof, Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane and Bifidobacterium longum CD11 cell content postbiotic components can reduce the mRNA gene expression level of inflammatory cytokines IL-1β and TNF-α in RAW264.7 macrophages. Preferably, the postbiotic component is preferably a Bifidobacterium longum CD11 cell wall postbiotic component.

[0056] Preferably, the postbiotic component is heat-killed bacteria, cell wall postbiotic components, cell membrane components and / or Bifidobacterium longum CD11 cell contents.

[0057] Preferably, the postbiotic component can 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 lipoteichoic acid (LTA) and Bifidobacterium longum CD11 cell wall surface protein (SLP) prepared from heat-killed Bifidobacterium longum CD11.

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

[0059] Preferably, when the concentrations of postbiotic components of 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 lipoteichoic acid (LTA) and Bifidobacterium longum CD11 cell wall surface protein (SLP) are 1×108CFU / mL, 5×108CFU / mL and 1×109CFU / mL, each postbiotic can significantly inhibit the relative expression 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 of the present invention was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) on June 25, 2012 (the abbreviation of the depository is CGMCC), and the deposit number is CGMCC No. 6273. The strain is named CD11 in this application.

[0061] Compared with the prior art, the beneficial effects of the postbiotic preparation of the present invention include but are not limited to:

[0062] (1) The present invention selects Bifidobacterium longum CD11 in the late logarithmic period with the strongest microbial metabolic activity and biological activity through growth curve detection to conduct experiments, clarify the growth cycle and suitable growth conditions of Bifidobacterium longum CD11, and explore the acquisition of highly active postbiotic products;

[0063] (2) Heat treatment of Bifidobacterium longum CD11 was performed using different heat treatment temperature gradients and duration gradients to investigate the effects of heat treatment temperature and duration on the immune-related properties of probiotic bacteria and probiotic cell surfaces;

[0064] (3) The postbiotic Bifidobacterium longum CD11 metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components and Bifidobacterium longum CD11 cell content components of the present invention are not only non-toxic to RAW264.7 macrophages, but also promote cell proliferation at a certain concentration. The postbiotic Bifidobacterium longum CD11 cell wall component has the best effect on RAW264.7 macrophage proliferation and immunoregulatory effects.

[0065] (4) The present invention extracts components from the cell wall composition of Bifidobacterium longum CD11 with the best immunomodulatory effect, and the extracted components include 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 lipoteichoic acid (LTA) and Bifidobacterium longum CD11 cell wall surface protein (SLP) and other postbiotic components are non-toxic to RAW264.7 macrophages and promote the proliferation of RAW264.7 macrophages at a certain concentration; the postbiotic component of Bifidobacterium longum CD11 cell wall peptidoglycan has the best effect on cell proliferation;

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

[0067] (6) The heat-killed Bifidobacterium longum CD11 bacterial postbiotics and their metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, Bifidobacterium longum CD11 cell content 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 lipoteichoic acid (LTA), and Bifidobacterium longum CD11 cell wall surface protein (SLP) and other postbiotic components can promote the immune activity of RAW264.7 macrophages. Therefore, Bifidobacterium longum CD11 can be used as a potential candidate strain for immunomodulatory postbiotics;

[0068] (7) The preparation method of the Bifidobacterium longum CD11 postbiotic of the present invention is simple, low-cost, and can be mass-produced; because the postbiotic has strain specificity, and Bifidobacterium longum is the main beneficial bacteria in the gastrointestinal microbial composition of the body, the Bifidobacterium longum CD11 postbiotic is highly safe and has no side effects, and is a potential immunomodulator. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0070] Figure 2 This is a transmission electron micrograph of the effects of different heat treatment temperatures and durations on the cells of Bifidobacterium longum CD11 in an embodiment of the present invention. Red circle: intracellular substances flow out; blue circle: cell breakage / depression.

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

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

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

[0074] Figure 6 This is a bar graph showing the effects of the postbiotic components of the cell wall of Bifidobacterium longum CD11 on the cell viability of RAW264.7 macrophages in an embodiment of the present invention.

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

[0076] Figure 8 This is a bar graph showing the effects of the postbiotic components of the cell wall of Bifidobacterium longum CD11 on the mRNA gene expression of the inflammatory factor IL-1β in RAW264.7 macrophages in an embodiment of the present invention. DETAILED DESCRIPTION

[0077] Before further describing the 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 terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0078] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

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

[0080] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with implementation cases. It should be understood that the specific implementation cases described herein are only used to explain the present invention and are not used to limit the present invention.

[0081] The bacterial strain sources, reagents and basal culture medium involved in the present invention include:

[0082] Bifidobacferium longum CD11 (Bifidobacferium longum CD11) was isolated from the feces of a healthy centenarian and preserved in the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC No.6273 and a preservation date of June 25, 2012.

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

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

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

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

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

[0088] Preparation of lipopolysaccharide (LPS) solution:

[0089] Accurately weigh 0.001 g of LPS powder in 10 mL of sterile PBS buffer, vortex to mix, and prepare a 1 mg / mL LPS stock solution, which is stored at -20°C for later use. When used, 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 cryopreserved cell line, thaw quickly, put it in a 15 mL sterile centrifuge tube, add 5 mL of 10% fetal bovine serum culture medium (10% FBS) and mix well, centrifuge at 1500 rpm / min for 3 min, remove the supernatant and transfer the cell pellet to a 100 mm cell culture dish, and culture it at 37°C and saturated humidity.

[0092] When the cells grew to about 80%, they were gently washed twice with PBS, digested with 0.25% trypsin-EDTA digestion solution for 3-4 minutes, and the cells at the bottom were blown with serum-free culture medium to make the cells fall off.

[0093] Take the cell suspension and centrifuge at 1500rpm / min for 3min, discard the supernatant, add fresh culture medium to suspend the cell sediment at the bottom in the culture medium. Take the cell suspension and place it in 3 culture dishes at a ratio of 1:3, and add culture medium respectively, and continue to culture at 37℃ under saturated humidity.

[0094] Other reagents can be purchased from commercial sources or prepared according to biological experimental manuals.

[0095] Example 1 Bacteria culture

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

[0097] Pick pure colonies and inoculate them in MRS broth medium. Place in a 37°C constant temperature incubator and culture for 24 hours to activate the strain. Subculture three times in the same way with an inoculum volume of 2% (v / v%), and store in a 4°C refrigerator for later use.

[0098] Example 2 Preparation of postbiotics and postbiotic components

[0099] The postbiotics of the present application include heat-killed Bifidobacterium longum CD11 cells and their metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components and Bifidobacterium longum CD11 cell content 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) and other postbiotic components.

[0100] The concentration of the heat-killed Bifidobacterium longum CD11 postbiotic components was 1.0×10 8 CFU / mL, 5.0×10 8 CFU / mL and 1.0×10 9 CFU / mL.

[0101] The specific preparation method of postbiotics in this application comprises the following steps:

[0102] a. Cultivate Bifidobacterium longum CD11.

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

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

[0105] The activation steps are as follows:

[0106] a1. Inoculate the activated Bifidobacterium longum CD11 at a rate of 2% (v / v%) in an anaerobic test tube of MRS broth medium, vortex and mix well, and culture in a 37°C constant temperature incubator for 24 hours. The obtained first generation strain is placed in a 4°C refrigerator for standby use.

[0107] a2. Inoculate the first-generation strain at a 2% (v / v%) inoculum into an anaerobic test tube containing fresh MRS broth medium, vortex mix, and culture in a 37°C constant temperature incubator for 24 h to obtain the second-generation strain, which is then stored in a 4°C refrigerator for later use.

[0108] a3. Inoculate the second-generation strain into an anaerobic test tube filled with fresh MRS broth medium at an inoculum size of 2% (v / v%), vortex mix, and culture in a 37°C constant temperature incubator for 10 h to obtain a late logarithmic phase bacterial solution of Bifidobacterium longum CD11, which is then stored in a 4°C refrigerator for later use.

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

[0110] b. Heat inactivate the Bifidobacterium longum CD11 cultured in step a to obtain Bifidobacterium longum CD11 postbiotics.

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

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

[0113] b3. Use sterile distilled water to resuspend the Bifidobacterium longum CD11 bacterial precipitate washed with PBS to prepare a Bifidobacterium longum CD11 bacterial suspension. The final concentration of the bacterial suspension is 10 10 CFU / mL.

[0114] b4. Heat the Bifidobacterium longum CD11 bacterial suspension in a water bath to inactivate the strains in each bacterial suspension to obtain Bifidobacterium longum CD11 bacterial postbiotics, which can be stored in a 4°C refrigerator for later use.

[0115] The water bath heating temperature and duration were 65℃, 30min; 75℃, 10min; 80℃, 10min; 100℃, 10min and 121℃, 10min, respectively, and the heat treatment was carried out under five different temperature gradients and two time gradients.

[0116] The morphology of Bifidobacterium CD11 cells was observed under a transmission electron microscope at different heat inactivation temperatures and durations. If the cells were sunken and intracellular fluids were flowing out, it indicated that the cells were ruptured.

[0117] The heat-killed Bifidobacterium longum CD11 bacterial suspension at each temperature was mixed evenly in MRS solid medium by the pour plate method and cultured for 48 hours. If no colonies appeared in the culture medium, it indicated that the bacteria were completely inactivated.

[0118] c. The postbiotics of Bifidobacterium longum CD11 cells that have been heat-killed at a thermal lethal temperature are placed in a non-contact ultrasonic disruptor for disruption.

[0119] c1. The crushing conditions of non-contact ultrasonic crusher are: time: 1h; temperature: 4℃; amplitude: 35%; energy: 900000 Joules; frequency: crushing 9 seconds, pause 9 seconds, 200× cycles. (Time: 1h, Temperature: 4℃, Amplitude: 35%, Energy: 9000000 Joules), Pulse on: 9sec; Pulse off: 9sec, 200xcycles)

[0120] c2. Use differential centrifugation to separate the cell wall component, cell membrane component and cell content component of Bifidobacterium longum CD11.

[0121] c3. The cell disruption liquid of Bifidobacterium longum CD11 was centrifuged at 4℃10000×g for 10min, and the supernatant and precipitate were collected respectively. The precipitate was resuspended in distilled water to obtain the cell wall component of Bifidobacterium longum CD11; the supernatant was further centrifuged at 4℃25000×g for 90min, and the precipitate was collected to obtain the cell membrane component of Bifidobacterium longum CD11, and the supernatant was the postbiotic components such as the cell contents of Bifidobacterium longum CD11.

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

[0123] d1. Extraction method of Bifidobacterium longum CD11 peptidoglycan (PGN): The 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 for 5 min at room temperature, and washed with distilled water until there was no SDS, at least 3 times.

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

[0126] The pellet 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) at 37°C with a shaker at 140 rpm for 2 h.

[0127] Trypsin (10 μL, 10 mg / mL) and CaCL2 (10 mM) were added and incubated at 37°C on a shaker at 140 rpm for 12 h.

[0128] After centrifugation at 6000×g for 5 min, the pellet was boiled in 1% SDS for 10 min, and the SDS-insoluble cell wall components were washed free of SDS and resuspended in trichloroacetic acid (10%) at 4°C for 24 h to remove cell wall lipoteichoic acid (LTA).

[0129] The water-insoluble components were washed with pre-cooled distilled water until the pH was neutral. The PGN was collected, freeze-dried, and stored at -20°C until use.

[0130] d2. Bifidobacterium longum CD11 capsular polysaccharide (CPS) extraction method: The bacterial cells cultured in step a were collected by centrifugation, resuspended in digestion buffer (PBS supplemented with 5 mM MgCL2, 1 mM CaCL2), and mutanolysin and lysozyme (concentration of 100 μg / mL) were added and digested at 37°C for 18 h.

[0131] Insoluble materials were removed by centrifugation at 6000×g for 5 min at 4°C, and the supernatant was incubated with nucleases (DNase I and RNase A, 100 μg / mL) at 37°C for 4 h.

[0132] Proteinase K (100 μg / mL) was added and incubated at 56°C for 18 h.

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

[0134] The precipitate was resuspended in distilled water, transferred to a prepared dialysis bag (8000-14000Da), and dialyzed at 4°C for 4 days. During this period, the dialysis water was replaced every 8 hours to completely remove solvent molecules and small molecule impurities.

[0135] d3. Bifidobacterium longum CD11 surface protein (SLP) extraction method: 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 cells were resuspended in 4 mol / L LiCL (pH 2.0), protease inhibitors were added, and incubated for 1 h at 37° C. The samples were centrifuged at 12800×g for 30 min at 4° C., and the supernatant containing SLP was collected.

[0137] SLP was placed in a prepared dialysis bag (8000-14000Da) and dialyzed at 4°C for 4 days. During this period, the dialysis water was replaced every 8 hours to completely remove solvent molecules and small molecule impurities.

[0138] d4. Bifidobacterium longum CD11 wall teichoic acid (WTA) extraction method: The bacterial cells cultured in step a were centrifuged at 2,000×g for 10 min to collect the bacterial cells.

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

[0140] The precipitate was collected by centrifugation at 6000×g for 5 min at 4°C and resuspended in 30 mL of buffer 2 (4% [wt / vol] sodium dodecyl sulfate [SDS], 50 mM MES, pH 6.5). The sample was placed in a boiling water bath for 1 h.

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

[0142] The pellet was washed once with buffer 2, once with buffer 3 (2% NaCl, 50 mM MES, pH 6.5), and finally with buffer 1. After the final wash, the sample was treated with proteinase K digestion solution (1 mL solution of 20 mM 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 samples once with buffer 3 and at least three times with distilled water to remove SDS.

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

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

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

[0147] d4. Extraction of lipoteichoic acid (LTA) of Bifidobacterium longum CD11: The bacterial cells cultured in step a were centrifuged at 2,000×g for 10 min at room temperature to collect Bifidobacterium longum CD11 bacterial cells.

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

[0149] The bacterial solution was treated with an ultrasonic processor at 700 W for 10 min and centrifuged at 6000 × g for 5 min at 4 °C.

[0150] The broken cell wall sample was mixed with an equal volume of water-saturated n-butanol at room temperature and stirred continuously for 60 min.

[0151] The mixture was divided into centrifuge tubes and centrifuged at 13,000 × g for 30 min at 4 °C.

[0152] The aqueous layer was collected and centrifuged again under the same conditions to completely remove the precipitate to obtain a crude LTA extract.

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

[0154] Example 3 Functional verification of postbiotics and postbiotic components

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

[0156] The microbial growth curve of Bifidobacterium longum CD11 was analyzed to determine the late logarithmic growth time.

[0157] S11 was inoculated at 2% (v / v%) in fresh MRS broth and cultured in a 37°C constant temperature incubator;

[0158] S12 uses a UV spectrophotometer at 600 nm Detect absorbance OD 600 Value, draw the growth curve;

[0159] S13 The colony number of Bifidobacterium longum CD11 in the late logarithmic phase in S12 was counted using the pour plate method.

[0160] Experimental Example 2 Effects of heat inactivation temperature and duration on the activity of Bifidobacterium longum CD11

[0161] S11 was set to heat treat at five different temperature gradients and two time gradients: 65°C, 30 min; 75°C, 10 min; 80°C, 10 min; 100°C, 10 min and 121°C, 10 min;

[0162] S12 will be inoculated at a 2% (v / v%) inoculum into an anaerobic test tube of fresh MRS broth medium. After culturing to the late logarithmic stage, it will be placed in the above-mentioned S11 temperature and duration conditions for heat inactivation. Then, the bacterial solution heat-treated at each temperature will be cultured in MRS solid medium for 48 hours using the pour plate method. If no colonies appear in the culture medium, it means that the bacteria are completely inactivated, and the temperature and duration without viable bacteria are determined to be the thermal lethal temperature and time.

[0163] Experimental Example 3 Observation of the Morphology of Heat-Killed Bifidobacterium longum CD11 by Transmission Electron Microscopy

[0164] Here are the steps:

[0165] The cultured Bifidobacterium longum CD11 in the late logarithmic growth period was vortexed and mixed, and then distributed into 2 mL sterile enzyme-free centrifuge tubes, and heat-treated at different temperatures and durations; centrifuged at 4°C, 12000 rpm, for 5 min; the supernatant was discarded and the precipitate was collected, and the precipitate was washed 3 times with sterile PBS, and 200 μL of fresh 25% glutaraldehyde was added to fix it at 4°C overnight; washed 3 times with sterile PBS, 10 min / time;

[0166] Cryosectioning: Liquid nitrogen rapidly freezes the sample, and then taps it gently with a hammer to cause it to break naturally.

[0167] Gradient dehydration was performed using 30%, 50%, 70%, 90%, and 100% ethanol, with each gradient dehydration lasting 10 min;

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

[0169] Supercritical drying: using CO 2 Supercritical instrument drying for 1.5 h at 32 °C and 73 atmospheres (7.373 MPa);

[0170] Coating: Platinum film is coated by ion sputtering;

[0171] The samples were photographed using a transmission electron microscope.

[0172] The results of Experimental Examples 1-3 are as follows: Figure 1 , 2It can be seen that by drawing the growth curve of Bifidobacterium longum CD11, it is determined that the late logarithmic growth period obtained according to the above culture method is 10h. After heat treatment at 100℃ and 121℃ for 10min, no live bacteria appear in the inverted plate culture, indicating that Bifidobacterium longum CD11 cells die after heat treatment at 100℃ for 10min. Transmission electron microscopy observed that the bacteria ruptured during this period, the bacteria were concave, and the cell contents flowed out. When heat treated at 121℃ for 10min, the degree of bacteria rupture increased and the number of bacteria concave increased. Therefore, Bifidobacterium longum CD11 can be heat treated at 100℃ for 10min to produce postbiotic products for subsequent experiments.

[0173] Experimental Example 4 Effect of Bifidobacterium longum CD11 postbiotics on the proliferation ability of RAW264.7 macrophages

[0174] 1×10 RAW264.7 macrophages in the late logarithmic growth phase were collected. 5 The cells were seeded at a density of 10 cells / well in a 96-well plate, 200 μL / well, and cultured in a 37°C saturated humidity incubator for 24 h.

[0175] The culture medium in the 96-well plate was removed, and the non-adherent cells were washed with sterile PBS. Bifidobacterium longum CD11 postbiotics were added to the 96-well plate and cultured in a 37°C saturated humidity incubator for 24 h.

[0176] Different concentration groups were set up: heat-killed 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 content components. The concentration of different functional components was 1×10 8 CFU / mL, 5×10 8 CFU / mL, 1×10 9 CFU / mL, 200 μL per well, and continue culturing for 24 h.

[0177] The postbiotic liquid in the 96-well plate was removed, and the plates were washed twice with sterile PBS. 10% CCK8 solution was added to each well, and the plates were incubated at 37°C in the dark for 1-4 h for color development. The absorbance was measured at 450 nm using a multifunctional microplate reader, and a blank control group was used.

[0178] The cell viability calculation formula is as follows:

[0179]

[0180] Where: OD 0 For zero adjustment hole; OD 1 For blank wells; OD 2 For the experimental hole.

[0181] Experimental Example 5: Establishment of an Inflammatory Model by Inducing RAW264.7 Macrophages with Lipopolysaccharides (LPS)

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

[0183] Take RAW264.7 macrophage suspension and adjust the density to 1.2×10 6 / mL, seeded in 12-well cell culture plates, after cell adherence culture for 12 h, 1 μg / mL LPS was added to each well of cells in the other groups except the NC group for stimulation for 24 h for cell polarization treatment.

[0184] For cells with successful LPS polarization modeling, except for the NC group and the LPS group, the other groups were added with heat-killed Bifidobacterium longum CD11 bacteria, Bifidobacterium longum CD11 metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components, and Bifidobacterium longum CD11 cell content components, and the concentration of 1×10 8 CFU / mL, 5×10 8 CFU / mL, 1×10 9 CFU / mL of different postbiotic functional components.

[0185] The results are as follows Figure 3 As shown, except for the heat-killed Bifidobacterium longum CD11 bacterial component, the other postbiotic components of the present invention are not only non-toxic to RAW264.7 macrophages, but also can promote the proliferation of RAW264.7 macrophages at a certain concentration.

[0186] like Figure 4 , 5 As shown in the figure, compared with the blank control group (NC group), lipopolysaccharide (LPS) induced RAW264.7 macrophages to secrete inflammatory cytokines IL-1β and TNF-α. Heat-killed Bifidobacterium longum CD11 bacteria and its metabolites, Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane and Bifidobacterium longum CD11 cell contents and other postbiotic components can significantly reduce 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 (Positive) group, heat-killed Bifidobacterium longum CD11 bacteria, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components and Bifidobacterium longum CD11 cell contents had an inhibitory effect on the secretion of TNF-α and IL-1β by RAW264.7 macrophages.

[0188] Compared with the heat-killed Bifidobacterium longum CD11 bacterial fraction, the cell wall postbiotic fraction of Bifidobacterium longum CD11 had a better inhibitory effect on the secretion of IL-1β and TNF-α by RAW264.7 macrophages.

[0189] Compared with the metabolites of Bifidobacterium longum CD11, the postbiotic components such as heat-killed Bifidobacterium longum CD11 bacterial components, Bifidobacterium longum CD11 cell wall postbiotic components, Bifidobacterium longum CD11 cell membrane components and Bifidobacterium longum CD11 cell contents had the best inhibitory effect on the secretion of TNF-α and IL-1β by RAW264.7 macrophages.

[0190] In summary, combined with Experimental Examples 4 and 5, Figure 3-5 As shown in the figure, the heat-inactivated Bifidobacterium longum CD11 bacteria, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components and Bifidobacterium longum CD11 cell contents of the present invention have certain immunomodulatory abilities, and the Bifidobacterium longum CD11 cell wall components have the strongest inhibitory effect on TNF-α and IL-1β secreted by RAW264.7 macrophages. 8 CFU / mL, 5×10 8 CFU / mL and 1×10 9 The mRNA gene expression of inflammatory cytokines TNF-α and IL-1β was inhibited in a dose-dependent manner under the CFU / mL concentration gradient.

[0191] Experimental Example 6 Effects of Bifidobacterium longum CD11 cell wall postbiotic components on the proliferation ability of RAW264.7 macrophages

[0192] The difference from Experimental Example 4 is that the Bifidobacterium longum CD11 postbiotics are replaced with Bifidobacterium longum CD11 cell wall postbiotic 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 lipoteichoic acid (LTA) and Bifidobacterium longum CD11 cell wall surface protein (SLP) and other postbiotic components. The other experimental steps remain unchanged.

[0193] Experimental Example 7 Effects of Bifidobacterium longum CD11 postbiotics 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 taken and the density was adjusted to 1.2×10 6 The cells were inoculated into 12-well cell culture plates and placed in a 37°C saturated humidity constant temperature incubator for 24 h.

[0195] The culture medium in the 12-well plate was aspirated with a pipette, washed twice with sterile PBS buffer, and Bifidobacterium longum CD11 postbiotics were added at a volume of 1 mL / well, and cultured in a 37°C constant temperature incubator for 24 h.

[0196] Different concentration groups were set: the concentrations of heat-killed Bifidobacterium longum CD11 and its metabolites, Bifidobacterium longum CD11 cell wall fraction, Bifidobacterium longum CD11 cell membrane fraction and Bifidobacterium longum CD11 cell content fraction were 1×10 8 CFU / mL, 5×10 8 CFU / mL, 1×10 9 CFU / mL.

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

[0198] qRT-PCR kit 5×All In One RT-MasterMix kit and PowerUp TM SYBR TM Reverse transcription and real-time fluorescence quantitative PCR were performed using the PCR product kit and a blank control group was used.

[0199] There were 4 parallel experiments and the reaction system was 20 μL. The reverse transcription amplification conditions are shown in Table 1.

[0200] Table 1 qRT-PCR amplification conditions

[0201]

[0202] See Table 2 for real-time fluorescence quantitative qRT-PCR primers, and the internal reference is GAPDH.

[0203] Adoption 2 ^(-△△Ct) The data were analyzed by the method to calculate the relative fold changes of gene expression.

[0204] Table 2 Real-time fluorescence quantitative qRT-PCR primer sequence list

[0205]

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

[0207] Different from Experimental Example 7, the heat-killed Bifidobacterium longum CD11 and its metabolites, Bifidobacterium longum CD11 cell wall components, Bifidobacterium longum CD11 cell membrane components and Bifidobacterium longum CD11 cell content components are replaced with postbiotic 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).

[0208] like Figure 6 As shown, compared with the blank control group (NC group), the peptidoglycan (PGN) component of Bifidobacterium longum CD11 cell wall, the capsular polysaccharide (CPS) component of Bifidobacterium longum CD11 cell wall, the wall teichoic acid component (WTA) component of Bifidobacterium longum CD11 cell wall, the lipoteichoic acid (LTA) component of Bifidobacterium longum CD11 cell wall and the surface protein (SLP) component of Bifidobacterium longum CD11 cell wall were not only non-toxic to RAW264.7 macrophages, but also promoted the proliferation of RAW264.7 macrophages at a certain concentration.

[0209] Compared with the positive drug dexamethasone (Positive) group, the cell wall peptidoglycan (PGN) fraction of Bifidobacterium longum CD11, the cell wall capsular polysaccharide (CPS) fraction of Bifidobacterium longum CD11, the cell wall wall teichoic acid (WTA) fraction of Bifidobacterium longum CD11, the cell wall lipoteichoic acid (LTA) fraction of Bifidobacterium longum CD11, and the cell wall surface protein (SLP) fraction of Bifidobacterium longum CD11 significantly promoted the cell proliferation of RAW264.7 macrophages (P<0.05). Moreover, the cell wall peptidoglycan (PGN) fraction of Bifidobacterium longum CD11 and the cell wall surface protein (SLP) fraction of Bifidobacterium longum CD11 promoted the cell proliferation of RAW264.7 macrophages in a dose-dependent manner. That is, 1×10 8 , 5×10 8 and 1×10 9 At the CFU / mL dose concentration, as the concentration of PGN postbiotics increased, the proliferation ability of RAW264.7 macrophages and the inhibitory effect on inflammatory cytokines increased. The higher the concentration of PGN postbiotics, the better the effect.

[0210] like Figure 7-8It can be seen that compared with the LPS model group (NC group), the postbiotic components of the cell wall of Bifidobacterium longum CD11 can significantly reduce 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 (Positive) group, postbiotic components such as the peptidoglycan (PGN) component of the cell wall of Bifidobacterium longum CD11, the capsular polysaccharide (CPS) component of the cell wall of Bifidobacterium longum CD11, the wall teichoic acid (WTA) component of the cell wall of Bifidobacterium longum CD11, the lipoteichoic acid (LTA) component of the cell wall of Bifidobacterium longum CD11 and the surface protein (SLP) of Bifidobacterium longum CD11 had an inhibitory effect on the relative mRNA expression of TNF-α and IL-1β genes in RAW264.7 macrophages.

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

[0213] In summary, in combination with Experimental Examples 3-5, the heat-killed Bifidobacterium longum CD11 and postbiotics of the present application, including heat-killed Bifidobacterium longum cells, metabolites, cell walls, cell membranes and cell contents, can significantly inhibit the relative mRNA expression of TNF-α and IL-1β genes in RAW264.7 macrophages. It can be seen that the heat-killed Bifidobacterium longum CD11 postbiotics of the present invention have certain immunomodulatory abilities, and the Bifidobacterium longum CD11 cell wall components dose-dependently inhibit the relative mRNA expression of TNF-α and IL-1β genes in RAW264.7 macrophages.

[0214] Combined with Experimental Examples 6-8, Bifidobacterium longum CD11 cell wall postbiotic components, including peptidoglycan (PGN), capsular polysaccharide (CPS), wall teichoic acid (WTA), lipoteichoic acid (LTA) and surface protein (SLP) postbiotic components can inhibit the relative expression of mRNA of TNF-α and IL-1β genes in RAW264.7 macrophages. It can be seen that the postbiotic components of the Bifidobacterium longum CD11 cell wall of the present invention have certain immunomodulatory abilities, and the postbiotic Bifidobacterium longum CD11 cell wall peptidoglycan (PGN) component significantly inhibits the relative expression of mRNA of TNF-α and IL-1β genes in RAW264.7 macrophages, restoring inflammation to healthy (NC group) levels.

[0215] Conclusion: Through the analysis of the results of in vitro assay of the proliferation ability of RAW264.7 macrophages, when the concentration of the postbiotic components of heat-killed Bifidobacterium CD11 cells was 1×10 8 CFU / mL, 5×10 8 CFU / mL and 1×10 9 CFU / mL, it has a certain toxicity to RAW264.7 macrophages and reduces cell activity. However, the effects of Bifidobacterium longum CD11 metabolites, Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane and Bifidobacterium longum CD11 cell intracellular substances are better than those of the postbiotic components of heat-killed Bifidobacterium longum CD11 bacteria.

[0216] When the concentration of the metabolites of Bifidobacterium longum CD11, cell wall of Bifidobacterium longum CD11, cell membrane of Bifidobacterium longum CD11 and intracellular substances of Bifidobacterium longum CD11 was 1×10 8 CFU / mL, 5×10 8 CFU / mL and 1×10 9 CFU / mL, all postbiotics could significantly promote the cell activity of RAW264.7 macrophages, but the intracellular postbiotic components of Bifidobacterium longum CD11 cells were 9 At high CFU / mL concentrations, it was toxic to RAW264.7 macrophages and reduced cell activity. Bifidobacterium longum CD11 cell wall postbiotic components had no toxicity to RAW264.7 macrophages and promoted the proliferation of RAW264.7 macrophages in a dose-dependent manner.

[0217] Furthermore, for cell wall component postbiotics, the peptidoglycan (PGN), capsular polysaccharide (CPS), wall teichoic acid (WTA), lipoteichoic acid (LTA) and surface protein (SLP) components of Bifidobacterium longum CD11 cell wall components had no toxicity to RAW264.7 macrophages, and at a concentration of 1×10 8 CFU / mL~1×10 9CFU / mL, it can significantly promote the proliferation of RAW264.7 macrophages (P<0.05). However, the promotion effect of Bifidobacterium longum CD11 cell wall peptidoglycan (PGN) and surface protein (SLP) postbiotic components is better than that of Bifidobacterium longum CD11 capsular polysaccharide (CPS), wall teichoic acid (WTA) and lipoteichoic acid (LTA) postbiotic components.

[0218] Under LPS induction, RAW264.7 macrophages turn from anti-inflammatory M2 macrophages to pro-inflammatory M1 macrophages, secreting inflammatory cytokines such as TNF-α and IL-1β, leading to immune imbalance. Therefore, the in vitro immune activity of each postbiotic was evaluated by detecting the relative expression of TNF-α and IL-1β mRNA genes secreted by RAW264.7 macrophages.

[0219] When heat-killed Bifidobacterium longum CD11 cells and their metabolites, Bifidobacterium longum CD11 cell wall, Bifidobacterium longum CD11 cell membrane and Bifidobacterium longum CD11 cell intracellular components were added at a concentration of 1×10 8 CFU / mL~1×10 9 CFU / mL, each postbiotic could significantly inhibit the relative expression of TNF-α and IL-1β mRNA genes secreted by RAW264.7 macrophages, and the postbiotic components of 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 cell wall postbiotic components of Bifidobacterium longum CD11, 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 lipoteichoic acid (LTA) and Bifidobacterium longum CD11 cell wall surface protein (SLP). The concentration of postbiotic components was 1×10 8 CFU / mL, 5×10 8 CFU / mL and 1×10 9 CFU / mL, each postbiotic could significantly inhibit the relative expression of TNF-α and IL-1β mRNA genes secreted by RAW264.7 macrophages, and the postbiotic component of Bifidobacterium longum CD11 cell wall peptidoglycan (PGN) extremely 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 being cultured in a 37°C constant temperature incubator for 10 h, and the colony count at this stage was 2.0×10 10CFU / mL. At 100°C, the bacteria ruptured after heat treatment for 10 minutes, the intracellular fluid flowed out, and no live bacteria were present. At 121°C, when heat treated for 10 minutes, the bacteria depression increased and the degree of bacteria rupture increased. In addition, in addition to the heat-killed bacteria of Bifidobacterium longum CD11, the metabolites, cell walls, cell membranes and postbiotic components of the heat-killed Bifidobacterium longum CD11 can promote the immune activity of RAW264.7 macrophages, among which the cell wall components dose-dependently promoted the proliferation of RAW264.7 macrophages and inhibited the relative expression of inflammatory factors TNF-α and IL-1β mRNA genes. In addition, the peptidoglycan (PGN) postbiotics from the cell wall components can significantly inhibit 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 be used as a potential immunomodulatory postbiotic 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 pointed out that for ordinary technicians in this technical field, any modifications, equivalent replacements and improvements made without departing from the technical principles of the present invention should be included in the protection scope of the present invention.

[0223] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A postbiotic, characterized in that: Obtained by inactivating Bifidobacterium longum CD11 after culture.

2. The postbiotic according to claim 1, characterized in that The postbiotics are one or more combinations of inactivated Bifidobacterium longum CD11 bacteria, metabolites of Bifidobacterium longum CD11, cell walls of Bifidobacterium longum CD11, cell membranes of Bifidobacterium longum CD11 and / or intracellular substances of Bifidobacterium longum CD11 cells.

3. The postbiotic according to claim 1 or 2, characterized in that The postbiotics are one or more combinations of peptidoglycan (PGN), capsular polysaccharide (CPS), wall teichoic acid (WTA), lipoteichoic acid (LTA) and / or surface protein (SLP) of the cell wall components of Bifidobacterium longum CD11.

4. The postbiotic according to any one of claims 1 to 3, characterized in that The concentration of postbiotic components was 1×10 6 CFU / mL~1×10 11 CFU / mL, preferably, the concentration is 1×10 8 CFU / mL, 5×10 8 CFU / mL or 1×10 9 CFU / mL.

5. A composition comprising the postbiotics according to any one of claims 1 to 4, characterized in that: The composition can promote macrophage proliferation and is used for immune regulation.

6. The postbiotic according to any one of claims 1 to 4 or the composition according to claim 5, characterized in that: The macrophages are RAW264.7 macrophages; preferably, the immunomodulation is to inhibit the expression of cytokine TNF-α and IL-1β mRNA genes and alleviate inflammation.

7. The method for preparing the postbiotics according to any one of claims 1 to 4, characterized in that: a) the postbiotics are obtained by culturing Bifidobacterium longum CD11 to the logarithmic growth phase; preferably, the postbiotics are obtained by culturing MRS broth medium at 37° C. under an anaerobic environment to the logarithmic growth phase; b) the postbiotics are inactivated by inactivation conditions of 65°C for 30 min; 75°C for 10 min; 80°C for 10 min; 100°C for 10 min or 121°C for 10 min; c) separating the cell wall component, cell membrane component and cell content component of Bifidobacterium longum CD11 from the inactivated postbiotic bacteria by a non-contact ultrasonic disruptor and differential centrifugation; d) extracting peptidoglycan (PGN), capsular polysaccharide (CPS), wall teichoic acid (WTA), lipoteichoic acid (LTA) and / or surface protein (SLP) from the cell wall of postbiotic bacteria as postbiotic components.

8. Use of the postbiotic according to any one of claims 1 to 4, or the composition according to claim 5 or 6, or the product obtained by the preparation method of claim 7 in the preparation of an immunomodulatory product, characterized in that: Preferably, the product is a food or a medicine.

9. The use according to claim 8, characterized in that The immunomodulatory product refers to a product that can promote the proliferation of macrophages in mammals; or inhibit the relative expression of inflammatory factors TNF-α and IL-1β mRNA genes. Preferably, the mammal is selected from mice, pigs, rabbits, monkeys, sheep or humans.

10. The use according to claim 8 or 9, characterized in that The drug further comprises a pharmaceutically acceptable carrier; The drug is in the form of a pill, powder, capsule, tablet (e.g., effervescent tablet), film-coated tablet, orally disintegrating granules, liquid, suppository or enema; The food has one or more characteristics selected from the following: (1) The food is a dietary supplement; (2) The food further comprises prebiotics; Preferably, the prebiotic is selected from fructooligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharide, carrot nitrogen-containing polysaccharide, casein hydrolyzate, α-lactalbumin, lactoferrin, or any combination thereof; (3) The food is selected from solid beverages, candies or juices, or the food is a dairy product (e.g., yogurt, flavored fermented milk, lactic acid bacteria beverage, cheese); (4) The food is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), film-coated tablets, orally disintegrating granules, or liquids; Preferably, the amount of postbiotics added to the medicine or food is 0.001g-0.1g; more preferably, the amount of postbiotics added to the medicine or food is 0.001-0.005g, 0.005-0.01g, 0.01-0.05g or 0.05-0.1g.

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