Application of metabiotics of fermented lactobacillus mucus in preparation of immune activator

Abstract

The invention relates to the field of microorganisms, in particular to an application of a metagen of fermented lactobacillus mucus in preparation of an immune activator, the fermented lactobacillus mucus is Limosilicobacterium fermentum B44, and the metagen is a substance prepared by the following method: centrifugally collecting the fermented lactobacillus mucus in fermented lactobacillus mucus fermentation liquor, washing and then resuspending, and drying to obtain the immune activator. And inactivating the thalli, cooling, centrifuging, retaining the precipitate, and freeze-drying to obtain the metabiotics. The immune activator is a macrophage activator, the food safety can be guaranteed, the preparation method is simple and convenient, the applicability is high, the immune activator has very good immune activation ability, has the characteristics of inactivity and stability, can be widely applied to foods, dietary supplements, fermented and non-fermented milk products and the like, and has very good product processability.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to the use of metagenes of fermenting *Lactobacillus mucinus* in the preparation of immune activators. Background Technology

[0002] Immunity is the cornerstone of human health. Disruptions to the homeostasis of the immune system can affect immune responses and lead to various diseases. A damaged immune system can cause the body to become less responsive to pathogens. Immunosuppression is a temporary or long-term state of immune dysfunction. Immunosuppression can occur in individuals who have undergone organ transplantation, suffer from cancer (tumor), autoimmune diseases, are infected with viruses, or are in a state of chronic sub-health with weakened immune function. Excessive immunosuppression can be fatal and damage the body's immune system, potentially exposing patients to various infections and complications, thus delaying diagnosis and treatment outcomes.

[0003] Probiotics are generally defined as live microbial additives that provide health benefits to host animals by improving the balance of microbes in the gut. In recent years, the use of probiotics to improve the immune response in immunocompromised patients has received sustained attention. Increasing evidence supports the important role of Lactobacillus and its components (such as metabolites, peptidoglycans, and / or surface proteins) in regulating immune responses. Probiotics stimulate the host's immune system and may be a potential therapeutic alternative to chemotherapy. *Lactobacillus fermentum* exhibits excellent tolerance to the gut environment and has long been suitable for industrial and food fermentation. *Lactobacillus fermentum* is a strain included in my country's "List of Microbial Strains that Can Be Used in Food," and is also certified by the EU's Microbial Strains Safety Assessment System and the US Food and Drug Administration. Domestic and international studies have shown that *Lactobacillus fermentum* interacts with immune cells and regulates specific pathways involved in innate and adaptive immune processes in various inflammatory diseases. *Lactobacillus fermentum* has been reported to effectively regulate the immunity and microbiota of immunocompromised mice; however, studies on in vitro immune activation by *Lactobacillus fermentum* are limited, and immune regulation is strain-dependent. Further evidence is needed to clarify the specific immune responses produced by each *Lactobacillus fermentum* strain.

[0004] The International Society for the Study of Probiotics and Prebiotics (ISAPP) defines postbiotics as "preparations of non-living microorganisms and / or components thereof that are beneficial to the health of the host." These postbiotics are inactivated through specific methods such as high temperature, ultraviolet light, or chemical treatment, rendering previously active microorganisms unable to grow and reproduce while retaining, to some extent, their structure, characteristics, and efficacy. They exhibit significant advantages in industrial and product applications: ① In terms of stability, because there is no need to maintain microbial activity, the sensitivity of probiotics to oxygen and heat can be overcome, easily resulting in products with long shelf lives, making them more suitable for geographical areas with inadequate cold chains or where environmental temperatures easily lead to storage problems; ② In terms of intellectual property, since the microorganisms derived from postbiotics cannot be isolated from commercial products, it helps product developers maintain ownership of the ingredients; ③ In terms of safety, inactivated microorganisms lose their ability to replicate, completely avoiding the risk of bacteremia. Based on these favorable characteristics, postbiotics are regarded by the industry as the fourth generation of microecological products after probiotics, prebiotics, and synbiotic preparations. Driven by increased health awareness and demand for health foods, they are gradually gaining popularity due to their diverse potential effects and good stability, and are considered by the industry as a potential alternative to probiotics.

[0005] Therefore, screening for stable and highly applicable metabiotics with independent intellectual property rights that possess immune activation capabilities has great value and prospects. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide the use of post-biotics of fermented Lactobacillus mucinus in the preparation of immune activators, in order to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention provides the use of postgenes of fermenting *Lactobacillus mucinus* in the preparation of immune activators.

[0008] In some embodiments of the present invention, the fermenting *Lactobacillus fermentum* is *Limosilactobacillus fermentum* B44, with accession number CGMCC No. 17321.

[0009] In some embodiments of the present invention, the metagene is a substance prepared by the following method: collecting fermenting *Lactobacillus myxoides* from the fermentation broth by centrifugation, washing and resuspending, inactivating the bacterial cells, cooling and centrifuging to retain the precipitate, and freeze-drying to obtain the metagene.

[0010] In some embodiments of the present invention, the viable cell count in the fermentation broth of *Lactobacillus mucinus* is 1 × 10⁻⁶. 5 CFU / mL or higher, preferably 1×10⁻⁶. 5 CFU / mL ~9×10 9 CFU / mL.

[0011] In some embodiments of the present invention, the inactivation method is boiling water bath inactivation for 2 to 5 minutes, preferably inactivation for 2 to 3 minutes.

[0012] In some embodiments of the present invention, the immune activator is a macrophage activator. The macrophage activator has one or more of the following effects:

[0013] 1) Enhance macrophage cell viability;

[0014] 2) Enhance the phagocytic capacity of macrophages;

[0015] 3) Increase the production of pro-inflammatory factors or NO by macrophages.

[0016] 4) Induces macrophages to polarize into the M1 type and perform corresponding functions.

[0017] In some embodiments of the present invention, the pro-inflammatory factor is selected from TNF-α, IL-6 and / or IL-1β.

[0018] In some embodiments of the present invention, the immune activator is a food or dietary supplement.

[0019] The food products are selected from fermented dairy products or non-fermented dairy products.

[0020] As described above, the use of the postgeneric form of *Lactobacillus mucinus* of the present invention in the preparation of immune activators has the following beneficial effects: food safety can be guaranteed, the preparation method is simple, it has strong applicability, it has very good immune activation ability, it has the characteristics of inactivity and stability, it can be widely used in food, dietary supplements, fermented and non-fermented milk products, etc., and it has very good product processability.

[0021] From a social benefit perspective, this invention utilizes inactivated fermented Lactobacillus mucinus B44 to promote the proliferation, phagocytosis, NO production, M1 polarization, and increase the expression of cytokines TNF-α, IL-1β, and IL-6 in RAW264.7 macrophages, demonstrating that B44 possesses in vitro macrophage immune activation capabilities. As a safe and accessible immune intervention, its value lies not only in "passive defense" (such as reducing infection) but also in "active regulation" (such as correcting immune imbalance). In the future, with the development of precision medicine and synthetic biology, probiotics are expected to become one of the core tools for personalized immune management, but their application must always be based on evidence-based medicine and individual health needs.

[0022] From an economic perspective, due to its stability, probiotics can be widely used in health supplements, food, and fermented products to enhance their efficacy. It can also save costs for the healthcare system, as improved immunity may reduce disease incidence, thereby lowering medical expenses. Furthermore, it benefits the labor market; fewer sick employees and lower absenteeism rates lead to increased corporate productivity, which can be calculated as economic value. Additionally, the production and sales of probiotic products drive related industrial chains, such as agriculture (probiotic raw material production), manufacturing (processing), and retail, creating jobs and income. It can also stimulate the development of related service industries, such as health consultation and nutritional guidance, which may also benefit from increased demand for probiotics. Attached Figure Description

[0023] Figure 1 The flowchart shown is an embodiment of the present invention.

[0024] Figure 2 The image shown is a result of inducing macrophages to polarize to the M1 type in Example 2.

[0025] Figure 3 The image shown is a graph illustrating the cell viability results from Example 2.

[0026] Figure 4 The image shows the results of measuring NO release from macrophages in Example 2.

[0027] Figure 5 The image shown is a graph illustrating the results of macrophage phagocytic capacity measurements in Example 2.

[0028] Figure 6 The figure shown is a graph illustrating the measurement results of TNF-α release from macrophages in Example 2.

[0029] Figure 7 The image shows the results of measuring the amount of IL-6 released by macrophages in Example 2.

[0030] Figure 8 The image shows the results of measuring the amount of IL-1β released by macrophages in Example 2.

[0031] In the figure, a, b, c, and d represent statistical differences. If the letters are the same, it means there is no statistical difference, p ≥ 0.05. Detailed Implementation

[0032] This invention provides the use of postgenes of fermented Lactobacillus mucinus in the preparation of immune activators.

[0033] In some embodiments of the present invention, the fermenting *Lactobacillus fermentum* is *Limosilactobacillus fermentum* B44, with accession number CGMCC No. 17321. This strain was deposited on March 8, 2019, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. At the time of deposit, this strain was classified and named *Lactobacillus fermentum*, and in 2020 it was renamed *Lactobacillus fermentum*. The fermenting *Lactobacillus fermentum* B44 is an edible strain, ensuring food safety.

[0034] In some embodiments of the present invention, the metagene is a substance prepared by the following method: collecting fermenting *Lactobacillus myxoides* from the fermentation broth by centrifugation, washing and resuspending, inactivating the bacterial cells, cooling and centrifuging to retain the precipitate, and freeze-drying to obtain the metagene.

[0035] The fermentation broth of *Lactobacillus mucinus* of the present invention can be cultured anaerobically in a commonly used lactobacillus culture medium (e.g., MRS medium). The fermentation temperature is 35-38°C, and the fermentation time can be adjusted according to specific needs, for example, 7-24 hours.

[0036] In some embodiments of the present invention, the washing method is washing with water, and / or the resuspension method is resuspension with water, and the cooling method is cooling to 0-28°C.

[0037] In some embodiments of the present invention, the viable cell count in the fermentation broth of *Lactobacillus mucinus* is 1 × 10⁻⁶. 5 CFU / mL or higher, preferably 1×10⁻⁶. 5 CFU / mL ~9×10 9 CFU / mL, more preferably 1×10⁻⁶. 5 CFU / mL ~9×10 7 CFU / mL. For example, the viable count is 1×10⁻⁶. 5 ~5×10 5 CFU / ml, 5×10 5 ~1×10 6 CFU / ml, 1×10 6 ~5×10 6 CFU / ml, 5×10 6 ~1×10 7 CFU / ml, 1×10 7 ~5×107 CFU / ml, 5×10 7 ~1×10 8 CFU / ml, 1×10 8 ~5×10 8 CFU / ml, 5×10 8 ~1×10 9 CFU / ml, 1×10 9 ~9×10 9 CFU / ml.

[0038] In some embodiments of the present invention, the inactivation method is boiling water bath inactivation for 2-15 minutes, preferably 2-5 minutes; more preferably 2-3 minutes. This inactivation method is simple to operate, convenient to process and apply, and has strong industrial applicability.

[0039] In some embodiments of the present invention, the freeze-drying method involves freezing at -20 to -90°C for at least 2 hours, followed by vacuum freeze-drying for 6 to 12 hours. In some embodiments of the present invention, freezing at -20 to -90°C until the bacterial cells solidify is performed before vacuum freeze-drying. In one embodiment, freezing at -20 to -90°C for 2 to 24 hours, for example, 2 to 5 hours, 5 to 10 hours, 10 to 15 hours, 15 to 20 hours, or 20 to 24 hours, the lower the temperature, the shorter the freeze-drying time can be, as long as the bacterial cells are frozen until solidification.

[0040] In some embodiments of the present invention, the immune activator is a macrophage activator. The macrophage activator has one or more of the following effects:

[0041] 1) Enhance macrophage cell viability;

[0042] 2) Enhance the phagocytic capacity of macrophages;

[0043] 3) Increase the production of pro-inflammatory factors or NO by macrophages;

[0044] 4) Induces macrophages to polarize into the M1 type and perform corresponding functions.

[0045] "Improvement" refers to the group that did not use immune activators.

[0046] In some embodiments of the present invention, the pro-inflammatory factor is selected from TNF-α, IL-6 and / or IL-1β.

[0047] In some embodiments of the present invention, the immune activator is a food, dietary supplement, or vaccine adjuvant.

[0048] The food product is selected from fermented dairy products or non-fermented dairy products. The food product may be, for example, a solid beverage or pasteurized milk. The solid beverage may be, for example, the solid beverage described in patent application number 202411574014.4. The pasteurized milk may be, for example, the pasteurized modified milk described in patent application number 202411574004.0.

[0049] The macrophage activator possesses excellent immune-activating capabilities, inducing macrophage polarization to the M1 type. When used in combination with LPS, it can enhance LPS-induced macrophage phagocytosis, pro-inflammatory factor production, and NO production. This macrophage activator is suitable for daily intake by healthy, sub-healthy, or diseased individuals. The dosage of this macrophage activator is not specifically limited and can be adjusted according to actual needs. By specifically activating the immune response pathways of macrophages, this macrophage activator can play an important role in the fields of immune regulation-related foods, health foods, vaccine adjuvants, and biomedicine.

[0050] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0051] 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; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0052] 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.

[0053] The flowchart of the embodiment is as follows Figure 1 As shown.

[0054] Example 1: Culture of fermented Lactobacillus mucinus

[0055] Step 1: Cultivation of Lactobacillus mucinus

[0056] Freeze-dried tubes of *Limosilactobacillus fermentum* B44 strain, stored at -80℃ (CGMCC No. 17321), were deposited on March 8, 2019, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. After activation on B44 plates and anaerobic culture at 37℃ for 48 hours, single strains were selected and inoculated into MRS liquid medium. The medium was then statically cultured at 37℃ for 16 hours, followed by 5% inoculation for activation. The activated *Limosilactobacillus fermentum* B44 was then inoculated into seed culture and fermentation medium. The formulations of the solid and liquid fermentation media are as follows:

[0057] Medium-density slabs (MRS)

[0058] 10g peptone, 8g beef extract, 4g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1g Tween 80, pH=5.7, add 12g agar, autoclave at 121℃ for 20min, cool to about 50℃, plate, and store for later use.

[0059] Seed culture medium and fermentation broth (MRS)

[0060] 10g peptone, 8g beef extract powder, 4g yeast extract powder, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1g Tween 80, add distilled water to a final volume of 1L, pH=5.7, autoclave at 121℃ for 20min, cool and store for later use.

[0061] Step 2: Collect fermented mucinous Lactobacillus strains, inactivate and freeze-dry them.

[0062] Collect fermented *Lactobacillus mucinus* by centrifugation at 7000 rpm and wash with double-distilled water. Resuspend the bacterial cells in double-distilled water and inactivate them by boiling in a water bath for 2 min. After cooling to room temperature, centrifuge to discard the supernatant and retain the bacterial cells. Freeze-dry at -80℃ for 2 hours and then freeze-dry overnight under vacuum.

[0063] Example 2 Cell Experiment

[0064] Weigh out 10 units of the corresponding live bacteria count before inactivation. 5 CFU / mL, 10 7 CFU / mL of freeze-dried fermented Lactobacillus mucinus was added to DMEM cell culture medium containing 10% FBS and 1% penicillin and streptomycin.

[0065] (1) Fermentation of Lactobacillus mucin B44 promotes macrophage M1 polarization.

[0066] 5×10 5 RAW264.7 was seeded in 12-well plates and incubated at 37°C with 5% CO2 for 24 h. The plates were washed twice with PBS, and then seeded with 2 mL of 10... 5 10 7 Cells were cultured in DMEM complete medium at 37°C with 5% CO2 for 24 h using CFU / mL B44. After trypsin digestion and neutralization with DMEM complete medium, the cells were washed twice with PBS. FcRblock cells were incubated at room temperature in the dark for 10 min. CD11b-FITC and CD86-PE antibodies were incubated at 4°C in the dark for 30 min. After washing with PBS, the cells were fixed in 4% paraformaldehyde at room temperature in the dark for 20 min. Cells were then permeated by centrifugation and washing three times with Intracellular Staining Permeabilization Wash Buffer. CD206-APC antibody cells were incubated at room temperature in the dark for 30 min. Cells were then washed three times with Staining Permeabilization Wash Buffer. Cells were resuspended in 1% FBS cell preservation medium, dispersed in a cell strainer, and analyzed using Cytoflex.

[0067] The results are as follows Figure 2 As shown, the results indicate that 10 7 CFU / mL B44 significantly induced M1 polarization in RAW264.7 macrophages, with the M1 polarization rate reaching 30.3–35.5% (range obtained from 3 parallel samples).

[0068] (2) Cell proliferation

[0069] RAW264.7 macrophages were 1×10 4 Cells were seeded at a density of / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. After washing twice with PBS, the lyophilized B44 cells were resuspended in DMEM medium to a final concentration of 10 μL. 5 10 7 CFU / mL was added to 96-well plates as the experimental group, and a negative control group (DMEM culture medium without lyophilized bacteria) and a positive control group (1 μg / mL LPS) were also set up. After culturing at 37℃ and 5% CO2 for 12 h, 10 μl of CCK-8 solution was added to each well, and the cells were incubated at 37℃ for 1.5 h. The absorbance was measured at 450 nm to assess the effect of B44 on the proliferation of RAW264.7 cells.

[0070] The components of each group are as follows:

[0071] Negative control group (containing cell culture medium, CCK-8, and no test substance), sample group (either experimental or positive group, containing cell culture medium, CCK-8, and test sample), and blank wells (containing culture medium and CCK-8 without cells and test substance).

[0072]

[0073] The results of cell viability are as follows Figure 3 As shown. By Figure 3 It can be seen that the cell viability of the experimental group containing the freeze-dried fermented Lactobacillus mucilaginosus of the present invention was significantly higher than that of the negative control group and the positive group. 5 10 7 The activity of the CFU / mL lyophilized fermented Lactobacillus mucinus experimental group was increased by 32.32% and 60.80% respectively compared with the positive group.

[0074] (3) Determination of NO

[0075] NO plays a crucial role in immune responses, inflammation, and tissue damage repair. Appropriate release of NO by the body has a defensive effect against microbial invasion.

[0076] On the first day, mouse macrophages RAW264.7 cells from passages 5 to 8 were processed at a dose of 1 × 10⁻⁶. 4 Cells were seeded in 96-well plates and incubated at 37°C for 24 h with 5% CO2. The culture medium was discarded, and the cells were washed with PBS. DMEM complete medium containing inactivated *Lactobacillus fermentatus* and 1 μg / mL LPS (Ecoli 0111:B4) was added to the cells. For the positive control, 1 μg / mL LPS (Ecoli 0111:B4) in DMEM complete medium was added. For the blank control, DMEM complete medium was used. Cells were incubated at 37°C for 12 h with 5% CO2. The cell supernatant was collected and centrifuged at 8000 rpm for 5 min. The NO content was determined using a NO assay kit. Standard curves were prepared using serial dilutions with NaNO2. 50 μL of sample and standard curve were added to each well of the 96-well plate, along with 50 μL of Griess Reagent I and 50 μL of Griess Reagent II. The absorbance at 540 nm was measured within 10 min. The NO content in the cell supernatant was calculated using the standard curve.

[0077] The results are as follows Figure 4As shown, the experimental group containing the freeze-dried fermented *Lactobacillus mucinus* and LPS of the present invention released significantly more NO than the blank group and the positive group. Since NO plays a crucial role in immune responses, inflammation, and tissue damage repair, appropriate release of NO by the body has a defensive effect against microbial invasion. It also possesses anti-tumor, anti-angiogenic, and anti-pathogen effects. Therefore, the conclusion of this embodiment indicates that the freeze-dried fermented *Lactobacillus mucinus* of the present invention helps the body defend against microbial invasion and has anti-tumor, anti-angiogenic, and anti-pathogen effects.

[0078] (4) Measurement of phagocytic capacity

[0079] Pathogens and damaged cells can be cleared by macrophages through phagocytosis to maintain homeostasis.

[0080] On the first day, mouse macrophages RAW264.7 cells from passages 5 to 8 were injected at 100 μL with 1 × 10⁻⁶ cells / mL. 4 RAW264.7 was seeded in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. After washing twice with PBS, the lyophilized B44 was resuspended in DMEM medium to a final volume of 10⁻⁶. 5 10 7 In DMEM medium containing CFU / mL, LPS was added to each well to ensure a concentration of 1 μg / mL, and the cells were incubated at 37°C with 5% CO2 for 12 h. After washing with PBS, 100 μL of neutral red (0.1%, w / w, 0.9% NaCl) solution was added to each well and the cells were incubated for 1 h. After removing the neutral red, the cells were washed twice with PBS to remove any unabsorbed neutral red and external dyes. 200 μL of cell lysis buffer (glacial acetic acid: ethanol = 1:1) was added. After incubation at room temperature for 2 h, the absorbance was measured at 540 nm.

[0081]

[0082] The results are as follows Figure 5 As shown, compared to the LPS group, adding 10 5 10 7 The experimental groups with CFU / mL lyophilized fermented Lactobacillus mucin increased by 4.91% and 9.65%. Pathogens and damaged cells can be cleared by macrophages through phagocytosis to maintain homeostasis; therefore, assessing the phagocytic capacity of fermented Lactobacillus mucin B44 on RAW264.7 cells can reflect its impact on immune function.

[0083] (5) Measurement of cytokines TNF-α, IL-6, and IL-1β

[0084] Cytokines are primarily produced by macrophages and lymphocytes, and they are crucial for macrophage function. They mediate the release of effective immune responses, link innate and adaptive immunity, and influence the macrophage microenvironment.

[0085] On the first day, mouse macrophages RAW264.7 cells from passages 5 to 8 were processed at a dose of 5 × 10⁻⁶. 5 Cells were seeded in 12-well cell culture plates. The next day, DMEM complete medium containing inactivated *Lactobacillus fermentatus* and 1 μg / mL LPS (Ecoli 0111:B4) was added to the cells. Positive controls were prepared with 1 μg / mL LPS (Ecoli 0111:B4) in DMEM complete medium, and blank controls were prepared with DMEM complete medium. Cells were incubated at 37°C for 24 h with 5% CO2. The cell supernatant was collected, centrifuged at 8000 rpm for 5 min, aliquoted, and stored at -80°C for ELISA assay of inflammatory cytokines TNF-α, IL-6, and IL-1β.

[0086] The results are as follows Figures 6 to 8 As shown, the experimental group showed increased levels of TNF-α, IL-1β, and IL-6 compared to the positive group. Cytokines are crucial for macrophage function. When macrophages are exposed to inflammatory stimuli, they secrete cytokines such as tumor necrosis factor (TNF), IL-1β, and IL-6. Increased levels of TNF-α, IL-1β, and IL-6 can promote macrophage activation and function, thereby mobilizing the body's immunity.

[0087] In summary, the results of cell viability, phagocytic capacity, NO levels, and the determination of cytokines TNF-α, IL-6, and IL-1β clearly demonstrate that inactivated fermenting Lactobacillus mucinus B44, as a metabiotic, has excellent immune-activating effects.

[0088] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. The use of post-biotics of fermented Lactobacillus mucin in the preparation of immune activators.

2. The use according to claim 1, characterized in that, The fermenting *Lactobacillus fermentum* was identified as Limosilactobacillus fermentum B44, with accession number CGMCC No. 17321.

3. The use according to claim 1, characterized in that, The postbiotic is a substance prepared by the following method: collecting fermenting *Lactobacillus myxoides* from the fermentation broth by centrifugation, washing and resuspending, inactivating the bacterial cells, cooling and centrifuging to retain the precipitate, and freeze-drying to obtain the postbiotic.

4. The use according to claim 3, characterized in that, The washing method is washing with water, and / or the resuspension method is resuspension with water, and / or the cooling method is cooling to 0-28°C.

5. The use according to claim 3, characterized in that, The viable cell count in the fermentation broth of *Lactobacillus mucinus* was 1×10⁻⁶. 5 CFU / mL or higher, preferably 1×10⁻⁶. 5 CFU / mL ~9×10 9 CFU / mL; or, the inactivation method is boiling water bath inactivation for 2–15 min, preferably 2–5 min.

6. The use according to claim 3, characterized in that, The freeze-drying method involves freezing at -70 to -90°C for more than 2 hours, followed by vacuum freeze-drying for 6 to 12 hours; preferably, freezing at -70 to -90°C until the bacterial cells solidify is followed by vacuum freeze-drying.

7. The use according to claim 1, characterized in that, The immune activator is a macrophage activator.

8. The use according to claim 1, characterized in that, The macrophage activator has one or more of the following effects: 1) Enhance macrophage cell viability; 2) Enhance the phagocytic capacity of macrophages; 3) Increase the production of pro-inflammatory factors or NO by macrophages; 4) Induces macrophage polarization to M1 type.

9. The use according to claim 8, characterized in that, The pro-inflammatory factors are selected from TNF-α, IL-6 and / or IL-1β.

10. The use according to claim 1, characterized in that, The immune activator is a food, dietary supplement, or vaccine adjuvant; preferably, the food is selected from fermented dairy products or non-fermented dairy products.

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