Lactobacillus muci genosus with anti-inflammatory effect and use thereof

Abstract

The present application relates to the field of microorganisms, and particularly relates to a fermented Lactobacillus muiyi and application thereof. The preservation number of the fermented Lactobacillus muiyi strain BD8047 is CGMCC No. 37551. A new fermented Lactobacillus muiyi with anti-inflammatory effect is isolated from pickles and preserved, the bacteria can simultaneously significantly reduce the content of TNF-alpha and IL-6 produced by macrophages, and the effect of promoting macrophage proliferation is significant, and the comprehensive anti-inflammatory effect is optimal, and can be widely applied to food, dietary supplements, fermented and non-fermented milk products and the like, and used for preparing medicines for treating and / or relieving diseases related to inflammation.

Description

Technical Field

[0001] This invention relates to the field of food microbiology and functional food technology, and in particular to a fermentable Lactobacillus mucinus with anti-inflammatory effects and its uses. Background Technology

[0002] Chronic low-grade inflammation is a common pathological basis for chronic non-communicable diseases such as obesity, type 2 diabetes, cardiovascular disease, and inflammatory bowel disease. Lipopolysaccharide (LPS) is a core component of the cell wall of Gram-negative bacteria. It activates the NF-κB pathway through the TLR4 receptor, driving macrophages to polarize towards the M1 pro-inflammatory phenotype and secrete large amounts of pro-inflammatory cytokines such as TNF-α and IL-6. It is a standard model for evaluating immunomodulatory efficacy in vitro.

[0003] Limosilactobacillus fermentum, formerly known as Lactobacillus fermentum, is a lactic acid bacterium with a long history of safe use. It has been included in the "List of Microbial Strains that Can Be Used in Food" by my country's health authorities and has also obtained QPS certification from the European Food Safety Authority (EFSA) and GRAS recognition from the U.S. Food and Drug Administration (FDA).

[0004] In recent years, research on *Lactobacillus fermentum* has mainly focused on its probiotic functions, including regulating intestinal flora and alleviating infectious diarrhea. CN118480471B discloses a *Lactobacillus fermentum* strain BN11 that improves oral health and its applications, demonstrating that *Lactobacillus fermentum* can promote the proliferation of probiotics, regulate the oral microecological balance, and promote oral mucosal repair. CN119770527A discloses the application of *Lactobacillus fermentum* in the preparation of antitumor drugs; this strain can activate the immune system and increase CD8 in spleen tissue, thereby treating melanoma or colon cancer. CN118516260A discloses a strain of *Lactobacillus fermentum* HGProbio 028 and its applications; this bacterium has the effect of effectively degrading uric acid and purine nucleosides. CN118995465A discloses a *Lactobacillus fermentum* Lferm-1 and its applications; this bacterium has the effects of resisting gastric acid, inhibiting various pathogenic bacteria, inhibiting the expression of pro-inflammatory factors, and improving intestinal barrier damage, and can significantly improve chemotherapy-related diarrhea.

[0005] However, no good anti-inflammatory properties have yet been found in fermenting *Lactobacillus mucinus*. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a fermented Lactobacillus mucinus with anti-inflammatory effects and its uses, in order to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention provides a fermenting Lactobacillus mucinus strain BD8047 with anti-inflammatory effects, which has the accession number CGMCC No. 37551.

[0008] The present invention also provides a fermentation culture of Lactobacillus mucinus, obtained by inoculating the strain BD8047 as described above into a culture medium and culturing it.

[0009] The present invention also provides the use of strain BD8047 as described above or fermentation culture as described above in the preparation of products with anti-inflammatory effects.

[0010] The present invention also provides a product with anti-inflammatory effects, wherein the product with anti-inflammatory effects contains strain BD8047 as described above, or fermentation culture as described above.

[0011] As described above, the fermented Lactobacillus mucinus with anti-inflammatory effects and its uses according to the present invention have the following beneficial effects:

[0012] 1) Food-grade safety: Fermenting Lactobacillus mucinus is listed in my country's "List of Microbial Strains that can be Used in Food" and has GRAS / QPS certification background, ensuring full safety.

[0013] 2) Dual-targeted anti-inflammatory (core advantage): Among five strains of the same fermenting Lactobacillus strain, this strain is the only one that simultaneously and significantly reduces TNF-α (p<0.001) and IL-6 (p<0.01), resulting in the best overall anti-inflammatory effect.

[0014] 3) Clear mechanism: NF-κB-dependent targeted regulation with a clear target, which is significantly different from the compensatory activation risk mediated by bypass pathways.

[0015] 4) Polarization silence, no excessive immunosuppression: IL-10 does not increase compensatorily and does not induce excessive M2 polarization, making it suitable for long-term consumption.

[0016] 5) Postbiotic stability advantage: Heat-inactivated products do not require maintaining live bacteria, overcome cold chain limitations, extend shelf life, avoid the risk of bacteremia, and have a wider range of applications. Attached Figure Description

[0017] Figure 1 The images shown are actual photographs of the fermented Lactobacillus mucinus BD8047 and B44 as described in Example 1 of this invention.

[0018] Figure 2 The images shown are actual photographs of the fermenting Lactobacillus mucinus BD8047 and 7570 as described in Example 1 of this invention.

[0019] Figure 3 The images shown are actual photographs of fermenting Lactobacillus mucinus 5088 and 9984, which are examples of the present invention.

[0020] Figure 4 The image shows Gram staining patterns of BD8047, B44, 7570, 5088, and 9984 of *Lactobacillus fermentans* as described in Example 1 of this invention. Detailed Implementation

[0021] This invention provides a fermentation strain of Lactobacillus mucinus BD8047 with anti-inflammatory effects, which has the preservation number CGMCC No. 37551.

[0022] This invention isolates and preserves a strain of *Limosilactobacillus fermentum* BD8047 with anti-inflammatory properties from Kunming pickled vegetables. The preservation number is CGMCC No. 37551. This strain was deposited on January 27, 2026, 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 preservation, this strain was classified and named *Limosilactobacillus fermentum*. The *Limosilactobacillus fermentum* BD8047 of this invention is an edible strain, ensuring food safety.

[0023] This invention isolated and screened four novel *Lactobacillus fermentans* strains (9984, BD8047, 7570, and 5088) from fermented yogurt, kimchi fermentation broth, fermented sourdough, and fresh raw milk, respectively. In vitro anti-inflammatory activity screening results showed that strain BD8047 significantly inhibited LPS-induced macrophage inflammatory responses, and its effects on reducing TNF-α and IL-6 protein secretion and downregulating NF-κB p65, iNOS, and TGF-β mRNA expression were superior to the control strain B44. Furthermore, in human THP-1 and mouse RAW 264.7 cell models, the secretion levels of TNF-α and IL-6 in the BD8047 treatment group were significantly lower than those in the LPS model group, and there was no statistically significant difference compared to the dexamethasone positive control group. The above results indicate that fermented Lactobacillus mucinus BD 8047 has broad-spectrum and potent anti-inflammatory activity. Its mechanism may be through inhibiting the activation of the NF-κB signaling pathway, blocking macrophage polarization to the pro-inflammatory M1 phenotype, thereby effectively alleviating LPS-induced inflammatory responses.

[0024] In some embodiments, the 16S rRNA sequence of the strain BD8047 comprises the sequence shown in SEQ ID NO.1.

[0025] In this invention, strain BD8047 and strain 8047 are different ways of representing the same strain. Unless otherwise specified, both refer to the Lactobacillus fermentans strain with accession number CGMCC No. 37551.

[0026] The present invention also provides a fermentation culture of Lactobacillus mucinus, obtained by inoculating the strain BD8047 as described above into a culture medium and culturing it.

[0027] In some embodiments, the culture temperature is 8–37°C, or it can be 8–16°C, or it can be 14–22°C, or it can be 20–34°C, or it can be 26–37°C, or it can be 10°C, 12°C, 15°C, 26°C, or 37°C.

[0028] In some embodiments, the culture medium comprises a carbon source, a nitrogen source, and inorganic salts.

[0029] In some embodiments, the carbon source is selected from one or more of glucose, sucrose, and maltose. Preferably, it is glucose.

[0030] In some embodiments, the nitrogen source is selected from one or more of peptone, beef extract, and yeast extract. Preferably, it is peptone, beef extract, and yeast extract.

[0031] In some embodiments, the inorganic salt is selected from one or more of diammonium citrate, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, and manganese sulfate.

[0032] In some specific embodiments, the culture medium contains the following components in parts by weight, based on the total volume of the culture medium: peptone 5-15 g / L, beef extract 2.5-15 g / L, yeast extract 2-10 g / L, glucose 10-30 g / L, diammonium citrate 1-5 g / L, sodium acetate 1-10.0 g / L, dipotassium hydrogen phosphate 1-4 g / L, magnesium sulfate 0.1-1 g / L, and manganese sulfate 0.01-0.1 g / L.

[0033] In one specific embodiment, the culture medium comprises the following components in parts by weight: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 2.0 g / L diammonium citrate, 5.0 g / L sodium acetate, 2.0 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, and 0.05 g / L manganese sulfate.

[0034] The present invention also provides the use of strain BD8047 as described above or fermentation culture as described above in the preparation of products with anti-inflammatory effects.

[0035] In some embodiments, the product with anti-inflammatory effects contains at least 10 μL of strain BD8047. 8 CFU / mL or 1×10 8 CFU / g, for example 1×10 8 CFU / g or 1×10 8 CFU / mL, 4×10 8 CFU / g or 4×10 8 CFU / mL, 8×10 8 CFU / g or 8×10 8 CFU / mL, 2×10 9 CFU / g or 2×10 9 CFU / mL, 5×10 9 CFU / g or 5×10 9 CFU / mL, 8×10 9 CFU / g or 8×10 9 CFU / mL, 1×10 10 CFU / g or 1×10 10 CFU / mL, 5×10 10 CFU / g or 5×10 10 CFU / mL, 8×10 10 CFU / g or 8×10 10 CFU / mL, 1×10 11 CFU / g or 1×10 11 CFU / mL, 4×10 11 CFU / g or 4×10 11 CFU / mL, 5×10 11 CFU / g or 5×10 11 CFU / mL, 8×10 11 CFU / g or 8×10 11 CFU / mL, etc., those skilled in the art can select the range of values ​​according to actual needs.

[0036] In some embodiments, the product with anti-inflammatory properties includes one or more of the following: food, medicine, daily chemical products, or microbial agents.

[0037] In some embodiments, the product with anti-inflammatory properties has one or more of the following effects:

[0038] B1) Inhibits macrophages from producing pro-inflammatory factors;

[0039] B2) Regulates macrophage polarization balance, which manifests as the inhibition of M1 marker expression.

[0040] In some embodiments, the pro-inflammatory factor is selected from TNF-α and IL-6.

[0041] In some implementations, the M1 type marker is selected from iNOS.

[0042] In the aforementioned products, the fermenting Lactobacillus mucin strain BD8047 can exist in either a live or inactivated state. It is preferred to use it in the inactivated state (postbiotic) because postbiotics are safer and more stable than live bacteria, giving them greater advantages in processing, transportation, and storage. Compared to live probiotics, the longer shelf life ensures that postbiotic products can reach many areas with poor transportation, making it easier to promote and expand their use.

[0043] The present invention also provides a product with anti-inflammatory effects, wherein the product with anti-inflammatory effects contains strain BD8047 as described above, or fermentation culture as described above.

[0044] In some embodiments, the content of strain BD8047 in the product with anti-inflammatory effect is not less than 10. 8 CFU / mL or 1×10 8 CFU / g, for example 1×10 8 CFU / g or 1×10 8 CFU / mL, 4×10 8 CFU / g or 4×10 8 CFU / mL, 8×10 8 CFU / g or 8×10 8 CFU / mL, 2×10 9 CFU / g or 2×10 9 CFU / mL, 5×10 9 CFU / g or 5×10 9 CFU / mL, 8×10 9 CFU / g or 8×10 9 CFU / mL, 1×10 10 CFU / g or 1×10 10 CFU / mL, 5×10 10 CFU / g or 5×10 10 CFU / mL, 8×10 10 CFU / g or 8×10 10 CFU / mL, 1×10 11 CFU / g or 1×10 11 CFU / mL, 4×10 11 CFU / g or 4×10 11 CFU / mL, 5×10 11 CFU / g or 5×10 11 CFU / mL, 8×1011 CFU / g or 8×10 11 CFU / mL, etc., those skilled in the art can select the range of values ​​according to actual needs.

[0045] In some embodiments, the strain BD8047 or the fermentation culture is in live or inactivated form.

[0046] In some embodiments, inactivation is performed by boiling water bath for 2–15 minutes. This inactivation method is simple to operate, convenient to process and apply, and has strong industrial applicability.

[0047] In some implementations, the inactivation method is boiling water bath inactivation for 2 to 5 minutes.

[0048] In some embodiments, the product with anti-inflammatory properties comprises one or more of the following: food, pharmaceutical, daily chemical products, and microbial agents.

[0049] In some embodiments, the food is a health food, a food for special medical purposes, or a functional food. In this application, the functional food refers to a food in food form that is intended to supplement specific nutrients or regulate bodily functions and claims to have specific health benefits.

[0050] In some embodiments, the daily chemical product is a skin care preparation, which is one or more selected from solutions, suspensions, masks, lotions, creams, ointments, gels, dry powders, wet powders, and sprays. Preferably, it is a solution. The daily chemical product is used to soothe irritated skin and reduce discomfort such as redness and stinging.

[0051] In some embodiments, the bacterial agent is a probiotic, a synbiotic, or a metabiotic. The probiotic is a live microorganism, the synbiotic comprises a live strain BD8047 and its available substrates, and the metabiotic is an inactivated strain BD8047 and / or its metabolites. In some specific embodiments, the metabiotic is a substance prepared by the following method: collecting *Lactobacillus myxoidus* fermentation broth by centrifugation, washing and resuspending, inactivating the bacterial cells, cooling and centrifuging to retain the precipitate, and then freeze-drying to obtain the metabiotic. The *Lactobacillus myxoidus* fermentation broth of the present invention can be cultured anaerobically in a commonly used lactobacillus culture medium (e.g., MRS medium) using *Lactobacillus myxoidus* fermentation. The fermentation temperature is 35-38°C, and the fermentation time can be adjusted according to specific needs, for example, 7-24 hours. In some embodiments of the present invention, washing is performed with water, and / or resuspension is performed with water, and cooling is performed at 0-28°C. 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 24 to 36 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. Preferably, the dosage form of the product with anti-inflammatory effect includes powder and solution. The dosage form of the product with anti-inflammatory effect of the present invention is not limited, including the most commonly used powder and solution, or further processed capsules, tablets, granules, etc.

[0052] The present invention also provides the use of strain BD8047 as described above, or fermentation culture as described above, or product with anti-inflammatory activity as described above, in the preparation of articles for the prevention or treatment of respiratory diseases.

[0053] In some implementations, the respiratory disease includes infectious respiratory diseases and allergic respiratory diseases.

[0054] In some embodiments, the infectious respiratory disease includes viral, bacterial, mycoplasmal / chlamydial, and tuberculous diseases. The bacterial diseases include Streptococcus pneumoniae pneumonia and purulent tonsillitis.

[0055] In some embodiments, the allergic respiratory disease includes allergic rhinitis, bronchial asthma, and allergic bronchopulmonary aspergillosis (ABPA).

[0056] The fermentation strain 8047 of *Lactobacillus mucilaginosus* of this invention can be widely used in the following product types to perform corresponding functions:

[0057] 1) Functional fermented milk or yogurt

[0058] Heat-inactivated bacterial powder of strain 8047 (10) 7 ~10 9 Adding CFU / mL equivalent amounts to fermented milk base (whole or skim milk) to prepare dairy products that combine the flavor of traditional fermented milk with anti-inflammatory functions, suitable for relieving chronic low-grade inflammation and maintaining intestinal immune homeostasis, and can be used as a functional food for daily intake by sub-healthy individuals. Product forms: flavored fermented milk, low-temperature yogurt, functional room-temperature yogurt.

[0059] 2) Probiotics or postbiotic supplements

[0060] Using freeze-dried strain 8047 as the active ingredient, it is prepared into dosage forms such as capsules, tablets, powders or granules using microencapsulation technology. It is used as a dietary supplement for chronic inflammatory diseases such as inflammatory bowel disease in remission or metabolic syndrome. It can also be used in combination with prebiotics (such as inulin and fructooligosaccharides) to form synergistic preparations to exert intestinal immune regulation function.

[0061] 3) Solid beverages or foods for special medical purposes (foods for special medical purposes)

[0062] Using strain 8047 postbiotic as a functional ingredient, solid beverages or special medical purpose formula foods with claims of intestinal health or immune regulation functions can be prepared, which are suitable for consumers who need immune management, such as those in the postoperative immune recovery period or chemotherapy adjuvant period. Since the postbiotic form does not contain live bacteria, it can avoid the risk of bacteremia, and has a wider range of applicable populations and higher safety.

[0063] 4) Functional dairy ingredients

[0064] Inactivated bacterial powder of strain 8047 can be added to milk powder, whey protein powder or sports nutrition foods to enrich product functions. It is suitable for products for the management of chronic inflammation in middle-aged and elderly people or for the relief of exercise-induced inflammation. This bacterial powder is compatible with conventional processing technology and is suitable for dairy products stored and transported at room temperature.

[0065] 5) Health food

[0066] With "enhancing immunity" or "aiding in improving gastrointestinal function" as the health care function direction, functional evaluation (rat / mouse immune function test) will be carried out in accordance with the requirements for health food application, and the health food registration application of strain 8047 post-biotic will be promoted to achieve compliance of functional claims.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Example 1: Isolation and purification of fermenting Lactobacillus mucinus

[0071] 1.1 Sample Sources and Preprocessing

[0072] 1) Cheri Village Yogurt (Homemade Yogurt) from Tibet: Under aseptic conditions, take 5 g of well-mixed yogurt, add 45 mL of sterile physiological saline, vortex thoroughly for 10 min, and prepare 10... -1 The bacterial suspension was allowed to stand for 3 minutes. The upper layer of homogeneous suspension was then collected for use in subsequent step 1.5 for gradient dilution and separation.

[0073] 2) Kunming traditional pickled vegetable fermentation liquid (farmhouse fermentation): Take 5 mL of the pickled vegetable fermentation supernatant, centrifuge at 8000 r / min for 3 min to remove solid residue, and take the supernatant as the initial stock solution for subsequent step 1.5 for gradient dilution to separate.

[0074] 3) Tianshui Old Dough Fermentation Water: Shake the old dough water thoroughly, aseptically take 5 mL of the original liquid, no centrifugation required, and use it directly as the initial bacterial solution for subsequent step 1.5 for gradient dilution and separation.

[0075] 4) Fresh raw milk from Tibet: Aseptically extract 5 mL of fresh raw milk and use it directly as the stock solution for subsequent step 1.5 for gradient dilution and separation.

[0076] 1.2 Culture medium formulation (MRS universal lactic acid bacteria culture medium)

[0077] MRS liquid culture medium (1 L): 10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 2.0 g diammonium citrate, 5.0 g sodium acetate, 2.0 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate monohydrate, 1.0 mL Tween 80, bring the volume to 1000 mL with distilled water, and adjust the pH to 6.2–6.4.

[0078] Solid MRS medium (1 L): Add 12 g of agar powder to the liquid MRS medium.

[0079] 1.3 Auxiliary reagents and equipment

[0080] Sterile saline (0.85% NaCl), sterile PBS buffer, 75% medical alcohol, sterile spreader, sterile pipette tips, sterile centrifuge tubes, petri dishes, anaerobic culture jars, anaerobic gas generators, autoclave, laminar flow hood, and constant temperature incubator.

[0081] 1.4 Sterilization parameters

[0082] Sterilize with high-pressure steam at 121 ℃ and 0.1 MPa for 15 min. After sterilization, remove and place in a clean bench to cool for later use.

[0083] 1.5 Separation

[0084] 1) Prepare several sterile 9 mL saline test tubes.

[0085] 2) Take 1 mL of the initial bacterial culture after the above treatment (i.e., the sample after pretreatment in step 1.1), add it to 9 mL of sterile physiological saline, vortex thoroughly to mix, and prepare 10 -1 Diluent.

[0086] 3) Replace with a sterile pipette tip and aspirate 10 -1 Add 1 mL of diluent to the next tube of 9 mL of physiological saline, mix well to obtain 10 -2 Diluent. Continuously dilute in a gradient until a solution of 10 is prepared. -3 10 -4 10 -5 10 -6 Gradient dilution solution.

[0087] 4) Take 100 μL of each of the 10 -3 10-4 10 -5 Three gradients were applied using a sterile spreader to spread the culture evenly onto sterile solid MRS medium, ensuring an appropriate number of single colonies (30–300 CFU / plate). After spreading, the culture was allowed to stand for 3–5 minutes to absorb.

[0088] 5) Invert the plate and incubate it at a constant temperature of 37 ℃ for anaerobic incubation for 48 h.

[0089] The typical characteristics of fermenting *Lactobacillus mucinus* are:

[0090] Colony morphology: round, with neat edges, smooth and moist surface, milky white, translucent, and plump.

[0091] Cell morphology: Gram-positive, non-spore-forming, short rod-shaped or slender rod-shaped, arranged singly or in pairs.

[0092] Biochemical characteristics: acid-resistant, ferments glucose to produce acid, does not produce gas, and is suitable for the acid-selective culture environment of MRS.

[0093] 1.6 Purification

[0094] To eliminate contaminating bacteria and obtain a pure culture of fermenting *Lactobacillus mucilaginosus*, three consecutive streak purification cycles must be performed, as follows:

[0095] 1) Select a single colony with a typical morphology obtained in step 1.6 and streak it in four zones on a fresh MRS solid plate for isolation.

[0096] 2) Incubate anaerobicly at 37℃ for 48 h and observe the growth of single colonies.

[0097] 3) Pick a single colony again and repeat the streak purification process. Repeat the purification process three times until the colony morphology on the plate is completely uniform and free of contaminants.

[0098] 4) After purification, Gram staining and microscopic examination showed that all bacteria in the field of view were Gram-positive bacilli, with no other bacteria or coccal contamination, and the strain was determined to be purified.

[0099] Colony morphology standards for purified strains: 1) Size: 1-3 mm in diameter, slightly larger after 48 h of culture; 2) Shape and edge: perfectly round, with smooth and neat edges (entire); 3) Elevation: clearly raised, hemispherical (convex); 4) Color: milky white / milky white, light beige; a few strains are slightly light gray; 5) Transparency: translucent to slightly opaque; 6) Surface texture: smooth and moist, most strains have a slightly mucous texture (mucoid) (from which the name "Mucobacterium mucosae" originates), not dry, and without wrinkles.

[0100] Strain 8047 was isolated from Kunming pickled vegetables, strain 9984 was isolated from Cheri Village yogurt in Tibet, strain 7570 was isolated from Tianshui fermented vegetable juice, and strain 5088 was isolated from raw milk in Tibet. Figure 1 See the colony morphology diagrams for 8047 and B44. Figure 1 Colony morphology diagrams for 7570 and 8047 are shown below. Figure 2 See the colony morphology diagrams for 5088 and 9984. Figure 3 . Figure 1-3 In the middle, the top is the lid of the dish, and the bottom is the base.

[0101] After purification, genomic DNA of the strain was extracted, and the 16S rRNA gene was amplified using the bacterial universal primer 27F / 1492R.

[0102] The obtained sequences were compared with the database of the National Center for Biotechnology Information (NCBI) in the United States (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).

[0103] Among them, the 16S rRNA sequence of strain 8047 has 99.93% homology with the model strain Limosilactobacillus fermentum in GenBank.

[0104] Among them, the 16S rRNA sequence of strain 9984 has 99.80% homology with the model strain Limosilactobacillus fermentum in GenBank.

[0105] Among them, the 16S rRNA sequence of strain 7570 has 99.93% homology with the model strain Limosilactobacillus fermentum in GenBank.

[0106] Among them, the 16S rRNA sequence of strain 5088 has 99.87% homology with the model strain Limosilactobacillus fermentum in GenBank.

[0107] The 16S rRNA of strain 8047 is shown in SEQ ID No. 1.

[0108]

[0109] The 16S rRNA of strain 9984 is shown in SEQ ID No. 2; the 16S rRNA of strain 7570 is shown in SEQ ID No. 3; and the 16S rRNA of strain 5088 is shown in SEQ ID No. 4.

[0110] Based on the overall morphology and 16S rRNA analysis, the strain is identified as *Limosilactobacillus fermentum*. The strain's taxonomic name is *Limosilactobacillus fermentum*, accession number CGMCC No. 37551, and it was deposited on January 27, 2026, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0111] Smears of various bacterial suspensions in the logarithmic growth phase were taken, heat-fixed, and then subjected to sequential staining with crystal violet (1 min), iodine mordant (1 min), 95% ethanol destaining (30 s), and safranin counterstaining (1 min). Each step was followed by gentle rinsing with distilled water. After staining, the color and morphology of the bacteria were observed under an optical microscope. Those exhibiting a purple color were Gram-positive bacteria (G). + Those that appear red are Gram-negative bacteria (G). - Simultaneously, Gram staining was performed on strain B44. Results are shown below. Figure 4 .

[0112] from Figure 4 It can be seen that strains 8047, 9984, 7570, 5088 and 44 are all Gram-positive bacteria.

[0113] Example 2: Preparation of heat-inactivated Lactobacillus mucinus BD8047 cells by fermentation

[0114] In this embodiment, the preparation and acquisition of heat-sterilized Lactobacillus fermentans BD8047 cells includes the following steps:

[0115] 1) Activation and culture: Take the -80℃ frozen L. fermentum BD8047 lyophilized tubes and MRS agar plates (10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 2.0 g diammonium citrate, 5.0 g sodium acetate, 2.0 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate monohydrate, 1.0 mL Tween 80, and distilled water to a final volume of 1000 mL, adjusting the pH to 6.2–6.4. The agar addition amount is 10–15 g / L) (37℃ anaerobic workstation, 48 h) for activation. Pick a single colony and inoculate it into MRS liquid medium (same as step 1.2 in implementation 1), and incubate at 37℃ for anaerobic culture for 15 h.

[0116] 2) Bacterial cell collection and heat inactivation: Centrifuge at 6000 g for 10 min, discard the supernatant, wash twice with sterile physiological saline, resuspend the bacterial cell in an appropriate amount of sterile ultrapure water, and heat in a 100℃ boiling water bath for 5 min to complete inactivation, obtaining a heat-inactivated bacterial cell suspension; plate verification (MRS agar plates, anaerobic at 37℃ for 48 h) confirms no colony growth. In addition, before heat inactivation, bacterial suspension samples were serially diluted, plated on MRS agar plates for counting (anaerobic at 37℃ for 48 h), and the equivalent viable cell count (CFU / mg) per unit mass of lyophilized powder was calculated for subsequent experimental dosage conversion.

[0117] 3) Freeze-drying treatment: The inactivated bacterial solution was pre-frozen at -80℃ overnight, and then freeze-dried in a vacuum freeze dryer (-50℃, 24 h) to obtain a grayish-white freeze-dried powder (i.e., heat-inactivated bacterial powder). After being packaged, it was sealed and stored at -20℃ for later use.

[0118] Meanwhile, the other strains 9984, 7570 and 50885 obtained from Example 1 were prepared into corresponding heat-inactivated bacterial cells using the same method as BD8047, and then freeze-dried to obtain the corresponding heat-inactivated bacterial powder.

[0119] In addition, strain B44 was prepared in the same way as BD8047 to obtain the corresponding heat-inactivated bacterial cells, which were then freeze-dried to obtain the corresponding heat-inactivated bacterial powder.

[0120] Example 3: Validation of in vitro anti-inflammatory efficacy (RAW264.7 cell inflammation model)

[0121] In this embodiment, the in vitro anti-inflammatory function of five heat-inactivated fermentation *Lactobacillus mucinus* strains (B44, 9984, BD8047, 7570, and 5088) was verified.

[0122] 3.1 Determination of cell viability

[0123] The cell viability of five heat-inactivated fermenting *Lactobacillus mucinus* cells obtained in Example 2 against RAW264.7 cells was determined using the CCK-8 assay.

[0124] Divided into 6 groups:

[0125] Positive control group (i.e. LPS group): After RAW264.7 cells adhered to the culture medium, they were added to complete culture medium containing only 1 μg / mL LPS and no BD8047 heat-inactivated cells.

[0126] Control group: Only complete culture medium was added, without RAW264.7 cells.

[0127] Group B44 (Experimental Group 1): After RAW264.7 cells adhered to the culture medium, they were added to complete culture medium containing 10... 8 CFU / mL equivalent of fermenting Lactobacillus mucin B44 postbiotic and 1 μg / mL LPS.

[0128] Group 9984 (Experimental Group 2): After RAW264.7 cells adhered, they were added to complete culture medium containing 10... 8 CFU / mL equivalent of Lactobacillus fermentum 9984 postbiotic and 1 μg / mL LPS.

[0129] BD8047 group (experimental group 3): After RAW264.7 cells adhered, they were added to complete culture medium containing 10 8 CFU / mL equivalent of fermentation Lactobacillus mucinus BD8047 postbiotic and 1 μg / mL LPS.

[0130] Group 7570 (Experimental Group 4): After RAW264.7 cells adhered, they were added to complete culture medium containing 10 8 CFU / mL equivalent of Lactobacillus fermentum 7570 postbiotic and 1 μg / mL LPS.

[0131] Group 5088 (Experimental Group 5): After RAW264.7 cells adhered, they were added to complete culture medium containing 10 8 CFU / mL equivalent of Lactobacillus fermentum 50885 postbiotic and 1 μg / mL LPS.

[0132] RAW264.7 macrophages were 1×10 4 Cells were seeded at a density of 10 cells / well in 96-well plates and then incubated at 37°C and 5% CO2 for 24 h. After adhesion, each group was treated with the corresponding heat-inactivated bacterial cells obtained in Example 2 (10 cells / well). 8Complete culture medium (DMEM + 10% FBS) containing CFU / mL equivalent and LPS (1 μg / mL) was incubated for 6 h. Then, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2 h. The absorbance was read at 450 nm to assess the effect of five fermented Lactobacillus species on the proliferation of RAW264.7 cells. Triple replicates were set up for each concentration.

[0133] CCK8 calculation formula: Cell viability (%) = (OD sample group − OD blank) ÷ (OD negative control − OD blank) × 100 Where, negative control refers to negative control group with added CCK-8; sample group refers to B44 group, 9984 group, BD8047 group, 7570 group and 50885 group; blank well refers to blank control group without added CCK-8.

[0134] The results are shown in Table 1.

[0135] Table 1. Viability of RAW264.7 macrophages after co-incubation of five heat-inactivated Lactobacillus fermentation cultures with LPS for 6 h. (CCK-8, n=3, mean±SD, compared with LPS group)

[0136] Note: *p<0.05, **p<0.01, ****p<0.0001, ns=no significant difference (Welch t test, compared with LPS group); ↑ indicates increase, ↓ indicates decrease.

[0137] Table 1 shows that the cell viability of the LPS group decreased to 82.29%, and the addition of *Lactobacillus fermentum* strains B44, 9984, BD8047, 7570, and 5088 all improved cell viability. Among them, strain 5088 showed the highest cell viability, followed by B44, BD8047, 9984, and 7570. The cell viability of the *Lactobacillus fermentum* BD8047 group was ≥84%, which was statistically different from that of the LPS group (p>0.05), indicating that *Lactobacillus fermentum* BD8047 had no additional cytotoxicity.

[0138] 3.2 Measurement of cytokines TNF-α, IL-6, and IL-10

[0139] Cytokines, primarily secreted by macrophages and lymphocytes, are key mediators regulating immune responses. They not only bridge innate and adaptive immunity but also directly shape the macrophage microenvironment and functional state. When macrophages are stimulated by inflammation, they rapidly release pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Therefore, this experiment constructed an LPS-induced in vitro anti-inflammatory efficacy evaluation model. By adding five strains of fermenting *Lactobacillus mucinus*, the anti-inflammatory capabilities of each strain were assessed by detecting the levels of TNF-α, IL-6, and IL-10.

[0140] Macrophages RAW 264.7 at 5×10 5 Cells were seeded into 12-well cell culture plates and cultured at 37°C and 5% CO2 for 24 h. After adhesion, each group was treated with the corresponding heat-inactivated bacterial cells (10 cells / well). 7 The cells were incubated with DMEM medium containing 10% FBS (1 μg / mL) and LPS (1 μg / mL) (DMEM medium contains 1% ps penicillin and streptomycin) for 5 h. The cell culture supernatant was collected, centrifuged at 1000 rpm for 5 min, and the precipitate was used for detection. The protein concentrations of cytokines TNF-α, IL-6, and IL-10 were detected according to the ELISA kit (Pronos, Elibscience) instructions (quantitative at 450 nm).

[0141] Simultaneously, two groups were established: a Control group and an LPS group. In the LPS group, only LPS was added after cell adhesion. In the Control group, only culture medium was added after cell adhesion, without LPS or heat-sterilized cells.

[0142] Table 2. Contents of pro-inflammatory cytokines TNF-α, IL-6 and anti-inflammatory cytokine IL-10 in RAW264.7 cells after co-incubation with LPS for 5 h (n=6, mean±SD, with LPS group as reference).

[0143]

[0144] Note: Values ​​in parentheses are mean ± SD; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant (Welch t test, compared with the LPS group).

[0145] Table 2 shows that, compared with the LPS group, in this experiment, the baseline levels of inflammatory factors in the blank control group were extremely low when LPS-induced macrophages were used to construct an inflammation model. After LPS stimulation, TNF-α, IL-6, and IL-10 all significantly increased. Pretreatment results of the five Lactobacillus strains showed that none of the strains significantly upregulated the anti-inflammatory factor IL-10, indicating that anti-inflammatory regulation was independent of the IL-10 pathway. Fermented *Lactobacillus mucinus* B44 significantly increased TNF-α and IL-6, significantly exacerbating inflammation. Strains 9984, BD8047, 7570, and 5088 all significantly downregulated the pro-inflammatory factor IL-6. Among them, only BD8047 could simultaneously and significantly reduce TNF-α, exhibiting the best overall anti-inflammatory effect. 9984 and 5088 had no significant regulatory effect on TNF-α, while 7570 significantly increased TNF-α, showing a pro-inflammatory tendency.

[0146] 3.3 Detection of expression levels of anti-inflammatory factors

[0147] To further investigate the anti-inflammatory effects of fermented Lactobacillus mucinus, this study investigated the expression levels of LPS-induced inflammatory pathway factors NF-κB p65, iNOS, and the M2-type marker TGF-β.

[0148] After 5 h of treatment as described above (step 3.2), cells were collected, total RNA was extracted using the TRIzol method, and cDNA was synthesized by reverse transcription. Using GAPDH as an internal control, the relative mRNA expression levels of NF-κB (p65), iNOS, and TGF-β were quantitatively detected using the 2^-ΔΔCt method.

[0149] Table 3. Relative mRNA expression of inflammatory pathways and M1 / M2 polarization-related genes in RAW264.7 cells after co-incubation with LPS for 5 h (RT-qPCR, n=3, mean±SD, with LPS group as reference).

[0150]

[0151] Note: Parentheses contain mean ± SD (relative expression level) and direction labels; ↑ indicates upregulation, ↓ indicates downregulation, → indicates no significant change; statistics are the same as in Table 2. ***p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns=not significant (Welch t-test, compared with the LPS group).

[0152] Table 3 shows that LPS stimulation of RAW264.7 cells significantly increased TGF-β expression levels, indicating that LPS induced inflammatory stress and immune regulatory responses in the cells. Compared with the LPS model group, the expression levels of NF-κB mRNA (inhibiting the core inflammatory pathway), iNOS mRNA (a marker inhibiting M1 polarization of pro-inflammatory macrophages), and TGF-β mRNA (reducing inflammation-related damage factors) in each fermented Lactobacillus treatment group (B44, 9984, 8047, 7570, and 5088) were significantly reduced (p < 0.05). The results indicate that different species of *Lactobacillus fermentans* can effectively inhibit LPS-induced inflammatory responses. Their common characteristics include: downregulating the gene expression of the core inflammatory transcription factor NF-κB p65, blocking the initiation of the inflammatory cascade signaling; reducing the transcriptional level of the M1 pro-inflammatory polarization marker iNOS, thus decreasing the production of pro-inflammatory mediators such as nitric oxide; and downregulating TGF-β mRNA expression, suggesting a potential reduction in tissue fibrosis caused by excessive inflammation. Overall, *Lactobacillus fermentans* exhibits significant anti-inflammatory activity, capable of inhibiting macrophage polarization towards the pro-inflammatory M1 type at the transcriptional level and alleviating LPS-mediated inflammatory damage.

[0153] Among the five tested strains, the *Lactobacillus fermentum* 8047 treatment group showed the best overall effect. This indicates that the anti-inflammatory effect of *Lactobacillus fermentum* is strain-specific, and different strains of *Lactobacillus fermentum* exhibit significant differences in regulating inflammatory pathways.

[0154] Overall efficacy: BD8047 fermented Lactobacillus mucinus can effectively block the NF-κB inflammatory pathway, inhibit M1 and promote M2 repair macrophage polarization, and has excellent anti-inflammatory regulatory effects.

[0155] Example 4: Studies in different inflammation models

[0156] 4.1. An inflammation model was constructed using human THP-1 cells as a model.

[0157] Take THP-1 cells in logarithmic growth phase and use 5 × 10⁻⁶ cells. 5 Cells / mL were seeded in RPMI-1640 complete medium containing 10% FBS, and phorbol 12-myristate 13-acetate (PMA) was added to a final concentration of 100 ng / mL. The cells were induced at 37°C and 5% CO2 for 24-48 h until they adhered and differentiated. The old medium was discarded, and the cells were washed 2-3 times with sterile PBS. The medium was then replaced with fresh RPMI-1640 complete medium containing 10% FBS and allowed to rest for 24 h.

[0158] After cell resting, cells were divided into a normal control group (Control), an LPS model group (LPS), a dexamethasone positive control group, and a *Lactobacillus fermentum* 8047 treatment group. All groups were treated with fresh RPMI-1640 complete medium containing 10% FBS. The normal control group received no stimulation. The LPS model group received LPS at a final concentration of 1 μg / mL. The dexamethasone positive control group received LPS at a final concentration of 1 μg / mL and dexamethasone at a final concentration of 1 μM (approximately 0.392 μg / mL). The *Lactobacillus fermentum* 8047 treatment group received LPS at a final concentration of 1 μg / mL and dexamethasone at a final concentration of 10% FBS. 8 The heat-inactivated fermentation Lactobacillus mucinus 8047 cell suspension obtained in step 2) of Example 1 (CFU / mL) was treated with equal volume in each group and then incubated together at 37 °C and 5% CO2 for 6 h.

[0159] After incubation, cell supernatant was collected, centrifuged at 4 ℃ and 1000 rpm for 15 min, and the secretion levels of inflammatory factors (TNF-α and IL-6) were detected at 450 nm using a human TNF-α and IL-6 ELISA kit (Pronos, Elibscience).

[0160] 4.2. An inflammation model was constructed using mouse-derived RAW 264.7 cells.

[0161] RAW 264.7 at 5×10 5 Cells / mL were inoculated into DMEM complete medium containing 10% FBS at 37°C and 5% CO2 for 24 hours.

[0162] The grouping and processing of cells after resting are the same as in step 4.1.

[0163] After 6 hours of incubation, the secretion level of inflammatory factors was detected, as in step 4.1.

[0164] 4.3 Results

[0165] The results of fermenting *Lactobacillus mucinus* 8047 in an inflammation model constructed using mouse RAW 264.7 macrophages are shown in Tables 4 and 5. The results of fermenting *Lactobacillus mucinus* 8047 in an inflammation model constructed using human THP-1 mononuclear cells are shown in Tables 6 and 7.

[0166] Table 4. Effect of fermented Lactobacillus mucinus 8047 on TNF-α expression level in mouse macrophages RAW264.7 (N=6, TNF-α unit is ng / mL)

[0167] LPS 6275.67±53.46 - - - Control 2.60±0.17 99.96% <0.0001 **** Dexamethasone positive + LPS 1584.33±21.94 74.75% <0.0001 **** 8047+LPS 2126.67±6.13 66.11% <0.0001 ****

[0168] Table 5. Effect of fermented Lactobacillus mucinus 8047 on IL-6 expression levels in mouse macrophages RAW264.7 (N=6, IL-6 units are pg / mL)

[0169] LPS 4033.33±819.22 - - - Control 517.00±31.61 87.18% <0.0001 **** Dexamethasone positive + LPS 588.00±89.71 85.42% <0.0001 **** 8047+LPS 676.00±188.17 83.24% <0.0001 ****

[0170] Table 6. Effect of fermented Lactobacillus mucinus 8047 on TNF-α expression levels in human monocytes THP-1 (N=6, TNF-α unit is ng / mL)

[0171] LPS 538.00±6.24 - - - Control 1.94±0.10 99.64% <0.0001 **** Dexamethasone positive + LPS 126.33±0.58 76.52% <0.0001 **** 8047+LPS 231.67±13.57 56.93% <0.0001 ****

[0172] Table 7. Effect of fermented Lactobacillus mucinus 8047 on IL-6 expression levels in human monocytes THP-1 (N=6, IL-6 units are pg / mL)

[0173] LPS 676.67±114.61 - - - Control 19.33±7.57 97.14% <0.0001 **** Dexamethasone positive + LPS 70.00±7.94 89.66% <0.0001 **** 8047+LPS 89.67±13.01 86.75% <0.0001 ****

[0174] Tables 4-7 show that, regardless of whether human THP-1 macrophages or mouse RAW 264.7 macrophages were used as models, treatment with fermented Lactobacillus mucinus 8047 significantly reduced the secretion levels of TNF-α and IL-6 compared to the LPS model group (p < 0.05), and showed no statistically significant difference compared to the positive control dexamethasone group (p > 0.05). These results indicate that this strain can effectively inhibit LPS-induced inflammatory responses in immune cells from two different species, exhibiting broad-spectrum anti-inflammatory activity, and its anti-inflammatory potency is comparable to that of the classic anti-inflammatory drug dexamethasone, suggesting its promising development potential in the field of anti-inflammatory applications.

[0175] Example 5: Preparation of Functional Fermented Milk

[0176] Whole milk (protein ≥ 3.2%) was homogenized (20 MPa), pasteurized (85°C, 15 min), and then cooled to 43°C. A commercial starter culture (a mixed powder of Streptococcus thermophilus and Lactobacillus bulgaricus, inoculated at 0.02%) and the heat-inactivated bacterial powder prepared in Example 2 (i.e., inactivated bacterial powder obtained by freeze-drying, inoculated at 10g) were added. 8 After thorough mixing (CFU / mL equivalent), ferment at 43℃ until the final pH reaches 4.6. After fermentation, transfer to 4℃ for 24 hours of post-fermentation maturation to obtain the functional fermented milk product, which combines the flavor of traditional fermented milk with anti-inflammatory and immunomodulatory functions. The BD8047 cell count is ≥10 per 100 mL of product. 8 CFU equivalent dose

[0177] Example 6: Postbiotic Microcapsule Formulation of Fermented Lactobacillus mucinus BD8047 and Its Preparation

[0178] This embodiment provides a post-fermentation microcapsule formulation of Lactobacillus mucinus BD8047, the preparation method of which includes the following steps:

[0179] (1) Gradual expansion of bacterial strain: BD8047 of fermenting mucin lactobacillus was inoculated into the fermentation medium at an inoculation rate of 2%-5% and cultured at a constant temperature of 37°C. During the fermentation process, the pH was maintained at 6.5-7.0. After large-scale anaerobic fermentation, a high concentration of fermentation liquid was obtained.

[0180] (2) Collection and heat inactivation of bacterial cells: The fermentation liquid obtained in step (1) is centrifuged at 4000-6000 r / min to enrich the wet bacterial cells. After collecting the bacterial cells, heat inactivation treatment is performed.

[0181] (3) Addition of freeze-drying protectant: After inactivation, add 5%-20% of the wet bacterial cell mass of a compound freeze-drying protectant (containing trehalose and skim milk) and mix thoroughly. Since the bacterial cells have been completely heat-inactivated, the freeze-drying protectant is not used to maintain the activity of the strain, but to protect the intact cell structure, functional proteins and active metabolic components of the inactivated bacterial cells, so as to avoid the collapse of the bacterial cell structure and the denaturation and loss of effective components caused by low temperature and negative pressure during vacuum freeze-drying, thereby maximizing the preservation of the anti-inflammatory activity of the inactivated bacterial cells.

[0182] (4) Vacuum freeze-drying: The mixed bacterial suspension obtained in step (3) is subjected to vacuum freeze-drying. Pre-freezing stage: rapidly cool down to below -40°C and keep warm for a sufficient period of time; primary drying stage: vacuum degree ≤5 Pa, plate temperature gradient rise to -20-0°C, dry for 24-48 h; analytical drying stage: heat up to 25-30°C, dry until the final powder residual moisture ≤1%, and obtain inactivated bacterial powder.

[0183] (5) Microcapsule encapsulation: Using 0.75-3% sodium alginate solution and 1.62-3% calcium chloride solution as wall material and crosslinking agent respectively, microcapsulation was carried out by ion gelation method: The inactivated bacterial powder obtained in step (4) was dispersed in sodium alginate solution, and then dropped into crosslinking liquid containing calcium chloride for solidification. After washing and sieving, microcapsules encapsulating the inactivated bacterial powder were obtained.

[0184] (6) Formulation: The encapsulated inactivated bacterial powder obtained in step (5) is mixed with sugar alcohol excipients at a mass ratio of (90-98): (2-10) at low temperature and then quantitatively filled into ordinary hard capsules or acid-resistant enteric capsule shells to obtain the fermented Lactobacillus mucinus BD8047 post-biotic microcapsule formulation.

[0185] (7) Packaging and storage: The finished product is packaged in nitrogen-filled sealed packaging. The water activity of the powder is controlled at 0.2-0.3. It is stored in the dark at 2-8℃ to maintain the stability of the formulation for a long time.

[0186] Example 7: Capsule formulation containing heat-inactivated Lactobacillus fermentum BD8047 powder and its preparation.

[0187] Example 7 provides a capsule formulation containing heat-inactivated Lactobacillus fermentum BD8047 powder, the preparation method of which includes the following:

[0188] 1) Obtain heat-inactivated freeze-dried bacterial powder:

[0189] Under aseptic conditions, strain BD8047 was inoculated into fermentation medium and cultured at a constant temperature of 37°C. The pH was controlled between 6.5 and 7.0 during fermentation. A concentration of 1×10⁻⁶ was obtained after fermentation. 9 -2×10 10 The bacterial suspension was prepared at CFU / mL. The resulting bacterial suspension was centrifuged at 4000-6000 r / min, the bacterial precipitate was collected, resuspended, mixed, and then subjected to heat inactivation. After inactivation, vacuum freeze-drying was performed with the following process parameters: Pre-freezing stage: rapid cooling to below -40℃; First drying stage: vacuum degree ≤5Pa, plate temperature gradient increased to -20~0℃, continuous drying for 24~48 h; Desorption drying stage: temperature increased to 25~30℃, drying until residual moisture content ≤1%, thus obtaining heat-inactivated freeze-dried bacterial powder.

[0190] (2) Capsule filling

[0191] The heat-inactivated freeze-dried bacterial powder obtained in step (1) is passed through an 80-100 mesh sieve and quantitatively filled into No. 0 gelatin hollow capsule shells using a capsule filling machine under ambient temperature ≤25℃ and relative humidity ≤45%, with each capsule containing 500mg of inactivated bacterial powder. After the capsules are locked, they are polished to remove surface powder and then packaged in aluminum-plastic blister packs or high-barrier film bags filled with nitrogen and sealed to obtain the capsule formulation.

[0192] In the obtained capsule formulation, each capsule contains an equivalent live bacteria count of ≥5×10⁻⁶ corresponding to the inactivated bacterial powder. 9 CFU. The finished product has a water activity <0.3. Store in a cool, dry, and dark place at ≤25℃, sealed. Shelf life is 18 months. It is recommended to use within one month of opening. Dosage: 1-2 capsules daily, taken with meals or after meals with warm water.

[0193] 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. A fermenting Lactobacillus mucinus strain BD8047 with anti-inflammatory properties, its preservation number is CGMCC No. 37551.

2. The strain BD8047 as described in claim 1, characterized in that, The 16S rRNA sequence of strain BD8047 is as shown in SEQ ID NO.

1.

3. A fermentation culture of *Lactobacillus mucinus*, characterized in that, It is obtained by inoculating the strain BD8047 as described in claim 1 or 2 into a culture medium and culturing it.

4. The fermentation culture as described in claim 3, characterized in that, The culture temperature is 30–37°C; And / or, the culture medium contains a carbon source, a nitrogen source, and inorganic salts; Preferably, the carbon source is selected from one or more of glucose, sucrose, and maltose; Preferably, the nitrogen source is selected from one or more of peptone, beef extract, and yeast extract; Preferably, the inorganic salt is selected from one or more of diammonium citrate, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, and manganese sulfate.

5. Use of strain BD8047 as described in claim 1 or 2, or fermentation culture as described in claim 3 or 4, in the preparation of products with anti-inflammatory effects.

6. The use as described in claim 5, characterized in that, The product with anti-inflammatory properties has one or more of the following effects: B1) Inhibits macrophages from producing pro-inflammatory cytokines; B2) Regulates macrophage polarization balance, which manifests as inhibition of M1 marker expression; Preferably, the pro-inflammatory cytokines are selected from TNF-α and / or IL-6; Preferably, the M1 type marker is selected from iNOS.

7. A product with anti-inflammatory properties, characterized in that, The product with anti-inflammatory properties contains strain BD8047 as described in claim 1 or 2, or the fermentation culture as described in claim 3 or 4.

8. The product with anti-inflammatory effect as described in claim 7, characterized in that, The products with anti-inflammatory effects include one or more of the following: food, pharmaceuticals, daily chemical products, and microbial agents.

9. The product with anti-inflammatory effect as described in claim 7, characterized in that, The strain BD8047 or the fermentation culture is in an inactivated form; And / or, the content of strain BD8047 in the product with anti-inflammatory effect is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g; Preferably, the inactivation method is boiling water bath inactivation for 2 to 15 minutes.

10. The use of strain BD8047 as described in claim 1 or 2, or the fermentation culture as described in claim 3 or 4, or the product with anti-inflammatory activity as described in any one of claims 7-9, in the preparation of articles for the prevention or treatment of respiratory diseases.

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