A b. longum lyophilized preparation, a preparation method and use thereof

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a Bifidobacterium longum freeze-dried preparation, a preparation method and application thereof. The Bifidobacterium longum strain has a preservation number of CGMCC No. 25684. The Bifidobacterium longum freeze-dried preparation can significantly regulate immunity, stimulate mouse macrophage RAW264.7 to produce cytokines TNF-alpha and IL-6, reduce the production of inflammatory factors NO, TNF-alpha and IL-6 of RAW264.7 cells induced by LPS, and enhance cell immune function. The production process parameters are simple, easy to control, short in cycle, and the obtained product can be stored for a long time, and the product quality is stable.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a freeze-dried preparation containing Bifidobacterium longum, its preparation method, and its uses. Background Technology

[0002] A weakened or deficient immune system often leads to various local or systemic infections. Mild cases include the common cold; secondary cases involve hepatitis B or C virus infections that damage the liver and can lead to liver cancer; the most serious consequence is HIV infection, which deprives the body of its ability to fight pathogens. A weakened immune system can also cause pharyngitis, gastritis, enteritis, pneumonia, bronchitis, rhinitis, otitis media, hepatitis, mastitis, cancer, and skin infections. Conversely, an overactive immune system can treat normal cells and tissues as invaders, attacking them and damaging the immune system, increasing the risk of developing conditions such as allergic purpura, systemic lupus erythematosus, scleroderma, and dermatomyositis. Ideally, the body should maintain a stable immune system; even if the immune system is neither too high nor too low, health will remain in a balanced state.

[0003] Bifidobacterium longum subsp. longum is a genus of Gram-positive, non-motile, non-spore-forming, rod-shaped, sometimes bifurcated, strictly anaerobic bacteria that are widely distributed in the human digestive tract, vagina, and oral cavity, and is an important component of the human gut microbiota.

[0004] CN114728028A discloses a synbiotic composition comprising *Bifidobacterium longum* subsp. *longum* CR15 and *Bifidobacterium pseudocatenulatum*, which is used to regulate an individual's gastrointestinal microbiota. CN114728028A does not disclose that this composition can be used to enhance immunity, nor does it disclose a lyophilized formulation comprising said composition.

[0005] Therefore, there is a need for a freeze-dried preparation of Bifidobacterium longum subsp. longum that can be stored for a long time, has stable product quality, and can regulate immunity and maintain immune balance. Summary of the Invention

[0006] One object of the present invention is to provide a lyophilized preparation containing Bifidobacterium longum, which can significantly regulate immunity, stimulate mouse macrophages RAW264.7 to produce cytokines TNF-α and IL-6, reduce LPS-induced production of inflammatory factors NO, TNF-α and IL-6 in RAW264.7 cells, enhance cellular immune function, humoral immune function and monocyte-macrophage function, maintain immune balance, and the strain can produce high levels of lactic acid and short-chain fatty acids, thus exhibiting good production performance.

[0007] Another object of the present invention is to provide a freeze-dried preparation containing Bifidobacterium longum, which can significantly enhance cellular immune function, humoral immune function and mononuclear-macrophage function, thereby maintaining normal human immune function.

[0008] Another objective of this invention is to provide a method for preparing a lyophilized formulation containing Bifidobacterium longum, wherein the method has simple production process parameters, is easy to control, has a short cycle, and the resulting product can be stored for a long time with stable product quality.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] On one hand, the present invention provides a freeze-dried formulation comprising a strain of Bifidobacterium longum and a freeze-drying protectant, wherein the preservation number of the Bifidobacterium longum strain is CGMCC No. 25684.

[0011] In one example, the Bifidobacterium longum strain contains the 16S rRNA gene represented by SEQ ID NO: 1.

[0012] Preferably, the freeze-drying protectant comprises: 80-100 g / L of skim milk powder, 40-50 g / L of trehalose, 2-3 g / L of vitamin C, and 4-5 g / L of monosodium glutamate.

[0013] On the other hand, the present invention provides the use of the lyophilized formulation described above in the preparation of a medicament for enhancing immunity and maintaining immune balance.

[0014] Preferably, the Bifidobacterium longum strain is used to bidirectionally regulate the secretion of the cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

[0015] Preferably, the lyophilized formulation is used to reduce the inflammatory factor NO.

[0016] Preferably, the lyophilized formulation is used to stimulate mouse macrophages to produce cytokines TNF-α and IL-6, and to reduce LPS-induced macrophage production of inflammatory factors NO, TNF-α, and IL-6.

[0017] Preferably, the lyophilized formulation is used to enhance cellular immune function, humoral immune function, and mononuclear-macrophage function, thereby maintaining normal human immune function.

[0018] Furthermore, the present invention provides a method for preparing the lyophilized formulation as described above, comprising the following steps:

[0019] (1) Culture of strains: Inoculate the Bifidobacterium longum strain into sterile liquid culture medium at an inoculation amount of 1-3% of the total culture medium and culture for 16-24 hours to obtain seed culture solution. Then, inoculate the obtained seed culture solution into fermentation medium at an inoculation amount of 1-3% of the total culture medium and carry out fermentation culture to obtain Bifidobacterium longum fermentation liquid.

[0020] (2) Drying.

[0021] Preferably, the fermentation medium comprises: glucose 40-60 g / L, yeast extract 60-100 g / L, sodium acetate trihydrate 3-10 g / L, magnesium sulfate 0.1-0.2 g / L, manganese sulfate 0.05-0.1 g / L, dipotassium hydrogen phosphate 1-2 g / L, triammonium citrate 2-4 g / L, Tween 80 1-2 g / L, calcium chloride 0.05-0.1 g / L, and L-cysteine ​​salt 0.5-1 g / L.

[0022] Preferably, during fermentation, sodium hydroxide solution is automatically added to maintain a constant pH of 5.5-6.5 until acid production stops, and fermentation is terminated when sodium hydroxide is no longer added.

[0023] Preferably, the freeze-dried formulation is a live bacterial formulation, and the method further includes preparing a freeze-drying protectant after the strain culture step and before drying, and performing freeze-drying in the drying step; optionally, the freeze-drying protectant comprises: 80-100 g / L of skim milk powder, 40-50 g / L of trehalose, 2-3 g / L of vitamin C, and 4-5 g / L of L-glutamate as a protectant.

[0024] Preferably, the freeze-drying conditions are as follows: pre-freezing temperature is -40~-45℃, pre-freezing time is 4~5 h, primary drying temperature is -20~-15℃, primary drying time is 20~25 h, secondary drying temperature is 30~35℃, secondary drying time is 6~10 h.

[0025] Preferably, the freeze-dried formulation is a dead bacterial formulation, and the drying step is to first perform heat inactivation, and then dry it through a spray drying tower to obtain the dead bacterial formulation; optionally, the heat inactivation conditions are 80~100℃ for 10~40min.

[0026] The present invention has the following beneficial effects:

[0027] The freeze-dried formulation of the present invention containing Bifidobacterium longum strain can significantly regulate immunity, stimulate mouse macrophages RAW264.7 to produce cytokines TNF-α and IL-6, reduce LPS-induced production of inflammatory factors NO, TNF-α and IL-6 in RAW264.7 cells, enhance cellular immune function, humoral immune function and monocyte-macrophage function, maintain immune balance, and this strain can produce high levels of lactic acid and short-chain fatty acids, exhibiting good production performance.

[0028] The method for preparing lyophilized formulations containing Bifidobacterium longum according to the present invention has simple production process parameters, is easy to control, has a short cycle, and the resulting product can be stored for a long time with stable product quality. Attached Figure Description

[0029] Figure 1 A biological evolutionary tree of Bifidobacterium strains is shown.

[0030] Figure 2 The diagram shows the RAPD clustering analysis of the *Bifidobacterium longum* subspecies HOM1190 strain constructed using the UPGMA method.

[0031] Figure 3 The effect of the Bifidobacterium longum subspecies HOM1190 strain of the present invention on the viability of mouse macrophage RAW264.7 cells is shown.

[0032] Figure 4 The effect of the Bifidobacterium longum subspecies HOM1190 strain of the present invention on the secretion level of the inflammatory mediator NO in LPS-induced mouse macrophage RAW264.7 cells is shown.

[0033] Figure 5 The effect of the Bifidobacterium longum subspecies HOM1190 strain of the present invention on the secretion level of the inflammatory mediator TNF-α in LPS-induced mouse macrophage RAW264.7 cells is shown.

[0034] Figure 6 The effect of the *Bifidobacterium longum* subsp. *HOM1190* strain of the present invention on the secretion level of the inflammatory mediator IL-6 in LPS-induced mouse macrophage RAW264.7 cells is shown. Note: Compared with the positive control group, p<0.05. Compared with the positive control group, p<0.01. Compared with the positive control group, p<0.001.

[0035] Figure 7The effect of the Bifidobacterium longum subspecies HOM1190 strain of the present invention on the secretion of the cytokine TNF-α in mouse macrophages RAW264.7 is shown.

[0036] Figure 8 The effect of the present invention, *Bifidobacterium longum* subspecies HOM1190, on the secretion of the cytokine IL-6 in mouse macrophages RAW264.7 is shown.

[0037] Figure 9 The results of the present invention showing the enhancement of cellular immune function by the Bifidobacterium longum subspecies HOM1190 strain are illustrated.

[0038] Figure 10 The results of the present invention on enhancing humoral immune function by the Bifidobacterium longum subspecies HOM1190 strain are shown.

[0039] Figure 11 The results of enhancing mononuclear-macrophage function by the Bifidobacterium longum subspecies HOM1190 strain of the present invention are shown.

[0040] Figure 12 The results show that the active bacterial powder of Bifidobacterium longum subsp. HOM1190 of the present invention significantly enhances the cellular immune function, humoral immune function, and mononuclear-macrophage function in mice.

[0041] Microbial Preservation Instructions

[0042] The Bifidobacterium longum HOM1190 strain of the present invention was deposited on September 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; accession number CGMCC No. 25684.

[0043] The Bifidobacterium longum strain HOM1190 of this invention was submitted to the Institute of Microbiology, Chinese Academy of Sciences for identification in May 2023.

[0044] The detection and identification conclusions are as follows: Under the conditions of this laboratory, based on the comprehensive analysis of experimental data such as cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, and tuf gene sequence of the submitted bacterial strain, and with reference to relevant research papers in "Bergey's Manual of Systematic Bacteriology" and the International Journal of Systematic and Evolutionary Microbiology, the identification result of the submitted bacterial strain (strain number: HOM1190) is: Bifidobacterium longum subsp. Longum.

[0045] The cell morphology of this strain is: pleomorphic rod-shaped; its physiological and biochemical characteristics are: Gram-positive, catalase-negative (-), oxidase-negative (-); the 16S rRNA gene sequence is shown in SEQ ID NO: 1, and the tuf gene sequence is shown in SEQ ID NO: 9. Detailed Implementation

[0046] This invention discloses the strains, characteristics, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0047] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the technical solution of the present invention will be further described below in conjunction with specific embodiments, but this is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are implemented according to conventional methods and conditions in the art.

[0048] As identified above, the Bifidobacterium longum strain HOM1190 in this invention is identified as Bifidobacterium longum subsp. Longum. Therefore, the Bifidobacterium longum used in this invention refers to Bifidobacterium longum subsp. Longum.

[0049] Nitric oxide (NO) is an inflammatory factor that is a small molecule mediator produced by endothelial cells, epithelial cells and inflammatory cells. Its concentration is highly correlated with the number of inflammatory cells and can be used as a marker of inflammation.

[0050] Tumor necrosis factor-α (TNF-α) is mainly produced by activated macrophages, NK cells, and T lymphocytes, and can regulate the body's immune response. On the one hand, when the body's immunity is too low, it can activate various immune cells such as lymphocytes and macrophages, enhancing their killing activity and playing a role in anti-infection and anti-tumor activity; on the other hand, when immunity is too high and inflammation occurs, it is over-activated and also serves as an inflammatory marker.

[0051] Interleukin-6 (IL-6) is mainly produced by monocytes / macrophages, Th2 cells, vascular endothelial cells, and fibroblasts, and it regulates the body's immune response. On the one hand, IL-6 enables B cell precursors to become antibody-producing cells; synergistically with colony-stimulating factors, it promotes the growth and differentiation of primitive bone marrow-derived cells and enhances the lytic function of natural killer cells. On the other hand, overexpression or regulation of IL-6 can disrupt the normal function of multiple organ systems in the human body and can cause many diseases. In inflammatory responses, the increase of IL-6 occurs earlier than other cytokines and lasts for a longer period, thus making it a key inflammatory factor.

[0052] TNF-α and IL-6 are both inflammatory factors and cytokines.

[0053] The inventors of this application have also discovered that the *Bifidobacterium longum* subspecies *HOM1190* strain of this invention has a bidirectional regulatory effect on the secretion of cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), specifically stimulating mouse macrophages RAW264.7 to produce cytokines TNF-α and IL-6, and reducing LPS-induced production of inflammatory factors NO, TNF-α, and IL-6 by RAW264.7 cells. Furthermore, this strain can significantly enhance the body's immunity, significantly enhancing cellular immune function, humoral immune function, and monocyte-macrophage function, thus helping to maintain normal human immune function.

[0054] To make the technical problem to be solved by the present invention, the technical solution adopted, and the advantages clearer, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0055] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods.

[0056] Unless otherwise specified, all reagents and materials used in this invention are prepared using conventional methods or obtained commercially.

[0057] The *Bifidobacterium longum* subsp. strain used in this invention was isolated from the feces of a healthy infant containing the desired strain, and the specific isolation method is as described in Example 1.

[0058] Example 1: Isolation and Identification of Bifidobacterium longum subsp. HOM1190

[0059] (1) Preparation of artificial gastrointestinal fluid

[0060] Artificial gastric fluid: Take 16.4 mL of dilute hydrochloric acid (1 mol / L), add 800 mL of water, adjust the pH to 3.0, add 10 g of pepsin, shake well, add water to 1000 mL, centrifuge at 5000 rpm for 5 min, take the supernatant, filter it through a 0.22 µm filter membrane for sterilization, and store at -20℃ for later use.

[0061] Artificial intestinal fluid: Take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water, and adjust the pH to 6.8 with 0.4% (0.1 mol / L) sodium hydroxide solution. Separately, take 10 g of pancreatic enzyme and 3 g of porcine bile salt, add an appropriate amount of water to dissolve them, mix the two solutions, add water to 1000 mL, centrifuge at 5000 rpm for 5 min, take the supernatant, filter it through a 0.22 µm filter membrane for sterilization, and store at -20℃ for later use.

[0062] (2) Preparation of isolation culture medium

[0063] MRS-Cys liquid medium: Add 0.5 g of L-cysteine ​​hydrochloride to each liter of MRS broth medium (catalog number: CM1163, OXOID, UK), sterilize at 121℃ for 15 min, and store at 2-8℃ protected from light for one week.

[0064] MRS-Cys solid medium containing bromocresol purple: Add 0.04 g of bromocresol purple to each liter of MRS solid medium (catalog number: CM1175, OXOID, UK) and mix thoroughly. Sterilize at 121℃ for 15 min. In a laminar flow hood, pour approximately 15 mL into each petri dish and allow to solidify before use.

[0065] (3) Isolation and screening of Bifidobacterium longum subsp. HOM1190 strain

[0066] The *Bifidobacterium longum* subsp. of this invention was isolated from the feces of healthy infants.

[0067] Ex vivo fecal samples were collected using sterile anaerobic tubes and stored at low temperature under anaerobic conditions. The experiment began on the day of sampling. Approximately 1 g of fecal sample was added to an anaerobic tube containing 9 mL of MRS-Cys liquid medium and anaerobically incubated at 37°C for 24 h. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded, and 10 mL of artificial gastric fluid was added. The mixture was then incubated anaerobically at 37°C for 3 h, centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. After adding 10 mL of artificial intestinal fluid, the mixture was incubated anaerobically at 37°C for 3 h. The sample was then diluted 10-fold using the 10-fold dilution method to a final concentration of 10. -6 From the original solution to 10 -6 Take 100 µL of each sample and spread it onto MRS-Cys solid medium plates containing bromocresol purple. Incubate anaerobically at 37°C for 72 h.

[0068] Select single colonies with yellowing edges, streak them, and purify them 3-4 times until the colonies are uniform. Simultaneously, perform Gram staining and microscopic examination of colony morphology. Transfer the single colonies to liquid culture medium for pure culture, preserve the culture with glycerol, and store at -80°C.

[0069] (4) Morphological observation of Bifidobacterium longum subspecies HOM1190 strain

[0070] Bifidobacterium longum subspecies HOM1190 was anaerobically cultured on MRS agar at 37°C for 48 h. Colonies were milky white, round, approximately 2–5 mm in diameter, with a moist, smooth surface, regular edges, and a mucous appearance. Under a light microscope, the bacteria were long and curved, resembling club-shaped or Y-shaped rods, approximately 0.3–0.8 μm × 4–10 μm in size, and did not form spores. Two other strains with the same morphology were also screened and named BH14-1 and BH18-7.

[0071] (5) Identification of Bifidobacterium longum subsp. HOM1190 strain

[0072] 16S rRNA gene identification: DNA was extracted from preserved strains HOM1190, BH14-1, and BH18-7 for 16S rRNA gene amplification. PCR amplification and agarose gel electrophoresis were performed using universal primers 27F and 1492R. The resulting fragments were then excised, recovered, and sequenced. The isolated strains were then sequenced for their 16S rRNA genes. Based on the 16S rRNA gene sequences, they were compared against the NCBI database using a BLAST tool. A phylogenetic tree was constructed using Mega 7.0 software. Figure 1As shown, the three strains HOM1190, BH14-1, and BH18-7 were all identified as *Bifidobacterium longum* subsp. *longum*. The 16S rRNA gene sequence of strain HOM1190 is shown in SEQ ID NO: 1.

[0073]

[0074] Based on its 16S rRNA gene sequence, a BLAST algorithm was used to compare it against the NCBI database, and a phylogenetic tree was constructed using Mega 7.0 software. Figure 1 As shown, the identification result of strain HOM1190 is Bifidobacterium longum subsp. longum, and it is named HOM1190.

[0075] (6) Identification of strains by random amplified polymorphic DNA (RAPD): DNA was extracted from the preserved strains. Using the strain DNA as a template, PCR was performed using five primers: OPA-02, OPA-18, OPL-07, OPL-16, and OPM-05, as shown in Table 1. PCR amplified polymorphic DNA fragments, which exhibited different DNA differences after gel electrophoresis. These differences were analyzed using cluster analysis software. Figure 2 As shown, Bifidobacterium longum subspecies HOM1190 is unique and differs from other Bifidobacterium longum subspecies strains.

[0076] Table 1

[0077]

[0078] Example 2: Test on the ability to inhibit common pathogenic bacteria

[0079] (1) Activation of indicator bacteria

[0080] The indicator strains Escherichia coli ATCC8739, Staphylococcus aureus ATCC6538, Salmonella typhimurium ATCC14028, Pseudomonas aeruginosa ATCC9027, Listeria monocytogenes ATCC19111, and Clostridium difficile ATCC9689 were all purchased from the China Industrial Microbial Culture Collection Center. Indicator bacteria (Escherichia coli ATCC8739; Staphylococcus aureus ATCC6538; Salmonella typhimurium ATCC14028; Pseudomonas aeruginosa ATCC9027; Listeria monocytogenes ATCC19111) were inoculated into TSB medium at 1% of the total culture medium volume and cultured aerobically at 37°C and 180 rpm for 24 h for later use. Clostridium difficile ATCC9689 was inoculated into BHI medium at 1% of the total culture medium volume and cultured anaerobically at 37°C for 24 h for later use.

[0081] (2) Activation of Bifidobacterium longum subsp.

[0082] The *Bifidobacterium longum* subsp. *HOM1190*, cryopreserved at -80℃ as prepared in Example 1, was inoculated into sterilized MRS-Cys liquid medium as described in Example 1 at an inoculum of 1% of the total culture medium. The medium was then anaerobically cultured at 37℃ for 24 hours, and activated twice to obtain the fermentation broth. The broth was then centrifuged at 8000 rpm for 10 min, and the supernatant was used for an antibacterial test.

[0083] (3) Plate preparation

[0084] Heat the sterilized TSA medium until completely melted, pour it into a petri dish, and place it on a horizontal platform to allow the agar layer to form a uniform thickness. Allow it to solidify. Add the indicator bacteria to the TSA medium, shake well, and then pour it into a pre-prepared TSA blank agar plate. Let it stand to solidify.

[0085] (4) Antibacterial test

[0086] Using sterile forceps, gently place the Oxford cups onto the agar plate, maintaining a certain distance between them. Add 0.2 mL of the supernatant of the lactic acid bacteria fermentation broth to be tested to each cup. After diffusion at 4°C for 24 hours, incubate at 37°C for at least 18 hours and observe the appearance of inhibition zones. Measure the inhibition zones with a ruler after they form. Use the liquid culture medium (MRS-Cys) from Example 1 as a negative control, and *Bifidobacterium longum* subsp. *longum* BH14-1 and BH18-7 as positive control strains. Perform three replicates for each sample. The inhibition results are shown in Table 2.

[0087] Table 2. Inhibitory effect of Bifidobacterium longum subsp. HOM1190 on pathogenic bacteria.

[0088]

[0089] Note: "-" indicates no antibacterial activity; "+" indicates 11-16mm; "++" indicates 17-22mm; "+++" indicates ≥23mm

[0090] As shown in Table 2, compared with the positive control strains BH14-1 and BH18-7, Bifidobacterium longum subsp. HOM1190 has a strong inhibitory effect on all six pathogenic bacteria, and has a better inhibitory effect on Pseudomonas aeruginosa and Listeria monocytogenes, indicating that this strain has a good ability to inhibit pathogenic bacteria.

[0091] Example 3: Intestinal Epithelial Cell Adhesion Test

[0092] The purpose of this experiment was to evaluate the adhesion properties of Bifidobacterium longum HOM1190 to small intestinal epithelial cells. Human colon cancer cells (Caco-2) are a commonly used cell model; they are organelles that can be continuously passaged in vitro. After a certain period of culture, they can become homologous with small intestinal epithelial cells.

[0093] (1) Preparation of cell culture medium

[0094] Complete culture medium: high-glucose DMEM medium supplemented with 10% inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin, 100 µg / mL streptomycin), and stored at 4°C after mixing.

[0095] Incomplete culture medium: high-glucose DMEM medium, mixed with 10% inactivated fetal bovine serum (FBS) and stored at 4°C.

[0096] (2) Cell resuscitation and culture

[0097] Human colorectal adenocarcinoma cells (Caco-2) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Caco-2 cells were resuspended in fresh culture medium and evenly dispersed in culture flasks. They were cultured at 37°C under a gas atmosphere of 5% CO2 and 95% air, with the culture medium changed every 48 hours during recovery. When the cells showed good growth (80% confluence), they were digested with trypsin-EDTA solution at 37°C. After digestion, the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 / mL and cultured in 24-well plates until they reached 80% confluence.

[0098] (3) Activation of Bifidobacterium longum subsp.

[0099] The Bifidobacterium longum subsp. HOM1190, BH14-1 and BH18-7, which were frozen at -80℃ as prepared in Example 1, were inoculated into the sterilized MRS-Cys liquid medium described in Example 1 at an inoculation amount of 1% of the total culture medium. After static anaerobic culture at 37℃ for 24 hours, the fermentation broth of the strains was obtained after activation twice.

[0100] (4) Adhesion test

[0101] Collect bacterial cells grown in the appropriate culture medium by centrifugation at 8000 rpm for 10 min; wash the bacterial cells three times with DPBS, resuspend the cells in incomplete culture medium, and adjust the bacterial concentration to 10. 7cfu / mL; 1 mL of the above bacterial suspension was added to a 24-well plate containing a monolayer of Caco-2 cells and incubated at 37°C for 2 h in a 5% CO2 incubator; after incubation, the cells were washed three times with sterile DPBS; Caco-2 cells were digested with trypsin-EDTA solution at 37°C, and the number of cells and the number of viable bacteria before and after adhesion were counted. Each sample was performed in triplicate, and the results are shown in Table 3.

[0102] Table 3. Adhesion ability of Bifidobacterium longum subsp. HOM1190 to Caco-2 cells.

[0103]

[0104] Note: Different capital letters in the same column indicate highly significant differences (p<0.01).

[0105] The results showed that the adhesion index of Bifidobacterium longum subspecies HOM1190 to human colon cancer cells Caco-2 was 1.17, which significantly (p<0.01) enhanced the adhesion ability of small intestinal epithelial cells compared with the other two strains of Bifidobacterium longum subspecies BH14-1 and BH18-7.

[0106] Adhesion index = Number of bacteria after adhesion / Number of cells per plate

[0107] Adhesion rate = Number of bacteria after adhesion / Number of bacteria before adhesion

[0108] Example 4: Lactic acid and short-chain fatty acid production capacity test

[0109] Bifidobacterium longum subsp. HOM1190, BH14-1, and BH18-7, prepared and frozen at -80℃ as described in Example 1, were inoculated into sterilized MRS-Cys liquid medium as described in Example 1 at an inoculum volume of 1% of the total culture medium. The cultures were then anaerobically cultured at 37℃ for 24 hours and activated twice to obtain the fermentation broth. The broth was then centrifuged at 8000 rpm for 10 min, and the supernatant was analyzed by gas chromatography to determine the content of lactic acid and short-chain fatty acids. The MRS-Cys liquid medium described in Example 1 served as a negative control. Each sample was performed in triplicate. Data were analyzed using SPSS 25.0 software via one-way ANOVA and Duncan's multiple comparisons. The results are shown in Table 4.

[0110] Table 4. Lactic acid and short-chain fatty acid production capacity of Bifidobacterium longum subsp. HOM1190

[0111]

[0112] Note: Different capital letters in the same column indicate highly significant differences (p<0.01).

[0113] As shown in Table 4, compared with the other two strains of Bifidobacterium longum subsp. BH14-1 and BH18-7, Bifidobacterium longum subsp. HOM1190 can significantly (p<0.01) increase the production capacity of lactic acid and short-chain fatty acids (formic acid, acetic acid and butyric acid).

[0114] Example 5: In vitro anti-inflammatory test

[0115] (1) Preparation of cell culture medium

[0116] Complete culture medium: high-glucose DMEM medium supplemented with 10% inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin, 100 µg / mL streptomycin), and stored at 4°C after mixing.

[0117] Incomplete culture medium: high-glucose DMEM medium, mixed with 10% inactivated fetal bovine serum (FBS) and stored at 4°C.

[0118] (2) Preparation of test bacterial samples

[0119] The *Bifidobacterium longum* subsp. *HOM1190*, cryopreserved at -80℃ as prepared in Example 1, was inoculated into sterilized MRS-Cys liquid medium as described in Example 1 at an inoculum of 1% of the total culture medium. The medium was then anaerobically cultured at 37℃ for 24 hours. After two generations of activation, the cells were used for viable cell counting and cell experiments. The cells were centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected. The concentration was then adjusted to the working concentration (viable cell group) using antibiotic-free DMEM complete medium. Additionally, after cell collection, the cells were heated at 100℃ for 30 min, and the bacterial count was adjusted to the working concentration (dead cell group).

[0120] (3) Culture of mouse macrophage RAW264.7 cells

[0121] Mouse macrophages RAW264.7 (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were seeded in complete culture medium and passaged at a ratio of 1:3 (v / v). Cells from passages 3 to 10 were selected for experiments.

[0122] (4) Cell viability detection

[0123] The cell number was adjusted to 5 × 10⁶ using incomplete culture medium. 5 Cells / mL, 0.1 mL of cell culture was aspirated from each well and seeded into a 96-well plate. The cells were allowed to mature for 4 hours. After culturing, the cell culture medium was removed from the 96-well plate, and 0.1 mL of the test bacterial cells (5 × 10⁶ cells / mL) prepared with incomplete culture medium was added to each well. 5 1×10 6 5×10 6 1×10 75×10 7 1×10 8 5×10 8 1×10 9 Cells were cultured at 37℃ in a 5% CO2 incubator for 1 hour, then 1 μg / mL LPS was added, and the cells were co-cultured for 24 hours. Cell viability was then assessed using the MTS Cell Proliferation and Cytotoxicity Assay Kit (Prologis). The procedure was as follows: 20 μL of MTS was added to each well, and the cells were incubated in the dark for 1 hour. The absorbance was read at 490 nm using a microplate reader. Each treatment was performed in triplicate. Normally cultured cells were used as a control, and the relative cell viability was calculated. The results are shown below. Figure 3 As shown. By Figure 3 The results show that when the concentration of live bacteria is no greater than 1×10⁻⁶, 8 CFU / mL, dead bacteria concentration greater than 5×10 8 At CFU / mL, the viability of RAW264.7 macrophages was consistently above 100%; therefore, 1×10⁻⁶ live bacteria were selected. 8 CFU / mL and 1×10 7 CFU / mL, dead cells selected at 5×10⁻⁶ 8 CFU / mL and 1×10 8 An experiment was conducted to evaluate the anti-inflammatory effect of cells using CFU / mL.

[0124] (5) Anti-inflammatory capacity test

[0125] Mouse macrophages RAW264.7 (approximately 5 × 10⁻⁶) 5 1 mL of cells / mL was added to each well of a 24-well culture plate. After 4 hours of adhesion, the culture medium was discarded, and 1 mL of bacterial culture medium (with a cell viability of at least 100% selected as the experimental group) was added to each well. After incubation for 1 hour, LPS was added to each well to a final concentration of 1 μg / mL. 1 mL of incomplete culture medium was used as a negative control, and 1 mL of incomplete culture medium + 1 μg / mL LPS was used as a positive control. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. After centrifugation at 3000 rpm for 10 minutes, the supernatant was collected, and the levels of NO, TNF-α, and IL-6 in the cell supernatant were determined according to the kit instructions. Each treatment was performed in triplicate. Data were analyzed using GraphPad Prism 9 software for one-way ANOVA and Dunnett's t-test (pairwise comparison of means between multiple experimental groups and a control group). The results are shown below. Figure 4 , Figure 5 and Figure 6As shown. Compared with the negative control group, LPS stimulation of RAW264.7 cells led to a highly significant increase (p<0.01) in the secretion of NO, TNF-α, and IL-6. Compared with LPS treatment alone, the secretion of live Bifidobacterium longum subsp. HOM1190 cells (1×10⁻⁶) was significantly increased. 8 CFU / mL) or dead bacteria (5×10⁻⁶) 8 Co-treatment with LPS (CFU / mL) significantly reduced the secretion of NO, TNF-α, and IL-6 (p<0.01). Compared with the LPS group, both live and dead *Bifidobacterium longum* subsp. *longum* HOM1190 significantly (p<0.01) inhibited the production of inflammatory factors NO, TNF-α, and IL-6 in RAW264.7 cells stimulated by LPS, exhibiting a dose-dependent effect, with higher doses showing greater efficacy than lower doses. The innate immune system can recognize shared, structurally conserved molecular structures in many microorganisms through pattern recognition receptors (PRRs), known as pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs) are important cell surface PRRs, mainly expressed on the surface of immune-functional cells such as macrophages. Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria and a major pathogenic molecule for Gram infections. LPS is a potent antigen that can be recognized by TLR4. Upon binding, it initiates the MyD88t IRAK signaling cascade, thereby activating the nuclear transcription factor NF-κB and its target genes. This leads to the overexpression of pro-inflammatory factors and induces inflammation-related diseases. Bifidobacterium longum subsp. HOM1190 reduces the expression of the LPS receptor TLR4, thereby inhibiting the production and release of inflammatory mediators and reducing the inflammatory response.

[0126] Example 6: In vitro cytokine secretion assay

[0127] (1) Preparation of cell culture medium

[0128] Complete culture medium: High-glucose DMEM medium supplemented with 10% inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin, 100 µg / mL streptomycin), and stored at 4°C after mixing.

[0129] Incomplete culture medium: high-glucose DMEM medium, mixed with 10% inactivated fetal bovine serum (FBS) and stored at 4°C.

[0130] (2) Preparation of test bacterial samples

[0131] Bifidobacterium longum subsp. HOM1190, cryopreserved at -80℃ as prepared in Example 1, was inoculated into sterilized MRS-Cys liquid medium as described in Example 1 at an inoculum of 1% of the total culture medium. The medium was then anaerobically cultured at 37℃ for 24 hours. After two generations of activation, the cells were used for viable cell counting and cell experiments. After centrifugation at 8000 rpm for 10 min, the cells were collected and adjusted to the working concentration (5 × 10⁻⁶) using antibiotic-free DMEM complete medium. 5 The live bacteria group consisted of cells collected at 5 × 10⁻⁵ CFU / mL and heated at 100°C for 30 minutes. The cells were then adjusted to different working concentrations to form the dead bacteria group. 5 CFU / mL represents the low-dose group for dead bacteria, 5×10⁻⁶. 6 CFU / mL represents the medium-dose group for dead bacteria, 5×10⁻⁶. 7 CFU / mL represents the high-dose group of dead bacteria.

[0132] (3) Culture of mouse macrophage RAW264.7 cells

[0133] Mouse macrophage RAW264.7 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were seeded in complete culture medium 1 and passaged at a ratio of 1:3. Cells from passages 3 to 10 were selected for experiments.

[0134] (4) Cytokine activity detection

[0135] Activated mouse macrophages RAW264.7 (approximately 5 × 10⁻⁶) 5 1 mL of cells / mL was added to each well of a 24-well culture plate. After 4 hours of cell adhesion, the culture medium was discarded, and 1 mL of bacterial culture medium was added to each well. A blank control group was included, with 1 mL of DMEM culture medium added. After co-culturing for 24 hours, the supernatant was collected, and the levels of TNF-α and IL-6 in the cell supernatant were determined by enzyme-linked immunosorbent assay (ELISA) according to the kit instructions. The MRS-Cys liquid culture medium described in Example 1 served as a negative control. Each sample was performed in triplicate. Data were analyzed using GraphPad Prism 9 software for one-way ANOVA and Dunnett's t-test, i.e., pairwise comparisons of means between multiple experimental groups and a control group. The results are shown in [Figure 1]. Figure 7 and Figure 8 As shown.

[0136] The results showed that both live and dead *Bifidobacterium longum* subsp. *HOM1190* significantly increased the secretion of TNF-α and IL-6 by RAW264.7 cells, and the ability of RAW264.7 cells to secrete TNF-α and IL-6 increased with the increase of dead bacteria. The mechanism of action is as follows: the main components of the cell wall of *Bifidobacterium longum* subsp. *HOM1190*, peptidoglycan (WPG) and teichoic acid (LTA), act on RAW264.7 macrophages, bind to Toll receptor 2, activate the NF-κB signaling pathway, and induce macrophages to release tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), thereby enhancing the body's immunity. Based on the results of Example 6, it can be concluded that *Bifidobacterium longum* subsp. *HOM1190*, under normal circumstances, maintains intestinal epithelial cells in a moderate state of inflammation without causing harm to the body, and is used to regulate immune function and clear pathogens. On the other hand, when the body is in a state of excessive inflammation, it can also suppress inflammation and maintain immune balance by regulating TLR-mediated signals, thus maintaining a healthy state.

[0137] Example 7 Antibiotic susceptibility test

[0138] The drug susceptibility test was conducted in accordance with ISO 10932-2010 Milk and dairy products - Determination of the minimum inhibitory concentration (MIC) of antibiotics applicable to Bifidobacteria and non-enterococcal lactic acid bacteria (LAB), as defined by the International Organization for Standardization.

[0139] (1) Preparation of culture medium

[0140] MRS-Cys liquid culture medium: Same as in Example 1

[0141] LSM-Cys liquid medium: Weigh 21.06g of Iso-Sensitest medium (catalog number: CM0473B, OXOID, UK), 5.2g of MRS Broth, and 0.3g of L-cysteine ​​hydrochloride. Add water to 0.5L, adjust the pH to 6.85±0.1, sterilize at 121℃ for 15min, the pH should be 6.7±0.1, and store at 2-8℃ protected from light for one week.

[0142] (2) Activation and proliferation of Bifidobacterium longum subsp.

[0143] The *Bifidobacterium longum* subsp. *HOM1190*, cryopreserved at -80℃ as prepared in Example 1, was inoculated into sterilized liquid culture medium (MRS-Cys) described in Example 1 at an inoculum of 1% of the total culture medium volume. The culture was then anaerobically incubated at 37℃ for 24 hours, and activated twice to obtain the fermentation broth. The activated *Bifidobacterium longum* subsp. *HOM1190* was then multiplied on MRS-Cys agar medium and anaerobically incubated at 37℃ for 48 hours.

[0144] (3) Preparation of antibiotic microdilution plates

[0145] Weigh 0.0512 g of antibiotics and add 10 mL of solvent. Dissolve chloramphenicol and erythromycin in ethanol (no filtration required). Dissolve ampicillin in phosphate buffer (pH 8.0, 0.1 mol / L). Dissolve other antibiotics in water. After shaking to dissolve, filter through a 0.22 µm filter membrane and aliquot into EP tubes at a concentration of 5120 µg / mL (5.12 mg / mL). Store at -20 °C for later use. Dilute the antibiotic stock solution with water (ampicillin with phosphate buffer) to the appropriate concentration range. Add 50 µL of the diluent to the wells of a microdilution plate.

[0146] (4) Preparation of bacterial suspension

[0147] Pick a single colony from an agar plate. Resuspend the obtained colony in a sterile culture tube containing 2 mL to 5 mL of sterile saline. Then, resuspend the obtained colony in pre-reconstituted LSM-Cys liquid medium. Resuspend the colony until the turbidity of the solution reaches McFarland standard 1 or the optical density at 625 nm is 0.16–0.2 using a spectrophotometer. The suspension is approximately equivalent to 3 × 10⁻⁶ cells / mL. 8 CFU / mL. Dilute the bacterial suspension with the recommended culture medium, diluting it 500-fold with MRS-Cys liquid medium, as the antibiotic solution will dilute the medium by two times. Dispense the diluted bacterial suspension within 30 minutes of preparation. When using a cryoplate, thaw the frozen antibiotic solution under anaerobic conditions immediately before use. Dispense 50 µL of the diluted suspension into each well of the microdilution plate (approximately 3 × 10⁻⁶). 4 CFU / well). Incubate plates under static anaerobic conditions at 37°C for 48 hours. When using an anaerobic jar, cover each plate with a lid to create a homogeneous environment within the jar. Each experiment is performed in triplicate, with both positive and negative control groups included. Positive control wells do not contain antibiotics but contain the test strain and culture medium containing the solvent for dissolving the highest concentration of antibiotic. Negative control wells do not contain the test strain or antibiotics but contain culture medium.

[0148] (5) Read the MIC results

[0149] After 48 hours of incubation, visually read the MIC. After incubation, check the negative control wells for visible bacterial growth. If contamination is found, reject all data produced for that strain. Note: If no growth is observed in the positive control wells, it indicates that the tested strain is sensitive to the solvent used to dissolve the antibiotic. In this case, reading the MIC for that specific antibiotic is meaningless. If the negative and positive control checks are normal, visually determine bacterial growth for each antibiotic by comparing it with the positive control. It is best to place the microdilution plate on top of a stand with a magnifying glass and a desk lamp providing indirect light for easy reading. Bacterial growth is easily detected under a magnifying glass as a deposit at the bottom of the well. Discard any series of wells where discontinuous growth is observed (e.g., growth at 16 µg / mL and 64 µg / mL, but no growth at 32 µg / mL). The endpoint is defined as the lowest antibiotic concentration at which no visual growth is observed. This concentration is the MIC of that antibiotic for that specific strain. Perform three replicates for each sample, with *Lactobacillus plantarum* ATCC14917 (purchased from the China Industrial Microbiological Culture Collection Center) as the positive control strain. The results of antibiotic susceptibility testing for Bifidobacterium longum subspecies HOM1190 are shown in Table 5.

[0150] Table 5. Antibiotic susceptibility results of Bifidobacterium longum subsp. HOM1190

[0151]

[0152] Table 5 shows that the MIC results of *Lactobacillus plantarum* ATCC14917 are consistent with those shown in Annex to ISO 10932-2010, indicating that the experimental method is accurate. According to the antibiotic resistance standards for microorganisms used in food established by the European Food Safety Authority (EFSA) in 2012, *Bifidobacterium longum* subsp. *longum* HOM1190 is sensitive to erythromycin, chloramphenicol, tetracycline, vancomycin, ampicillin, clindamycin, gentamicin, streptomycin, and kanamycin. Therefore, products containing *Bifidobacterium longum* subsp. *longum* HOM1190 are relatively safe.

[0153] Example 8 Preparation process of active bacterial powder of Bifidobacterium longum subsp. longum HOM1190 1

[0154] (1) Culture of microbial strains

[0155] The -80℃ cryopreserved *Bifidobacterium longum* subsp. *HOM1190* prepared in Example 1 was inoculated into sterile MRS-Cys liquid medium at an inoculum of 1% of the total culture medium volume and cultured at 37℃ for 16-24 hours. This process was repeated twice to obtain an activated seed culture. The seed culture was then inoculated into fermentation medium M447 at an inoculum of 3% of the total culture medium volume and cultured anaerobically at 37℃. During fermentation, sodium hydroxide solution was automatically added to maintain a constant pH of 5.5 until acid production ceased. Fermentation was terminated when sodium hydroxide was no longer added, resulting in a high-density culture of *Bifidobacterium longum* subsp. *HOM1190* with a viable count of up to 13 billion CFU / mL.

[0156] (2) Preparation of freeze-drying protectant

[0157] A preservative containing 100 g / L skim milk powder, 50 g / L trehalose, 3 g / L vitamin C, and 5 g / L monosodium glutamate was prepared by mixing sterile water with the preservative raw materials.

[0158] (3) Freeze-drying

[0159] The fermentation broth of *Bifidobacterium longum* subsp. *longum* HOM1190, after culture, was centrifuged at 6000 rpm for 10 min at 2–8 °C. The supernatant was discarded, and the bacterial sludge was collected. The sludge was washed 1–2 times with 0.9% sterile physiological saline. The washed sludge was then mixed with the aforementioned protective agent to achieve a bacterial concentration of 10. 10 The mycelium was freeze-dried in a freeze dryer with a concentration of CFU / mL or higher. Pre-freezing was performed at -45°C for 4 hours, followed by vacuum drying. The temperature was then raised to -15°C for a first drying of 20 hours, and then raised to 30°C for a second drying of 10 hours. After freeze-drying, the mycelium cake was pulverized using a fine grinder to obtain the freeze-dried mycelium powder. The viable cell count of the freeze-dried mycelium powder reached 1.9 × 10⁻⁶. 11 CFU / g. The formulation of fermentation medium M447 is shown in Table 6.

[0160] Table 6 Fermentation medium M447 formulation

[0161]

[0162] Example 9: Preparation process of active bacterial powder of Bifidobacterium longum subsp. longum HOM1190 2

[0163] (1) Culture of microbial strains

[0164] The -80℃ cryopreserved *Bifidobacterium longum* subsp. *HOM1190* prepared in Example 1 was inoculated into sterile MRS-Cys liquid medium at an inoculum volume of 3% of the total culture medium volume and cultured at 37℃ for 16-24 hours. This process was repeated twice to obtain an activated seed culture. The seed culture was then inoculated into fermentation medium M425 at an inoculum volume of 3% of the total culture medium volume and cultured anaerobically at 35℃. During fermentation, sodium hydroxide solution was automatically added to maintain a constant pH of 6.0 until acid production ceased. Fermentation was terminated when sodium hydroxide was no longer added, resulting in a high-density culture of *Bifidobacterium longum* subsp. *HOM1190* with a viable count of up to 9.5 billion CFU / mL. The formulation of fermentation medium M425 is shown in Table 7.

[0165] Table 7 Fermentation medium M425 formulation

[0166]

[0167] (2) Preparation of freeze-drying protectant

[0168] A preservative containing 80 g / L skim milk powder, 40 g / L trehalose, 2 g / L vitamin C, and 4 g / L monosodium glutamate was prepared by mixing sterile water with the preservative raw materials.

[0169] (3) Freeze-drying

[0170] The fermentation broth of *Bifidobacterium longum* subsp. *longum* HOM1190, after culture, was centrifuged at 6500 rpm for 15 min at 2–8 °C. The supernatant was discarded, and the bacterial sludge was collected. The sludge was washed 1–2 times with 0.9% sterile physiological saline. The washed sludge was then mixed with the aforementioned protective agent to achieve a bacterial concentration of 10-1 in the mixed broth. 10 The mycelium was freeze-dried in a freeze dryer with a CFU / mL concentration of -40°C for 5 hours, then vacuum-dried for 25 hours at -20°C, followed by a second drying at 35°C for 6 hours. After freeze-drying, the mycelium cake was pulverized using a fine grinder to obtain the freeze-dried mycelium powder. The viable cell count of the freeze-dried mycelium powder was higher than 1.7 × 10⁻⁶. 11 CFU / g.

[0171] Example 10: Preparation process of inactivated Bifidobacterium longum subsp. longum HOM1190 powder

[0172] (1) Culture of microbial strains

[0173] Microbial culture is as described in Example 8 or Example 9.

[0174] (2) Preparation of mycelium powder

[0175] The fermentation broth of *Bifidobacterium longum* subsp. *longum* HOM1190 obtained by high-density fermentation is treated at 90°C for 30 minutes, 100 g of maltodextrin adjuvant is compounded per liter of the fermentation broth, and the product can be obtained by drying through a spray drying tower. When counting dead bacteria in the dead bacteria powder under a microscope via a hemocytometer, the number of dead bacteria reaches 2.1×10 11 CFU / g.

[0176] Example 11 Immunity-enhancing animal experiment

[0177] (1) Experimental animals and grouping: 192 female KM mice of SPF grade with a weight of 18 g-20 g, bred by Beijing Huafukang Biotechnology Co., Ltd. [license number: SCXK (Jing) 2020-0004], were randomly divided into four batches after acclimatization feeding observation, with 4 groups in each batch and 12 mice in each group. The experimental group included three dose groups: low-dose group (5×10 9 CFU / kg BW), medium-dose group (2.5×10 10 CFU / kg BW) and high-dose group (5×10 10 CFU / kg BW). The active *Bifidobacterium longum* subsp. *longum* HOM1190 powder prepared in Example 8 was administered by intragastric gavage once daily via oral route, with normal saline as solvent. Each indicator was measured after 30 consecutive days of intragastric gavage. The intragastric gavage volume for mice was 10 mL / kg BW. The control group was given an intragastric gavage of a normal saline solution containing the same mass of bacterial powder adjuvant as that in the high-dose group. All dose groups were fed maintenance feed. The first batch of experiment was carried out for carbon clearance test; the second batch for organ / body weight ratio determination and delayed-type hypersensitivity experiment; the third batch for mouse peritoneal macrophage phagocytosis test of chicken red blood cells; the fourth batch for ConA-induced mouse lymphocyte transformation test.

[0178] (2) Experimental methods

[0179] A. Determination of organ to body weight ratio

[0180] After weighing, the mice were sacrificed by cervical dislocation. The spleen and thymus were taken, all fascia was removed, blood stains on the organ surface were absorbed dry with filter paper, then weighed, and the spleen / body weight ratio and thymus / body weight ratio were calculated.

[0181] B. Delayed-type hypersensitivity (DTH) experiment (footpad thickening method)

[0182] Sheep blood was collected and washed three times with physiological saline. Each mouse was intraperitoneally injected with 0.2 mL of 2% (v / v, prepared with physiological saline) sheep red blood cell (SRBC) suspension (2000 r / min, 10 min). Four days after sensitization, the thickness of the left hind paw was measured. Then, 20 µL of 20% (v / v, prepared with physiological saline) SRBC suspension was subcutaneously injected at the measurement site, and the thickness of the left hind paw was measured 24 h after injection. Both measurements were taken three times at the same site, and the average value was used. The difference in paw thickness before and after challenge was used to represent the degree of DTH. A significantly higher difference in the tested sample group compared to the control group indicated a positive result for this experiment.

[0183] C. ConA-induced mouse lymphocyte transformation assay (MTT assay)

[0184] Spleens were aseptically harvested and placed in a small petri dish containing an appropriate amount of sterile Hank's solution. The spleen was gently ground with forceps to prepare a single-cell suspension. The suspension was then filtered through a 200-mesh sieve to prepare a cell suspension. The cells were washed twice with Hank's solution, centrifuged for 5 minutes each time (1000 rpm). The cells were then resuspended in 1 mL of complete culture medium, counted under a microscope, and the cell concentration was adjusted to 3 × 10⁻⁶ cells / mL. 6 Spleen cell suspension was then added to two wells of a 24-well plate, 1.0 mL per well. One well contained 75 µL of ConA solution (equivalent to 7.5 µg / mL), and the other served as a control. The plates were incubated at 37°C with 5% CO2 for 72 hours. Four hours before the end of the incubation period, 0.7 mL of supernatant was gently aspirated from each well, and 0.7 mL of RPMI 1640 medium (without fetal bovine serum) was added. Simultaneously, 50 µL of MTT (5 mg / mL) per well was added, and the plates were incubated for another 4 hours. After the incubation period, 1.0 mL of acidic isopropanol was added to each well, and the mixture was pipetted to dissolve the purple crystals completely. This solution was then transferred to a cuvette and measured colorimetrically at 570 nm using a spectrophotometer. The lymphocyte proliferation capacity was calculated by subtracting the optical density value of the well containing ConA from the value of the well without ConA. A significantly higher optical density difference in the tested sample group compared to the control group indicates a positive result for this experiment.

[0185] D. Determination of antibody-producing cell count (PFC) (Jerne modified slide method)

[0186] Sheep blood was collected and washed three times with physiological saline. Each mouse was intraperitoneally injected with 0.2 mL of 2% (v / v, prepared with physiological saline) SRBC suspension. Five days after SRBC immunization, mice were euthanized by cervical dislocation. The spleen was removed and placed in a small petri dish containing an appropriate amount of sterile Hank's solution. The spleen was gently ground to prepare a cell suspension. The suspension was centrifuged (1000 rpm) for 5 min, washed twice with Hank's solution, and finally resuspended in 8.0 mL of Hank's solution. After dissolving agarose by heating, mix it with an equal volume of twice the concentration of Hank's solution. Dispense 0.5 mL into small test tubes, then add 50 µL of 10% (v / v, prepared with SA solution) SRBC suspension and 8 µL of spleen cell suspension to each tube. Mix quickly and pour onto a glass slide coated with a thin layer of agarose, making parallel slides. After the agarose solidifies, place the slide horizontally on a slide holder and incubate at 37°C for 1 h in a CO2 incubator. Then, add complement diluted with SA buffer (1:8) to the grooves of the slide holder and continue incubation for 1.5 h. Count the number of hemolytic plaques. Express the result as plaque count / whole spleen cells. The number of plaques in the test sample group was significantly higher than that in the control group, indicating a positive result for this experiment.

[0187] E. Determination of the half-hemolytic value (HC50)

[0188] Sheep blood was collected and washed three times with physiological saline. Each mouse was immunized by intraperitoneal injection of 0.2 mL of 2% (v / v, prepared with physiological saline) SRBC suspension. Five days later, the eyeballs were removed, and blood was collected in centrifuge tubes. After standing for about 1 hour, the clotted blood was peeled off the tube wall to allow the serum to be fully separated. The tubes were centrifuged at 3000 rpm for 10 minutes, and the serum was collected. The serum was diluted 300 times with SA buffer, and 1.0 mL was placed in a test tube. 0.5 mL of 10% (v / v, prepared with SA buffer) SRBC suspension and 1.0 mL of complement (diluted with SA buffer at a ratio of 1:8) were added sequentially. A control tube without serum was prepared (using SA buffer instead). The tubes were incubated at 37°C for 15 minutes, and then the reaction was stopped by ice bath. The tubes were centrifuged at 2000 rpm for 10 minutes, and 1.0 mL of the supernatant was collected and 3.0 mL of Hb diluent was added. Simultaneously, take 0.25 mL of 10% (v / v, prepared with SA buffer), add Hb dilution to 4.0 mL in another test tube, mix thoroughly, and let stand for 10 min. Measure the optical density of each tube at 540 nm, using the control tube as a blank. The amount of hemolysin is expressed as the half-hemolysis value (HC50), calculated using the following formula: Sample half-hemolysis value = Sample optical density value / Optical density value at half-hemolysis of SRBC × Dilution factor. If the HC50 of the test sample group is significantly higher than that of the control group, the result of this experiment can be considered positive.

[0189] F. Mouse carbon clearance assay

[0190] Inject 4-fold diluted Indian ink (0.05 mL / 10 g BW) into the tail vein of mice according to body weight. Timing begins immediately after injection. 2 and 10 minutes after injection, 20 µL of blood is collected from the internal canthal venous plexus and added to 2.0 mL of 0.1% Na₂CO₃ solution. The optical density (OD) is measured at 600 nm using a spectrophotometer, with 0.1% Na₂CO₃ solution as a blank control. Mice are then sacrificed, and their livers and spleens are weighed. The phagocytic index (a), representing the carbon clearance capacity of the mice, is calculated using the following formula:

[0191] k = (lgOD1 - lgOD2) / (t2 - t1) a = body weight ÷ (liver weight + spleen weight) × k 1 / 3

[0192] The phagocytic index of the test sample group was significantly higher than that of the control group, which indicates that the result of this experiment was positive.

[0193] G. Experiment of mouse peritoneal macrophages phagocytosing chicken red blood cells (semi-in vivo method)

[0194] Mice were intraperitoneally injected with 1 mL of 20% (v / v, prepared with physiological saline) chicken erythrocyte suspension (2000 r / min, 10 min). After 30 min intervals, the mice were euthanized by cervical dislocation, fixed in a supine position on a mouse board, and 2 mL of physiological saline was injected intraperitoneally. The abdomen was gently massaged 20 times. 1 mL of peritoneal macrophage washings were collected and dropped onto two separate glass slides, placed in an enamel box lined with damp gauze, and incubated at 37°C for 30 min. After incubation, the slides were rinsed in physiological saline to remove unattached cells. The slides were air-dried, fixed with 1:1 acetone-methanol solution, stained with Giemsa-phosphate buffer, rinsed with distilled water, and air-dried again. Macrophages were counted under an oil immersion microscope, 100 cells per slide. The phagocytic rate and phagocytic index were calculated using the following formula:

[0195] Phagocytosis percentage (%) = (Number of macrophages phagocytosing chicken red blood cells / Total number of macrophages counted) × 100%

[0196] Phagocytic index = Total number of phagocytosed chicken red blood cells / Number of macrophages counted

[0197] The obtained phagocytosis percentage is then transformed using the following formula. In the formula, P represents the phagocytic percentage, expressed as a decimal. The obtained data are quantitative data. The phagocytic percentage and phagocytic index of the test sample group are significantly higher than those of the control group, indicating that the experimental result is positive.

[0198] H. Assay for NK cell activity (Lactate dehydrogenase (LDH) assay)

[0199] 24 hours before the experiment, the target cells YAC-1 were subcultured, washed 3 times with Hank's solution before use, and the cell concentration was adjusted to 4×10 5 cells / mL with RPMI1640 complete culture medium containing 10% calf serum. Test mice were euthanized by cervical dislocation, spleens were harvested aseptically, and spleen cell suspension was prepared, washed 2 times with Hank's solution, and centrifuged for 10 min (1000 r / min) each time. The supernatant was discarded, the cell pellet was loosened, 0.5 mL of sterilized water was added for 20 s to lyse red blood cells, then 0.5 mL of 2×Hank's solution and 8.0 mL of Hank's solution were added, followed by centrifugation at 1000 r / min for 10 min. The cells were resuspended in 1.0 mL of RPMI1640 complete culture medium containing 10% calf serum, counted under microscope, and the cell concentration was adjusted to 2×10 7 cells / mL to achieve an effector-to-target ratio of 50:1. 100 µL of target cells and 100 µL of effector cells were taken and added to a U-shaped 96-well culture plate; 100 µL of target cells and 100 µL of culture medium were added to the target cell spontaneous release wells, and 100 µL of target cells and 100 µL of 1% NP40 were added to the target cell maximum release wells; all the above treatments were set with three parallel wells. The culture plate was placed in a carbon dioxide incubator and cultured at 37°C with 5% CO2 for 4 h. The 96-well plate was centrifuged at 1500 r / min for 5 min, 100 µL of supernatant from each well was aspirated and transferred to a flat-bottom 96-well culture plate, 100 µL of LDH substrate solution was added, the reaction was allowed to proceed for 10 min, then 30 µL of 1 mol / L HCl solution was added to each well to terminate the reaction, and the optical density (OD) was measured at 490 nm with a microplate reader. NK cell activity was calculated as: NK cell activity (%) = (OD of reaction well - OD of spontaneous release well) / (OD of maximum release well - OD of spontaneous release well) × 100%

[0200] The obtained NK cell activity was converted according to the following formula, wherein P is NK cell activity, expressed as a decimal. The obtained data are measurement data, if the NK cell activity of the test sample group is significantly higher than that of the control group, the test result can be determined as positive.

[0201] (3) Data processing

[0202] Data processing was performed with SPSS software. Analysis of variance was used, but homogeneity of variance test was performed first according to the procedure of analysis of variance. If the variance was homogeneous, the F value was calculated. If F value < F0.05, the conclusion was that there was no significant difference among the mean values of all groups; if F value ≥ F0.05 and p ≤ 0.05, Duncan's test was used for multiple comparison. Appropriate variable transformation was performed for non-normally distributed or non-homogeneous variance data, and statistics were performed with the transformed data after the requirements of normality and homogeneity of variance were satisfied; if the normality or homogeneity of variance was still not achieved after variable transformation, rank sum test was used for statistics instead.

[0203] (4) Experimental results

[0204] Table 8 shows the effect of the live Bifida longibrane subsp. longibrane HOM1190 powder prepared in Example 8 on mouse body weight.

[0205] Table 8. Effects of Bifidobacterium longum subsp. longum HOM1190 bacterial powder on mouse body weight ( ±S)

[0206]

[0207] As shown in Table 8, compared with the control group, there was no significant difference in body weight before and after the experiment among the high, medium and low dose groups of Bifidobacterium longum subsp. HOM1190 (p > 0.05), indicating that the active bacterial powder of Bifidobacterium longum subsp. HOM1190 had no effect on the body weight of mice.

[0208] Table 9 shows the effect of the live Bifida longum subsp. longum HOM1190 powder prepared in Example 8 on the organ / body ratio of mice.

[0209] Table 9. Effects of Bifidobacterium longum subsp. longum HOM1190 bacterial powder on the visceral / body ratio in mice. ±S)

[0210]

[0211] Note: p>0.05 compared with the negative control group.

[0212] As shown in Table 9, compared with the control group, there were no significant differences in spleen / body and thymus / body in mice in the high, medium and low dose groups of Bifidobacterium longum subsp. HOM1190 (p > 0.05). This indicates that the active bacterial powder of Bifidobacterium longum subsp. HOM1190 had no effect on the weight of spleen and thymus in mice.

[0213] Table 10 and Figure 9 The effects of the live Bifida longifolia subsp. HOM1190 powder prepared in Example 8 on sheep erythrocyte (SRBC)-induced DTH in mice and on the proliferation capacity of ConA-induced mouse spleen lymphocytes were shown.

[0214] Table 10 Effects of Bifidobacterium longum subsp. HOM1190 bacterial powder on sheep erythrocyte (SRBC)-induced DTH in mice and on ConA-induced proliferation of mouse spleen lymphocytes. ±S)

[0215]

[0216] Note: Compared with the control group, p<0.05. Compared with the control group, p<0.01. Compared with the control group, p<0.001.

[0217] From Table 10 and Figure 9 The results showed that, compared with the control group, the medium-dose group of Bifidobacterium longum subsp. HOM1190 active bacterial powder significantly improved the ConA-induced proliferation of mouse spleen lymphocytes (p < 0.05), and the high-dose group of Bifidobacterium longum subsp. HOM1190 active bacterial powder significantly improved the ConA-induced proliferation of mouse spleen lymphocytes (p < 0.001). This indicates that the active bacterial powder of Bifidobacterium longum subsp. HOM1190 has a significant effect on enhancing the cellular immune function of mice.

[0218] Table 11 and Figure 10 The effect of the live Bifida longum subsp. longum HOM1190 powder prepared in Example 8 on the number of antibody-producing cells and the serum half-hemolysis value is shown.

[0219] Table 11 Effects of Bifidobacterium longum subsp. longum HOM1190 bacterial powder on antibody-producing cell count and serum half-hemolytic value ( ±S)

[0220]

[0221] From Table 11 and Figure 10 The results showed that, compared with the control group, the medium-dose group of Bifidobacterium longum subsp. HOM1190 active bacterial powder significantly increased the serum half-hemolytic value in mice (p < 0.05); the high-dose group significantly increased the number of hemolytic plaques in mice (P < 0.05), and extremely significantly increased the serum half-hemolytic value in mice (p < 0.001). This indicates that the active bacterial powder of Bifidobacterium longum subsp. HOM1190 has a significant effect on enhancing the humoral immune function of mice.

[0222] Table 12 and Figure 11 The effects of the live Bifida longibrane subsp. longibrane HOM1190 powder prepared in Example 8 on carbon clearance capacity and on the phagocytic rate and phagocytic index of mouse macrophages phagocytizing chicken erythrocytes are shown.

[0223] Table 12 Effects of Bifidobacterium longum subsp. longum HOM1190 bacterial powder on carbon clearance capacity and on phagocytic rate and phagocytic index of mouse macrophages phagocytosis of chicken erythrocytes. ±S)

[0224]

[0225] From Table 12 and Figure 11The results showed that, compared with the control group, the low-dose group of *Bifidobacterium longum* subsp. *longum* HOM1190 significantly improved the carbon clearance capacity of mice (p < 0.05). Among *Bifidobacterium longum* subsp. *longum* HOM1190, the high-dose group significantly increased the phagocytic rate (p < 0.001) and phagocytic index (p < 0.001) of mouse macrophages phagocytosis of chicken erythrocytes; the low-dose group also significantly increased the phagocytic rate (p < 0.01) and phagocytic index (p < 0.01) of mouse macrophages phagocytosis of chicken erythrocytes. This indicates that the active bacterial powder of *Bifidobacterium longum* subsp. *longum* HOM1190 significantly enhances the function of mouse mononuclear-macrophages.

[0226] Table 13 shows the effect of the live Bifida longum subsp. longum HOM1190 powder prepared in Example 8 on the assay of NK cell activity.

[0227] Table 13. Effects of Bifidobacterium longum subsp. HOM1190 bacterial powder on NK cell activity assay ( ±S)

[0228]

[0229] As shown in Table 13, there was no significant difference in NK cell activity among the different dose groups of Bifidobacterium longum subsp. HOM1190 compared with the control group (p > 0.05).

[0230] Figure 12 The results show that the active bacterial powder of Bifidobacterium longum subsp. HOM1190 of the present invention significantly enhances the cellular immune function, humoral immune function, and mononuclear-macrophage function in mice.

[0231] Depend on Figure 12 It was found that, compared with the control group, the active bacterial powder of *Bifidobacterium longum* subsp. *longum* HOM1190 significantly enhanced the cellular immune function, humoral immune function, and mononuclear-macrophage function of mice, while having no effect on mouse body weight, thymus and spleen weight, or NK cell activity. In conclusion, the active bacterial powder of *Bifidobacterium longum* subsp. *longum* HOM1190 has the function of enhancing immunity.

[0232] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0233] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details shown and described herein.

Claims

1. A lyophilized formulation comprising a strain of Bifidobacterium longum and a lyophilization protectant, wherein the Bifidobacterium longum strain has the accession number CGMCC No. 25684, and wherein the Bifidobacterium longum strain contains a 16S rRNA gene represented by SEQ ID NO: 1, which has the ability to bidirectionally regulate the secretion of the cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

2. The lyophilized formulation according to claim 1, characterized in that, The freeze-drying protectant contains: 80-100 g / L skim milk powder, 40-50 g / L trehalose, 2-3 g / L vitamin C, and 4-5 g / L L-monosodium glutamate.

3. The lyophilized formulation according to claim 1 or 2, characterized in that, The freeze-dried formulation is used to enhance immunity and maintain immune balance.

4. The lyophilized formulation according to claim 3, characterized in that, The lyophilized formulation is used to reduce the inflammatory factor NO.

5. The lyophilized formulation according to claim 3, characterized in that, The lyophilized formulation is used to stimulate mouse macrophages to produce cytokines TNF-α and IL-6, and to reduce LPS-induced macrophage production of inflammatory factors NO, TNF-α, and IL-6.

6. The lyophilized formulation according to claim 3, characterized in that, The lyophilized formulation is used to enhance cellular immune function, humoral immune function, and mononuclear-macrophage function, thereby maintaining normal human immune function.

7. A method for preparing the lyophilized formulation according to claim 1 or 2, comprising the following steps: (1) Culture of strains: Inoculate the Bifidobacterium longum strain into sterile liquid culture medium at an inoculation amount of 1-3% of the total culture medium and culture for 16-24 hours to obtain seed culture solution. Then, inoculate the obtained seed culture solution into fermentation medium at an inoculation amount of 1-3% of the total culture medium and carry out fermentation culture to obtain Bifidobacterium longum fermentation liquid. (2) Drying.

8. The method according to claim 7, characterized in that, The fermentation medium comprises: glucose 40-60 g / L, yeast extract 60-100 g / L, sodium acetate trihydrate 3-10 g / L, magnesium sulfate 0.1-0.2 g / L, manganese sulfate 0.05-0.1 g / L, dipotassium hydrogen phosphate 1-2 g / L, triammonium citrate 2-4 g / L, Tween 80 1-2 g / L, calcium chloride 0.05-0.1 g / L, and L-cysteine ​​salt 0.5-1 g / L.

9. The method according to claim 7, characterized in that, During fermentation, sodium hydroxide solution is automatically added to maintain a constant pH of 5.5-6.5 until acid production stops and fermentation is terminated when sodium hydroxide is no longer added.

10. The method according to any one of claims 7 to 9, characterized in that, The lyophilized formulation is a live bacterial formulation, and the method further includes preparing a lyophilization protectant after the strain culture step and before drying, and performing freeze-drying in the drying step.

11. The method according to claim 10, characterized in that, The freeze-drying protectant contains: 80-100 g / L of skim milk powder, 40-50 g / L of trehalose, 2-3 g / L of vitamin C, and 4-5 g / L of monosodium glutamate.

12. The method according to claim 10, characterized in that, The freeze-drying conditions are as follows: pre-freezing temperature is -40~-45℃, pre-freezing time is 4~5 h, primary drying temperature is -20~-15℃, primary drying time is 20~25 h, secondary drying temperature is 30~35℃, secondary drying time is 6~10 h.

13. The method according to any one of claims 7 to 9, wherein, The freeze-dried formulation is a dead bacterial formulation, and the drying step involves first performing heat inactivation, and then drying it in a spray drying tower to obtain the dead bacterial formulation.

14. The method according to claim 13, wherein, The heat inactivation conditions are 80~100℃ for 10~40 min.

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