Bifidobacterium bifidum bb36 with improved antigen presentation ability and enriched calcium iron selenium, postbiotics and application thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to bifidobacterium BB36 with improved antigen presentation ability and enriched calcium, iron and selenium, probiotics and application thereof. Bifidobacterium bifidum The present application provides bifidobacterium BB36 with activated macrophages, up-regulated immune factor expression level, and activated dendritic cells, enhanced antigen presentation ability in immune process, and enriched calcium, iron and selenium, and can be used as a biological carrier of mineral elements, in addition, can inhibit the growth of escherichia coli and staphylococcus aureus, and protect intestinal health of the body.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bifidobacterium bifidum BB36 that enhances antigen presentation and enriches calcium, iron, and selenium, as well as post-biotics and their applications. Background Technology

[0002] Probiotics, as an important microbial resource for maintaining host health, have become a research hotspot in the field of nutrition and health due to their immunomodulatory functions. Among them, Bifidobacteria are widely considered to have the potential to enhance the body's immune response due to their excellent intestinal colonization ability and host interaction characteristics. Existing research shows that some Bifidobacteria can indirectly enhance non-specific immunity by activating macrophages and promoting cytokine release, or affect specific immunity by regulating T cell and B cell function. However, the immune mechanisms and effects of different strains vary significantly, especially those strains that can directly activate dendritic cells and enhance antigen presentation capabilities, which are still relatively rare, limiting their application in immune synergistic regulation.

[0003] Currently, probiotic strains with single immunomodulatory functions have been reported, but most strains have relatively simple pathways of action, making it difficult to achieve multi-level, multi-target synergistic immune enhancement. Furthermore, with the development of the interdisciplinary field of nutrition and immunology, probiotics with mineral loading capacity are gradually gaining attention. They can supplement essential trace elements (such as calcium, iron, and selenium) while exerting probiotic functions, showing promising application prospects of dual "nutrition-immunity" benefits. However, existing technologies lack probiotic strains that possess both highly efficient immune activation capabilities (especially targeting macrophages and dendritic cells) and mineral loading functions, limiting the effectiveness of related products in synergistic immune regulation and nutritional supplementation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidum BB36 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37593 and deposit date of February 2, 2026.

[0005] The present invention also provides a fermentation broth, which is obtained by fermentation of Bifidobacterium bifidum BB36 as described above.

[0006] This invention also provides a metabiotic product, which is prepared by inoculating Bifidobacterium bifidum BB36 into a culture medium and then fermenting and inactivating it;

[0007] The Bifidobacterium BB36 is the Bifidobacterium BB36 as described above.

[0008] The present invention also provides a composition comprising one or more of the following: Bifidobacterium bifidum BB36 fermentation broth, Bifidobacterium bifidum BB36 fermentation supernatant, and Bifidobacterium bifidum BB36 postbiotic.

[0009] The present invention also provides a Bifidobacterium bifidum BB36 powder, the components of which include Bifidobacterium bifidum BB36 as described above.

[0010] The present invention also provides a Bifidobacterium bifidum BB36 probiotic product, the components of which include Bifidobacterium bifidum BB36 as described above.

[0011] This invention also provides an application of the above-described Bifidobacterium bifidum BB36 in the preparation of any of the following functional products:

[0012] (1) Functional products used to activate macrophages and upregulate the expression level of immune factors;

[0013] (2) Functional products used to activate dendritic cells and enhance antigen presentation capabilities during the immune process;

[0014] (3) Functional products that have the ability to enrich calcium, iron and selenium and serve as carriers of mineral elements;

[0015] (4) Functional products used to inhibit the growth of Escherichia coli and Staphylococcus aureus.

[0016] The application of Bifidobacterium bifidum BB36 as described above in the preparation of functional products that enhance the body's immunity or protect intestinal health. Furthermore, the functional products include one of capsules, probiotic powder, or pellets.

[0017] Furthermore, the functional products include functional foods or health supplements.

[0018] Compared with the prior art, the Bifidobacterium bifidum BB36 provided by the present invention, which enhances antigen presentation ability and enriches calcium, iron, and selenium, has the following beneficial effects:

[0019] The Bifidobacterium bifidum BB36 provided by this invention has outstanding dual immune activation advantages: it can not only effectively activate macrophages and enhance the body's non-specific immune response, but also directly activate dendritic cells, significantly improving antigen presentation efficiency, thereby simultaneously strengthening innate and adaptive immunity, achieving multi-level, multi-target synergistic immune regulation, and a more comprehensive and efficient mechanism of action. While exerting its immunomodulatory function, this strain also has the ability to load essential minerals such as calcium, iron, and selenium, breaking through the limitations of traditional probiotics with their single function. By combining mineral delivery with immune activation, it can synergistically enhance the body's immunity while supplementing trace elements and promoting nutrient absorption, providing an innovative solution for developing products with combined nutritional and immune functions. With its highly efficient immune activation advantages and enhanced nutrient absorption capacity, the Bifidobacterium bifidum BB36 of this invention can be widely used in products that improve immunity, providing a safer and more effective solution for people pursuing health. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 : Colony morphology of Bifidobacterium bifidum BB36;

[0022] Figure 2 Microscopic morphology of Bifidobacterium bifidum BB36;

[0023] Figure 3 Effects of different concentrations of Bifidobacterium bifidum BB36 fermentation broth on macrophage proliferation;

[0024] Figure 4 Effects of Bifidobacterium bifidum BB36 on the expression of immune factors in macrophages;

[0025] Figure 5 Effects of different concentrations of Bifidobacterium bifidum BB36 fermentation broth on dendritic cell proliferation;

[0026] Figure 6 Activation effect of Bifidobacterium bifidum BB36 on dendritic cells;

[0027] Figure 7 Growth of Bifidobacterium bifidum BB36 under different iron concentrations;

[0028] Figure 8 Determination of the conversion rate of Bifidobacterium bifidum BB36 at different iron concentrations;

[0029] Figure 9 Growth of Bifidobacterium bifidum BB36 under different calcium concentrations;

[0030] Figure 10 Determination of the conversion rate of Bifidobacterium bifidum BB36 at different calcium concentrations;

[0031] Figure 11 Growth of Bifidobacterium bifidum BB36 at different selenium concentrations;

[0032] Figure 12 Determination of the conversion rate of Bifidobacterium bifidum BB36 at different selenium concentrations;

[0033] Figure 13 Results of antibiotic resistance in Bifidobacterium bifidum BB36;

[0034] Figure 14 Results of hemolytic activity test for Bifidobacterium bifidum BB36;

[0035] Figure 15 The antibacterial effect of Bifidobacterium bifidum BB36 on Escherichia coli and Staphylococcus aureus. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] This invention provides a Bifidobacterium bifidum ( Bifidobacterium bifidum BB36 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37593 and deposit date of February 2, 2026.

[0038] Source of raw materials: The Bifidobacterium bifidum was isolated from the intestines of healthy infants in Xiamen, Fujian Province. After sequencing analysis and Blast sequence alignment, this strain was found to be highly homologous to Bifidobacterium bifidum, and was named Bifidobacterium bifidum BB36.

[0039] Colony morphology: In MRS+cysteine ​​solid medium, the colonies are white, round, and have smooth and neat edges.

[0040] Functions: It can activate macrophages, upregulate the expression of immune factors, activate dendritic cells, enhance antigen presentation ability, enrich calcium, iron and selenium, and inhibit Escherichia coli and Staphylococcus aureus.

[0041] Example 1: Isolation and identification of Bifidobacterium bifidum BB36

[0042] 1. Separation:

[0043] The plate coating method was used. 5 g of sample was placed in a sterile homogenizing bag, and 45 mL of 0.85% physiological saline was added. The mixture was then homogenized to obtain the sample dilution. After thorough mixing, 100 μL of sample was sequentially diluted 10-fold. A dilution factor of 10 was selected. -3 10 -4 10 -5 The sample dilution solution was pipetted into a 200 μL container and spread onto a solid medium containing MRS and cysteine. The container was then incubated anaerobically at 37°C for 48 h.

[0044] Different strains were initially selected based on their morphology and color. They were repeatedly isolated and purified using the streak plate method until all colonies on the MRS+cysteine ​​solid medium maintained a single morphology. Single colonies were then picked and cultured in MRS+cysteine ​​liquid medium at 37°C for 48 h. The fermentation broth of the strains was then mixed with 50% glycerol in equal proportions and stored in glycerol tubes and preserved in a bacterial bank at -80°C.

[0045] The colony morphology of the isolated and purified Bifidobacterium bifidum BB36 was as follows: in MRS + cysteine ​​solid medium, the colonies were white, round, with smooth and regular edges, as shown in the figure. Figure 1 As shown.

[0046] The formula for MRS liquid medium with cysteine ​​is as follows: 10.0 g beef meal, 20.0 g glucose, 10.0 g tryptone, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g ammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, 0.5 g cysteine, 1.0 L deionized water, pH 6.5 (adding 2% agar makes it MRS solid medium).

[0047] 2. Strain identification:

[0048] The screened and purified strains were Gram-stained to check whether their morphology was uniform. Figure 2 As shown.

[0049] The bacterial genome was extracted according to the instructions of the bacterial DNA extraction kit, and 16S rDNA was amplified. The amplified product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing, and the isolated strain was identified as Bifidobacterium bifidum. Bifidobacterium bifidum BB36. Its gene sequence is shown in SEQ ID NO.1.

[0050] SEQ ID NO.1

[0051] ATTTGATAAGTGTCTTGTGGGTGAGATTCTATCGGCGTGGGATGGGGTCGCGTCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTTGGGAGCAAGCCTTCGGGTGAGTGTACCTTTCGAATAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGCTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCGATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGACGCTGGATGTGGGGCACGTTCCACGTGTTCCGTGTCGGAGCTAACGCGTTAAGCGTCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTCCCGACGACGCCAGAGATGCGTATTAGGAAC

[0052] Example 2: Determination of the intestinal colonization ability of the strain

[0053] 1. Hydrophobicity test

[0054] After activating the strain for 2 - 3 generations, wash the bacterial cells 2 - 3 times with PBS (Phosphate Buffered Saline), and adjust the bacterial count to 1.9 The absorbance (A0) of the bacterial suspension at 600 nm was measured using cfu / mL. 3 mL of bacterial suspension was added to 1 mL of xylene, pre-cultured at room temperature for 10 min, then rapidly vortexed for 2 min. The mixture was allowed to stand at room temperature for 15 min until the solution separated into layers. The absorbance (A0) of the lower aqueous phase was measured at 600 nm. Each sample was tested in triplicate.

[0055] The hydrophobicity of the cell surface of the strain was calculated according to formula (1). Among them, the hydrophobicity of Bifidobacterium bifidum BB36 was calculated to be 36.48±0.07%, which is moderately hydrophobic.

[0056] Equation (1)

[0057] 2. Cell adhesion rate determination: the ability of the strain to adhere to HT-29 cells.

[0058] HT-29 cells were seeded into 6-well cell culture plates and cultured until a monolayer was formed. The cells were washed twice with sterile PBS, and 0.5 mL of 10⁻⁶ PBS was added. 5 CFU / mL Bifidobacterium bifidum BB36 was incubated at 37°C for 3 h. The cells were washed three times with sterile PBS to remove unadhered strains. 500 μL of 0.25% trypsin digestion solution was added, followed by digestion in a 37°C, 5% CO2 incubator for 3-5 min until the cells were completely detached. 500 μL of MEM complete medium was added to terminate the digestion. The bacterial suspension was collected, diluted, and spread. The viable number of Lactobacillus was determined by plate count. The adhesion rate of Bifidobacterium bifidum BB36 to HT-29 cells was calculated to be (47.37±0.13)% according to Equation 2.

[0059] Adhesion rate (%) = (Number of viable bacteria adhered / Total number of bacteria initially added) × 100% Equation (2)

[0060] Example 3: Acid and bile salt resistance test

[0061] 1. Tolerance in artificial gastric fluid

[0062] The activated Bifidobacterium bifidum BB36 was inoculated into MRS + cysteine ​​liquid medium and cultured at 37°C for 48 h to obtain the fermentation broth.

[0063] Inoculate 10% of the bacterial culture into MRS+cysteine ​​medium at pH 3 and incubate at 37°C. At 0 h and 1 h, take 1 mL of the bacterial culture, dilute it to the appropriate concentration with sterile physiological saline, and spread it onto MRS+cysteine ​​agar plates. After anaerobic incubation at 37°C for 36-48 h, perform plate counting.

[0064] The viable count of Bifidobacterium bifidum BB36 was determined by plate count method. The viable count measured at 0 h was taken as the initial viable count (N0), and the viable count measured after 1 h of treatment was taken as the post-treatment viable count (N1). The survival rate was calculated according to formula (3).

[0065] The results showed that the survival rate of Bifidobacterium bifidum BB36 was 86.06% after 1 h of culture at pH 3.

[0066] Survival rate (%) = (N1 / N0) × 100% Equation (3)

[0067] The results of this experiment show that after being treated in a simulated acidic environment of the human stomach (pH 3.0) for 1 hour, the viable bacterial survival rate of Bifidobacterium bifidum BB36 reached as high as 86.06%. This demonstrates that this strain has strong tolerance to gastric acid and can pass through the stomach with a high survival rate, providing a key guarantee for its subsequent colonization in the intestine and exerting its probiotic function.

[0068] 2. Tolerance in artificial pancreatic juice

[0069] Trypsin was added to MRS+cysteine ​​liquid culture medium to prepare 0.2% simulated artificial pancreatic juice. The mixture was thoroughly dissolved and mixed, then filtered through a 0.22 μm filter membrane for sterilization and set aside for later use.

[0070] The activated Bifidobacterium bifidum BB36 was inoculated into MRS + cysteine ​​liquid medium and cultured at 37°C for 48 h to obtain the fermentation broth. The Bifidobacterium bifidum BB36 fermentation broth was then inoculated into the above-mentioned 0.2% simulated artificial pancreatic juice at a 10% inoculation rate and incubated at 37°C for 1 h.

[0071] 1 mL of bacterial culture was taken at 0 h and 1 h respectively, diluted with sterile physiological saline to a suitable dilution and spread on MRS+cysteine ​​solid plates. After anaerobic culture at 37℃ for 36-48 h, the viable number of Bifidobacterium bifidum BB36 was determined by plate colony counting method. The viable number measured at 0 h was taken as the initial viable number (N0), and the viable number measured after 1 h of treatment was taken as the post-treatment viable number (N1). The survival rate was calculated according to formula (3).

[0072] The results showed that the survival rate of Bifidobacterium bifidum BB36 was 70.61%.

[0073] The results of this experiment show that after treatment in a simulated human intestinal pancreatic juice environment (containing 0.2% pancreatic enzyme) for 1 hour, the survival rate of Bifidobacterium bifidum BB36 was approximately 70.61%. This indicates that the strain has a certain tolerance to trypsin and can maintain high activity in intestinal digestive fluids.

[0074] Example 4: Effects of Bifidobacterium bifidum BB36 on the proliferation and immunomodulatory activity of RAW264.7 macrophages

[0075] 1. Cell resuscitation and culture

[0076] RAW264.7 macrophages were revived by a 37°C water bath and then subjected to 1500 r·min - Centrifuge for 3 min and discard the supernatant containing the cryopreservation solution. Resuspend the cells in 5 mL of complete culture medium (DEME, containing 10% fetal bovine serum and 1% penicillin-streptomycin), transfer to a T25 culture flask, and incubate at 37°C in a 5% CO2 incubator. When the cell confluence reaches approximately 80%, discard the culture medium, gently pipette the complete culture medium to collect the cells, centrifuge, and discard the supernatant. Seed the cell suspension into 96-well plates, 200 μL per well, and incubate for 24 h until the cells adhere. After the cells have filled the 96-well plates, discard the culture medium and wash with PBS. The following groups were set up: blank group (complete culture medium only), LPS group (complete culture medium + LPS), and experimental group (complete culture medium + 10% penicillin-streptomycin). -1 -10 -4 (Gradually diluted Bifidobacterium bifidum BB36 fermentation broth). The cells were further cultured at 37°C and 5% CO2 for 24 h.

[0077] 2. Cell proliferation and activity detection

[0078] After the cells were cultured, 1% CCK-8 solution was added to each well, and the cells were incubated at 37°C and 5% CO2 for 1 h. The absorbance of each well was measured at 450 nm using a microplate reader. The results are as follows: Figure 3 The dilution gradient of Bifidobacterium bifidum BB36 fermentation broth was 10. -3 The relative cell proliferation rate was 105.73%, indicating that this concentration of fermentation broth had no damaging effect on cell proliferation. Therefore, the dilution gradient was set at 10... -3 As the experimental concentration for the following experiments.

[0079] 3. Neutral red phagocytosis detection

[0080] After culturing the cells as described in step 1 above, discard the culture medium, wash with PBS, and add 100 μL of neutral red solution (1 mg·mL⁻¹) to each well. -1 Incubate for 1 h. Discard the supernatant, wash with PBS, and add 100 μL of cell lysis buffer (ethanol: glacial acetic acid = 1:1). Incubate at 37°C and 5% CO2 for 4 h. Measure absorbance at 540 nm to evaluate the phagocytic activity of RAW264.7 macrophages.

[0081] The blank group received only 100 μL of DMEM medium, with all other treatments the same as the other groups. The control group (untreated group) was inoculated with RAW264.7 cells and inoculated with 100 μL of blank DMEM medium without Bifidobacterium bifidum BB36 fermentation broth, without adding any sample. The BB36 treatment group was inoculated with RAW264.7 cells and inoculated with 100 μL of DMEM medium to a final concentration of 10. - The fermentation broth of Bifidobacterium bifidum BB36 was diluted 3 times. The relative phagocytic capacity was calculated according to formula (4).

[0082] Relative phagocytic capacity (%) = (BB36 treatment group OD) 540 - Blank group OD 540 ) / (Control group OD 540 - Blank group OD 540 Formula (4) × 100%

[0083] The results are shown in Table 1 below. Adding 10... -3 The fermentation broth of Bifidobacterium bifidum BB36 increased the phagocytic capacity of macrophages to 140.16% of the untreated group. This indicates that the addition of 10... -3 The fermentation broth of Bifidobacterium bifidum BB36 can enhance the body's ability to defend against and eliminate foreign substances by macrophages.

[0084] Table 1: Results of detecting the phagocytic capacity of Bifidobacterium bifidum BB36 on RAW264.7 macrophages

[0085]

[0086] 4. NO production detection

[0087] After culturing cells according to the above method, the supernatant was collected, and the absorbance was measured at 540 nm using a microplate reader according to the Beyotime NO kit. The results are shown in Table 2. The experimental group (complete culture medium + 10...) -3 The NO production of the Bifidobacterium bifidum BB36 fermentation broth (+cells) was 2.33 times that of the control group (complete culture medium + cells) and lower than that of the positive control group (complete culture medium + LPS). This indicates that the Bifidobacterium bifidum BB36 fermentation broth can enhance the NO production of macrophages, exhibiting bactericidal and antitumor activities, thereby strengthening the body's defense capabilities, while avoiding inflammatory damage and cellular exhaustion caused by excessive immune responses.

[0088] Table 2. Effects of Bifidobacterium bifidum BB36 on NO production in macrophages.

[0089]

[0090] 5. Relative expression levels of cellular immune factors test

[0091] After culturing cells according to the above method, cells were collected, and mRNA was extracted and reverse transcribed. The relative expression levels of TNF-α, COX-2, IL-6, and IL-1β were tested using qPCR. -ΔΔCt The relative expression level of the target gene is calculated using this method.

[0092] The results are as follows Figure 4 As shown, compared with the blank control group, the relative mRNA expression levels of four immune factors, TNF-α, COX-2, IL-6, and IL-1β, were significantly upregulated in the Bifidobacterium bifidum BB36 experimental group. P <0.05). This indicates that the fermentation broth of this strain can effectively activate RAW264.7 macrophages, promote their synthesis and secretion of immune effector molecules, thereby enhancing the body's innate immune response.

[0093] The upregulation of expression of each factor in the experimental group was significantly lower than that in the LPS-positive control group. P <0.05). LPS, as a potent pathogen-associated molecular model, typically triggers a severe inflammatory response. In this study, Bifidobacterium bifidum BB36 exhibited a mild yet effective immune activation model. It promoted factor expression to a degree sufficient to initiate a beneficial immune response, while avoiding the excessive inflammatory response and potential tissue damage risks associated with LPS.

[0094] Example 5: Effects of Bifidobacterium bifidum BB36 on the proliferation and immunomodulatory activity of dendritic cells (BMDCs)

[0095] The experiment further tested whether Bifidobacterium bifidum BB36 could directly activate BMDCs (bone marrow-derived dendritic cells), that is, to deeply elucidate the precise molecular mechanism by which probiotics exert their immunomodulatory effects from the perspective of innate immune recognition and cellular immune activation.

[0096] 1. Cell resuscitation and culture

[0097] After reviving dendritic cells (BMDCs) in a 37°C water bath, they were subjected to 1500 r·min -Centrifuge for 3 min and discard the supernatant containing the cryopreservation solution. Resuspend the cells in 5 mL of complete culture medium (DEME, containing 10% fetal bovine serum and 1% penicillin-streptomycin), transfer to a T25 culture flask, and incubate at 37°C in a 5% CO2 incubator. When the cell confluence reaches approximately 80%, discard the culture medium, gently pipette the complete culture medium to collect the cells, centrifuge, and discard the supernatant. Seed the cell suspension into 96-well plates, 200 μL per well, and incubate for 24 h until the cells adhere. After the cultured cells have filled the 96-well plates, discard the culture medium and wash with PBS. The following groups were set up: blank group (complete culture medium only), LPS group (complete culture medium + LPS), and experimental group (complete culture medium + 10% penicillin-streptomycin). -1 -10 -4 (Gradually diluted Bifidobacterium bifidum BB36 fermentation broth). The cells were further cultured at 37°C and 5% CO2 for 24 h.

[0098] 2. Cell proliferation and activity detection

[0099] Add 1% CCK-8 solution to each well of the cultured cells from step 1 above, incubate at 37°C and 5% CO2 for 1 h, and then measure the absorbance of each well at 450 nm using a microplate reader. The results are as follows: Figure 5 As shown, the dilution gradient of the Bifidobacterium bifidum fermentation broth was 10. -3 The relative cell proliferation rate was 104.78%, indicating that this concentration of fermentation broth promoted cell proliferation. Therefore, the dilution gradient was set at 10... -3 As the experimental concentration for the following experiments.

[0100] 3. Dendritic cell activation test

[0101] After culturing cells according to method 1 above, cells were collected, and mRNA was extracted, reverse transcribed, and qPCR was performed to test the relative expression levels of CD80, CD86, IL-10, and TNF-α. Experimental results are as follows: Figure 6 As shown, Bifidobacterium bifidum BB36 significantly upregulated the mRNA expression of dendritic cell co-stimulatory molecules CD80 and CD86, and simultaneously promoted the production of the pro-inflammatory cytokine TNF-α. This clearly indicates that dendritic cells successfully initiated the maturation and immune activation process, laying the molecular foundation for their effective presentation of antigens to T cells and the provision of key co-stimulatory signals. However, compared with the strong response induced by the typical potent stimulant LPS, the activation signals induced by Bifidobacterium bifidum BB36 were at a relatively mild intensity level. While effectively enhancing the antigen presentation and immune initiation capabilities of dendritic cells, it may have avoided the excessive inflammatory storm caused by LPS, demonstrating a highly efficient and controlled immune regulatory model.

[0102] Example 6: Accumulation capacity of Bifidobacterium bifidum BB36 for inorganic iron

[0103] 1. Activation of the strain

[0104] Bifidobacterium bifidum BB36 was cultured anaerobically at 37°C for 48 h in test tubes containing MRS + cysteine ​​medium. Then, it was transferred to a fresh Erlenmeyer flask containing MRS + cysteine ​​medium at an inoculum of 3% (v / v) and cultured until the late logarithmic phase for later use.

[0105] 2. Growth of Bifidobacterium bifidum BB36 under different iron concentrations

[0106] Logarithmic-phase Bifidobacterium bifidum BB36 was inoculated at a rate of 3% into MRS + cysteine ​​culture medium containing different iron concentrations (FeCl3 concentrations of 0, 30, 60, 90, and 120 μg / mL), and cultured at 37℃ for 48 h to obtain fermentation broths for different treatments. The OD values ​​of the different fermentation broths were then measured. 600nm The growth of Bifidobacterium bifidum BB36 under different iron concentrations was analyzed. The results are as follows: Figure 7 As shown, FeCl3 concentrations of 30-120 μg / mL did not have a significant impact on the growth of Bifidobacterium bifidum BB36.

[0107] 3. Iron conversion rate determination

[0108] The iron-enriched solutions under different culture conditions were centrifuged at 6000 r / min for 10 min, and the supernatant was collected. The concentration of residual inorganic iron in the supernatant was measured by atomic absorption spectrometry. The iron conversion rate of Bifidobacterium bifidum BB36 was calculated using Formula 5, where:

[0109] C 添加 The initial FeCl3 concentration (μg / mL) added to the culture medium, i.e. 30, 60, 90, and 120 μg / mL;

[0110] C 残留 : The residual inorganic iron concentration (μg / mL) measured in the supernatant.

[0111] The results are as follows Figure 8 As shown, the lactic acid bacteria exhibited strong enrichment and transformation capabilities for iron within the experimental concentration range (30–120 μg / mL), with the inorganic iron conversion rate consistently remaining at a high level. This indicates that the lactic acid bacteria strain can effectively perform organic iron transformation under different iron stress environments, possessing potential application value as a microbial iron carrier or iron fortifier.

[0112] Equation (5)

[0113] Example 7: Accumulation capacity of Bifidobacterium bifidum BB36 for inorganic calcium

[0114] 1. Activation of the strain

[0115] Bifidobacterium bifidum BB36 was cultured anaerobically at 37°C for 48 h in test tubes containing MRS + cysteine ​​medium. Then, it was transferred to a fresh Erlenmeyer flask containing MRS + cysteine ​​medium at an inoculum of 3% (v / v) and cultured until the late logarithmic phase for later use.

[0116] 2. Growth of Bifidobacterium bifidum BB36 under different calcium concentrations

[0117] Logarithmic-phase Bifidobacterium bifidum BB36 was inoculated at a rate of 3% into MRS + cysteine ​​culture medium containing different calcium concentrations (CaCl2 concentrations of 0, 0.5, 1.0, 1.5, and 2.0 mg / mL), and cultured at 37℃ for 48 h. Fermentation broths of different treatments were obtained, and the OD values ​​of the different fermentation broths were measured. 600nm The growth of Bifidobacterium bifidum BB36 was analyzed under different calcium concentrations.

[0118] The results are as follows Figure 9 As shown, CaCl2 concentrations of 0.5–2.0 mg / mL did not significantly affect the growth of Bifidobacterium bifidum BB36.

[0119] 3. Calcium conversion rate determination

[0120] The calcium-enriched solutions under different culture conditions were centrifuged at 6000 r / min for 10 min, and the supernatant was collected. The concentration of residual inorganic calcium in the supernatant was measured by atomic absorption spectrometry. The calcium conversion rate of Bifidobacterium bifidum BB36 was calculated using Equation 5, where:

[0121] C 添加 The initial concentration of CaCl2 added to the culture medium (μg / mL) is 0.5, 1.0, 1.5, and 2.0 μg / mL.

[0122] C 残留 : The residual inorganic calcium concentration (μg / mL) measured in the supernatant.

[0123] The results are as follows Figure 10As shown, within the experimentally defined concentration range (0.5-2.0 mg / mL), lactic acid bacteria exhibited excellent enrichment and conversion capabilities for calcium, converting inorganic calcium into organic calcium, with the conversion rate showing a significant concentration-dependent increase. This result indicates that within a certain substrate concentration range, increasing the calcium source concentration can effectively promote the enrichment efficiency of lactic acid bacteria, and the strain demonstrates good potential as a microbial method for producing organic calcium or developing calcium biofortifiers.

[0124] Example 8: Enrichment capacity of Bifidobacterium bifidum BB36 for inorganic selenium

[0125] 1. Activation of the strain

[0126] Bifidobacterium bifidum BB36 was cultured anaerobically at 37°C for 48 h in test tubes containing MRS + cysteine ​​medium. Then, it was transferred to a fresh Erlenmeyer flask containing MRS + cysteine ​​medium at an inoculum of 3% (v / v) and cultured until the late logarithmic phase for later use.

[0127] 2. Growth of Bifidobacterium bifidum BB36 under different selenium concentrations

[0128] Logarithmic-phase Bifidobacterium bifidum BB36 was inoculated at a rate of 3% into MRS + cysteine ​​culture medium containing different selenium concentrations (sodium selenite concentrations of 0, 30, 60, 90, and 120 μg / mL), and cultured at 37℃ for 48 h to obtain fermentation broths for different treatments. The OD values ​​of the different fermentation broths were then measured. 600nm The growth of Bifidobacterium bifidum BB36 was analyzed under different selenium concentrations.

[0129] The results are as follows Figure 11 As shown in the experimental results, sodium selenite exhibits a typical "low-promoting, high-inhibiting" dual effect on the growth of Bifidobacterium bifidum BB36. At lower concentrations (0-90 μg / mL), sodium selenite, as an essential trace element, promotes bacterial growth, possibly due to its involvement in or activation of certain intracellular metabolic pathways. However, when the concentration is increased to 90-120 μg / mL, sodium selenite exhibits significant toxicity, inhibiting bacterial growth. This is usually due to excessive inorganic selenium (Se). + It can trigger oxidative stress, interfere with normal enzyme function and damage cell structure, leading to growth inhibition.

[0130] 3. Selenium conversion rate determination

[0131] The selenium-enriched solutions under different culture conditions were centrifuged at 6000 r / min for 10 min, and the supernatant was collected. The concentration of residual inorganic selenium in the supernatant was measured by atomic absorption spectrometry. The conversion rate of selenium by Bifidobacterium bifidum BB36 was calculated using Equation 5, where:

[0132] C 添加 The initial concentration (μg / mL) of sodium selenite added to the culture medium, i.e., 30, 60, 90, and 120 μg / mL;

[0133] C 残留 : The residual inorganic selenium concentration (μg / mL) measured in the supernatant.

[0134] The results are as follows Figure 12 As shown, Bifidobacterium bifidum BB36 has a certain capacity for selenium enrichment and biotransformation. Among them, the conversion rate of selenium by Bifidobacterium bifidum BB36 is the highest when the sodium selenite concentration is 60 μg / mL.

[0135] Example 9: Antibiotic Resistance Analysis of Bifidobacterium bifidum BB36

[0136] Bifidobacterium bifidum BB36 was activated for two generations and cultured in MRS + cysteine ​​liquid medium at 37°C until a McFarland turbidity of 0.5 was reached. 200 μL of the bacterial culture was added dropwise to the surface of MRS + cysteine ​​solid medium and evenly spread using a spreader. Within 15 min after inoculation, antibiotic susceptibility testing discs were laid flat on the medium surface, allowed to dry, and then incubated upside down at 37°C for 8–24 h. The diameter of the complete inhibition zone was measured to assess the antibiotic susceptibility of the strain. The results are shown below. Figure 13 As shown in Table 3;

[0137] The specifications of the drug sensitivity test strips are as follows: tetracycline (TET, 30 μg / tablet), amoxicillin (AMX, 10 μg / tablet), azithromycin (AZI, 15 μg / tablet), chloramphenicol C (30 μg / tablet), erythromycin E (15 μg / tablet), and ampicillin (AMP, 10 μg / tablet).

[0138] According to the interpretation criteria of the inhibition range of the paper disc method for antibiotic susceptibility testing, tetracycline (TET, 30 μg / tablet) with an inhibition zone diameter ≥18 mm is considered sensitive (S); amoxicillin (AMX, 10 μg / tablet) with an inhibition zone diameter ≥18 mm is considered sensitive (S); azithromycin (AZI, 15 μg / tablet) with an inhibition zone diameter of 14~17 mm is considered intermediate (I); chloramphenicol C (30 μg / tablet) with an inhibition zone diameter ≥18 mm is considered sensitive (S); erythromycin E (15 μg / tablet) with an inhibition zone diameter ≥23 mm is considered sensitive (S); and ampicillin (AMP, 10 μg / tablet) with an inhibition zone diameter ≥17 mm is considered sensitive (S).

[0139] Table 3: Results of resistance analysis of Bifidobacterium bifidum BB36 to common antibiotics

[0140]

[0141] Example 10: Hemolytic activity test of Bifidobacterium bifidum BB36

[0142] Take Bifidobacterium bifidum BB36 glycerol-preserved bacteria from the bacterial bank and activate it for two generations. Add 20% sterile defibrinated sheep blood to the MRS+cysteine ​​solid medium preparation solution at 50-55℃, mix well, pour into a petri dish, and obtain blood agar medium after solidification. Pick a single colony of activated Bifidobacterium bifidum BB36 and streak it on the blood agar medium. Incubate at 37℃ for 24 h. Observe whether there is a hemolytic clear zone around the colony. If there is, it is a positive hemolysis test.

[0143] After 24 hours of blood agar incubation, the experimental results are as follows: Figure 14 As shown, no hemolytic zone was observed on the streak plate of Bifidobacterium bifidum BB36, while Staphylococcus aureus, as a positive control, formed a clear transparent hemolytic zone around the colony. This result indicates that the Bifidobacterium bifidum BB36 strain does not exhibit hemolysis and is safe for use in food.

[0144] Example 11 Antibacterial activity test of Bifidobacterium bifidum BB36

[0145] Bifidobacterium bifidum BB36 was inoculated at a ratio of 2% into MRS + cysteine ​​liquid medium for activation, and then statically cultured at 37°C for 48 h to activate the first-generation strain. The bacterial culture was then transferred to MRS + cysteine ​​liquid medium at a 2% inoculum and cultured at 37°C for 48 h to activate the second-generation strain, thus obtaining the fermentation broth.

[0146] Centrifuge the fermentation broth at 1000 rpm for 2 min, discard the bacterial cells, and obtain the supernatant.

[0147] The fermentation broth was sterilized in a high-pressure steam sterilizer at 121°C for 15 minutes to obtain post-biotic.

[0148] Escherichia coli and Staphylococcus aureus grown to the logarithmic growth phase were inoculated into MRS + cysteine ​​solid medium at a 2% (v / v) inoculum concentration, mixed well, and approximately 10 mL was poured into each culture dish. After the medium solidified, Oxford cups were placed on each solid medium plate and gently pressed to ensure complete contact between the cups and the medium. 200 μL of Bifidobacterium bifidum BB36 fermentation broth, supernatant, and post-genetic agent were added to the Oxford cups, respectively, and the plates were incubated upright at 37°C for 12 h.

[0149] The results are as follows Figure 15 As shown, the fermentation broth, supernatant, and metabiotics of Bifidobacterium bifidum BB36 all inhibited the growth of Escherichia coli and Staphylococcus aureus.

[0150] Example 12: Preparation of bacterial powder using Bifidobacterium bifidum BB36

[0151] Bifidobacterium bifidum BB36 was fermented at high density and the bacterial sludge was collected by centrifugation. The bacterial sludge and a protective agent were mixed in a 1:1 mass ratio and then freeze-dried under vacuum. The protective agent was a mixture of trehalose and skim milk in a 1:1 ratio.

[0152] Example 13: Bifidobacterium bifidum BB36 probiotic product

[0153] This embodiment provides a functional solid beverage with Bifidobacterium bifidum BB36 as its core. Each 100g of the finished product contains: 10.0g of active bacterial powder (obtained by high-density fermentation of the strain, centrifugation to collect bacterial sludge, and vacuum freeze-drying with a freeze-drying protectant [trehalose:skim milk = 1:1]); 5.0g of fermented astragalus powder; 3.0g of fermented angelica powder; dietary fiber carrier: inulin (72.0g, accounting for 72.0%); flavor modifiers: erythritol (8.0g) and citric acid (2g). Preparation process: The above components are mixed evenly under conditions of <10% humidity and 25℃, and packaged into 2g / bags.

[0154] It should be noted that the above concept is based on the preparation of probiotic products by Bifidobacterium bifidum BB36 with other ingredients. The selection of ingredients and the ratio of ingredients can be adjusted adaptively, including but not limited to the example scheme.

[0155] Based on the results of the above embodiments, the Bifidobacterium bifidum BB36 provided by the present invention has the following properties and effects:

[0156] 1. Activate macrophages and upregulate the expression level of immune factors.

[0157] 2. Directly activates dendritic cells, enhancing antigen presentation capabilities.

[0158] 3. It is enriched with calcium, iron, and selenium, and can serve as a biological carrier of mineral elements.

[0159] 4. Inhibits Escherichia coli and Staphylococcus aureus, protecting intestinal health.

[0160] 5. It has strong resistance to acid and bile salts, and is well tolerated in artificial gastric and intestinal fluids, allowing it to reach the human intestine smoothly.

[0161] It should be noted that:

[0162] (1) Definition:

[0163] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders, tablets, and gels, and also to disperse in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0164] (2) The relevant prior art means or prior art terms involved in this application:

[0165] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through a analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. "OD625" is the optical density value measured at a wavelength of 625 nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is commonly used to indicate bacterial cell density. The method for measuring "OD" values ​​is existing technology, and its principles and methods will not be elaborated here.

[0166] Application of strains:

[0167] The example illustrates that Bifidobacterium bifidum BB36 can be applied to immune-enhancing products. Based on the above design concept, this strain can be applied to capsules, probiotic powders, crystal balls, etc.

[0168] Although this document frequently uses terms such as Bifidobacterium bifidum, probiotics, postbiotics, and bacterial powder, the possibility of using other terms is not excluded. These terms are used merely for the convenience of describing and explaining the essence of this invention; interpreting them as any additional limitation would contradict the spirit of this invention.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Bifidobacterium ( Bifidobacterium bifidum BB36, characterized in that: Its accession number is CGMCC No.37593.

2. A fermentation broth, characterized in that: It is obtained by fermentation of Bifidobacterium bifidum BB36 as described in claim 1.

3. An epigenetic product, characterized in that... It is prepared by fermentation and high-pressure steam sterilization of Bifidobacterium bifidum BB36 as described in claim 1, wherein the high-pressure steam sterilization conditions are 121°C for 15 min.

4. A composition, characterized in that: The composition includes Bifidobacterium bifidum BB36 fermentation broth and / or Bifidobacterium bifidum BB36 fermentation supernatant.

5. A Bifidobacterium bifidum BB36 bacterial powder, characterized in that... Its components include Bifidobacterium bifidum BB36 as described in claim 1.

6. A Bifidobacterium bifidum BB36 probiotic product, characterized in that... Its components include Bifidobacterium bifidum BB36 as described in claim 1.

7. A Bifidobacterium bifidum BB36 capsule, characterized in that... Its components include Bifidobacterium bifidum BB36 as described in claim 1.

8. A type of Bifidobacterium bifidum BB36 crystal ball, characterized in that... Its components include Bifidobacterium bifidum BB36 as described in claim 1.

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