Bifidobacterium breve BBr69 with immunomodulatory function, its applications, products and methods

Abstract

The present invention relates to a Bifidobacterium breve BBr69 with immunomodulatory function, its applications, products and methods, belonging to the field of microbial technology. The preservation number of the Bifidobacterium breve BBr69 with immunomodulatory function is CGMCC NO. 32425. Based on this strain, the present invention also provides its uses and related products. The BBr69 strain has broad-spectrum antibacterial properties, can regulate the intestinal flora, and also has an enhanced immune function. It enhances the immunity by proliferating RAW264.7 cells, increasing the cytokine content, inhibiting weight loss, increasing the immune organ index, and reducing ileum tissue damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Bifidobacterium breve BBr69 with immunomodulatory function, and its applications, products and methods. Background Art

[0002] The immune system is composed of immune molecules, immune cells and immune organs, and can protect the body from foreign pathogens. Cyclophosphamide (CTX) is one of the most commonly used anti-cancer drugs in clinical practice and has been used to treat various cancers and autoimmune diseases, such as liver cancer, rectal cancer, breast cancer and rheumatoid arthritis. However, the treatment effect of CTX on patients is accompanied by serious adverse reactions, including immunosuppression and intestinal flora disorder.

[0003] Immunosuppression will reduce the body's ability to fight bacteria and viruses. With the increasing number of immune diseases globally, improving immune protection has become an urgent problem to be solved. Therefore, developing new and safer immunomodulators is one of the most effective methods for preventing and treating immunosuppressive diseases.

[0004] The intestine, as an important immune organ of the human body, plays an important role in immune regulation. At present, the Bifidobacterium breve provided on the market cannot meet the multiple selection needs of industrial production, the market and consumers, etc., and the available selection space is small, especially in terms of enhancing immunity. The existing Bifidobacterium breve strains provided in the art are few, and few can increase the abundance of the genus Lactobacillus in the intestine. Therefore, there is still a need to develop more beneficial new Bifidobacterium breve strains in this field. Summary of the Invention

[0005] Based on the above needs existing in the prior art in this field, the present invention provides a Bifidobacterium breve BBr69 with immunomodulatory function, and its applications, products and methods.

[0006] The technical solution of the present invention is as follows:

[0007] A Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with immunomodulatory function, and its preservation number is CGMCC NO.32425.

[0008] Use of Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with preservation number CGMCC NO.32425 in the preparation of food with enhanced immune function, and / or, food for regulating intestinal flora, and / or, bacteriostatic agent, and / or, bacteriostasis for non-therapeutic purposes.

[0009] The said enhancement of immunity includes: proliferating RAW264.7 cells, increasing the content of cytokines, inhibiting weight loss, increasing the immune organ index, and reducing ileum tissue damage;

[0010] Preferably, the cytokines include: IL-6, IL-10, IL-17, IL-1β, TNF-α, SIgA;

[0011] Preferably, the immune organ indices include: thymus index, spleen index;

[0012] Preferably, the regulation of intestinal flora includes: increasing the abundances of Clostridia_UCG_014_unclassified genus ( unclassified_Clostridia_UCG_014 ), Prevotellaceae_NK3B31_group ( Alloprevotella ), Alistipes ( Alistipes ), Helicobacteraceae_NK4A136_group ( Lachnospiraceae_NK4A136_group ), Ligilactobacillus ( Ligilactobacillus ), Lactobacillus ( Lactobacillus ), and decreasing the abundances of Desulfovibrio ( Desulfovibrio ), Desulfovibrionaceae_unclassified ( unclassified_Desulfovibrionaceae );

[0013] Preferably, the antibacterial spectrum of the bacteriostatic agent includes: Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus paris, Streptococcus pasteurianus.

[0014] An immune function enhancing food, comprising functional active ingredients; the functional active ingredients include: Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with the preservation number of CGMCC NO. 32425.

[0015] The above-mentioned immune function enhancing food further comprises: excipients.

[0016] A food for regulating intestinal flora, comprising functional active ingredients; the functional active ingredients include: Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with the preservation number of CGMCC NO. 32425.

[0017] The above-mentioned food for regulating intestinal flora further comprises: excipients.

[0018] A bacteriostatic agent, comprising bacteriostatic active ingredients; the bacteriostatic active ingredients include: Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with the preservation number of CGMCC NO. 32425.

[0019] The above-mentioned bacteriostatic agent comprises: excipients.

[0020] An in vitro bacteriostatic method, which uses Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with the preservation number of CGMCC NO. 32425 for bacteriostasis.

[0021] The beneficial effects of the present invention are as follows:

[0022] A strain of bacteria was isolated from an infant fecal sample in the present invention, and it was found to have a relatively broad-spectrum antibacterial ability, showing good inhibitory effects on numerous pathogenic bacteria including Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus parasanguinis, and Streptococcus pasteurianus. After classifying and identifying this strain, it was known to be a strain of Bifidobacterium breve ( Bifidobacterium breve ), and it was named BBr69. Through a large number of experiments, the present invention has proven that in addition to its broad-spectrum antibacterial ability, the BBr69 strain also has excellent immune-enhancing and intestinal flora-regulating functions. The BBr69 strain can significantly proliferate RAW264.7 cells, increase the contents of cytokines including IL-6, IL-10, IL-17, IL-1β, TNF-α, and SIgA, inhibit weight loss, increase the indexes of immune organs such as thymus index and spleen index, and reduce ileal tissue damage; at the same time, the BBr69 strain can also significantly increase the abundances of Clostridia_UCG_014_unclassified genus ( unclassified_Clostridia_ UCG_014 ), Alloprevotella ( Alloprevotella ), Alistipes ( Alistipes ), Helicobacteraceae_NK4A136_group ( Lachnospiraceae_NK4A136_group ), Ligilactobacillus ( Ligilactobacillus ), and Lactobacillus ( Lactobacillus ), and reduce the abundances of Desulfovibrio ( Desulfovibrio ) and unclassified Desulfovibrionaceae ( unclassified_Desulfovibrionaceae ).

[0023] The preservation information of the Bifidobacterium breve ( Bifidobacterium breve ) BBr69 of the present invention is as follows:

[0024] Preservation number: CGMCC NO.32425;

[0025] Taxonomic name: Bifidobacterium breve Bifidobacterium breve ;

[0026] Preservation date: October 30, 2024;

[0027] Preservation unit: China General Microbiological Culture Collection Center;

[0028] Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the Drawings

[0029] Figure 1 It is a line graph showing the effect of Bifidobacterium breve BBr69 on the body weight of immunodeficient mice in Experimental Example 6 of the present invention.

[0030] Figure 2Bar chart showing the effect of Bifidobacterium breve BBr69 in Experimental Example 6 of the present invention on the immune organs of immunosuppressed mice. A is the bar chart of thymus index, and B is the bar chart of spleen index.

[0031] Figure 3 Bar chart showing the effect of Bifidobacterium breve BBr69 in Experimental Example 7 of the present invention on various immune factors in the serum of immunosuppressed mice.

[0032] Figure 4 Pathological section diagram showing the effect of Bifidobacterium breve BBr69 in Experimental Example 8 of the present invention on the ileum tissue of immunosuppressed mice.

[0033] Figure 5 Bar chart showing the results of α-diversity analysis of the intestinal flora of each group of mice in Experimental Example 9 of the present invention.

[0034] Figure 6 Diagram showing the results of β-diversity analysis of the intestinal flora of each group of mice in Experimental Example 9 of the present invention.

[0035] Figure 7 Bar chart showing the results of genus-level species diversity analysis of the intestinal flora of each group of mice in Experimental Example 9 of the present invention.

[0036] Figure 8 Diagram showing the results of OTUs clustering analysis of the intestinal flora of each group of mice in Experimental Example 9 of the present invention.

[0037] The labels in the figure are listed as follows: NC: normal control group, MC: immunosuppressive model group, BBr69: Bifidobacterium breve BBr69 group. Detailed implementation manners

[0038] The present invention will be further described in detail below with reference to the drawings and embodiments. However, the implementation manners of the present invention are not limited thereto, nor does it limit the protection scope of the present invention.

[0039] I. Sources of biological materials

[0040] (I) Escherichia coli, Salmonella and Staphylococcus aureus used in Experimental Example 3 of the present invention are commercially available. Porphyromonas gingivalis ATCC BAA-308 (W83) was purchased from the Guangdong Microbial Culture Collection Center. Streptococcus parasanguinis 12758, this strain has been registered in NCBI, and its accession number is MW445225; Streptococcus paris is Streptococcus paris 21178, this strain has been registered in NCBI, and its accession number is OM403351. The above strains are all preserved in the applicant's laboratory. The applicant undertakes to distribute them to the public for verifying the technical effects of the present invention within 20 years from the filing date of the present invention.

[0041] (II) RAW264.7 cells used in Experimental Examples 4 and 5 of the present invention are commercially available.

[0042] (3) The BALB / c male mice (20 - 22 g) used in Experimental Examples 6, 7, 8, and 9 of the present invention were purchased from Beijing Speywood Biotechnology Co., Ltd.

[0043] II. Culture media used in the experimental examples of the present invention:

[0044] (1) MRS solid medium: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium hydrogen citrate, 20 g of glucose, 1 mL of Tween 80, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 15 g of agar, 1000 mL of distilled water, sterilized at 121 °C for 20 min, and the pH was adjusted to 6.8.

[0045] (2) MRS liquid medium: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium hydrogen citrate, 20 g of glucose, 1 mL of Tween 80, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 1000 mL of distilled water, sterilized at 121 °C for 20 min, and the pH was adjusted to 6.8.

[0046] (3) LMRS medium: 0.1% L-cysteine hydrochloride was added to the MRS medium.

[0047] (4) BHI medium was purchased from Qingdao Hope Bio-Technology Co., Ltd., and sterile defibrinated sheep blood was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0048] Unless otherwise specified, various reagents and consumables used in the examples and experimental examples of the present invention are commercially available, and the relevant experimental steps are common operations in the art and have technical meanings that can be routinely understood by those skilled in the art.

[0049] Group 1 Examples, Bifidobacterium breve BBr69 of the present invention

[0050] This group of examples provides a Bifidobacterium breve with immunomodulatory function ( Bifidobacterium breve ) BBr69, and its preservation number is CGMCC NO.32425.

[0051] Any act of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, reconstructing, selling, or offering to sell a strain of Bifidobacterium breve BBr69 with the deposit number CGMCC NO. 32425, and / or any act of combining a strain of Bifidobacterium breve BBr69 with the deposit number CGMCC NO. 32425 with other probiotics, and / or any act of antagonizing various pathogenic bacteria with a strain of Bifidobacterium breve BBr69 with the deposit number CGMCC NO. 32425, and / or preparing an antibacterial agent, and / or preparing a food for enhancing immune function, and / or preparing a food for regulating intestinal flora function falls within the protection scope of the present invention.

[0052] The other probiotics include, but are not limited to: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces cerevisiae, Torulaspora delbrueckii, Candida spp., Wickerhamomyces spp., Pichia spp., Saccharomyces boulardii, Torulopsis candida, Schizosaccharomyces pombe, Rhodotorula rubra, Schizosaccharomyces pombe, Bacillus thuringiensis, Brevibacillus laterosporus, Bacillus megaterium, Bacillus mucilaginosus, Bacillus azotofixans, Bacillus sphaericus, Clostridium butyricum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium infantis (i.e., Bifidobacterium longum subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis subsp. lactis), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium thermophilum, and Bifidobacterium thermophilum acidophilus.

[0053] Those skilled in the art can, according to actual production needs, combine conventional technical means or basic common sense in the production process of the pharmaceutical field (such as "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Inoculants", etc.) to make a conventional selection or adjustment of pharmaceutical excipients, and then make a strain of Bifidobacterium breve BBr69 with the deposit number CGMCC NO. 32425 into products with different dosage forms, different storage conditions, and different shelf lives. This is not a technical obstacle for those skilled in the art and can be easily achieved.

[0054] Group 2 Examples, Uses of Bifidobacterium breve BBr69 of the Present Invention

[0055] This group of examples provides Bifidobacterium breve with the deposit number CGMCC NO. 32425 ( Bifidobacterium breveUse of BBr69 in the preparation of food for enhancing immune function, and / or food for regulating intestinal flora function, and / or bacteriostatic agent, and / or bacteriostasis for non-therapeutic purposes.

[0056] In a specific embodiment, the enhancement of immunity includes: proliferation of RAW264.7 cells, increase in cytokine content, inhibition of weight loss, elevation of immune organ index, and reduction of ileum tissue damage;

[0057] Preferably, the cytokines include: IL-6, IL-10, IL-17, IL-1β, TNF-α, SIgA;

[0058] Preferably, the immune organ indices include: thymus index, spleen index;

[0059] Preferably, the regulation of intestinal flora includes: increasing the abundances of Clostridia_UCG_014_unclassified genus ( unclassified_Clostridia_UCG_014 ), Alloprevotella ( Alloprevotella ), Alistipes ( Alistipes ), Lachnospiraceae_NK4A136_group ( Lachnospiraceae_NK4A136_group ), Ligilactobacillus ( Ligilactobacillus ), Lactobacillus ( Lactobacillus ), and decreasing the abundances of Desulfovibrio ( Desulfovibrio ), Desulfovibrionaceae_unclassified ( unclassified_Desulfovibrionaceae );

[0060] Preferably, the bacteriostatic spectrum of the bacteriostasis includes: Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus paris, Streptococcus pasteurianus.

[0061] Group 3 embodiments, food for enhancing immune function of the present invention

[0062] This group of embodiments provides a food for enhancing immune function. All embodiments in this group have the following common features: the food for enhancing immune function includes functional active ingredients; the functional active ingredients include: Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with the preservation number of CGMCC NO.32425.

[0063] In a further embodiment, the food for enhancing immune function further includes: excipients.

[0064] In a more specific embodiment, the auxiliary materials are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0065] According to the content of the present invention, for different requirements in actual production applications, combined with the conventional technical means in the field of preparation (for example, "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Bacterial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned auxiliary materials, and make Bifidobacterium breve BBr69 with the preservation number CGMCC NO. 32425 into different dosage forms, such as powder, tablet, oral liquid, gel, patch, spray, lotion, granule, etc.

[0066] In a specific embodiment, the dosage form of the bacteriostatic agent is selected from one or more of: powder, tablet, liquid, capsule.

[0067] Group 4 Examples, Functional Food for Regulating Intestinal Flora of the Present Invention

[0068] This group of examples provides a functional food for regulating intestinal flora. All examples in this group have the following common characteristics: The functional food for regulating intestinal flora includes functional active ingredients; the functional active ingredients include: Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with the preservation number CGMCC NO. 32425.

[0069] In a further embodiment, the functional food for regulating intestinal flora further includes: auxiliary materials.

[0070] In a more specific embodiment, the auxiliary materials are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0071] According to the content of the present invention, for different requirements in actual production applications, combined with the conventional technical means in the field of food preparation (such as "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Inoculant Technology", etc.), those skilled in the art can select and formulate the above excipients, and make Bifidobacterium breve BBr69 with the preservation number CGMCC NO. 32425 into different functional food dosage forms, such as solid beverages, milk beverages, powders, tablets, oral liquids, gels, patches, sprays, granules, etc.

[0072] In a specific embodiment, the dosage form of the functional food for regulating intestinal flora is selected from one or more of the following: solid beverage, milk beverage, tablet, liquid agent, and capsule.

[0073] Group 5 Examples, the bacteriostatic agent of the present invention

[0074] This group of examples provides a bacteriostatic agent. All examples in this group have the following common characteristics: the bacteriostatic agent includes a bacteriostatic active ingredient; the bacteriostatic active ingredient includes: Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with the preservation number CGMCC NO. 32425.

[0075] In a further embodiment, the bacteriostatic agent further includes: excipients.

[0076] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0077] According to the content of the present invention, for different requirements in actual production applications, combined with the conventional technical means in the field of food preparation (such as "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Inoculant Technology", etc.), those skilled in the art can select and formulate the above excipients, and make Bifidobacterium breve BBr69 with the preservation number CGMCC NO. 32425 into different functional food dosage forms, such as solid beverages, milk beverages, powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0078] In a specific embodiment, the dosage form of the immunopotentiating functional food is selected from one or more of solid beverages, milk beverages, tablets, liquid preparations, and capsules.

[0079] Example Group 6, in vitro antibacterial method of the present invention

[0080] This group of embodiments provides an in vitro antibacterial method. It is characterized in that Bifidobacterium breve ( Bifidobacterium breve ) BBr69 with the preservation number of CGMCC NO.32425 is used for antibacterial.

[0081] In a specific embodiment, the antibacterial spectrum of the antibacterial includes: Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus parasanguinis, Streptococcus pasteurianus.

[0082] The behavior of using a strain of Bifidobacterium breve BBr69 with the preservation number of CGMCC NO.32425 in combination with other probiotics for in vitro antibacterial falls within the protection scope of the present invention.

[0083] The other probiotics include but are not limited to: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces cerevisiae, Torulaspora delbrueckii, Candida spp., Wickerhamomyces spp., Pichia spp., Saccharomyces boulardii, Torulopsis glabrata, Schizosaccharomyces pombe, Rhodotorula rubra, Schizosaccharomyces pombe, Bacillus thuringiensis, Brevibacillus laterosporus, Bacillus megaterium, Bacillus mucilaginosus, Bacillus azotofixans, Bacillus sphaericus, Clostridium butyricum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium infantis (i.e., Bifidobacterium longum subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis subsp. lactis), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium thermophilum, and Bifidobacterium thermophilum acidophilus.

[0084] Experimental Example 1, strain isolation and screening

[0085] The collected infant fecal samples were placed in a sterile sampling tube and transported in an ice box. Under sterile conditions, they were serially diluted with 0.85% saline and spread on LMRS agar plates supplemented with 5% (V / V) lithium mupirocin. They were cultured under anaerobic conditions at 37°C for 48 h. Suspected single colonies were picked by observing their colony morphology with the naked eye, and then examined by microscopy and subjected to preliminary screening and purification culture. After purification, they were cultured in MRS liquid anaerobic tubes containing 0.1% L-cysteine hydrochloride at 37°C for 8 - 12 hours. After centrifugation to remove the supernatant, they were resuspended in a sterile 30% glycerol aqueous solution and stored in the strain bank of Wuhan Microkang Probiotics Research Institute.

[0086] Experimental Example 2: Strain Identification

[0087] The target strains screened were cultured in liquid for expansion, and the cells were collected. Genomic DNA was extracted, and the 16S rDNA fragment was amplified using the universal primers 27F and 1492R described in paragraph 58 of Chinese Invention Patent ZL202210478937.4. The PCR amplification products were detected by agarose gel electrophoresis, and the PCR products were sequenced. The PCR reaction system was as follows: 10×buffer 10 μL, 10 mM dNTP 2 μL, 1 μL each of the upstream and downstream primers, 2 μL of DNA template, 0.5 μL of Taq enzyme, and ddH 2 O 34 μL. Pre-denaturation was carried out at 95°C for 10 min; then 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min were performed, and after completion, extension was carried out at 72°C for 5 min. The PCR products were sent to Wuhan Kingcare Bioengineering Co., Ltd. for sequencing after detection by gel electrophoresis. The identified gene sequences were aligned in the NCBI database using the BLAST tool. According to the results of molecular biological identification, the Latin name of the strain was Bifidobacterium breve determined to be Bifidobacterium breve. This strain was named Bifidobacterium breve BBr69 and was deposited, and its deposit information is as follows:

[0088] Deposit number: CGMCC NO.32425;

[0089] Taxonomic name: Bifidobacterium breve Bifidobacterium breve ;

[0090] Deposit date: October 30, 2024;

[0091] Depositary institution: China General Microbiological Culture Collection Center;

[0092] Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0093] Example 3: Inhibitory Ability of Bifidobacterium breve BBr69 against Pathogenic Bacteria

[0094] (1)Preparation of Bifidobacterium breve BBr69 fermentation broth

[0095] Inoculate into L-MRS anaerobic tube medium at an inoculation amount of 2%, and culture at 37 °C for 16 hours.

[0096] (2)Preparation of pathogenic bacteria

[0097] Escherichia coli: Inoculate into LB liquid medium at 2% (v / v), culture at 37 °C for 16 - 20 h, and then adjust the bacterial liquid concentration to 10 8 CFU / mL.

[0098] Salmonella: Inoculate into LB liquid medium at 2% (v / v), culture at 37 °C for 16 - 20 h, and then adjust the bacterial liquid concentration to 10 8 CFU / mL.

[0099] Staphylococcus aureus: Inoculate into LB liquid medium at 2% (v / v), culture at 37 °C for 16 - 20 h, and then adjust the bacterial liquid concentration to 10 8 CFU / mL.

[0100] Streptococcus parasanguinis: Inoculate into BHI plate containing 5% defibrinated sheep blood, culture at 37 °C for 16 - 20 h, and then adjust the bacterial liquid concentration to 10 8 CFU / mL.

[0101] Streptococcus pasteurianus: Inoculate into BHI plate containing 5% defibrinated sheep blood, culture at 37 °C for 16 - 20 h, and then adjust the bacterial liquid concentration to 10 8 CFU / mL.

[0102] Porphyromonas gingivalis: Inoculate into TSA plate containing 5% defibrinated sheep blood, anaerobically culture at 37 °C for 48 - 72 h, and then adjust the bacterial liquid concentration to 10 8 CFU / mL.

[0103] (3)Antibacterial experiment

[0104] Cool the MRS medium containing 1.5% agar (BHI medium containing 5% defibrinated sheep blood for Streptococcus parasanguinis and Streptococcus pasteurianus, TSA medium containing 5% defibrinated sheep blood for Porphyromonas gingivalis) to about 55 °C, mix it with the indicator bacteria suspension in a certain proportion so that the viable count of the indicator bacteria is at the order of 10 6 CFU / mL, and then quickly pour it into the plate pre-placed with Oxford cups. After the medium cools and solidifies, take out the Oxford cups, inject 200 μL of the strain fermentation broth (the viable count is at the order of 10 8 CFU) into each well, culture overnight at 37 °C, and then measure the diameter of the inhibition zone.

[0105] Table 1. Antagonistic ability of BBr69 against pathogenic bacteria

[0106]

[0107] The experimental results are shown in Table 1. Bifidobacterium breve BBr69 has strong inhibitory effects on Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus paris, Streptococcus pasteurianus, and Porphyromonas gingivalis.

[0108] Example 4. Effect of Bifidobacterium breve BBr69 on the proliferation ability of RAW264.7 cells

[0109] RAW264.7 cells were inoculated into a 96-well cell culture plate at a density of 1×10 5 cells / mL (100 μL per well) and cultured for 24 hours. After the cells adhered, the original culture medium was replaced with DMEM cell culture medium containing bacteria. The experiment was set up with a control group (DMEM culture medium) and a probiotic group (the bacterial solution was centrifuged, washed twice with PBS, and then resuspended with DMEM). Each group had 5 replicates. They were cultured in an incubator at 37°C with 5% CO 2 for 24 h. After the culture ended, the old culture medium was aspirated, and 100 μL of DMEM medium containing 10% CCK-8 was added to each well. It was placed in the dark and continued to be cultured in the CO 2 incubator for 2 h; the OD values of each well were measured at 450 nm, and the relative cell proliferation rate was calculated according to the following formula.

[0110] Relative cell proliferation rate % = (OD 1 – OD 0 ) / (OD 2 – OD 0 )

[0111] where OD 1 is the OD of the cells after treatment with the experimental strain, OD 0 is the OD of the culture medium, and OD 2 is the OD of the cells and the culture medium.

[0112] The experimental results showed that the proliferation rate of Bifidobacterium breve BBr69 on RAW264.7 cells was 178.68 ± 0.35%, indicating that BBr69 has the potential for immunomodulation.

[0113] Example 5. Effect of Bifidobacterium breve BBr69 on cytokine secretion of RAW264.7 cells

[0114] Adjust the concentration of RAW264.7 cells to 1×10 6cells / mL, inoculate 0.5 mL per well into a 24-well plate and culture for 24 h. The experiment was divided into a blank group and a probiotic group. Blank group: aspirate the supernatant, wash twice with PBS, add only 0.5 mL of DMEM medium per well, without any other treatment, and place it in an incubator at 37 °C and 5% CO 2 and culture for 24 h; Probiotic group: aspirate the supernatant, wash twice with PBS, add 0.5 mL of bacterial suspension to the well plate (the bacterial solution was centrifuged, washed twice with PBS, and then resuspended with DMEM, diluted according to the ratio of the amount of bacteria to the amount of cells of 10:1), and continue to culture for 24 h. After the culture, take out the supernatant, then centrifuge at 1000 rpm / min for 5 min, and use a kit to measure the contents of TNF-α and IL-10 in the supernatant.

[0115] Table 2. Experimental results of the detection of immunomodulatory factors

[0116]

[0117] The experimental results are shown in Table 2. The results show that compared with the CK group, Bifidobacterium breve BBr69 can promote the production of cytokines TNF-α and IL-10, indicating that strain BBr69 has an immunomodulatory effect.

[0118] Experimental Example 6. Effects of Bifidobacterium breve BBr69 on the body weight and immune organs of mice

[0119] Establishment of a mouse immunosuppression model:

[0120] Specific pathogen-free male BALB / c mice (20 - 22 g) were housed in the animal room of Hubei Food and Drug Safety Evaluation Center. The breeding temperature was maintained at 22 ± 2 °C, the humidity was 50 ± 5%, and the light-dark cycle was 12 hours. Free access to drinking water and food was allowed.

[0121] Before the experiment, the mice were adaptively bred for one week, and then the mice were randomly divided into three groups, namely: normal control group (NC), immunosuppression model group (MC), and Bifidobacterium breve group (BBr69 group).

[0122] Throughout the experiment, the mice in the NC group and the MC group were gavaged with 0.2 mL of sterile normal saline every day, and the mice in the BBr69 group were gavaged with an equal volume of Bifidobacterium breve BBr69 bacterial suspension (the viable count was 10 9 CFU / mL). On days 7 - 9 of the experiment, the mice in the MC group and the BBr69 group were continuously injected with cyclophosphamide (CTX, 80 mg / kg / d) for 3 days to establish an immunosuppressed mouse model, and the NC group was injected with an equal volume of sterile normal saline. On the 15th day, the mice were sacrificed and the samples were collected for subsequent analysis. The detailed animal experiment is shown in Table 3.

[0123] Table 3. Grouping of animal experiment design

[0124]

[0125] After 1 week of adaptive feeding, the mice were randomly divided into 3 groups. The body weights of the experimental mice were measured on the 1st, 7th, 10th, and 14th days. At the time of dissection, the spleens and thymuses of the mice were collected, and the thymus index and spleen index of the mice were calculated.

[0126] The calculation formula for the immune organ index is as follows:

[0127] Immune organ index = mass of immune organ (mg) / body weight (g)

[0128] The experimental results are as Figure 1 shown. Compared with the NC group, the body weights of the mice in the model group decreased significantly after cyclophosphamide injection. Compared with the model group, the body weight of the mice in the BBr69 group decreased more slowly after cyclophosphamide injection. On the 14th day, the body weight of the mice in the BBr69 group was significantly higher than that in the MC group, indicating that BBr69 significantly inhibited the body weight loss of immunosuppressed mice induced by cyclophosphamide. In addition, from Figure 2 the thymus index and spleen index, it was found that compared with the NC group, the thymus and spleen indexes of the mice in the MC group decreased significantly, indicating that the thymus and spleen of the mice in the MC group atrophied. Compared with the MC group, the thymus and spleen indexes of the mice in the BBr69 group increased significantly, indicating that intragastric administration of BBr69 could relieve the atrophy of the thymus and spleen of mice induced by cyclophosphamide.

[0129] Experimental Example 7. Effect of Bifidobacterium breve BBr69 on serum cytokines of mice

[0130] The blood of the mice was collected into 2 mL centrifuge tubes and centrifuged at 4000 rpm / min for 10 minutes. The supernatant was stored in a -20°C refrigerator for subsequent experiments. According to the experimental method of the ELISA kit, the contents of IL-6, IL-10, IL-17, IL-1β, TNF-α, and SIgA in the serum of the mice were measured. The results are as Figure 3 shown. As an immunosuppressant, cyclophosphamide decreased the levels of various cytokines related to immunity in the serum of mice. Compared with the model group, intragastric administration of BBr69 could significantly increase the contents of IL-6, IL-10, IL-17, IL-1β, TNF-α, and SIgA in the serum.

[0131] Experimental Example 8. Effect of Bifidobacterium breve BBr69 on ileum tissue of mice

[0132] After the mice were sacrificed, 0.5 cm of the ileum tissue of the mice was collected, immersed and fixed in 10% formaldehyde solution, then stained with hematoxylin-eosin (HE), and finally the ileum structure was observed using an optical microscope. As Figure 4As shown, the ileal mucosa of the MC group was severely damaged, the intestinal muscular layer and intestinal glands were absent, the intestinal villi were destroyed and severely reduced, the ileal tissue structure was incomplete, and inflammatory cell infiltration was visible. Compared with the MC group, the intestinal injury of the BBr69 group of mice was restored, and the intestinal morphological structure was relatively complete, indicating that Bifidobacterium breve BBr69 could enhance the intestinal mucosal barrier and reduce ileal tissue damage.

[0133] Experimental Example 9. Effect of Bifidobacterium breve BBr69 on the intestinal flora of mice

[0134] The diversity of the intestinal flora was sequenced based on the Illumina Novaseq sequencing platform using the paired-end sequencing method to construct a small fragment library for sequencing. By splicing and filtering the reads, clustering or denoising, and performing species annotation and abundance analysis, the species composition of the samples could be revealed; further alpha diversity analysis, beta diversity analysis, species diversity analysis, etc. were carried out to explore the differences between the samples.

[0135] (1) Alpha-diversity analysis and beta-diversity analysis

[0136] The alpha-diversity analysis of the intestinal flora of mice showed that Figure 5 , compared with the control group, the ACE and Chao 1 indices of the intestinal flora of the model group mice were significantly reduced, indicating that CTX induced intestinal flora disorder in mice and caused a decrease in intestinal flora diversity. After feeding BBr69, the ACE and Chao 1 indices of the intestinal flora of mice were significantly higher than those of the model group, indicating that BBr69 could regulate the intestinal flora and increase the richness of the intestinal flora in immunocompromised mice. Beta-diversity measures the similarity of the flora composition between different samples, that is, it focuses on the differences in the flora composition between samples. The experimental results are as Figure 6 shown. The MC group was significantly separated from the NC group, indicating that there were significant differences in the microbial composition between the MC group and the NC group. The BBr69 group was adjacent to the NC group, indicating that gavage with BBr69 made the microbial community composition in the intestines of immunocompromised mice tend to be normal.

[0137] (2) Species diversity analysis at the genus level

[0138] Figure 7 What is shown is the relative abundance at the genus level of the cecal contents of mice in each group. Compared with the NC group, the relative abundances of Clostridia_UCG_014_unclassified genus ( unclassified_Clostridia_UCG_014 ), Alloprevotella ( Alloprevotella ), Alistipes ( Alistipes ), and Helicobacteraceae_NK4A136_group ( Lachnospiraceae_ NK4A136_group ) in the MC group were significantly reduced, and the relative abundances of Desulfovibrio ( Desulfovibrio ) and unclassified Desulfovibrionaceae ( unclassified_Desulfovibrionaceae)Relative abundance increased. Compared with the MC group, the relative abundances of Clostridia_UCG_014_unclassified genus ( unclassified_Clostridia_UCG_014 ), Alloprevotella ( Alloprevotella ), Alistipes ( Alistipes ), and Lachnospiraceae_NK4A136_group ( Lachnospiraceae_ NK4A136_group )were increased in the B. breve BBr69 group. Meanwhile, the relative abundances of Ligilactobacillus ( Ligilactobacillus )and Lactobacillus ( Lactobacillus )were significantly increased. In addition, compared with the MC group, the relative abundances of Desulfovibrio ( Desulfovibrio )and unclassified Desulfovibrionaceae ( unclassified_Desulfovibrionaceae )were significantly decreased in the B. breve BBr69 group. Alloprevotella ( Alloprevotella )can produce short-chain fatty acids (SCFAs) mainly composed of succinic acid and acetic acid, which can improve the intestinal epithelial barrier and prevent inflammation. Lactobacillus ( Lactobacillus )and its metabolites interact with immune receptors, leading to the production of immunomodulatory cytokines to resist the invasion of foreign pathogens. The results showed that B. breve BBr69 could increase the abundance of beneficial bacterial genera and decrease the abundance of harmful bacterial genera in the host intestine, promote the restoration of intestinal flora homeostasis, improve the intestinal barrier, and play an immunomodulatory role.

[0139] (3)OTUs clustering analysis

[0140] The experimental results were as Figure 8 shown. The number of shared OTUs between the MC group and the NC group was 582, and the number of shared OTUs between the BBr69 group and the NC group was 640, indicating that gavage with B. breve BBr69 could increase the shared intestinal flora between immunosuppressed mice and normally fed mice.

Claims

1. A Bifidobacterium breve with immunomodulatory function ( Bifidobacterium breve ) BBr69, characterized in that Its deposit number is CGMCC NO.32425.

2. Bifidobacterium breve with a deposit number of CGMCC NO.32425 ( Bifidobacterium breve ) Use of BBr69 in the preparation of foods for enhancing immune function, and / or foods for regulating intestinal flora, and / or antibacterial agents, and / or antibacterial agents for non-therapeutic purposes; the intestinal flora regulation is: increasing the number of Clostridium_UCG_014_Unclassified bacteria ( unclassified_ Clostridia_UCG_014 ), Pseudo-Prevotella ( Alloprevotella ), another genus ( Alistipes )、Helicobacter_NK4A136_group( Lachnospiraceae_NK4A136_group ), Lactobacillus spp. ( Ligilactobacillus ), Lactobacillus spp. Lactobacillus ) and reduced the abundance of Desulfovibrio ( Desulfovibrio ), unclassified Desulfovibrioides ( unclassified_Desulfovibrionaceae ) abundance; The antibacterial spectrum of the antibacterial agent is: Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus Parisi and Streptococcus pasteurianus.

3. The Bifidobacterium breve (CGMCC NO.32425) according to claim 2. Bifidobacterium breve ) Use of BBr69 in the preparation of foods for enhancing immune function, and / or foods for regulating intestinal flora, and / or antibacterial agents, and / or non-therapeutic antibacterial agents, characterized in that: The immunity enhancement includes: proliferating RAW264.7 cells, increasing cytokine content, inhibiting weight loss, increasing immune organ index, and reducing ileum tissue damage.

4. The Bifidobacterium breve (CGMCC NO.32425) according to claim 3. Bifidobacterium breve ) Use of BBr69 in the preparation of foods for enhancing immune function, and / or foods for regulating intestinal flora, and / or antibacterial agents, and / or non-therapeutic antibacterial agents, characterized in that: The cytokines include: IL-6, IL-10, IL-17, IL-1β, TNF-α, and SIgA.

5. The Bifidobacterium breve (CGMCC NO.32425) according to claim 3. Bifidobacterium breve ) Use of BBr69 in the preparation of foods for enhancing immune function, and / or foods for regulating intestinal flora, and / or antibacterial agents, and / or non-therapeutic antibacterial agents, characterized in that: The immune organ indexes include: thymus index and spleen index.

6. A food for enhancing immune function, comprising functional active ingredients; characterized in that: The functional active ingredients include: Bifidobacterium breve ( Bifidobacterium breve )BBr69.

7. The food for enhancing immune function according to claim 6, characterized in that: Also includes: Auxiliary materials.

8. A functional food for regulating intestinal flora, comprising functional active ingredients; characterized in that: The functional active ingredients include: Bifidobacterium breve ( Bifidobacterium breve )BBr69.

9. The functional food for regulating intestinal flora according to claim 8, characterized in that: Also includes: Auxiliary materials.

10. An antibacterial agent, comprising an antibacterial active ingredient; characterized in that: The antibacterial active ingredients include: Bifidobacterium breve ( Bifidobacterium breve )BBr69.

11. The antibacterial agent according to claim 10, characterized in that: include: Auxiliary materials.

12. An in vitro antibacterial method, characterized in that: The Bifidobacterium breve (CGMCC NO.32425) was used. Bifidobacterium breve )BBr69 was used for antibacterial effect.

Images