Bifidobacterium breve BBr69 with immune regulation function and application, product and method thereof

Abstract

The invention discloses bifidobacterium breve BBr69 with an immunoregulation function as well as application, a product and a method thereof, and belongs to the technical field of microorganisms. The preservation number of the bifidobacterium breve BBr69 with the immunoregulation function is CGMCC (China General Microbiological Culture Collection Center) NO. 32425. Based on the strain, the invention also provides application and related products of the strain. The BBr69 strain has broad-spectrum antibacterial performance, has the function of regulating intestinal flora and also has the function of enhancing immunity, and immunity is enhanced by proliferating RAW264.7 cells, increasing the content of cell factors, inhibiting weight loss, increasing the index of immune organs and reducing ileum tissue damage.

Description

Technical Field

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

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

[0003] Immunosuppression reduces the body's ability to fight bacteria and viruses. With the increase in the number of immune diseases worldwide, improving immune protection has become an urgent issue to be addressed. Therefore, the development of new and safer immunomodulators is one of the most effective ways to prevent and treat immunosuppressive diseases.

[0004] As an important immune organ of the human body, the intestine plays an important role in immune regulation. Currently, the Bifidobacterium breve provided on the market cannot meet the multiple selection requirements of industrial production, market and consumers, and there is little room for selection, especially in enhancing immunity. The prior art in the field provides fewer strains of Bifidobacterium breve, and rarely can improve the abundance of Lactobacillus in the intestine, so the field still needs to develop more beneficial new strains of Bifidobacterium breve. Summary of the invention

[0005] Based on the above-mentioned needs of the prior art in the art, the present invention provides Bifidobacterium breve BBr69 with immunomodulatory function and its application, product and method.

[0006] The technical solution of the present invention is as follows: A Bifidobacterium breve with immunomodulatory function ( Bifidobacterium breve )BBr69, its deposit number is CGMCC NO.32425.

[0007] Bifidobacterium breve (CGMCC NO.32425) Bifidobacterium breve ) Use of BBr69 in the preparation of foods that enhance immune function, and / or foods that regulate intestinal flora, and / or antibacterial agents, and / or antibacterial agents for non-therapeutic purposes.

[0008] The immunity enhancement includes: proliferating RAW264.7 cells, increasing cytokine content, inhibiting weight loss, increasing immune organ index, and reducing ileum tissue damage; Preferably, the cytokines include: IL-6, IL-10, IL-17, IL-1β, TNF-α, SIgA; Preferably, the immune organ index includes: thymus index, spleen index; Preferably, the intestinal flora regulation comprises: 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; Preferably, the antibacterial spectrum includes: Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus Parisi and Streptococcus pasteurianus.

[0009] A food for enhancing immune function, comprising functional active ingredients; the functional active ingredients include: Bifidobacterium breve ( Bifidobacterium breve )BBr69.

[0010] The food for enhancing immune function also includes auxiliary materials.

[0011] A functional food for regulating intestinal flora, comprising functional active ingredients; the functional active ingredients include: Bifidobacterium breve ( Bifidobacterium breve )BBr69.

[0012] The functional food for regulating intestinal flora also includes auxiliary materials.

[0013] An antibacterial agent, comprising an antibacterial active ingredient; the antibacterial active ingredient comprises: Bifidobacterium breve ( Bifidobacterium breve )BBr69.

[0014] The antibacterial agent comprises: auxiliary materials.

[0015] An in vitro antibacterial method, which uses Bifidobacterium breve (CGMCC NO.32425) Bifidobacterium breve )BBr69 was used for antibacterial effect.

[0016] The beneficial effects of the present invention are as follows: The present invention isolates a strain of bacteria from infant feces samples and finds that it has a relatively broad-spectrum antibacterial ability, and has a good inhibitory effect on many pathogens including Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus Parisi, and Streptococcus pasteurianus. The strain is classified and identified as a strain of Bifidobacterium breve ( Bifidobacterium breve ), and named it BBr69. The present invention has proved through a large number of experiments that in addition to its broad-spectrum antibacterial ability, the BBr69 strain also has excellent immune enhancement and intestinal flora regulation functions. The BBr69 strain can significantly proliferate RAW264.7 cells, increase the content of cytokines including IL-6, IL-10, IL-17, IL-1β, TNF-α, SIgA, inhibit weight loss, increase the thymus index, spleen index and other immune organ indexes, and reduce ileal tissue damage; at the same time, the BBr69 strain can also significantly increase the Clostridium class_UCG_014_Unclassified bacteria genus ( 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.

[0017] The Bifidobacterium breve of the present invention ( Bifidobacterium breve )The deposit information of BBr69 is as follows: Deposit number: CGMCC NO.32425; Classification name: Bifidobacterium breve Bifidobacterium breve ; Deposit date: October 30, 2024; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a line graph showing the effect of Bifidobacterium breve BBr69 of Experimental Example 6 of the present invention on the body weight of immunocompromised mice.

[0019] Figure 2 This is a bar graph showing the effects of Bifidobacterium breve BBr69 of Experimental Example 6 of the present invention on immune organs of immunocompromised mice, wherein A is a bar graph of thymus index, and B is a bar graph of spleen index.

[0020] Figure 3A bar graph showing the effects of Bifidobacterium breve BBr69 of Experimental Example 7 of the present invention on various immune factors in the serum of immunocompromised mice.

[0021] Figure 4 Pathological section diagram of the effect of Bifidobacterium breve BBr69 of Experimental Example 8 of the present invention on ileum tissue of immunocompromised mice.

[0022] Figure 5 This is a bar graph of the α-diversity analysis results of the intestinal flora of each group of mice in Experimental Example 9 of the present invention.

[0023] Figure 6 This is a graph showing the β-diversity analysis results of the intestinal flora of each group of mice in Experimental Example 9 of the present invention.

[0024] Figure 7 This is a bar graph of the species diversity analysis results of the intestinal flora of each group of mice in Experimental Example 9 of the present invention at the genus level.

[0025] Figure 8 This is a graph showing the OTUs clustering analysis results of the intestinal flora of each group of mice in Experimental Example 9 of the present invention.

[0026] The numbers in the figure are as follows: NC: normal control group, MC: immunosuppression model group, BBr69: Bifidobacterium breve BBr69 group. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the embodiments of the present invention are not limited thereto, and the protection scope of the present invention is not limited thereto.

[0028] 1. Sources of biological materials (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 Guangdong Microbiological Culture Collection Center. Streptococcus pasteurianus 12758, which has been registered in NCBI, and its registration number is MW445225; Streptococcus Parisi is Streptococcus Parisi 21178, which has been registered in NCBI, and its registration number is OM403351. The above strains are all preserved in the applicant's laboratory, and the applicant promises to release them to the public 20 years from the date of application of the present invention for verification of the technical effects of the present invention.

[0029] (ii) The RAW264.7 cells used in Experimental Examples 4 and 5 of the present invention are commercially available.

[0030] (III) BALB / c male mice (20-22 g) used in Experimental Examples 6, 7, 8, and 9 of the present invention were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0031] 2. Culture medium used in the experimental examples of the present invention: (I) MRS solid medium: peptone 10 g, beef extract 10 g, yeast extract 5 g, diammonium hydrogen citrate 2 g, glucose 20 g, Tween 80 1 mL, sodium acetate 5 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, agar 15 g, distilled water 1000 mL, sterilize at 121 °C for 20 min, adjust pH 6.8.

[0032] (ii) MRS liquid medium: peptone 10 g, beef extract 10 g, yeast extract 5 g, diammonium hydrogen citrate 2 g, glucose 20 g, Tween 80 1 mL, sodium acetate 5 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1000 mL, sterilize at 121°C for 20 min, adjust pH to 6.8.

[0033] (iii) LMRS medium: MRS medium supplemented with 0.1% L-cysteine ​​hydrochloride.

[0034] (IV) BHI medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., and sterile defibrinated sheep blood was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0035] Unless otherwise specified, all kinds of reagents and consumables used in the embodiments and experimental examples of the present invention are commercially available, and the relevant experimental steps are common operations in the field and have technical meanings that can be conventionally understood by those skilled in the art.

[0036] The first group of examples, Bifidobacterium breve BBr69 of the present invention This group of embodiments provides a Bifidobacterium breve having an immunomodulatory function ( Bifidobacterium breve )BBr69, its deposit number is CGMCC NO.32425.

[0037] Any act of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, or promising to sell a strain of Bifidobacterium breve BBr69 with a preservation number of CGMCC NO.32425, and / or using a strain of Bifidobacterium breve BBr69 with a preservation number of CGMCC NO.32425 in combination with other probiotics, and / or using a strain of Bifidobacterium breve BBr69 with a preservation number of CGMCC NO.32425 to antagonize various pathogens, and / or prepare antibacterial agents, and / or prepare foods that enhance immune function, and / or foods that regulate intestinal flora functions, all fall within the scope of protection of the present invention.

[0038] The other probiotics include but are not limited to: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii lactic acid subsp., Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces cerevisiae, Delb's spore-forming torula, Candida, Wickham's yeast, Pichia, Saccharomyces boulardii, Torulopsis spp., Saccharomyces cerevisiae, Rhodotorula, Schizosaccharomyces pombe , Saccharomyces spp., Bacillus thuringiensis, Bacillus laterosporus, Bacillus megaterium, Bacillus mucilaginosus, Bacillus azotobacterium, Bacillus sphaericus, Clostridium butyricum, Bifidobacterium adolescentis, Bifidobacterium angularis, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentate, Bifidobacterium infantis (i.e., Bifidobacterium longum infantis subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis subsp. cremoris), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, as well as Bifidobacterium thermophilum and Bifidobacterium acidothermophilum.

[0039] Those skilled in the art can make conventional selections or adjustments to pharmaceutical excipients according to actual production needs, combined with conventional technical means or basic common sense of pharmaceutical production processes (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), and then make a strain of Bifidobacterium breve BBr69 with a preservation number of CGMCC NO.32425 into products with different dosage forms, different storage conditions, and different shelf lives. This has no technical barriers for those skilled in the art and is possible and easy to do.

[0040] The second group of examples, use of Bifidobacterium breve BBr69 of the present invention This group of embodiments provides Bifidobacterium breve ( Bifidobacterium breve ) Use of BBr69 in the preparation of foods that enhance immune function, and / or foods that regulate intestinal flora, and / or antibacterial agents, and / or antibacterial agents for non-therapeutic purposes.

[0041] In a specific embodiment, the immunity enhancement includes: proliferating RAW264.7 cells, increasing cytokine content, inhibiting weight loss, increasing immune organ index, and reducing ileal tissue damage; Preferably, the cytokines include: IL-6, IL-10, IL-17, IL-1β, TNF-α, SIgA; Preferably, the immune organ index includes: thymus index, spleen index; Preferably, the intestinal flora regulation comprises: 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; Preferably, the antibacterial spectrum includes: Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus Parisi and Streptococcus pasteurianus.

[0042] The third group of embodiments, the food for enhancing immune function of the present invention This group of embodiments provides a food for enhancing immune function. All embodiments of 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.

[0043] In a further embodiment, the immune function enhancing food further includes: auxiliary materials.

[0044] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0045] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of preparation preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare Bifidobacterium breve BBr69 with a preservation number of CGMCC NO.32425 into different dosage forms, such as powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0046] In a specific embodiment, the dosage form of the antibacterial agent is selected from: one or more of powder, tablet, liquid, and capsule.

[0047] The fourth group of embodiments, functional food for regulating intestinal flora of the present invention This group of embodiments provides a functional food for regulating intestinal flora. All embodiments of this group have the following common features: the functional food for regulating intestinal flora includes functional active ingredients; the functional active ingredients include: Bifidobacterium breve ( Bifidobacterium breve )BBr69.

[0048] In a further embodiment, the functional food for regulating intestinal flora further includes: auxiliary materials.

[0049] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0050] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of food preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned auxiliary materials, and prepare Bifidobacterium breve BBr69 with a preservation number of CGMCC NO.32425 into different functional food dosage forms, such as solid beverages, milk beverages, powders, tablets, oral liquids, gels, patches, sprays, granules, etc.

[0051] In a specific embodiment, the dosage form of the functional food for regulating intestinal flora is selected from: one or more of: solid beverages, milk beverages, tablets, liquids, and capsules.

[0052] The fifth group of embodiments, the antibacterial agent of the present invention This group of embodiments provides an antibacterial agent. All embodiments of this group have the following common features: the antibacterial agent includes an antibacterial active ingredient; the antibacterial active ingredient includes: Bifidobacterium breve ( Bifidobacterium breve )BBr69.

[0053] In a further embodiment, the antibacterial agent further comprises: auxiliary materials.

[0054] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0055] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of food preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned auxiliary materials, and prepare Bifidobacterium breve BBr69 with a preservation number of 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.

[0056] In a specific embodiment, the dosage form of the food for enhancing immune function is selected from: one or more of: solid beverages, milk beverages, tablets, liquids, and capsules.

[0057] The sixth group of embodiments, the in vitro antibacterial method of the present invention This group of embodiments provides an in vitro antibacterial method. It is characterized in that the breve Bifidobacterium ( Bifidobacterium breve )BBr69 was used for antibacterial effect.

[0058] In a specific embodiment, the antibacterial spectrum includes: Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus Parisi, and Streptococcus pasteurianus.

[0059] The use of a strain of Bifidobacterium breve BBr69 with a preservation number of CGMCC NO.32425 in combination with other probiotics for in vitro bacteriostasis falls within the protection scope of the present invention.

[0060] The other probiotics include but are not limited to: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii lactic acid subsp., Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces cerevisiae, Delb's spore-forming torula, Candida, Wickham's yeast, Pichia, Saccharomyces boulardii, Torulopsis spp., Saccharomyces cerevisiae, Rhodotorula, Schizosaccharomyces pombe , Saccharomyces spp., Bacillus thuringiensis, Bacillus laterosporus, Bacillus megaterium, Bacillus mucilaginosus, Bacillus azotobacterium, Bacillus sphaericus, Clostridium butyricum, Bifidobacterium adolescentis, Bifidobacterium angularis, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentate, Bifidobacterium infantis (i.e., Bifidobacterium longum infantis subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis subsp. cremoris), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, as well as Bifidobacterium thermophilum and Bifidobacterium acidothermophilum.

[0061] Experimental Example 1: Strain Isolation and Screening The collected infant fecal samples were placed in sterile sampling tubes and transported in ice boxes. They were diluted with 0.85% saline gradients under sterile conditions, spread on LMRS agar plates supplemented with 5% (V / V) mupirocin lithium salt, and cultured at 37°C under anaerobic conditions for 48 hours. Suspected single colonies were picked by visual observation of their colony morphology, and they were observed under a microscope and preliminarily screened and purified. After purification, they were cultured at 37°C for 8-12 hours in MRS liquid anaerobic tubes containing 0.1% L-cysteine ​​hydrochloride, centrifuged to remove the supernatant, and resuspended in a sterile 30% glycerol aqueous solution and stored in the strain bank of Wuhan Weikang Probiotics Research Institute.

[0062] Experimental Example 2: Strain Identification The target strains were cultured in liquid, the cells were collected, the genomic DNA was extracted, and the universal primers 27F and 1492R recorded in paragraph 58 of Chinese invention patent ZL202210478937.4 were used to amplify the 16SrDNA fragment. The PCR amplification products were detected by agarose gel electrophoresis, and the PCR products were sequenced. The PCR reaction system: 10×buffer 10μL, 10mMdNTP 2μL, 1μL of upper and lower primers, 2μL of DNA template, 0.5μL of Taq enzyme, and 34μL of ddH2O. Pre-denaturation at 95℃ for 10min; then 94℃ 30s, 60℃ 30s, and 72℃ 1min for a total of 35 cycles, and then extended at 72℃ for 5min. The PCR products were detected by gel electrophoresis and sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. The identified gene sequences were compared in the NCBI database using the BLAST tool. According to the results of molecular biological identification, the Latin name of the strain isBifidobacterium breve The strain was determined to be Bifidobacterium breve. The strain was named Bifidobacterium breve BBr69 and sent for preservation. The preservation information is as follows: Deposit number: CGMCC NO.32425; Classification name: Bifidobacterium breve Bifidobacterium breve ; Deposit date: October 30, 2024; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0063] Example 3: Inhibitory effect of Bifidobacterium breve BBr69 on pathogenic bacteria (1) Preparation of Bifidobacterium breve BBr69 fermentation broth The culture medium was inoculated into L-MRS anaerobic tube culture medium at a rate of 2% and cultured at 37°C for 16 hours.

[0064] (2) Preparation of pathogens Escherichia coli: Inoculate into LB liquid medium at 2% (v / v), culture at 37°C for 16-20 h, and adjust the bacterial solution concentration to 10 8 CFU / mL.

[0065] Salmonella: Inoculate into LB liquid medium at 2% (v / v), culture at 37°C for 16-20 h, and adjust the bacterial solution concentration to 10 8 CFU / mL.

[0066] Staphylococcus aureus: inoculate into LB liquid medium at 2% (v / v), culture at 37°C for 16-20 h, and adjust the bacterial solution concentration to 10 8 CFU / mL.

[0067] Streptococcus Parisi: Inoculate into BHI plates containing 5% defibrinated sheep blood, incubate at 37°C for 16-20 h, and adjust the bacterial solution concentration to 10 8 CFU / mL.

[0068] Streptococcus pasteurianus: inoculate into BHI plates containing 5% defibrinated sheep blood, incubate at 37°C for 16-20 h, and adjust the bacterial solution concentration to 10 8 CFU / mL.

[0069] Porphyromonas gingivalis: inoculated into TSA plates containing 5% defibrinated sheep blood, cultured anaerobically at 37°C for 48-72 h, and then adjusted to a concentration of 10 8 CFU / mL.

[0070] (3) Antibacterial experiment Cool the MRS medium containing 1.5% agar (BHI medium containing 5% defibrinated sheep blood for Streptococcus Parisi and Streptococcus pasteurianus, TSA medium containing 5% defibrinated sheep blood for Porphyromonas gingivalis) to about 55°C, and mix it with the indicator bacteria suspension in a certain proportion to make the number of viable indicator bacteria reach 10 6 CFU / mL, and then quickly poured into the plate with the Oxford cup placed in advance. After the culture medium cooled and solidified, take out the Oxford cup and inject 200μL of the strain fermentation liquid (the number of viable bacteria is 10 8 CFU level), and the diameter of the inhibition zone was measured after overnight culture at 37°C.

[0071] Table 1. Antagonistic ability of BBr69 against pathogens

[0072] The experimental results are shown in Table 1. Bifidobacterium breve BBr69 has a strong inhibitory effect on Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus Parisi, Streptococcus pasteurianus, and Porphyromonas gingivalis.

[0073] Example 4: Effect of Bifidobacterium breve BBr69 on the proliferation ability of RAW264.7 cells RAW264.7 cells were collected at 1×10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well cell culture plate (100 μL per well) and cultured for 24 hours. After the cells adhered to the wall, the original culture medium was replaced with DMEM cell culture medium containing bacteria. The experiment set up a control group (DMEM culture medium) and a probiotic group (the bacterial solution was centrifuged, washed twice with PBS, and resuspended with DMEM), with 5 replicates in each group. Cultured in a 37°C, 5% CO2 incubator for 24 hours. After the culture was completed, the old culture medium was aspirated, and 100 μL of DMEM culture medium containing 10% CCK-8 was added to each well, protected from light, and placed in a CO2 incubator for further culture for 2 hours; the OD value of each well was detected at 450nm, and the relative cell proliferation rate was calculated according to the following formula.

[0074] Relative cell proliferation rate % = (OD1– OD0) / (OD2– OD0) OD1 is the OD of cells after treatment with the experimental strain, OD0 is the OD of the culture medium, and OD2 is the OD of cells and culture medium.

[0075] 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.

[0076] Example 5: Effect of Bifidobacterium breve BBr69 on cytokine secretion by RAW264.7 cells The concentration of RAW264.7 cells was adjusted to 1 × 10 6 cell / mL, 0.5mL per well was inoculated in a 24-well plate and cultured for 24h. The experiment was divided into a blank group and a probiotic group. Blank group: aspirate the supernatant, wash twice with PBS, add only 0.5mL DMEM medium to each well, without any other treatment, and place in a 37℃ and 5% CO2 incubator for 24h; Probiotic group: aspirate the supernatant, wash twice with PBS, add 0.5mL bacterial suspension to the well plate (centrifuge the bacterial liquid, wash twice with PBS, resuspend with DMEM, and dilute according to the ratio of bacterial volume: cell volume of 10:1), and continue to culture for 24h. After the culture, remove the supernatant, then centrifuge at 1000rpm / min for 5min, and use the kit to determine the content of TNF-α and IL-10 in the supernatant.

[0077] Table 2. Results of immunomodulatory factor detection experiments

[0078] 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.

[0079] Experimental Example 6: Effects of Bifidobacterium breve BBr69 on body weight and immune organs of mice Establishment of immunosuppression model in mice: Specific pathogen-free BALB / c male mice (20–22 g) were housed in the animal room of Hubei Food and Drug Safety Evaluation Center at a temperature of 22 ± 2 °C, a humidity of 50 ± 5%, a light-dark cycle of 12 h, and were allowed free access to water and food.

[0080] Before the experiment, the mice were adaptively fed for one week and then randomly divided into three groups: normal control group (NC), immunosuppression model group (MC), and Bifidobacterium breve group (BBr69 group).

[0081] During the whole experiment, mice in the NC and MC groups were gavaged with 0.2 mL of sterile saline every day, and mice in the BBr69 group were gavaged with an equal amount of Bifidobacterium breve BBr69 suspension (viable count was 10 9 CFU / mL). On days 7 to 9 of the experiment, mice in the MC and BBr69 groups were continuously injected with cyclophosphamide (CTX, 80 mg / kg / d) for 3 days to establish an immunosuppressive mouse model, and the NC group was injected with an equal volume of sterile saline. The mice were killed on day 15, and samples were collected for subsequent analysis. The detailed animal experiments are shown in Table 3.

[0082] Table 3. Animal experiment design and grouping

[0083] After one week of adaptive feeding, the mice were randomly divided into three groups. The body weights of the experimental mice were measured on the 1st, 7th, 10th and 14th days. During the autopsy, the spleen and thymus of the mice were collected, and the thymus index and spleen index of the mice were calculated.

[0084] The calculation formula of immune organ index is as follows: Immune organ index = immune organ mass (mg) / body weight (g) The experimental results are as follows Figure 1 As shown in the figure, compared with the NC group, the body weight of the model group mice decreased significantly after the injection of cyclophosphamide. Compared with the model group, the body weight of the BBr69 group mice decreased more slowly after the injection of cyclophosphamide. On the 14th day, the body weight of the BBr69 group mice was significantly higher than that of the MC group, indicating that BBr69 significantly inhibited the weight loss of immunocompromised mice induced by cyclophosphamide. Figure 2 The thymus index and spleen index of the mice in the MC group were significantly decreased compared with those in the NC group, indicating that the thymus and spleen of the mice in the MC group had atrophy. Compared with the MC group, the thymus and spleen index of the mice in the BBr69 group were significantly increased, indicating that oral administration of BBr69 can alleviate cyclophosphamide-induced atrophy of the thymus and spleen in mice.

[0085] Experimental Example 7: Effects of Bifidobacterium breve BBr69 on serum cytokines in mice The mouse blood was collected into a 2 mL centrifuge tube 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 levels of IL-6, IL-10, IL-17, IL-1β, TNF-α, and SIgA in mouse serum were determined. The results are shown in Figure 3 As shown, cyclophosphamide, as an immunosuppressant, reduced the levels of various immune-related cytokines in mouse serum. Compared with the model group, oral administration of BBr69 significantly increased the levels of IL-6, IL-10, IL-17, IL-1β, TNF-α, and SIgA in serum.

[0086] Experimental Example 8: Effects of Bifidobacterium breve BBr69 on ileum tissue in mice After killing the mice, 0.5 cm2 of ileum tissue was collected, fixed with 10% formaldehyde solution, and then stained with hematoxylin-eosin (HE). Finally, the ileum structure was observed using an optical microscope. Figure 4As shown in the figure, the ileum mucosa in the MC group was severely damaged, the intestinal muscle layer and intestinal glands were missing, the intestinal villi were destroyed and severely reduced, the ileum tissue structure was incomplete, and inflammatory cell infiltration was visible. Compared with the MC group, the intestinal damage of the mice in the BBr69 group was restored, and the intestinal morphology was relatively complete, indicating that Bifidobacterium breve BBr69 can enhance the intestinal mucosal barrier and reduce ileum tissue damage.

[0087] Experimental Example 9: Effects of Bifidobacterium breve BBr69 on intestinal flora of mice The diversity of intestinal flora is 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 reads, clustering or denoising, and performing species annotation and abundance analysis, the species composition of the sample can be revealed; further alpha diversity analysis, beta diversity analysis, species diversity analysis, etc. are performed to explore the differences between samples.

[0088] (1) α-diversity analysis and β-diversity analysis Analysis of α-diversity of mouse intestinal flora showed Figure 5 Compared with the control group, the ACE and Chao 1 indexes 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 indexes of the intestinal flora of mice were significantly higher than those of the model group, indicating that BBr69 can regulate the intestinal flora and increase the richness of the intestinal flora of immunocompromised mice. β-diversity is a measure of the similarity of the flora composition between different samples, that is, focusing on the differences in the flora composition between samples. The experimental results are as follows Figure 6 As shown in the figure, the MC group was clearly separated from the NC group, indicating that the microbial composition of the MC group was significantly different from that of the NC group. The BBr69 group was adjacent to the NC group, indicating that oral administration of BBr69 made the microbial composition of the intestine of immunosuppressed mice tend to be similar to that of the normal group.

[0089] (2) Species diversity analysis at the genus level Figure 7 The relative abundance of genus level in the cecal contents of mice in each group is shown. Compared with the NC group, the MC group has Clostridium_UCG_014_Unclassified bacteria ( unclassified_Clostridia_UCG_014 ), Pseudo-Prevotella ( Alloprevotella ), another genus ( Alistipes )、Helicobacter_NK4A136_group( Lachnospiraceae_ NK4A136_group ) decreased significantly, and the relative abundance of Desulfovibrio ( Desulfovibrio ), unclassified Desulfovibrioides ( unclassified_Desulfovibrionaceae) increased in relative abundance. Compared with the MC group, the relative abundance of Clostridium_UCG_014_Unclassified bacteria in the BBr69 group was higher than that in the MC group. unclassified_Clostridia_UCG_014 ), Pseudo-Prevotella ( Alloprevotella ), another genus ( Alistipes )、Helicobacter_NK4A136_group( Lachnospiraceae_ NK4A136_group ) increased in relative abundance, and at the same time, the combined Lactobacillus genus ( Ligilactobacillus ) and Lactobacillus spp. ( Lactobacillus ) relative abundance increased significantly. In addition, compared with the MC group, the relative abundance of Desulfovibrio spp. ( Desulfovibrio ), unclassified Desulfovibrioides ( unclassified_Desulfovibrionaceae ) relative abundance decreased significantly. Alloprevotella ) can produce short-chain fatty acids (SCFAs) mainly composed of succinate and acetate, which can improve the intestinal epithelial barrier and prevent inflammation. 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 Bifidobacterium breve BBr69 can increase the abundance of beneficial bacteria in the host intestine, reduce the abundance of harmful bacteria, promote the restoration of intestinal flora homeostasis, improve the intestinal barrier, and play an immunomodulatory role.

[0090] (3) OTUs cluster analysis The experimental results are as follows Figure 8 As shown, the number of OTUs shared by the MC group and the NC group was 582, and the number of OTUs shared by the BBr69 group and the NC group was 640, indicating that oral administration of Bifidobacterium breve BBr69 can increase the shared flora in the intestines of 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 that enhance immune function, and / or foods that regulate intestinal flora, and / or antibacterial agents, and / or antibacterial agents for non-therapeutic purposes.

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; And / or, the cytokines include: IL-6, IL-10, IL-17, IL-1β, TNF-α, SIgA; And / or, the immune organ index includes: thymus index, spleen index; And / or, the regulating intestinal flora comprises: increasing the 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; And / or, the antibacterial spectrum includes: Escherichia coli, Salmonella, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus Parisi, and Streptococcus pasteurianus.

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

5. The food for enhancing immune function according to claim 4, characterized in that: Also includes: Auxiliary materials.

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

7. The functional food for regulating intestinal flora according to claim 6, characterized in that: Also includes: Auxiliary materials.

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

9. An antibacterial agent according to claim 8, characterized in that: include: Auxiliary materials.

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

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