Bifidobacterium breve BBr37 with hypoglycemic capacity and application, product and method thereof

Abstract

The invention discloses bifidobacterium breve with hypoglycemic capacity and application, a product and a method thereof, and belongs to the technical field of microorganisms. The invention provides a bifidobacterium breve (Bifidobacterium breve) strain BBr37 with the blood sugar reducing capacity, and the preservation number of the bifidobacterium breve strain BBr37 is CGMCC (China General Microbiological Culture Collection Center) NO.32423. The invention also provides an application of the strain BBr37 in preparation of hypoglycemic drugs or health-care foods with an auxiliary hypoglycemic function or bacteriostatic agents or non-treatment-destination bacteriostasis, and a product and a method based on the strain BBr37. The strain BBr37 disclosed by the invention has a relatively strong inhibition effect on escherichia coli, staphylococcus aureus, salmonella, haemophilus parasuis, haemophilus parainfluenza, campylobacter jejuni and enterocolitis yarrowia, and can effectively achieve the effects of reducing blood sugar and regulating intestinal flora.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and particularly relates to Bifidobacterium breve BBr37 with blood sugar lowering ability, and applications, products and methods thereof. Background Art

[0002] Type II diabetes is a complex chronic metabolic disease caused by insufficient or abnormal insulin secretion in insulin-sensitive tissues, which leads to elevated blood sugar. It has become a serious global health problem, and exploring its treatment methods has also become the focus of attention of various countries.

[0003] Bifidobacterium breve ( Bifidobacterium breve ) is a type of Gram-positive bacillus, mainly found in breast milk and infant feces, and its abundance gradually decreases or even disappears with age. Bifidobacteria in the human intestine play an important role in human health. Bifidobacteria are a sign of human health and a beneficial flora that has many benefits for the human body.

[0004] Therefore, the art needs to develop Bifidobacterium breve that has multiple functions, such as strong α-glucosidase inhibition ability, hypoglycemic effect, and broad-spectrum antibacterial effect. Summary of the invention

[0005] Based on the above-mentioned needs of the prior art in the art, the present invention provides Bifidobacterium breve BBr37 with blood sugar lowering ability 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 )BBr37, its deposit number is CGMCC NO. 32423.

[0007] The preservation number is CGMCC NO. 32423 of Bifidobacterium breve ( Bifidobacterium breve ) Use of BBr37 in preparing functional foods for assisting in lowering blood sugar, and / or functional foods for assisting in lowering blood lipids, and / or functional foods for enhancing immunity, and / or functional foods for regulating intestinal flora, and / or antibacterial agents, and / or antibacterial agents for non-therapeutic purposes.

[0008] The blood sugar reduction includes: reducing blood sugar level, glycosylated serum protein, area below the oral glucose tolerance line, and insulin resistance index; Preferably, the lipid-lowering comprises: lowering triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol; Preferably, the immunity enhancement comprises: reducing the level of lipopolysaccharide, tumor necrosis factor TNF-α, interleukin-6, and / or increasing the level of interleukin-10; Preferably, the intestinal flora regulation comprises: increasing the number of Myxobacterium spp. Blautia ), Allobacterium ( Allobaculum ), Bifidobacterium spp. ( Bifidobacterium ), Akkermansia muciniphila ( Akkermansia ), Clostridium prausnitzii ( Faecalibaculum ), unnamed Bacteroidetes ( unclassified_Muribaculaceae ) and reduced the abundance of Bacteroides ( Bacteroides ), Aerococcus ( Aerococcus ) abundance; Preferably, the antibacterial spectrum includes: Escherichia coli, Salmonella, Staphylococcus aureus, Haemophilus parasuis, Haemophilus parainfluenzae, Campylobacter jejuni, and Yersinia enterocolitica.

[0009] A functional food for assisting in lowering blood sugar, comprising functional active ingredients; the functional active ingredients include: Bifidobacterium breve with a preservation number of CGMCC NO. 32423 ( Bifidobacterium breve )BBr37.

[0010] A functional food for assisting in lowering blood lipids, comprising functional active ingredients; the functional active ingredients include: Bifidobacterium breve with a preservation number of CGMCC NO. 32423 ( Bifidobacterium breve )BBr37.

[0011] A food for enhancing immune function, comprising functional active ingredients; the functional active ingredients include: Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

[0012] A functional food for regulating intestinal flora, comprising functional active ingredients; the functional active ingredients include: Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

[0013] An antibacterial agent, comprising an antibacterial active ingredient; the antibacterial active ingredient comprises: Bifidobacterium breve ( Bifidobacterium breve )BBr37.

[0014] The antibacterial agent also includes auxiliary materials.

[0015] An in vitro antibacterial method using Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37 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 the strain has a relatively broad-spectrum antibacterial ability, and has a good inhibitory effect on many pathogens including Escherichia coli, Salmonella, Staphylococcus aureus, Haemophilus parasuis, Haemophilus parainfluenzae, Campylobacter jejuni, and Yersinia enterocolitica. The strain is classified and identified as a strain of Bifidobacterium breve ( Bifidobacterium breve ), and named it BBr37. The present invention has proved through a large number of experiments that in addition to the broad-spectrum antibacterial ability, the BBr37 strain also has excellent immune enhancement and intestinal flora regulation functions. The BBr37 strain can significantly reduce the levels of lipopolysaccharide, tumor necrosis factor TNF-α, interleukin-6 and increase the level of interleukin-10; at the same time, the BBr37 strain can also significantly increase the level of Myxobacterium ( Blautia ), Allobacterium ( Allobaculum ), Bifidobacterium spp. Bifidobacterium ), Akkermansia muciniphila ( Akkermansia ), Clostridium prausnitzii ( Faecalibaculum ), Unnamed Bacteroidetes ( unclassified_Muribaculaceae ) and reduced the abundance of Bacteroides ( Bacteroides ), Aerococcus ( Aerococcus ). The BBr37 strain can assist in lowering blood sugar by reducing triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol, and can assist in lowering blood lipids by reducing triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol.

[0017] The Bifidobacterium breve of the present invention ( Bifidobacterium breve )The deposit information of BBr37 is as follows: Deposit number: CGMCC NO. 32423; Classification name: Bifidobacterium breve Bifidobacterium breve ; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Date of preservation: October 30, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a line graph showing the effect of Bifidobacterium breve BBr37 of Experimental Example 5 of the present invention on the food intake and water intake of type 2 diabetic mice.

[0019] Figure 2 This is a line graph showing the effect of Bifidobacterium breve BBr37 of Experimental Example 5 of the present invention on the body weight of type 2 diabetic mice.

[0020] Figure 3The line graph and bar graph are the effects of Bifidobacterium breve BBr37 of Experimental Example 6 of the present invention on blood glucose levels and glycosylated serum protein levels in type 2 diabetic mice.

[0021] Figure 4 This is a bar graph showing the effect of Bifidobacterium breve BBr37 of Experimental Example 6 of the present invention on oral glucose tolerance in type 2 diabetic mice.

[0022] Figure 5 This is a bar graph showing the effects of Bifidobacterium breve BBr37 of Experimental Example 6 of the present invention on type 2 diabetic mice and insulin resistance.

[0023] Figure 6 This is a bar graph of the effect of Bifidobacterium breve BBr37 of Experimental Example 8 of the present invention on inflammatory factors in type 2 diabetic mice; wherein, Figure a is a bar graph of lipopolysaccharide (LPS), Figure b is a bar graph of tumor necrosis factor (TNF-α), Figure c is a bar graph of interleukin-6 (IL-6), and Figure d is a bar graph of interleukin-10 (IL-10).

[0024] Figure 7 This is a bar graph showing the changes in Chao1 index and Shannon index of mouse intestinal microorganisms in Experimental Example 9 of the present invention.

[0025] Figure 8 This is a β-diversity analysis diagram of the mouse intestinal flora in Experimental Example 9 of the present invention.

[0026] Figure 9 This is a bar chart of the abundance of various types of bacterial flora in the mouse intestine of Experimental Example 9 of the present invention.

[0027] The markings in the figure are listed as follows: Figures 1 - 9 The group numbers are as follows: NC is the normal group mice, MC is the model group mice, MET is the drug treatment group mice, and BBr37 is the probiotic treatment group mice. DETAILED DESCRIPTION

[0028] The specific contents of the present invention are further described in detail below in conjunction with specific embodiments, experimental examples and drawings, but the protection scope of the present invention is not limited thereto.

[0029] Source of biological material 1. The Escherichia coli, Staphylococcus aureus and Salmonella used in Experimental Example 3 were purchased from Guangdong Microbiological Culture Collection Center; Haemophilus parasuis, Haemophilus parainfluenzae, Campylobacter jejuni and Yersinia enterocolitica were laboratory preserved strains.

[0030] 2. The mice used in Experimental Examples 5-9 are commercially available.

[0031] “Above” and “below” in this article include the original number.

[0032] In this article, "Bifidobacterium breve ( Bifidobacterium breve ) strain BBr37", "Bifidobacterium breve ( Bifidobacterium breve ) strain BBr37", "Bifidobacterium breve BBr37", "BBr37", "BBr37 strain", "strain BBr37" all refer to: Bifidobacterium breve (with a preservation number of CGMCC NO. 32423) Bifidobacterium breve ) strain BBr37.

[0033] The first group of examples, Bifidobacterium breve BBr37 of the present invention This group of embodiments provides a Bifidobacterium breve having an immunomodulatory function ( Bifidobacterium breve )BBr37, its deposit number is CGMCC NO. 32423.

[0034] 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 BBr37 with a preservation number of CGMCC NO. 32423, and / or using a strain of Bifidobacterium breve BBr37 with a preservation number of CGMCC NO. 32423 in combination with other probiotics, and / or using a strain of Bifidobacterium breve BBr37 with a preservation number of CGMCC NO. 32423 to antagonize various pathogens, and / or prepare antibacterial agents, and / or prepare foods that enhance immune function, and / or regulate intestinal flora functional foods all fall within the scope of protection of the present invention.

[0035] 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.

[0036] 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 production processes in the pharmaceutical field (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 BBr37 with a preservation number of CGMCC NO. 32423 into products with different dosage forms, different storage conditions, and different shelf lives. This is possible and easy to do without technical barriers for those skilled in the art.

[0037] The second group of examples, use of Bifidobacterium breve BBr37 of the present invention This group of embodiments provides a collection number of CGMCC NO. 32423 of Bifidobacterium breve ( Bifidobacterium breve ) Use of BBr37 in preparing functional foods for assisting in lowering blood sugar, and / or functional foods for assisting in lowering blood lipids, and / or functional foods for enhancing immunity, and / or functional foods for regulating intestinal flora, and / or antibacterial agents, and / or antibacterial agents for non-therapeutic purposes.

[0038] In a specific embodiment, the blood sugar reduction includes: reducing blood sugar level, glycosylated serum protein, area below the oral glucose tolerance line, and insulin resistance index; Preferably, the lipid-lowering comprises: lowering triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol; Preferably, the immunity enhancement comprises: reducing the level of lipopolysaccharide, tumor necrosis factor TNF-α, interleukin-6, and / or increasing the level of interleukin-10; Preferably, the intestinal flora regulation comprises: increasing the number of Myxobacterium spp. Blautia ), Allobacterium ( Allobaculum ), Bifidobacterium spp. Bifidobacterium ), Akkermansia muciniphila ( Akkermansia ), Clostridium prausnitzii ( Faecalibaculum ), Unnamed Bacteroidetes ( unclassified_Muribaculaceae ) and reduced the abundance of Bacteroides ( Bacteroides ), Aerococcus ( Aerococcus ) abundance; Preferably, the antibacterial spectrum includes: Escherichia coli, Salmonella, Staphylococcus aureus, Haemophilus parasuis, Haemophilus parainfluenzae, Campylobacter jejuni, and Yersinia enterocolitica.

[0039] The third group of embodiments, the blood sugar lowering functional food of the present invention This group of embodiments provides a blood sugar lowering functional food. All embodiments of this group have the following common features: the blood sugar lowering functional food comprises a functional active ingredient; the functional active ingredient comprises: Bifidobacterium breve ( Bifidobacterium breve )BBr37.

[0040] In a further embodiment, the blood sugar lowering auxiliary functional food further includes: auxiliary materials.

[0041] 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.

[0042] According to the content of the present invention, for different needs in actual production applications, 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 BBr37 with a preservation number of CGMCC NO. 32423 into different dosage forms, such as powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0043] In a specific embodiment, the dosage form of the food having the auxiliary blood sugar lowering function is selected from: one or more of powder, tablet, liquid and capsule.

[0044] The fourth group of embodiments, the functional food for assisting in lowering blood lipids of the present invention This group of embodiments provides a functional food that assists in lowering blood lipids. All embodiments of this group have the following common features: the functional food that assists in lowering blood lipids includes functional active ingredients; the functional active ingredients include: Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

[0045] In a further embodiment, the functional food for assisting in lowering blood lipids further comprises: auxiliary materials.

[0046] 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.

[0047] According to the content of the present invention, for different needs in actual production applications, 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 BBr37 with a preservation number of CGMCC NO. 32423 into different dosage forms, such as powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0048] In a specific embodiment, the dosage form of the functional food for assisting in lowering blood lipids is selected from: one or more of powder, tablet, liquid, and capsule.

[0049] The fifth 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 (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

[0050] In a further embodiment, the immune function enhancing food further includes: auxiliary materials.

[0051] 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.

[0052] According to the content of the present invention, for different needs in actual production applications, 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 BBr37 with a preservation number of CGMCC NO. 32423 into different dosage forms, such as powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0053] In a specific embodiment, the dosage form of the food for enhancing immune function is selected from: one or more of powder, tablet, liquid, and capsule.

[0054] The sixth 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 )BBr37.

[0055] In a further embodiment, the functional food for regulating intestinal flora further includes: auxiliary materials.

[0056] 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.

[0057] According to the content of the present invention, for different needs in actual production applications, 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 BBr37 with a preservation number of CGMCC NO. 32423 into different dosage forms, such as powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0058] In a specific embodiment, the dosage form of the functional food for regulating intestinal flora is selected from: one or more of powder, tablet, liquid, and capsule.

[0059] Group 7 Example, 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 (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

[0060] In a further embodiment, the antibacterial agent further comprises: auxiliary materials.

[0061] 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.

[0062] According to the content of the present invention, for different needs in actual production applications, 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 BBr37 with a preservation number of CGMCC NO. 32423 into different dosage forms, such as powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0063] In a specific embodiment, the dosage form of the antibacterial agent is selected from: one or more of powder, tablet, liquid, and capsule.

[0064] The eighth group of embodiments, the in vitro antibacterial method of the present invention This group of embodiments provides an in vitro antibacterial method. All embodiments of this group have the following common features: using Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37 for antibacterial effect.

[0065] Experimental Example 1: Isolation, screening and identification of Bifidobacterium breve (1) Isolation and screening of Bifidobacterium breve The collected infant feces were placed in a sterile sampling tube and transported in an ice box. They were diluted with 0.85% saline gradient under sterile conditions, and the appropriate dilution gradient was selected and spread on the LMRS + lithium salt agar plate, and cultured at 37°C for 48-72 hours. The suspected single colonies were picked by observing the colony morphology with the naked eye, and they were observed under a microscope and preliminarily screened and purified. After purification, LMRS liquid anaerobic tubes were used for culture at 37°C for 8-12 hours, and the supernatant was centrifuged and resuspended in a sterile 25% glycerol aqueous solution and stored in the strain bank of Wuhan Weikang Probiotics Research Institute.

[0066] (2) Identification of Bifidobacterium breve: 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 16S rDNA 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 category is Bifidobacterium breve The strain was determined to be Bifidobacterium breve. The strain was named Bifidobacterium breve BBr37 and deposited. The deposit information is as follows: Deposit number: CGMCC NO. 32423; Classification name: Bifidobacterium breve Bifidobacterium breve ; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Date of preservation: October 30, 2024.

[0067] Experimental Example 2: Artificial Gastric Juice and Intestinal Juice Tolerance Test Preparation of artificial gastric juice: prepare 0.5% sodium chloride solution, adjust the pH to 3 with 1 mol / L HCl, add 0.3% pepsin, and after fully dissolving, filter with a 0.22 μm microporous filter membrane for sterilization and set aside.

[0068] Preparation of artificial intestinal fluid: prepare 0.5% NaCl solution, adjust the pH to 8.0 with 0.1 mol / L NaOH, add 0.1% trypsin, and then filter with a 0.22 μm microporous filter membrane to sterilize after fully dissolving.

[0069] After the strain to be tested is activated for 2 generations, the concentration of the bacterial solution is adjusted to 10 8 CFU / mL. Take 1mL of bacterial suspension and centrifuge it, collect the bacteria, inoculate them into 1mL of prepared artificial gastric juice and intestinal juice, mix them, digest them at 37℃, and take the digestion fluid at 0h and 3h to detect the number of viable bacteria, calculate the survival rate, and the results are shown in Table 1. Among them, strain survival rate (%) = Nt / N0×100%, where N0 represents the number of viable bacteria of the strain at 0h, CFU / mL, and Nt represents the number of viable bacteria of the strain at 3h, CFU / mL.

[0070] Table 1. Survival rate of Bifidobacterium breve BBr37 tolerant to artificial gastric juice and intestinal juice

[0071] The experimental results showed that after 3 hours of gastric juice digestion, the survival rate of Bifidobacterium breve BBr37 was 87.96%, and after 3 hours of intestinal juice digestion, the survival rate of Bifidobacterium breve BBr37 was 96.83%, which indicates that Bifidobacterium breve BBr37 has good resistance to gastric acid and intestinal juice.

[0072] Experimental Example 3: Inhibitory ability of Bifidobacterium breve against pathogenic bacteria MRS broth containing 1.5% agar was cooled to about 55°C and mixed with Escherichia coli, Salmonella, Staphylococcus aureus, Haemophilus parasuis, Haemophilus parainfluenzae, Campylobacter jejuni, and Yersinia enterocolitica suspensions 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, the Oxford cup was taken out and 200 μL of Bifidobacterium breve BBr37 fermentation liquid was injected into each well. After overnight culture at 37°C, the diameter of the inhibition zone was measured. Table 2. Diameter of inhibition zone of Bifidobacterium breve against pathogens (mm)

[0073] It can be seen from the above table that Bifidobacterium breve can effectively inhibit intestinal pathogens, especially Campylobacter jejuni and Escherichia coli, with the diameters of the inhibition zones being 28.74 mm and 19.39 mm respectively, showing good inhibition ability.

[0074] Experimental Example 4: Inhibitory ability of Bifidobacterium breve on α-glucosidase Bifidobacterium breve was inoculated into liquid L-MRS medium and cultured overnight at 37°C. After the second generation was transferred, it was cultured for 8 hours. After that, the third generation was transferred and fermented for 14 hours. The fermentation liquid was centrifuged, the supernatant was discarded, and the liquid was resuspended with physiological saline and the concentration of the bacterial liquid was adjusted to 10 8 CFU / mL for later use. Add 75μL p-nitrophenol-α-D-pyranose glucoside (PNPG) solution and 25μL sample to be tested to 150μL PBS solution, place the mixture on ice for 3min, add 50μL α-glucosidase solution 0.2U / mL and react in a water bath at 37℃ for 15min, add 1mL 1mol / L Na2CO3 as the reaction stop solution, and measure the absorbance of the reaction solution at 405nm: A: add sample plus α-glucosidase; B: adding sample without α-glucosidase; C: no sample plus α-glucosidase; D: No sample or α-glucosidase added Table 3. Inhibition rate of Bifidobacterium breve BBr37 on α-glucosidase

[0075] As can be seen from the above table, Bifidobacterium breve has the ability to inhibit α-glucosidase, which indicates that the strain has the potential to lower blood sugar.

[0076] Experimental Example 5: Effects of Bifidobacterium breve on food intake, water intake, and body weight in type 2 diabetic mice Male C57BL / 6j mice (5-6 weeks; 19 ± 1 g) were housed in the animal room of Hubei Center for Disease Control and Prevention, with the license number for experimental animals: SCXK (E) 2020-0019. The animal experiments were maintained at 20-22°C, 40-60% humidity, and 12 h light / 12 h dark cycle. Mice had free access to food and water. After one week of adaptation, the mice were treated with the following methods according to references F. Yan, N. Li, J. Shi, H. Li, Y. Yue, W. Jiao, N. Wang, Y. Song, G. Huo and B. Li, Bifidobacterium breve alleviates Type 2 diabetes by regulating 2 hepaticglucose and lipid metabolism and gut microbiota in mice, Food Funct, 2019, Mice were modeled with type 2 diabetes. The mice were divided into 4 groups (n=12): normal group (NC group), model group (MC group), probiotic group (BBr37 group, 6*10 9CFU / d), and drug treatment group (MET, 0.2g / kg / d). The normal group and model group were gavaged with sterile saline, the probiotic group was gavaged with Bifidobacterium breve BBr37 once a day for 35 consecutive days, and the drug treatment group (MET group) was gavaged with metformin hydrochloride once a day for 35 consecutive days. The daily food intake, daily water intake, and weekly body weight of the mice were monitored.

[0077] The typical characteristics of type 2 diabetic mice are mainly manifested in increased daily food intake, increased water intake, increased urine output, and decreased body weight. After five weeks of intragastric administration of probiotics, the overall symptoms of diabetes were alleviated and improved. Figure 1 As shown. Treatment with the strain effectively controlled the increase in the amount of food and water consumed by mice. Daily monitoring of the humidity of the litter showed that the urine volume in the cages of the treatment group had a tendency to return to normal. Figure 2 The changes in the weight values ​​of mice indicate that the intervention of probiotics has a good stabilizing or restoring effect on the decreased weight values ​​of mice, and effectively improves the absorption of nutrients by type 2 diabetic mice, which overall show a trend of approaching the healthy group.

[0078] Experimental Example 6: Effect of Bifidobacterium breve on glucose metabolism in type 2 diabetic mice Before treatment and during the five-week treatment period, blood samples were collected from the tail vein of the mice every week to measure the fasting blood glucose level. The fasting blood glucose level (FBG) measurement results are shown in Figure 3 As shown on the left. After five weeks of treatment, the concentration of glycosylated serum protein and oral glucose tolerance of mice were measured. Each group of mice was given oral glucose, and the blood glucose level was measured at 0min, 15min, 30min, 60min, 90min, and 120min after oral glucose, and the area below the oral glucose tolerance line in the oral glucose tolerance test. At the end of the experiment, serum was taken to measure the serum insulin concentration of mice.

[0079] Experimental steps of oral glucose tolerance: The amount of glucose used in the mouse glucose tolerance test is 2 g per kilogram of body weight (2 g / kg). Glucose solution of appropriate concentration is prepared according to the experimental requirements, and 20% glucose solution is prepared with normal saline. The mice are fasted for 16 hours, during which time the mice maintain normal drinking water; before starting the glucose tolerance test, the weight of each mouse is weighed. Blood is collected from the tip of the mouse's tail, and fasting blood sugar is measured with a blood glucose meter. The measured value is considered as the blood sugar value at 0 min. Glucose is gavaged and the timing starts from the moment it is completed. The blood sugar value of each mouse at each time point is measured at 15, 30, 60, 90 and 120 min.

[0080] The results of the weekly blood glucose test of mice are as follows Figure 3As shown, it can be concluded that the strain can significantly reduce the blood sugar level of diabetic mice after five weeks of intervention. In the third week, the short bifidobacterium treatment group showed the effect of lowering blood sugar, and as the treatment time prolonged, the blood sugar lowering effect of the mice became more obvious. In the fifth week of treatment, the fasting blood sugar levels of the mice in the probiotic group reached a very significant difference with the model group, and there was no significant difference compared with the healthy group. This shows that the BBr37 strain can effectively lower the blood sugar level of mice and bring it closer to the healthy group. The glycated serum protein index reflects the average blood sugar level in the past 1 to 3 weeks, such as Figure 3 As shown on the right, it can be concluded that probiotic treatment effectively reduces the blood sugar level of mice. After five weeks of treatment, the oral glucose tolerance index of mice was measured. Figure 4 As shown, the area below the oral glucose tolerance line of the BBr37 treatment group reached a very significant level compared with the model group. The experimental results showed that the BBr37 strain not only lowered the blood sugar of mice, but also had a good effect on improving the glucose absorption and metabolism function of diabetic mice.

[0081] The insulin resistance index (HOMA-IR) is a value calculated based on the serum insulin level and fasting blood glucose value. It is used to determine the degree of insulin resistance. Figure 5 It can be concluded that the insulin resistance of the BBr37 strain treatment group and the drug treatment group was significantly improved and alleviated, and was close to that of the healthy group.

[0082] The above results indicate that treatment with the BBr37 strain can effectively improve and enhance the glucose metabolism ability of mice, thereby lowering the blood sugar levels of mice and controlling the development of diabetes in mice.

[0083] Experimental Example 7: Effect of Bifidobacterium breve on blood lipid levels in type 2 diabetic mice The indicators of mouse serum were measured: triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), as shown in Table 4: Table 4. Results of total cholesterol, total triglycerides, high-density lipoprotein, and low-density lipoprotein tests in serum

[0084] As shown in Table 4, oral administration of BBr37 strain has a certain effect on reducing the blood lipid level of type 2 diabetic mice. The indicators of total cholesterol and low-density lipoprotein showed a positive therapeutic effect, and reached a significant difference with the model group (MC) (p<0.05). In the treatment of diabetic dyslipidemia, the BBr37 strain is particularly effective in reducing total cholesterol and low-density lipoprotein in serum.

[0085] The above results indicate that the BBr37 strain can effectively reduce the blood lipid levels of type 2 diabetic mice and has a very good effect.

[0086] Experimental Example 8: Effects of Bifidobacterium breve on inflammatory factors in type 2 diabetic mice The onset of diabetes will cause systemic inflammation in the body. The occurrence of chronic inflammatory diseases will lead to an increase in lipopolysaccharide toxins in the patient's serum, while promoting the production of tumor necrosis factor and interleukin-6. At the end of the experiment, the whole blood of the mice was centrifuged to obtain serum. The ELISA kit was used to measure the inflammatory factors in the mouse serum, including the concentration levels of lipopolysaccharide (LPS), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10). The results are as follows: Figure 6 As shown in a, b, c, and d; compared with the model group (MC), oral administration of BBr37 strains reduced inflammation in mice and slowed down the occurrence of inflammation. Interleukin-10, as an anti-inflammatory factor in the body, participates in the regulation of immune cells, inflammatory cells, etc., such as Figure 6 As shown in middle d, oral administration of BBr37 effectively increased the concentration of interleukin-10 in mice and brought it close to the level of the healthy group.

[0087] The above data results show that oral administration of BBr37 bacteria can significantly reduce systemic inflammation in type 2 diabetic mice and enhance the immunity of mice.

[0088] Experimental Example 9: Effect of Bifidobacterium breve on intestinal flora of type 2 diabetic mice After the experiment, the mice were killed, the abdomen was disinfected and dissected, and the cecal contents of each group of mice were taken for microbial diversity analysis. The 16S rDNA V3 ~ V4 hypervariable regions of all bacteria in the samples were sequenced on the Mi Seq Illumina sequencing platform to determine the characteristics of the intestinal microbiota.

[0089] α-diversity reflects the species richness and diversity of the sample. Chao1 index and Shannon index are commonly used indicators for judging the diversity and abundance of intestinal microbial colonies. Figure 7As shown in the figure, the abundance and diversity of intestinal flora in type 2 diabetic mice were significantly decreased compared with those in the healthy group (p<0.01). According to the Chao1 index analysis, the treatment with BBr37 strain can increase and improve the abundance of intestinal flora, reaching a significant difference level compared with the model group. There was no significant difference compared with the drug treatment group (p>0.05). According to the Shannon index analysis, oral administration of BBr37 strain can significantly increase the diversity of intestinal flora in type 2 diabetic mice and approach that of the healthy group. The results show that five weeks of oral administration of BBr37 strain treatment can increase and improve the abundance and diversity of intestinal flora in type 2 diabetic mice.

[0090] β-diversity is used to compare the similarities between different samples in terms of species diversity. PCA is a common analysis method that can reflect the differences and distances of samples. PCA uses variance decomposition to reflect the differences between multiple groups of data on a two-dimensional coordinate graph, with the coordinate axes taking the two eigenvalues ​​that can best reflect the variance. The closer the distance between two samples, the more similar the composition of the two samples. Figure 8 As shown, both probiotics and drug treatments can make the intestinal flora of mice gradually show differences from the model group and move closer to the healthy group, indicating that the strain effectively improves the intestinal flora of type 2 diabetic mice and tends to normal.

[0091] Metformin is a first-line drug for the treatment of type 2 diabetes, and its mechanism of action is constantly being revealed by research. Many studies have shown that metformin can have an effect on type 2 diabetic mice by changing the intestinal flora. Figure 9 The results showed that Myxobacterium spp. ( Blautia ) and Allobacterium ( Allobaculum ) significantly increased. Literature reports that Myxobacterium can maintain glucose homeostasis and have anti-inflammatory effects, and Allobacterium ( Allobaculum ) is a bacterium that can use carbohydrates to produce butyric acid. Butyric acid can upregulate intestinal tight proteins and is closely related to human health. As shown in the figure, five weeks of oral gavage treatment significantly increased the number of Bifidobacterium ( Bifidobacterium ) abundance, indicating that BBr37 can play a good therapeutic effect by locating and metabolizing in the intestine. In addition, beneficial intestinal flora are expressed by Akkermansia muciniphila ( Akkermansia ), Clostridium prausnitzii ( Faecalibaculum ), unnamed Bacteroidetes ( unclassified_Muribaculaceae ), their abundance increased significantly in the BBr37 treatment group (p<0.05). Bacteroides ), Aerococcus ( Aerococcus ) showed significant decrease in the BBr37 treatment group and the drug treatment group (p<0.01).

[0092] The above results show that type 2 diabetic mice have intestinal flora disorders, and the treatment of Bifidobacterium breve BBr37 can regulate the intestinal flora of type 2 diabetic mice by effectively localizing in the mouse intestine, significantly reducing the abundance of harmful flora, significantly increasing the abundance of beneficial intestinal flora, and improving the species diversity and abundance of intestinal flora. It effectively controls and improves the sugar metabolism, inflammation, and blood lipid levels of type 2 diabetic mice.

Claims

1. A Bifidobacterium breve with immunomodulatory function ( Bifidobacterium breve ) BBr37, characterized in that Its deposit number is CGMCC NO. 32423.

2. Bifidobacterium breve with a deposit number of CGMCC NO. 32423 ( Bifidobacterium breve ) Use of BBr37 in preparing functional foods for assisting in lowering blood sugar, and / or functional foods for assisting in lowering blood lipids, and / or functional foods for enhancing immunity, and / or functional foods for regulating intestinal flora, and / or antibacterial agents, and / or antibacterial agents for non-therapeutic purposes.

3. The bifidobacterium breve (CGMCC NO. 32423) according to claim 2 Bifidobacterium breve ) Use of BBr37 in preparing blood sugar lowering functional foods, and / or blood lipid lowering functional foods, and / or immune enhancement functional foods, and / or intestinal flora regulating functional foods, and / or antibacterial agents, and / or non-therapeutic antibacterial agents, characterized in that: The blood sugar reduction includes: reducing blood sugar level, glycosylated serum protein, area below the oral glucose tolerance line, and insulin resistance index; And / or, the lipid-lowering includes: lowering triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol; And / or, the immunity enhancement includes: reducing the level of lipopolysaccharide, tumor necrosis factor TNF-α, interleukin-6, and / or increasing the level of interleukin-10; And / or, the intestinal flora regulation comprises: increasing the number of Myxobacterium spp. Blautia ), Allobacterium ( Allobaculum ), Bifidobacterium spp. Bifidobacterium ), Akkermansia muciniphila ( Akkermansia ), Clostridium prausnitzii ( Faecalibaculum ), Unnamed Bacteroidetes ( unclassified_Muribaculaceae ) and reduced the abundance of Bacteroides ( Bacteroides ), Aerococcus ( Aerococcus ) abundance; And / or, the antibacterial spectrum includes: Escherichia coli, Salmonella, Staphylococcus aureus, Haemophilus parasuis, Haemophilus parainfluenzae, Campylobacter jejuni, and Yersinia enterocolitica.

4. A functional food for assisting in lowering blood sugar, comprising functional active ingredients; characterized in that: The functional active ingredients include: Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

5. A functional food for assisting in lowering blood lipids, comprising functional active ingredients; characterized in that: The functional active ingredients include: Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

6. A food for enhancing immune function, comprising functional active ingredients; characterized in that: The functional active ingredients include: Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

7. A functional food for regulating intestinal flora, comprising functional active ingredients; characterized in that: The functional active ingredients include: Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

8. An antibacterial agent comprising an antibacterial active ingredient; characterized in that: The antibacterial active ingredients include: Bifidobacterium breve (CGMCC NO. 32423) Bifidobacterium breve )BBr37.

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

10. An in vitro antibacterial method, characterized in that: Adopt the short Bifidobacterium of CGMCC NO. 32423 ( Bifidobacterium breve )BBr37 for antibacterial effect.

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