Bifidobacterium animalis subsp. Lactis BLa65 capable of relieving constipation and regulating intestinal flora as well as application, product and method of bifidobacterium animalis subsp. Lactis BLa65

Abstract

The invention discloses bifidobacterium animalis subsp. Lactis BLa65 capable of relieving constipation and regulating intestinal flora as well as application, a product and a method thereof, and belongs to the technical field of microorganisms. The invention provides bifidobacterium animalis subsp. Lactis BLa65 capable of relieving constipation and regulating intestinal flora, and the preservation number of the bifidobacterium animalis subsp. Lactis BLa65 is CGMCC (China General Microbiological Culture Collection Center) No.32427. The invention also provides an application of the bifidobacterium animalis subsp. Lactis BLa65 in preparation of functional food for relaxing the bowels, and / or functional food for regulating intestinal flora, and / or a bacteriostatic agent, and / or non-treatment-destination in-vitro bacteriostasis. The BLa65 strain provided by the invention has excellent broad-spectrum antibacterial performance, a function of relieving constipation and a function of regulating intestinal flora at the same time.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to a strain of animal Bifidobacterium lactis subspecies BLa65 capable of relieving constipation and regulating intestinal flora, and an application, product and method thereof. Background Art

[0002] Constipation is one of the most common gastrointestinal diseases in clinical practice, with the main manifestations of difficult and infrequent defecation and dry stool, which seriously affects people's quality of life. Constipation is mainly treated with fiber supplements, laxatives and prescription drugs. However, due to the limited efficacy and potential side effects of these methods, nearly half of patients are dissatisfied with these methods. In addition, it is widely believed that long-term use of drugs will lead to drug resistance. Therefore, there is an urgent need to develop environmentally friendly and safer treatment alternatives.

[0003] The intestinal microecology is the most important and complex of all human microecological systems. It is composed of various intestinal flora and their living environment. Currently, more than 400 species of intestinal flora have been recognized, with a total of about 10 14 The intestinal flora is diverse and stable. Its taxonomic diversity, functional diversity and relatively stable living state constitute a healthy and balanced intestinal microecology. The intestinal flora plays a vital role in promoting intestinal health, and supplementing with probiotics has become a new strategy for preventing and treating constipation. The effectiveness of probiotics in relieving constipation symptoms has been verified by clinical trials and animal experiments. Bifidobacterium is one of the earliest microorganisms to colonize the human intestine, exerting various health-promoting probiotic effects, and changes in its abundance are correlated with the occurrence of constipation. As individuals age, the proportion of bifidobacteria in the human intestine decreases, and the probability of constipation increases. Supplementing with bifidobacteria is beneficial to relieve constipation.

[0004] However, the mechanism of action of bifidobacteria in relieving constipation is still unclear, and most strains have a single effect. It is very rare to have animal Bifidobacterium lactis strains that have the functions of laxative, balancing intestinal microecology and broad-spectrum antibacterial. Therefore, the field is in urgent need of developing new multifunctional and synergistic animal Bifidobacterium lactis strains. Summary of the invention

[0005] The present invention provides an animal Bifidobacterium lactis subspecies BLa65 capable of relieving constipation and regulating intestinal flora, and its application, product and method, aiming to solve the defects in the prior art that the action mechanism of the above-mentioned bifidobacteria in relieving constipation is still unclear and most strains have a single effect, and to fill the deficiency that animal Bifidobacterium lactis subspecies strains with the functions of moisturizing the intestine, balancing the intestinal microecology and having a broad spectrum of antibacterial effects are very rare, and to meet the urgent need to develop new multifunctional and synergistic animal Bifidobacterium lactis subspecies strains.

[0006] The technical solution of the present invention is as follows: A strain of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis) BLa65, its deposit number is CGMCC No.32427.

[0007] A strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427, which can relieve constipation and regulate intestinal flora, is used in the preparation of laxative functional foods, and / or intestinal flora regulating functional foods, and / or antibacterial agents, and / or in vitro antibacterial agents for non-therapeutic purposes.

[0008] The laxative method includes: shortening the time of first black stool, increasing small intestinal propulsion rate, regulating gastrointestinal regulatory peptides, and regulating intestinal flora; Preferably, the gastrointestinal regulatory peptides include: motilin, substance P, vasoactive intestinal peptide, somatostatin, endothelin; Preferably, the gastrointestinal regulating peptides include: increasing the content of motilin and substance P, and reducing the content of vasoactive intestinal peptide, somatostatin, and endothelin; Preferably, the intestinal flora regulation comprises: increasing the number of Prevotellaceae _UGG_001 ( Prevotellaceae_ UCG_001 ), Oscillibacterium spp. Oscillibacter ), Bifidobacterium spp. ( Bifidobacterium ) and another genus Mycobacterium ( Alistipes ) abundance, and / or, reducing the abundance of Desulfovibrio ( Desulfovibrio ) and Enterobacter (Enterorhabdus) abundance; Preferably, the antibacterial spectrum includes: Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Bacteroides fragilis, Streptococcus anginosus, and Escherichia fergusonii.

[0009] A laxative functional food comprises active ingredients; the active ingredients include: a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No. 32427, which can relieve constipation and regulate intestinal flora.

[0010] The laxative functional food also includes auxiliary materials.

[0011] A functional food for regulating intestinal flora comprises active ingredients; the active ingredients include: a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427, which can relieve constipation and regulate intestinal flora.

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

[0013] An antibacterial agent comprises an active ingredient; the active ingredient comprises: a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427, which can relieve constipation and regulate intestinal flora.

[0014] The antibacterial agent further comprises: auxiliary materials.

[0015] The invention discloses a non-therapeutic in vitro antibacterial method, which uses a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427, which can relieve constipation and regulate intestinal flora, to inhibit bacteria.

[0016] The beneficial effects of the present invention are as follows: The present invention isolates a strain from breast milk samples, which has excellent survival rates in artificial gastric juice and artificial intestinal juice, and has good adhesion to Caco-2 cells, and can also make good use of oligofructose, oligoxylose, oligogalactose, etc. More importantly, the strain also has broad-spectrum antibacterial properties, constipation relief function and intestinal flora regulation function. Molecular identification shows that the strain is Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ) strain and named it BLa65. The BLa65 strain inhibited the growth of Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Bacteroides fragilis, Streptococcus anginosus and Escherichia fergusonii, and at the same time increased the expression of Prevotella _UGG_001 ( Prevotellaceae_UCG_001 ), Oscillibacterium spp. Oscillibacter ), Bifidobacterium spp. ( Bifidobacterium ) and another genus Mycobacterium ( Alistipes ) abundance, and / or, reducing the abundance of Desulfovibrio ( Desulfovibrio ) and Enterobacter (Enterorhabdus) The abundance of Bifidobacterium lactis of the present invention is used to regulate the intestinal flora, and the effect of laxative and constipation relief is achieved by shortening the time of the first black stool, improving the propulsion rate of the small intestine, regulating gastrointestinal regulatory peptides, and regulating the intestinal flora. Bifidobacterium animalis subsp. lactis ) The deposit information of strain BLa65 is as follows: Deposit number: CGMCC No.32427 Classification and naming: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis Depository: China National Microbiological Culture Collection Administration General Microbiology Center 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

[0017] Figure 1 This is a bar graph comparing the number of stool particles, feces wet weight and small intestinal propulsion rate of each group of mice in Experimental Example 6 of the present invention.

[0018] Figure 2 This is a bar graph comparing motilin, substance P, vasoactive intestinal peptide, somatostatin, and endothelin in each group of mice in Experimental Example 6 of the present invention.

[0019] Figure 3 This is a bar graph of the α-diversity analysis results of each group of mice in Experimental Example 7 of the present invention.

[0020] Figure 4 This is a bar graph showing the relative abundance changes of intestinal flora phylum levels in the cecal contents of each group of mice in Experimental Example 7 of the present invention.

[0021] Figure 5 This is a bar graph showing the relative abundance changes of intestinal flora genus levels in the cecal contents of each group of mice in Experimental Example 7 of the present invention.

[0022] In the above figure, the meanings of each symbol are listed as follows: NC is the blank control group, MC is the model group, and BLa65 is the probiotic group. DETAILED DESCRIPTION

[0023] The detailed contents and technical effects 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.

[0024] 1. Sources of biological materials (I) The Caco-2 cells used in Experimental Example 3 of the present invention were purchased from the Culture Collection of Wuhan University; Lactobacillus rhamnosus LGG was purchased from the Guangdong Microbial Culture Collection Center.

[0025] (ii) The Escherichia coli ATCC25922, Salmonella enteritidis ATCC14028, Staphylococcus aureus ATCC25923, Streptococcus pyogenes ATCC19615, and Bacteroides fragilis ATCC25285 used in Experimental Example 5 of the present invention were purchased from Guangdong Microbiological Culture Collection Center; Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus anginosus, and Escherichia fergusonii were isolated and preserved in this laboratory. The Streptococcus mitis is the Streptococcus mitis strain 5013, which has been registered in NCBI with the accession number MT512114; the Streptococcus pseudopneumoniae is the Streptococcus pseudopneumoniae strain 5027, which has been registered in NCBI with the accession number MT512127; the Streptococcus anginosus is the Streptococcus anginosus 4997 with the accession number MT459365; the Escherichia fergusonii is the Escherichia fergusonii 7610, which has been registered in NCBI with the accession number MT516163; the above strains are all preserved in the applicant's laboratory, and the applicant promises to release them to the public for verification of the technical effects of the present invention within 20 years from the date of application of the present invention.

[0026] (III) The BALB / c male mice used in Experimental Examples 6 and 7 of the present invention were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and raised in the animal room of Hubei Provincial Center for Disease Control and Prevention. The animal experiment ethics number is: Safety Assessment Center Animal (Fu) No. 202310062.

[0027] 2. Culture medium and bacterial activation (I) MRSc medium (1L): peptone 10g, beef extract powder 5g, yeast extract powder 4g, K 2 HPO 4 2g, triammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1mL, MgSO 4 7H 2 O 0.58g, MnSO 4 ·4H 2 O 0.25g, L-cysteine ​​hydrochloride 1g, distilled water 1000mL; pH value 6.2±0.2. MRS medium preparation method refers to GB4789.35-2016, solid medium is added with 1.5% agar.

[0028] (ii) Modified MRS medium (1 L): peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, K 2 HPO 4 2 g, sodium citrate 2 g, MgSO 4 7H 2 O 0.2 g, MnSO 4 ·4H 2 O 0.2 g, Tween 80 1 ml, 1000 ml distilled water.

[0029] MRS broth (III) MRS medium (1 L): 10 g peptone, 10 g beef extract powder, 5 g yeast extract powder, 20 g glucose, 2 g diammonium citrate, 5 g sodium acetate anhydrous, 2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.19 g manganese sulfate monohydrate, 1 g Tween-80. When preparing the solid medium, add 2% agar powder and sterilize at 115°C for 20 min.

[0030] LB medium (1 L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, sterilized at 115°C for 20 min (V) Strain activation: The experimental strain Bifidobacterium lactis subsp. BLa65 was activated for three generations: the glycerol tube strain was inoculated in MRSc medium at 2% (v / v) and cultured at 37°C for 14 h; the second generation was transferred at 5% inoculation and cultured at 37°C for 8 h; the third generation was transferred at 2% inoculation and cultured at 37°C for 14 h to obtain the activated strain.

[0031] Group 1 Example, Bifidobacterium animalis subspecies lactis BLa65 of the present invention This group of embodiments provides a strain of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ) BLa65, characterized in that its preservation number is CGMCC No.32427.

[0032] Any act of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, or promising to sell a strain of Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC NO.32427, and / or using a strain of Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC NO.32427 in combination with other probiotics, and / or using a strain of Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC NO.32427 to antagonize various pathogens, and / or preparing laxative functional foods, and / or functional foods for regulating intestinal flora, and / or antibacterial agents, and / or in vitro antibacterial activities for non-therapeutic purposes all fall within the scope of protection of the present invention.

[0033] 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.

[0034] Those skilled in the art can make routine 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 animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC NO.32427 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.

[0035] The second group of examples, application of the present invention's Bifidobacterium lactis subspecies BLa65 This group of embodiments provides the use of a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427, which can relieve constipation and regulate intestinal flora, in the preparation of laxative functional foods, and / or intestinal flora regulating functional foods, and / or antibacterial agents, and / or in vitro antibacterial agents for non-therapeutic purposes.

[0036] In a specific embodiment, the laxative comprises: shortening the time to discharge the first black stool, increasing the propulsion rate of the small intestine, regulating gastrointestinal regulatory peptides, and regulating intestinal flora; Preferably, the gastrointestinal regulatory peptides include: motilin, substance P, vasoactive intestinal peptide, somatostatin, endothelin; Preferably, the gastrointestinal regulating peptides include: increasing the content of motilin and substance P, and reducing the content of vasoactive intestinal peptide, somatostatin, and endothelin; Preferably, the intestinal flora regulation comprises: increasing the number of Prevotellaceae _UGG_001 ( Prevotellaceae_ UCG_001 ), Oscillibacterium spp. Oscillibacter ), Bifidobacterium spp. ( Bifidobacterium ) and another genus Mycobacterium ( Alistipes ) abundance, and / or, reducing the abundance of Desulfovibrio ( Desulfovibrio ) and Enterobacter (Enterorhabdus) abundance; Preferably, the antibacterial spectrum includes: Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Bacteroides fragilis, Streptococcus anginosus, and Escherichia fergusonii.

[0037] The third group of embodiments, laxative functional food of the present invention This group of embodiments provides a laxative functional food. All embodiments in this group have the following common features: the laxative functional food comprises an active ingredient; the active ingredient comprises: a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427 that can relieve constipation and regulate intestinal flora.

[0038] In a further embodiment, the laxative functional food further includes: auxiliary materials.

[0039] 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, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0040] 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 make the animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC NO.32427 into different functional food dosage forms, such as solid beverages, milk beverages, powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, lotions, granules, etc.

[0041] In a specific embodiment, the dosage form of the laxative functional food is selected from: one or more of: solid beverages, milk beverages, tablets, liquids, and capsules.

[0042] 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 in this group have the following common features: the functional food for regulating intestinal flora includes active ingredients; the active ingredients include: a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427 that can relieve constipation and regulate intestinal flora.

[0043] In a further embodiment, the functional food for regulating intestinal flora 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 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 the animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC NO.32427 into different functional food dosage forms, such as solid beverages, milk beverages, powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, lotions, granules, etc.

[0046] 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.

[0047] The fifth group of embodiments, the antibacterial agent of the present invention This group of embodiments provides an antibacterial agent. All embodiments in this group have the following common features: the antibacterial agent comprises an active ingredient; the active ingredient comprises: a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427 that can relieve constipation and regulate intestinal flora.

[0048] In a further embodiment, the antibacterial agent further comprises: 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 applications, combined with conventional technical means in the field of formulation preparation (for example, "Encyclopedia of Formulation 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 the animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC NO.32427 into different dosage forms, such as powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, lotions, granules, etc.

[0051] In a specific embodiment, the dosage form of the antibacterial agent is selected from: one or more of powder, tablet, liquid, and capsule.

[0052] Group 6 Example, in vitro antibacterial method of the present invention This group of embodiments provides a non-therapeutic in vitro antibacterial method. All embodiments in this group have the following common features: using a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No. 32427 that can relieve constipation and regulate intestinal flora to inhibit bacteria.

[0053] The use of animal Bifidobacterium lactis subspecies BLa65, which is deposited with CGMCC No. 32427 and can relieve constipation and regulate intestinal flora, in combination with other probiotics for in vitro bacteriostasis falls within the protection scope of the present invention.

[0054] 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.

[0055] Experimental Example 1: Isolation, screening and identification of animal Bifidobacterium lactis subsp. (1) Isolation and screening of strains Breast milk samples were collected and placed in sterile sampling tubes for transportation in ice boxes. They were diluted with 0.85% saline gradients under sterile conditions, and the appropriate dilution gradients were selected and coated on MRS agar plates (MRSc) supplemented with 5% (V / V) mupirocin lithium salt and 0.1% L-cysteine ​​hydrochloride, and cultured at 37°C under anaerobic conditions for 48 hours. Suspected single colonies were selected by visual observation of their colony morphology, and opaque milky white, round and shiny, neatly edged, convex and moist single colonies were selected. After microscopic observation, preliminary screening and purification were performed. The purified strains were cultured anaerobically at 37°C in MRSc liquid for 14-16 hours, centrifuged to remove the supernatant, resuspended in a sterile 30% glycerol aqueous solution, and stored at -80°C.

[0056] (2) Molecular biological identification The genomic DNA of the bifidobacterium strain was extracted using a genomic DNA extraction kit, and the 16S rDNA sequence was amplified using the universal primers described in CN114574405A to obtain a PCR product. The PCR product was sequenced. The PCR reaction system included 20 μL of 10×Buffer, 4 μL of primer dNTP, 1 μL of upstream and downstream primers, 2 μL of DNA template, 0.5 μL of Taq enzyme, and ddH 2 O 34.5μL. PCR reaction conditions: 95°C 10min; 94°C 30s, 56°C 30s, 72°C 2min, 35 cycles; 72℃ 10min. The PCR product was detected by gel electrophoresis and sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. The identified gene sequence was submitted to the NCBI database (www.ncbi.nlm.nih.gov) for BLAST analysis and comparison. According to the results of molecular biological identification, the strain was determined to be animal Bifidobacterium lactis subsp., and it was named animal Bifidobacterium lactis subsp. BLa65. The strain was deposited, and its deposit information is as follows: Deposit number: CGMCC No.32427 Classification and naming: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis Depository: China National Microbiological Culture Collection Administration General Microbiology Center Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Date of preservation: October 30, 2024.

[0057] Experimental Example 2: Evaluation of gastrointestinal tolerance Simulated artificial gastric juice: prepare 0.5% NaCl solution, adjust the pH value to 2.5 with 1 mol / L HCL, add 0.3% pepsin, and then filter and sterilize with a 0.22 μm microporous filter membrane after fully dissolving.

[0058] Simulated artificial intestinal fluid: prepare 0.5% NaCl solution, adjust the pH value to 8.0 with 0.1 mol / L NaOH, add 0.1% trypsin, and then filter and sterilize with a 0.22 μm microporous filter membrane after fully dissolving.

[0059] BLa65 was activated and cultured for 3 generations under anaerobic conditions, and the cells were collected after centrifugation. 8 CFU / mL was inoculated into the prepared simulated artificial gastric juice of pH 2.5 and simulated artificial intestinal juice of pH 8.0, mixed evenly, and digested at 37°C. The number of viable bacteria was detected by taking the digestive juice at 0h and 3h, respectively. The survival rate of the strain in artificial gastric juice and artificial intestinal juice was calculated according to the following calculation formula:

[0060] Among them, N1 represents the number of viable bacteria (CFU / mL) after 3 hours of treatment; N0 represents the number of viable bacteria (CFU / mL) at 0 hours.

[0061] Table 1. Survival rate of Bifidobacterium animalis subsp. lactis BLa65 in artificial gastrointestinal fluid

[0062] As shown in Table 1, after 3 hours of digestion in artificial gastric juice or intestinal juice, the survival rates of animal Bifidobacterium lactis subsp. BLa65 were 99.16% and 100.45%, respectively, indicating that animal Bifidobacterium lactis subsp. BLa65 strain can tolerate the uncomfortable environment of the gastrointestinal tract, can smoothly pass through the gastrointestinal tract, and play a probiotic function.

[0063] Experimental Example 3: Adhesion ability of animal Bifidobacterium lactis subspecies BLa65 to Caco-2 cells Preparation of bacterial suspension: centrifuge the activated bacterial fermentation liquid, collect the bacteria, wash them three times with PBS, and then resuspend them in DMEM culture medium without double antibodies. 8 CFU / mL.

[0064] Caco-2 cell culture: Caco-2 cells were cultured in DMEM medium supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS), 1% penicillin and streptomycin at 37°C, 90% humidity, and 5% CO. 2 Culture in an incubator.

[0065] Cell adhesion experiment: ① Liquid culture of experimental strains: activated second-generation bacterial solution, 2% inoculation volume, 9 mL culture medium / 15 mL centrifuge tube, 37°C, static culture for 15 h. ② Preparation of single cell layer: Caco-2 cells were inoculated into DMEM culture medium supplemented with 20% (v / v) fetal bovine serum, transferred to 12-well cell culture plates, and the cell addition volume was 10 1 mL per well. 5 cell / ml, 5% CO 2 , culture at 37℃, change the medium every other day until a single cell layer is obtained for use; ③ Preparation of bacterial suspension: At the same time, take the cultured bacterial solution of the target strain, centrifuge at 10000 r / min at room temperature for 1 min to collect the bacteria, wash them twice with sterile PBS, resuspend them in DMEM medium, and adjust the bacterial suspension concentration to 10 8 CFU / mL; ④ Co-culture: Prepare the monolayer of Caco-2 cells, remove the culture medium, add PBS buffer to rinse twice, remove the buffer, and then add 1 mL / well of the prepared bacterial suspension, mix well, and incubate in 5% CO 2 , incubate at 37℃ for 2 h; ⑤ Carefully remove the culture supernatant and rinse 5 times with sterile PBS to remove non-adherent bacteria; ⑥ Add 0.2 mL / well of trypsin cell digestion solution and digest for 5 minutes to elute the cells from the culture plate wells. The collected solution is the sample; ⑦ Perform gradient dilution and count the viable bacteria on the collected samples. The above cell adhesion experiment uses Lactobacillus rhamnosus LGG as a control strain, and each experiment is performed in 3 parallels. Lactobacillus rhamnosus LGG is a commercial strain recognized in the industry as having good adhesion.

[0066] The adhesion capacity was calculated according to the following formula: adhesion capacity (CFU / cell) = number of bacteria adhering to cells (CFU) / number of cells in the well (cells).

[0067] Table 2. Cell adhesion ability of Bifidobacterium animalis subsp. lactis BLa65

[0068] The results are shown in Table 2. The adhesion ability of animal Bifidobacterium lactis subsp. BLa65 to Caco-2 cells was 8.70 CFU / cell, which was significantly higher than that of the control strain LGG. This indicates that BLa65 has good adhesion ability to Caco-2 cells and has the potential to colonize in the intestine, which helps it to exert its probiotic effect in the intestine.

[0069] Experimental Example 4: Oligosaccharide Utilization Ability MRSc-oligosaccharide medium: Add 0.5% (w / v) oligosaccharides (fructose, galactose, xylo-oligosaccharide) in equal amounts to replace glucose in MRSc. Inoculate the activated strain into the corresponding MRSc-oligosaccharide medium, and use MRSc medium with 0.5% glucose as a positive control. 600 Compared with the OD after growth in MRSc medium containing glucose 600 The ratio of is used to reflect the oligosaccharide utilization capacity, and the specific calculation formula is as follows: Oligosaccharide utilization capacity (%) = OD 600( MRSc-低聚糖培养基) / OD 600(MRSc-葡萄糖培养基) Table 3. The ability of animal Bifidobacterium lactis subsp. BLa65 to utilize different oligosaccharides

[0070] Functional oligosaccharides are indigestible and can reach the intestine to provide nutrition for the intestinal microbiota, selectively stimulating the growth and / or activity of microorganisms in the colon (usually bifidobacteria and lactobacilli). As shown in Table 3, animal Bifidobacterium lactis subspecies BLa65 can utilize oligofructose, galacto-oligosaccharide, and xylo-oligosaccharide, but the utilization capacity is different, among which the utilization degree is: oligofructose> galacto-oligosaccharide> xylo-oligosaccharide. The results show that animal Bifidobacterium lactis subspecies BLa65 can utilize oligosaccharides from the intestine to improve its competitiveness in the intestine.

[0071] Experimental Example 5: Experiment on antagonism of animal Bifidobacterium lactis subspecies BLa65 against pathogenic bacteria Preparation of pathogen suspension: (1) Escherichia coli ATCC25922, Salmonella enteritidis ATCC14028, and Staphylococcus aureus ATCC25923 were inoculated into LB liquid culture medium at 2% (v / v). After culturing at 37°C for 16-20 h, the bacterial solution concentration was adjusted to 10 8 CFU / mL.

[0072] (2) Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Bacteroides fragilis, Streptococcus anginosus, and Escherichia fergusonii were inoculated into a medium containing BHI + 5% sheep blood at 5% (v / v) and cultured at 37°C for 16-20 h. The bacterial solution concentration was adjusted to 10 8 CFU / mL.

[0073] Antagonistic pathogen experiment: (1) Cool the MRS broth containing 1.5% (w / v) agar to about 55°C and mix it with the indicator bacteria (Escherichia coli, Salmonella enteritidis, Staphylococcus aureus) suspension in a certain proportion so that the viable count of the indicator bacteria is 106 CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium cooled and solidified, the Oxford cup was taken out, and 200 μL of animal Bifidobacterium lactis subsp. BLa65 fermentation liquid was injected into each well. After overnight culture at 37°C, the diameter of the inhibition zone was measured.

[0074] (2) Cool the BHI broth medium (containing 5% sheep blood) containing 1.5% (w / v) agar to about 55°C and mix it with the indicator bacteria suspension (Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Bacteroides fragilis, Streptococcus anginosus, and Escherichia fergusonii) in a certain proportion so that the viable count of the indicator bacteria is 10 6 CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium cooled and solidified, the Oxford cup was taken out, and 200 μL of animal Bifidobacterium lactis subsp. BLa65 fermentation liquid was injected into each well. After overnight culture at 37°C, the diameter of the inhibition zone was measured.

[0075] Table 4. Inhibition zone of animal Bifidobacterium lactis subspecies BLa65 against different pathogens (mm)

[0076] As shown in Table 4, animal Bifidobacterium lactis subspecies BLa65 has an inhibitory effect on the growth of Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Bacteroides fragilis, Streptococcus anginosus and Escherichia fergusonii. This shows that animal Bifidobacterium lactis subspecies BLa65 can inhibit the growth of multiple pathogens, has a strong antibacterial ability, and is beneficial to maintaining intestinal health.

[0077] Experimental Example 6: Ability of Animal Bifidobacterium lactis subspecies BLa65 to Improve Stool Properties and Promote Intestinal Peristalsis 1) Preparation of ink: Mix 100 g of gum arabic with 800 mL of water, boil until the solution is transparent, add 50 g of activated carbon, boil three times, wait for the solution to cool, add water to make up to 1000 mL, store in a refrigerator at 4 ℃, and shake well before use. 2) Constipation model establishment: 7-8 week old BALB / c male mice (18-20 g) were housed in an environment with a room temperature of 25°C ± 2°C, a humidity of 50% ± 5%, and a 12-hour light-dark cycle. After a 7-day adaptation period, the mice were randomly divided into three groups (8 mice / group): blank control group (NC), model group (MC), and probiotic group (BLa65), and the formal experiment began. 14 days before the experiment, the NC group and the MC group were gavaged with 0.2 mL of normal saline every day, and the BLa65 group was gavaged with 0.2 mL of animal Bifidobacterium lactis subsp. BLa65 bacterial suspension (5×10 9CFU / mL). The 15th to 17th day of the experiment was the modeling period, during which the MC group and the BLa65 group were gavaged with 0.2 mL of 10 mg / kg.bw loperamide every day, and 4 hours later they were gavaged with an equal volume of saline or bacterial solution, while the NC group was only given an equal volume of saline in the same way.

[0078] 3) Index determination: a. Feces collection: On day 17, each mouse was transferred to a clean empty cage, and 3-h feces samples were collected, counted, and weighed.

[0079] b. Determination of the time of first black stool: On the 18th day (after 18 hours of fasting), all groups of mice except the normal group were gavaged with loperamide hydrochloride. One hour later, the blank control group and the constipation model were gavaged with ink, and the probiotic group was gavaged with 5×10 9 CFU / mL of ink of Bifidobacterium animalis subsp. lactis BLa65. The mice were transferred to clean, empty individual cages and provided with feed and water freely. The timing was started from the gavage of ink, and the time of the first black stool of each mouse was recorded. The time of the first black stool of the last mouse in the model group was the end time.

[0080] c. Determination of small intestinal propulsion rate: On the 19th day of gavage (after fasting for 18 hours), all groups of mice except the normal group were gavaged with loperamide hydrochloride. One hour later, the blank control group and the constipation model group were gavaged with ink, and the probiotic group was gavaged with 5×10 9 CFU / mL of ink containing animal Bifidobacterium lactis subsp. BLa65, the mice were killed 20 minutes later, the abdominal cavity was opened, the mesentery was separated, the intestinal tube from the pylorus at the upper end to the ileum and cecum at the lower end was cut, the small intestine was gently pulled into a straight line, the length of the intestinal tube was measured as the "total length of the small intestine", and the length from the pylorus to the front of the ink was the "ink advancement length" to calculate the small intestine advancement rate. The specific calculation formula is as follows: Small intestine advancement rate (%) = ×100% The results are as follows Figure 1 As shown in the figure, the number of stool particles, feces wet weight and small intestinal propulsion rate of mice in the MC group were significantly lower than those in the NC group ( p < 0.001), the time of first black stool was significantly higher than that of NC group, indicating that the constipation model of loperamide-induced mice was successfully established. The number of stool particles, feces wet weight and small intestinal propulsion rate of mice in BLa65 group were significantly higher than those in MC group ( p < 0.05), the time of first black stool was significantly lower than that of the model group ( p < 0.01). The above results show that intervention with animal Bifidobacterium lactis subspecies BLa65 can improve the fecal characteristics of mice, shorten the time to the first black stool, increase the small intestinal propulsion rate, and effectively relieve constipation in mice.

[0081] d. Determination of gastrointestinal regulatory peptides in mouse serum: Orbital blood was collected from mice, and serum was collected after centrifugation. The content of gastrointestinal regulatory peptides in mouse serum was determined using an ELISA kit according to the instructions. Figure 2 As shown in Figure 2, compared with the NC group, the levels of motilin and substance P in the MC group were significantly decreased ( p <0.01). On the contrary, the levels of vasoactive intestinal peptide, somatostatin, and endothelin in the MC group were significantly higher than those in the NC group ( p <0.05, p <0.01). Among the gastrointestinal regulatory peptides, motilin and substance P are excitatory transmitters, which can stimulate the production of pepsin and the contraction of the duodenum, thereby promoting gastrointestinal motility. Vasoactive intestinal peptide, somatostatin, and endothelin are inhibitory transmitters, which can inhibit the release of gastrointestinal hormones and the contraction of small intestinal smooth muscle, thereby leading to constipation. Animal Bifidobacterium lactis subspecies BLa65 can significantly promote the content of motilin and substance P in mice ( p <0.05, p <0.01), and significantly reduced the contents of three inhibitory neurotransmitters ( p <0.05, p <0.01), indicating that BLa65 can regulate the secretion of gastrointestinal regulatory peptides, promote gastrointestinal motility, and thus relieve constipation.

[0082] Experimental Example 7: Effect of Bifidobacterium lactis subspecies BLa65 on intestinal flora of mice The grouping and modeling of mice were the same as those in Experimental Example 6. After the experiment, the mice were killed and dissected, and the cecal contents were collected. The 16S rRNA amplicon sequencing analysis was performed on the cecal contents of the mice. The results of α-diversity analysis showed that ( Figure 3 ), loperamide caused disturbance of intestinal flora in mice. Compared with the NC group, the Chao1 index and Simpson index of the MC group were significantly reduced ( p <0.01). Compared with the MC group, the Chao1 index and Simpson index in the BLa65 group were significantly increased ( p < 0.01). This indicates that supplementation with animal Bifidobacterium lactis subsp. BLa65 can improve the richness and diversity of intestinal microbial flora and reduce the degree of damage to the intestinal microecology of mice induced by loperamide.

[0083] The changes in relative abundance at the phylum and genus levels in the cecal contents of mice among the groups were analyzed. The results at the phylum level showed that ( Figure 4 ), compared with the NC group, the relative abundance of Firmicutes and the ratio of Firmicutes to Bacteroidetes in the MC group were significantly reduced ( p(<0.001). Firmicutes and Bacteroidetes are the main microbiota in the intestine, and the ratio of Firmicutes / Bacteroidetes is considered an important indicator of intestinal flora balance. Its imbalance is often accompanied by the occurrence of diseases. Compared with the MC group, the relative abundance of Firmicutes and the ratio of Firmicutes / Bacteroidetes in the BLa65 group were significantly increased, indicating that supplementing BLa65 can restore the imbalance between Firmicutes and Bacteroidetes caused by loperamide and maintain intestinal microecological balance. The results at the genus level showed ( Figure 5 ), the relative abundances of Prevotellaceae _UGG_001 ( Prevotellaceae_UCG_001 ), Oscillibacter Oscillibacter ), Bifidobacterium Bifidobacterium ), and Alistipes Alistipes were significantly increased in the BLa65 group compared with the MC group ( p < 0.05, p < 0.001). The relative abundances of Desulfovibrio Desulfovibrio and Enterobacter (Enterorhabdus) in the MC group were significantly increased compared with the NC group ( p < 0.05), but BLa65 intervention could significantly reduce their relative abundances. Prevotellaceae _UGG_001 can produce short-chain fatty acids, and Alistipes can promote the production of saturated long-chain fatty acids, thus stimulating colonic muscle contraction and increasing the frequency of defecation. Bifidobacterium is generally considered a probiotic and has various functions such as regulating the intestinal flora structure, repairing the intestinal barrier, regulating immunity, and antioxidation. Desulfovibrio is one of the main members of Proteobacteria and can produce hydrogen sulfide to damage the intestinal mucosal barrier and cause intestinal inflammation. The results showed that Bifidobacterium animalis subsp. lactis BLa65 can significantly increase the abundance of beneficial bacteria, inhibit the colonization of harmful bacteria, promote the restoration of intestinal flora homeostasis, and is beneficial to enhancing the intestinal barrier function and promoting intestinal health.​​​​​​

Claims

1. A strain of Bifidobacterium animalis subsp. lactis that can relieve constipation and regulate intestinal flora ( Bifidobacterium animalis subsp. lactis ) BLa65, characterized in that Its deposit number is CGMCC No.32427.

2. Use of a strain of Bifidobacterium lactis subspecies BLa65, which is deposited with CGMCC No. 32427 and can relieve constipation and regulate intestinal flora, in the preparation of laxative functional foods, and / or intestinal flora regulating functional foods, and / or antibacterial agents, and / or in vitro antibacterial agents for non-therapeutic purposes.

3. The use of the strain of Bifidobacterium lactis subspecies BLa65 capable of relieving constipation and regulating intestinal flora with a deposit number of CGMCC No. 32427 according to claim 2 in the preparation of laxative functional foods, and / or intestinal flora regulating functional foods, and / or antibacterial agents, and / or in vitro antibacterial agents for non-therapeutic purposes, characterized in that: The laxative method includes: shortening the time of first black stool, increasing small intestinal propulsion rate, regulating gastrointestinal regulatory peptides, and regulating intestinal flora; And / or, the gastrointestinal regulatory peptides include: motilin, substance P, vasoactive intestinal peptide, somatostatin, endothelin; And / or, the regulating gastrointestinal regulatory peptides include: increasing the content of motilin and substance P, and reducing the content of vasoactive intestinal peptide, somatostatin, and endothelin; And / or, the intestinal flora regulation includes: increasing the number of Prevotellaceae _UGG_001 ( Prevotellaceae_UCG_ 001 ), Oscillibacterium spp. Oscillibacter ), Bifidobacterium spp. ( Bifidobacterium ) and another genus Mycobacterium ( Alistipes ) abundance, and / or, reducing the abundance of Desulfovibrio ( Desulfovibrio ) and Enterobacter (Enterorhabdus) abundance; And / or, the antibacterial spectrum includes: Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Bacteroides fragilis, Streptococcus anginosus, and Escherichia fergusonii.

4. A laxative functional food, comprising an active ingredient; characterized in that: The active ingredients include: a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427, which can relieve constipation and regulate intestinal flora.

5. The laxative functional food according to claim 4, characterized in that: Also includes: Auxiliary materials.

6. A functional food for regulating intestinal flora, comprising an active ingredient; characterized in that: The active ingredients include: a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427, which can relieve constipation and regulate intestinal flora.

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 active ingredient; characterized in that: The active ingredients include: a strain of animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No. 32427, which can relieve constipation and regulate intestinal flora.

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

10. A non-therapeutic in vitro antibacterial method, characterized in that: The animal Bifidobacterium lactis subspecies BLa65 with a preservation number of CGMCC No.32427, which can relieve constipation and regulate intestinal flora, is used for antibacterial purposes.

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