Bifidobacterium animalis subsp. Lactis BLa79 as well as application, product and method thereof

Abstract

The invention discloses bifidobacterium animalis subsp. Lactis BLa79 as well as application, a product and a method thereof, and belongs to the technical field of microorganisms. The preservation number of the bifidobacterium animalis subsp. Lactis BLa79 is CGMCC (China General Microbiological Culture Collection Center) NO. 32426, and the preservation number of the bifidobacterium animalis subsp. Lactis BLa79 is CGMCC NO. 32426. Based on the strain, the invention also provides application and related products of the strain. The BLa79 strain has broad-spectrum antibacterial performance, has the function of regulating intestinal flora, can effectively relieve ulcerative colitis, and can relieve colitis by increasing body weight, reducing DAI score, reducing spleen index, inhibiting colonic atrophy, reducing proinflammatory factor level and promoting SIgA secretion.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to animal Bifidobacterium lactis subspecies BLa79 and its application, product and method. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic inflammatory disease that affects the gastrointestinal tract, including Crohn's disease (CD) and ulcerative colitis (UC). UC usually directly affects the lining of the rectum or large intestine, causing diarrhea, abdominal pain, and rectal bleeding. 5-aminosalicylic acid (5-ASA), immunosuppressive drugs, and corticosteroids are the main drugs for the treatment of UC. However, they cannot eradicate UC, and long-term use has serious side effects. Surgical intervention is usually used for the treatment of acute or more severe UC, but there are more postoperative complications, such as diarrhea, intestinal adhesions, abdominal infections, etc. Therefore, new alternatives are needed for the prevention and clinical treatment of UC.

[0003] Probiotics are defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host". Probiotics exert beneficial effects on the host by inhibiting the growth of pathogenic microorganisms in the intestinal microbiota, promoting the production of short-chain fatty acids, balancing the intestinal microecological environment, and modulating the immune response. Bifidobacterium ) can promote the production of short-chain fatty acids, produce bacteriocins, and antagonize the adhesion and reproduction of intestinal pathogens. In addition, it can also regulate intestinal flora, effectively reduce the abundance of intestinal pathogens in UC patients, alleviate flora imbalance, and relieve intestinal inflammation.

[0004] However, the beneficial effects of probiotics are strain-specific. Animal Bifidobacterium lactis subspecies, which has multiple functions such as significantly inhibiting weight loss and colon atrophy caused by ulcerative colitis, regulating intestinal flora, enhancing immune function, significantly increasing the relative abundance of Bifidobacterium in the intestine, and having broad-spectrum antibacterial properties, is very rare. Therefore, the field urgently needs to develop new animal Bifidobacterium lactis subspecies strains. Summary of the invention

[0005] In order to solve the urgent need in the field to develop new strains of animal Bifidobacterium lactis subspecies, provide a new animal Bifidobacterium lactis subspecies that has multiple functions such as significantly inhibiting weight loss and colon atrophy caused by ulcerative colitis, regulating intestinal flora, enhancing immune function, significantly increasing the relative abundance of Bifidobacterium in the intestine, and having broad-spectrum antibacterial properties. The present invention provides an animal Bifidobacterium lactis subspecies BLa79 and its application, product and method.

[0006] The technical solution of the present invention is as follows: A Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis)BLa79, its deposit number is CGMCC NO.32426.

[0007] The invention discloses the use of animal Bifidobacterium lactis subspecies BLa79 with the preservation number of CGMCC NO.32426 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function, products for relieving ulcerative colitis or in vitro antibacterial effects for non-therapeutic purposes.

[0008] The antibacterial spectrum includes: Escherichia coli, Salmonella, Salmonella enteritidis, Staphylococcus aureus, Clostridium perfringens, Streptococcus Parisi, Streptococcus pharyngitis, Streptococcus pasteurianus; Preferably, the regulating intestinal flora includes: increasing the intestinal microbial diversity index, increasing the abundance of beneficial intestinal bacteria, and reducing the abundance of harmful intestinal bacteria; Preferably, the intestinal microbial diversity index includes: ACE index, Chao1 index, Shannon index and Simpson index; Preferably, the beneficial intestinal bacteria include: Ruminococcus ( Ruminococcus ), by Eubacterium mucosum ( Blautia ), Oscillibacterium spp. Oscillibacter ), Bifidobacterium spp. ( Bifidobacterium )and Butyricicoccus ; Preferably, the harmful intestinal bacteria include: Helicobacter , Candidatus_Saccharimonas and Erysipelatoclostridium ; Preferably, the immunity enhancement includes: reducing the level of pro-inflammatory factors and promoting SIgA secretion; Preferably, the relief of ulcerative colitis includes: increasing body weight, reducing DAI score, reducing spleen index, inhibiting colon atrophy, reducing the level of pro-inflammatory factors, and promoting SIgA secretion; Preferably, the pro-inflammatory factors include: TNF-α, IL-6, and MPO.

[0009] An antibacterial agent comprises: an antibacterial active ingredient; the antibacterial active ingredient comprises: animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCCNO.32426.

[0010] A product for relieving ulcerative colitis, comprising: an active ingredient; the active ingredient comprises: Bifidobacterium animalis subspecies lactis BLa79 with a preservation number of CGMCC NO.32426.

[0011] A functional food for regulating intestinal flora, comprising: an active ingredient; the active ingredient comprises: animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426.

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

[0013] A food for enhancing immune function, comprising: active ingredients; the active ingredients include: animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCCNO.32426.

[0014] The food for enhancing immune function also includes auxiliary materials.

[0015] The invention discloses an in vitro antibacterial method, which adopts animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426 to inhibit bacteria.

[0016] The beneficial effects of the present invention are as follows: The present invention screened a new strain from infant feces and identified it as Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ) and named it BLa79. The BLa79 strain has been proven by a large number of experiments to have broad-spectrum antibacterial properties, including Escherichia coli, Salmonella, Salmonella enteritidis, Staphylococcus aureus, Clostridium perfringens, Streptococcus Parisi, Streptococcus pharyngitis, Streptococcus pasteurianus and other pathogens. At the same time, the BLa79 strain also has the function of regulating intestinal flora, and can enhance the growth of rumencoccus ( Ruminococcus ), by Eubacterium mucosum ( Blautia ), Oscillibacterium spp. Oscillibacter ), Bifidobacterium spp. ( Bifidobacterium )and Butyricicoccus The abundance of beneficial intestinal bacteria, including Helicobacter ), Candidatus_ Saccharimonas and Erysipelatoclostridium The abundance of harmful intestinal bacteria such as TNF-α and IL-6; the BLa79 strain has the ability to enhance immunity, reduce the levels of pro-inflammatory factors including TNF-α, IL-6, MPO, and promote SIgA secretion; the strain can also effectively relieve ulcerative colitis by increasing body weight, reducing DAI score, reducing spleen index, inhibiting colon atrophy, reducing the level of pro-inflammatory factors, and promoting SIgA secretion to relieve colitis.

[0017] The deposit information of the animal Bifidobacterium lactis subspecies BLa79 of the present invention is as follows: Deposit number: CGMCC NO.32426 Classification and naming: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis Depository: China General Microbiological Culture Collection Management Center Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: October 30, 2024 BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a line graph of the body weight and DAI scores of the three groups of mice in Experimental Example 6 of the present invention; Figure A is a line graph of the body weight of mice in the model group and the normal feeding group, and Figure B is a line graph of the DAI scores of mice in the model group and the normal feeding group.

[0019] Figure 2 This is a bar graph of spleen index of three groups of mice in Experimental Example 7 of the present invention.

[0020] Figure 3 This is a bar graph of the colon lengths of three groups of mice in Experimental Example 8 of the present invention.

[0021] Figure 4 This is a bar graph showing the contents of TNF-α, IL-6, MPO and SIgA in the colon homogenates of three groups of mice in Experimental Example 9 of the present invention.

[0022] Figure 5 The bar graphs are the ACE index, Chao1 index, Shannon index and Simpson index of the three groups of mice in Experimental Example 10 of the present invention.

[0023] Figure 6 This is a bar graph of the relative abundance of species at the genus level in the cecal contents of the three groups of mice in Experimental Example 10 of the present invention.

[0024] Figures 1 - 6 The meanings of the symbols are listed as follows: NC is the normal group of mice, DSS is the model group of mice, and BLa79 is the animal Bifidobacterium lactis subsp. lactis treated group of mice. DETAILED DESCRIPTION

[0025] The details of the present invention are further described below in conjunction with specific embodiments and experimental examples, but the protection scope of the present invention is not limited thereto.

[0026] I. Source of Biological Material 1. The Caco-2 cells used in Experimental Example 3 were purchased from the Culture Collection of Wuhan University.

[0027] 2. Escherichia coli ATCC25922, Salmonella enteritidis ATCC14028, Staphylococcus aureus ATCC25923, Lactobacillus rhamnosus LGG, and Clostridium perfringens in Experimental Examples 4 and 5 were purchased from Guangdong Microbiological Culture Collection Center.

[0028] 3. The Streptococcus pasteurianus 12758, Streptococcus pharyngitis strain 4997, and Streptococcus Parisi 21178 of Experimental Example 4 have all been registered in NCBI, the accession number of Streptococcus pasteurianus 12758 is MW445225, the accession number of Streptococcus pharyngitis 4997 is MT459365, and the sequence number of Streptococcus Parisi 21178 is OM403351. Those skilled in the art can obtain the genetic information of these strains through the accession numbers and replicate the strains; these strains are currently preserved in the applicant's laboratory, and the applicant promises to release them to the public within 20 years from the date of application of the present invention for verification of the technical effects of the present invention.

[0029] 4. BALB / c male mice used in Experimental Examples 6-10 were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0030] 2. Culture medium and bacterial activation 1. MRSc medium (1L): peptone 10g, beef extract powder 5g, yeast extract powder 4g, K2HPO4 2g, triammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1mL, MgSO4·7H2O 0.58g, MnSO4·4H2O 0.25g, L-cysteine ​​hydrochloride 1g, distilled water 1000mL; pH 6.2±0.2. MRS medium preparation method refers to GB4789.35-2016, and 1.5% agar is added to the solid medium.

[0031] 2. Modified MRS medium (1L): peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, K2HPO4 2 g, sodium citrate 2 g, MgSO4·7H2O 0.2 g, MnSO4·4H2O 0.2 g, Tween 80 1 ml, distilled water 1000 ml.

[0032] MRS broth 3. 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 anhydrous sodium acetate, 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.

[0033] 4. LB medium (1 L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, sterilized at 115°C for 20 min 5. Strain activation: The experimental strain Bifidobacterium lactis subsp. BLa79 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.

[0034] Group 1 Example, Bifidobacterium animalis subspecies lactis BLa79 of the present invention This group of embodiments provides a kind of animal Bifidobacterium lactis subsp. Bifidobacterium animalis subsp. lactis )BLa79, its deposit number is CGMCC NO.32426.

[0035] 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 BLa79 with a preservation number of CGMCC NO.32426, and / or using a strain of Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426 in combination with other probiotics, and / or using a strain of Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426 to antagonize various pathogens, and / or preparing antibacterial products, and / or preparing products for relieving ulcerative colitis, and / or functional foods for regulating intestinal flora fall within the scope of protection of the present invention.

[0036] 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.

[0037] 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 BLa79 with a preservation number of CGMCC NO.32426 into products with different dosage forms, different storage conditions, and different shelf lives. This has no technical obstacles for those skilled in the art and is possible and easy to do.

[0038] The second group of examples, use of the present invention Bifidobacterium lactis subspecies BLa79 This group of embodiments provides the use of animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function, products for relieving ulcerative colitis or in vitro antibacterial effects for non-therapeutic purposes.

[0039] In some embodiments, the antibacterial spectrum includes: Escherichia coli, Salmonella, Salmonella enteritidis, Staphylococcus aureus, Clostridium perfringens, Streptococcus Parisi, Streptococcus pharyngitis, Streptococcus pasteurianus; Preferably, the regulating intestinal flora includes: increasing the intestinal microbial diversity index, increasing the abundance of beneficial intestinal bacteria, and reducing the abundance of harmful intestinal bacteria; Preferably, the intestinal microbial diversity index includes: ACE index, Chao1 index, Shannon index and Simpson index; Preferably, the beneficial intestinal bacteria include: Ruminococcus ( Ruminococcus ), by Eubacterium mucosum ( Blautia ), Oscillibacterium spp. Oscillibacter ), Bifidobacterium spp. ( Bifidobacterium )and Butyricicoccus ; Preferably, the harmful intestinal bacteria include: Helicobacter , Candidatus_Saccharimonas and Erysipelatoclostridium ; Preferably, the immunity enhancement includes: reducing the level of pro-inflammatory factors and promoting SIgA secretion; Preferably, the relief of ulcerative colitis includes: increasing body weight, reducing DAI score, reducing spleen index, inhibiting colon atrophy, reducing the level of pro-inflammatory factors, and promoting SIgA secretion; Preferably, the pro-inflammatory factors include: TNF-α, IL-6, and MPO.

[0040] The third group of examples, 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 antibacterial active ingredient; the antibacterial active ingredient comprises: animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426.

[0041] In a further embodiment, the antibacterial agent further comprises: auxiliary materials.

[0042] 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.

[0043] 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 the animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426 into different dosage forms, such as powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, lotions, granules, etc.

[0044] In a specific embodiment, the dosage form of the antibacterial agent is selected from: one or more of powder, tablet, liquid, and capsule.

[0045] The fourth group of embodiments, functional food for regulating intestinal flora of the present invention This group of embodiments provides a functional food for regulating intestinal flora. All embodiments of this group have the following common features: the functional food for regulating intestinal flora comprises: an active ingredient; the active ingredient comprises: animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426.

[0046] In a further embodiment, the functional food for regulating intestinal flora further includes: auxiliary materials.

[0047] 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.

[0048] 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 BLa79 with a preservation number of CGMCC NO.32426 into different functional food dosage forms, such as solid beverages, milk beverages, powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, lotions, granules, etc.

[0049] 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.

[0050] 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 comprises: an active ingredient; the active ingredient comprises: animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCCNO.32426.

[0051] In a further embodiment, the immune function enhancing food is characterized by further comprising: auxiliary materials.

[0052] 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.

[0053] 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 BLa79 with a preservation number of CGMCC NO.32426 into different functional food dosage forms, such as solid beverages, milk beverages, powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, lotions, granules, etc.

[0054] In a specific embodiment, the dosage form of the food for enhancing immune function is selected from: one or more of: solid beverages, milk beverages, tablets, liquids, and capsules.

[0055] The sixth group of embodiments, the product for relieving ulcerative colitis of the present invention This group of embodiments provides a product for relieving ulcerative colitis. All embodiments in this group have the following common features: the product for relieving ulcerative colitis comprises: an active ingredient; the active ingredient comprises: Bifidobacterium animalis subspecies lactis BLa79 with a preservation number of CGMCC NO.32426.

[0056] In a further embodiment, the product for relieving ulcerative colitis further includes: auxiliary materials.

[0057] 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.

[0058] 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", "Pharmaceutical Preparation Technology", "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 BLa79 with a preservation number of CGMCC NO.32426 into different dosage forms, such as powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, lotions, granules, etc.

[0059] In a specific embodiment, the dosage form of the product is selected from: one or more of powder, tablet, liquid, and capsule.

[0060] Group 7 Example, in vitro antibacterial method of the present invention This group of embodiments provides an in vitro antibacterial method. All embodiments in this group have the following common features: animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426 is used for antibacterial.

[0061] The use of a strain of Bifidobacterium animalis subspecies lactis BLa79 with a preservation number of CGMCC NO.32426 in combination with other probiotics for in vitro bacteriostasis falls within the protection scope of the present invention.

[0062] 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.

[0063] Experimental Example 1: Isolation, screening and identification of animal Bifidobacterium lactis subsp. (1) Isolation and screening of strains Infant fecal 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, with neat edges, and 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.

[0064] (2) Molecular biological identification The genomic DNA of the bifidobacterium strain was extracted using a genomic DNA extraction kit, and the 16S rDNA sequence of the strain was amplified using the universal primers described in CN114574405A to obtain PCR products. The PCR products were 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 34.5 μL of ddH2O. PCR reaction conditions: 95°C for 10 min; 94°C for 30 s, 56°C for 30 s, 72°C for 2 min, 35 cycles; 72°C for 10 min. 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 Bifidobacterium animalis subsp. lactis, and was named Bifidobacterium animalis subsp. lactis BLa79. The strain was deposited, and its deposit information is as follows: Deposit number: CGMCC NO.32426 Classification and naming: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis Depository: China General Microbiological Culture Collection Management Center Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: October 30, 2024 Experimental Example 2: Evaluation of gastrointestinal tolerance Simulate artificial gastric juice: prepare 0.5% NaCl solution, add 0.3% pepsin, adjust the pH value to 2.5 with 1 mol / L HCL, then filter and sterilize with a 0.22 μm microporous filter membrane after fully dissolving.

[0065] Bile salt solution: Add 0.2% (w / v) sodium thioglycolate to MRS liquid culture medium, then add 0.3% bile salt, fully dissolve and filter with a 0.22 μm microporous filter membrane for sterilization.

[0066] BLa79 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 gastric juice of pH 2.5 and 0.3% bile salt solution, and then digested at 37°C. The number of viable bacteria was detected by taking the digestive fluid at 0h and 3h, respectively. The survival rate of the strain in artificial gastric juice and bile salt was calculated according to the following calculation formula:

[0067] Among them, N1 represents the number of viable bacteria in the strain system after treatment, that is, the number of viable bacteria lgCFU / mL measured at 3h; N0 represents the initial number of viable bacteria in the strain system, that is, the number of viable bacteria lgCFU / mL measured at 0h.

[0068] Table 1. Tolerance of animal Bifidobacterium lactis subsp. BLa79 to gastric juice and bile salts

[0069] The results showed that after 3 hours of digestion with artificial gastric juice or bile salt, the survival rates of animal Bifidobacterium lactis subsp. BLa79 were 100.40% and 93.67%, respectively, indicating that animal Bifidobacterium lactis subsp. BLa79 strain has strong resistance to gastric acid and bile salts, can pass through the gastrointestinal tract smoothly, and exert its probiotic function.

[0070] Example 3: Adhesion ability of animal Bifidobacterium lactis subsp. BLa79 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.

[0071] 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 in a 37°C, 90% humidity, 5% CO2 incubator.

[0072] 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% CO2, 37℃ constant temperature culture, 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 twice with sterile PBS, resuspend in DMEM medium, and adjust the bacterial suspension concentration to 10 8CFU / mL; ④ Co-culture: Prepare the monolayer of Caco-2 cells, remove the culture medium, add PBS buffer to rinse twice, remove the buffer, add 1 mL / well of the prepared bacterial suspension, mix well, and incubate for 2 h at 5% CO2 and 37°C; ⑤ Carefully remove the culture supernatant, add sterile PBS to rinse 5 times 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 live bacteria count on the collected samples.

[0073] 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).

[0074] Table 2. Adhesion capacity of Bifidobacterium animalis subsp. lactis BLa79 to Caco-2 cells (CFU / cell)

[0075] The results are shown in Table 2. The adhesion ability of animal Bifidobacterium lactis subsp. lactis BLa79 to Caco-2 cells was 7.50 CFU / cell, which was better than the control strain LGG. This shows that BLa79 has good adhesion ability to Caco-2 cells and has the potential to colonize in the intestine.

[0076] Example 4: Experiment on antagonism of animal Bifidobacterium lactis subspecies BLa79 against pathogenic bacteria Preparation of pathogen suspension: Escherichia coli ATCC25922, Salmonella enteritidis ATCC14028, and Staphylococcus aureus ATCC25923 were inoculated into LB liquid culture medium at 2% (v / v), cultured at 37°C for 16-20 h, and then the bacterial solution concentration was adjusted to 10 8 CFU / mL.

[0077] Streptococcus Parisi, Streptococcus pasteurianus, and Streptococcus pharyngitis were inoculated into a medium containing BHI + 5% sheep blood at a rate of 5% (v / v). After culturing at 37°C for 16 to 20 h, the bacterial solution concentration was adjusted to 10 8 CFU / mL.

[0078] 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 10 6CFU / 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. BLa79 fermentation liquid was injected into each well. After overnight culture at 37°C, the diameter of the inhibition zone was measured.

[0079] (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 (Streptococcus Parisi, Streptococcus pharyngitis, Streptococcus pasteurianus) suspension 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. BLa79 fermentation liquid was injected into each well. After overnight culture at 37°C, the diameter of the inhibition zone was measured.

[0080] Table 3. Inhibition zone of animal Bifidobacterium lactis subspecies BLa79 against different pathogens (mm)

[0081] As shown in Table 3, the inhibition zones of animal Bifidobacterium lactis subspecies BLa79 against Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus Parisi, Streptococcus pharyngitis and Streptococcus pasteurianus are 22.3 mm, 33.83 mm, 29.17 mm, 21 mm, 16.83 mm and 20.67 mm, respectively, indicating that animal Bifidobacterium lactis subspecies BLa79 can inhibit the growth of different pathogens and has good antibacterial ability.

[0082] Example 5: Antibacterial experiment of co-culture of animal Bifidobacterium lactis subspecies BLa79 and intestinal pathogenic bacteria Escherichia coli, Salmonella enteritidis, and Clostridium perfringens were activated in the corresponding culture medium, and the bacterial quantity was adjusted to 10 8 CFU / ml. Escherichia coli and Salmonella Enteritidis were inoculated into modified MRS liquid medium at a 2% inoculation rate, and Clostridium perfringens was inoculated into BHI liquid medium at a 2% inoculation rate. The same amount of BLa79 was inoculated into the culture medium with pathogens and mixed cultured. At the same time, Escherichia coli, Salmonella Enteritidis, and Clostridium perfringens were inoculated into the corresponding liquid culture medium with the same inoculation rate as control culture. After static culture at 37 ℃ for 24 h, the inhibition of BLa79 on pathogens was observed after co-culture of BLa79 and pathogens. The results are shown in Table 4.

[0083] Table 4. Co-culture experiment of BLa79 and intestinal pathogenic bacteria

[0084] The results are shown in Table 4. After co-culture with pathogenic bacteria, BLa79 has a strong inhibitory effect on three intestinal pathogens. Its inhibition rates on Escherichia coli, Salmonella enteritidis and Clostridium perfringens are 94.13%, 99.78% and 97.69%, respectively. This shows that BLa79 has a broad-spectrum antibacterial property, can resist the invasion of multiple pathogens on the intestine, and protect the intestinal barrier function.

[0085] Example 6 Effects of Bifidobacterium animalis subsp. lactis BLa79 on body weight and DAI scores in colitis mice Colitis model establishment: The animal model was induced by DSS. Thirty SPF BALB / c male mice (19-20 g) were used in the experiment. The room temperature of the animals was 23±2 ℃, the humidity was 50%±10%, the artificial light was 12 h / d, and the diet was free. All mice were divided into 3 groups, 10 in each group, namely the normal group (NC), the model group (DSS), and the animal Bifidobacterium lactis subspecies treatment group (BLa79). The specific experimental process is as follows: one week of adaptive feeding, in the second week, all mice except the normal group were free to drink 3.5% DSS solution for 7 days, and the DSS solution was replaced every 2 days to ensure the concentration and quality of the DSS solution. At the same time, the experimental mice were gavaged with 0.2 ml of the corresponding solution every day. The normal group (NC) and the model group (DSS) were gavaged with PBS, and the animal Bifidobacterium lactis subspecies treatment group (BLa79) was gavaged with 5×10 9 CFU / ml concentration of animal Bifidobacterium lactis subsp. BLa79 bacterial suspension. The model group (DSS) and animal Bifidobacterium lactis subsp. BLa79 treated group (BLa79) freely drank 3.5% DSS solution for 7 days, and then replaced with normal drinking water. Gavage was performed once a day at a fixed time for 14 consecutive days, with a gavage volume of 0.2 ml each time; the normal group and the model group were gavaged with an equal volume of PBS every day. After the experiment, the mice were fasted for 12 hours, and the mice were killed and dissected, and samples were collected for subsequent index determination and analysis.

[0086] Changes in DAI scores of mice: The mice were weighed and recorded every day during the experiment, and the weight changes of the mice were counted. During the modeling period, the feces of the mice were collected every day, the fecal characteristics were observed, and the blood in the feces of the mice was detected with occult blood test paper to calculate the DAI score. The specific scoring criteria are shown in Table 5.

[0087] Table 5. DAI scoring criteria

[0088] Depend on Figure 1As shown in Figure A, the weight of mice decreased after DSS induction, while the weight of mice in the normal group gradually increased. By the end of the experiment, compared with before modeling, the weight of mice in the model group decreased by 17.42%, while the weight of mice in the animal Bifidobacterium lactis subsp. lactis treatment group decreased by only 2.96%, indicating that BLa79 intervention can significantly inhibit the weight loss of mice ( p < 0.001). The trend of DAI change is opposite to that of body weight change. Figure 1 Middle B shows that during the modeling period, the DAI score of the model group continued to rise. At the end of the experiment, the DAI score of the mice in the model group was significantly higher than that in the normal group ( p < 0.0001), while the DAI score of the group treated with animal Bifidobacterium lactis subsp. p < 0.001), indicating that animal Bifidobacterium lactis subsp. BLa79 effectively inhibited the occurrence of colitis and reduced the toxic effects of DSS on mice.

[0089] Example 7: Effect of Bifidobacterium lactis subsp. BLa79 on the spleen index of mice The grouping and modeling of mice were the same as in Example 6. After the mice were dissected, the spleens were collected and weighed, and the spleen index was calculated. Figure 2 It can be seen that DSS induced spleen enlargement in mice. Compared with the normal group, the spleen index of the model group was significantly increased ( p < 0.001), while the spleen index of the group treated with animal Bifidobacterium lactis subsp. p < 0.001), indicating that the intake of Bifidobacterium animalis subsp. lactis BLa79 could alleviate spleen damage in mice with DSS-induced colitis.

[0090] Example 8: Effect of Bifidobacterium animalis subsp. lactis BLa79 on colon length The grouping and modeling of mice were the same as in Example 6. After the mice were killed, the colon tissues of the mice were collected and the colon length was measured. Figure 3 As shown in the figure, the colon length of the model group was significantly lower than that of the normal group. After intervention with BLa79, the colon atrophy of mice was inhibited. Compared with the model group (4.83 cm), the colon length of the animal Bifidobacterium lactis subsp. lactis treated group (7.43 cm) was significantly increased, about 1.54 times that of the model group. These results indicate that animal Bifidobacterium lactis subsp. lactis BLa79 can effectively protect the mouse colon from damage caused by DSS.

[0091] Example 9: Effects of Bifidobacterium animalis subsp. lactis BLa79 on the content of myeloperoxidase (MPO) and cytokines in the colon of colitis mice The grouping and modeling of mice were the same as in Example 6. After the experiment, the mice were killed and dissected, and the colon was placed in phosphate buffered saline and homogenized. The content of TNF-α, IL-6, MPO and SIgA in the colon homogenate of each group of mice was determined according to the detection method in the manufacturer's instructions of the ELISA kit. The results are shown in Figure 4 Compared with the control group, the levels of pro-inflammatory factors TNF-α and IL-6 in the model group increased significantly ( p <0.05, p <0.01), MPO content increased significantly ( p <0.01). Compared with the model group, the concentrations of TNF-α, IL-6 and MPO in the colon of mice treated with animal Bifidobacterium lactis subsp. p <0.05). In addition, the intake of animal Bifidobacterium lactis subsp. BLa79 promoted the secretion of SIgA in the colon. Compared with the model group, the SIgA concentration in the animal Bifidobacterium lactis subsp. treated group was significantly increased ( p <0.001), which was approximately 187.02% of that in the model group. This shows that animal Bifidobacterium lactis subspecies BLa79 reduces the level of proinflammatory factors in the colon of DSS-induced colitis mice and alleviates neutrophil aggregation. In addition, it also promotes the production of SIgA and improves the immune function of the intestinal mucosa, thereby alleviating intestinal inflammatory response and maintaining intestinal homeostasis.

[0092] Example 10: Effect of Bifidobacterium lactis subsp. BLa79 on intestinal flora of mice The grouping and modeling of mice were the same as in Example 6. After the experiment, the mice were killed and dissected, and the cecal contents were collected. The 16S rRNA sequencing and analysis of the cecal contents of the mice were performed. The results showed that ( Figure 5 ), DSS caused intestinal flora disturbance in mice. Compared with the normal group, the ACE index, Chao1 index and Shannon index of the model group were significantly reduced ( p < 0.01). Compared with the model group, the ACE index, Chao1 index, Shannon index and Simpson index of intestinal microbial α-diversity in the animal Bifidobacterium lactis subsp. lactis treatment group were all increased, among which the ACE index, Chao1 index and Shannon index were extremely significantly increased ( p < 0.01). The above results indicate that supplementation with Bifidobacterium lactis subsp. BLa79 can improve the richness and diversity of intestinal microbial flora.

[0093] The changes in relative abundance of species at the genus level in the cecum contents of mice among the groups were analyzed, and the results showed that ( Figure 6 ), Ruminococcus in the group treated with Bifidobacterium animalis subsp. lactis ( Ruminococcus ), by Eubacterium mucosum ( Blautia ), Oscillibacterium spp. Oscillibacter), Bifidobacterium spp. ( Bifidobacterium )and Butyricicoccus The relative abundance was significantly higher than that of the model group ( p < 0.05, p < 0.001). Helicobacter genus ( Helicobacter ), Candidatus_Saccharimonas and Erysipelatoclostridium The relative abundance was significantly higher than that of the normal group ( p <0.05, p < 0.001), but BLa79 intervention can significantly reduce its relative abundance. Ruminococcus and Eubacterium vicissus are considered to be key flora in ulcerative colitis, and their abundance is usually reduced in UC patients. Ruminococcus is a genus of bacteria that produces propionic acid and butyric acid, which can participate in food digestion and maintain intestinal barrier function. Bifidobacterium is generally considered to be a probiotic, with multiple functions such as regulating intestinal flora structure, repairing intestinal barrier, regulating immune response, and anti-oxidation. This study found that BLa79 intervention can significantly increase the relative abundance of Bifidobacterium in the intestine of mice. The relative abundance of Bifidobacterium in the animal Bifidobacterium lactis subspecies treatment group was about 22 times that of the control group or model group. Eubacterium vicissus, Oscillibacterium and Butyricicoccus It can ferment carbohydrates to produce short-chain fatty acids, provide energy to host cells, and regulate immune responses. In addition, mucoid Eubacterium can also produce bacteriocins to prevent pathogens from colonizing in the intestine. Candidatus_Saccharimonas and Erysipelatoclostridium They are considered enteric pathogens that can adversely affect host health. Erysipelatoclostridium It is a potential pathogen associated with colon disease, and its abundance is significantly increased in mice with IBD. The results showed that animal Bifidobacterium lactis subspecies BLa79 can restore the structure of the intestinal flora, significantly increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, promote the restoration of intestinal flora homeostasis, and thus enhance the intestinal barrier function and promote intestinal health.

Claims

1. A Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ) BLa79, characterized in that Its deposit number is CGMCC NO.32426.

2. Use of animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function, products for relieving ulcerative colitis or in vitro antibacterial properties for non-therapeutic purposes.

3. The use of animal Bifidobacterium lactis subspecies BLa79 with a deposit number of CGMCC NO.32426 according to claim 2 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function, products for relieving ulcerative colitis or in vitro antibacterial effects for non-therapeutic purposes, characterized in that: The antibacterial spectrum includes: Escherichia coli, Salmonella, Salmonella enteritidis, Staphylococcus aureus, Clostridium perfringens, Streptococcus Parisi, Streptococcus pharyngitis, Streptococcus pasteurianus; And / or, the regulating intestinal flora includes: increasing the intestinal microbial diversity index, increasing the abundance of beneficial intestinal bacteria, and reducing the abundance of harmful intestinal bacteria; And / or, the intestinal microbial diversity index includes: ACE index, Chao1 index, Shannon index and Simpson index; And / or, the beneficial intestinal bacteria include: Ruminococcus ( Ruminococcus ), by Eubacterium mucosum ( Blautia ), Oscillibacterium spp. Oscillibacter ), Bifidobacterium spp. ( Bifidobacterium )and Butyricicoccus ; And / or, the harmful intestinal bacteria include: Helicobacter , Candidatus_Saccharimonas and Erysipelatoclostridium ; And / or, the immunity enhancement includes: reducing the level of pro-inflammatory factors and promoting SIgA secretion; And / or, the relief of ulcerative colitis includes: increasing body weight, reducing DAI score, reducing spleen index, inhibiting colon atrophy, reducing the level of pro-inflammatory factors, and promoting SIgA secretion; And / or, the pro-inflammatory factors include: TNF-α, IL-6, MPO.

4. An antibacterial agent, characterized in that include: Antibacterial active ingredients; The antibacterial active ingredients include: Bifidobacterium animalis subspecies lactis BLa79 with a preservation number of CGMCC NO.32426.

5. A product for relieving ulcerative colitis, characterized in that: include: Active ingredients; The active ingredients include: Bifidobacterium animalis lactis subspecies BLa79 with a preservation number of CGMCC NO.32426.

6. A functional food for regulating intestinal flora, characterized in that: include: Active ingredients; The active ingredients include: Bifidobacterium animalis lactis subspecies BLa79 with a preservation number of CGMCC NO.32426.

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

8. A food for enhancing immune function, characterized in that: include: Active ingredients; The active ingredients include: Bifidobacterium animalis lactis subspecies BLa79 with a preservation number of CGMCC NO.32426.

9. The food for enhancing immune function according to claim 8, characterized in that: Also includes: Auxiliary materials.

10. An in vitro antibacterial method, characterized in that: The animal Bifidobacterium lactis subspecies BLa79 with a preservation number of CGMCC NO.32426 was used for bacteriostasis.

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