Bifidobacterium animalis subsp. lactis BLa79, its applications, products and methods

Abstract

The present invention relates to Bifidobacterium animalis subsp. lactis BLa79, its applications, products and methods, belonging to the technical field of microbiology. The preservation number of the Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ) BLa79 is CGMCC NO. 32426. Based on this strain, the present invention also provides its uses and related products. The BLa79 strain has broad-spectrum antibacterial properties, can regulate the intestinal flora, and can also effectively relieve ulcerative colitis. It can relieve colitis by increasing body weight, reducing the DAI score, reducing the spleen index, inhibiting colon atrophy, reducing the levels of pro-inflammatory factors, and promoting the secretion of SIgA.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to Bifidobacterium animalis subsp. lactis BLa79, its application, product and method. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic inflammatory disease affecting the gastrointestinal tract, including Crohn's disease (CD) and ulcerative colitis (UC). UC usually directly affects the inner wall of the rectum or large intestine, resulting in diarrhea, abdominal pain, rectal bleeding, etc. 5-Aminosalicylic acid (5-ASA), immunosuppressive drugs, and corticosteroids are the main drugs for treating UC. However, they cannot eradicate UC and have serious side effects when used for a long time. Surgical intervention is usually used for the treatment of acute or severe UC, but there are many postoperative complications, such as diarrhea, intestinal adhesion, abdominal infection, etc. Therefore, new alternative solutions are needed for the prevention and clinical treatment of UC.

[0003] Probiotics are defined as "live microorganisms that are beneficial to the health of the host when ingested in sufficient quantities". Probiotics play beneficial roles in the host. They inhibit the growth of pathogenic microorganisms in the gut microbiota, promote the production of short-chain fatty acids, balance the intestinal microecological environment, and regulate the immune response. The genus Bifidobacterium ( Bifidobacterium ) can promote the production of short-chain fatty acids, produce bacteriocins, and antagonize the adhesion and reproduction of intestinal pathogenic bacteria. In addition, it can regulate the intestinal flora, effectively reduce the abundance of intestinal pathogenic bacteria in UC patients, alleviate dysbacteriosis, and relieve intestinal inflammation.

[0004] However, the beneficial effects of probiotics are strain-specific. It is very rare for Bifidobacterium animalis subsp. lactis to simultaneously have significant inhibitory effects on weight loss and colon atrophy caused by ulcerative colitis, regulate the intestinal flora, enhance immune function, significantly increase the relative abundance of the genus Bifidobacterium in the intestine, and have broad-spectrum antibacterial properties. Therefore, there is an urgent need in this field to develop new strains of Bifidobacterium animalis subsp. lactis. Summary of the Invention

[0005] In order to meet the need in this field to urgently develop new strains of Bifidobacterium animalis subsp. lactis, and to provide a new Bifidobacterium animalis subsp. lactis that simultaneously has significant inhibitory effects on weight loss and colon atrophy caused by ulcerative colitis, regulates the intestinal flora, enhances immune function, significantly increases the relative abundance of the genus Bifidobacterium in the intestine, and has broad-spectrum antibacterial properties, the present invention provides a Bifidobacterium animalis subsp. lactis BLa79, its application, product and method.

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

[0007] A Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis)BLa79, with the deposit number of CGMCC NO.32426.

[0008] Use of Bifidobacterium animalis subsp. lactis BLa79 with the deposit number of CGMCC NO.32426 in the preparation of bacteriostatic agents, functional foods for regulating intestinal flora, functional foods for enhancing immune function, products for relieving ulcerative colitis or in vitro bacteriostasis for non-therapeutic purposes.

[0009] The bacteriostatic spectrum of the bacteriostasis includes: Escherichia coli, Salmonella, Salmonella enteritidis, Staphylococcus aureus, Clostridium perfringens, Streptococcus paris, Streptococcus anginosus, Streptococcus pasteurianus;

[0010] Preferably, the regulation of intestinal flora includes: increasing the intestinal microbial diversity index, increasing the abundance of intestinal beneficial bacteria, and decreasing the abundance of intestinal harmful bacteria;

[0011] Preferably, the intestinal microbial diversity index includes: ACE index, Chao1 index, shannon index and simpson index;

[0012] Preferably, the intestinal beneficial bacteria include: Ruminococcus ( Ruminococcus ), Eubacterium coprostanoligenes ( Blautia ), Oscillibacter ( Oscillibacter ), Bifidobacterium ( Bifidobacterium ), and Butyricicoccus ;

[0013] Preferably, the intestinal harmful bacteria include: Helicobacter , Candidatus_Saccharimonas and Erysipelatoclostridium ;

[0014] Preferably, the enhancement of immunity includes: reducing the level of pro-inflammatory factors and promoting the secretion of SIgA;

[0015] Preferably, the relief of ulcerative colitis includes: increasing body weight, decreasing DAI score, decreasing spleen index, inhibiting colon atrophy, reducing the level of pro-inflammatory factors, and promoting the secretion of SIgA;

[0016] Preferably, the pro-inflammatory factors include: TNF-α, IL-6, MPO.

[0017] A bacteriostatic agent, which includes: a bacteriostatic active ingredient; the bacteriostatic active ingredient includes: Bifidobacterium animalis subsp. lactis BLa79 with the deposit number of CGMCC NO.32426.

[0018] A product for relieving ulcerative colitis, which includes: an active ingredient; the active ingredient includes: Bifidobacterium animalis subsp. lactis BLa79 with the deposit number of CGMCC NO.32426.

[0019] A functional food for regulating intestinal flora, comprising: active ingredients; the active ingredients include: Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO. 32426.

[0020] The above-mentioned functional food for regulating intestinal flora further comprises: excipients.

[0021] A functional food for enhancing immune function, comprising: active ingredients; the active ingredients include: Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO. 32426.

[0022] The above-mentioned functional food for enhancing immune function further comprises: excipients.

[0023] An in vitro bacteriostatic method using Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO. 32426 for bacteriostasis.

[0024] The beneficial effects of the present invention are as follows:

[0025] A new strain was screened from infant feces in the present invention and identified as Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ), and it was named BLa79. The BLa79 strain has been proven by a large number of experiments to have broad-spectrum antibacterial properties and can inhibit pathogenic bacteria including Escherichia coli, Salmonella, Salmonella enteritidis, Staphylococcus aureus, Clostridium perfringens, Streptococcus paris, Streptococcus anginosus, and Streptococcus pasteurianus; at the same time, the BLa79 strain also has the function of regulating intestinal flora and can increase the abundances of beneficial intestinal bacteria including Ruminococcus ( Ruminococcus ), Eubacterium rectale ( Blautia ), Oscillibacter ( Oscillibacter ), Bifidobacterium ( Bifidobacterium ), and Butyricicoccus , and reduce the abundances of harmful intestinal bacteria including Helicobacter ( Helicobacter ), Candidatus_ Saccharimonas and Erysipelatoclostridium ; the BLa79 strain has the function of enhancing immune function and can reduce the levels of pro-inflammatory factors including TNF-α, IL-6, MPO, etc., and promote the secretion of SIgA; the strain also has the ability to effectively relieve ulcerative colitis by increasing body weight, reducing the DAI score, reducing the spleen index, inhibiting colon atrophy, reducing the levels of pro-inflammatory factors, and promoting the secretion of SIgA to relieve colitis.

[0026] The preservation information of Bifidobacterium animalis subsp. lactis BLa79 of the present invention is as follows:

[0027] Preservation Number: CGMCC NO.32426

[0028] Taxonomic Name: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis

[0029] Preservation Institution: China General Microbiological Culture Collection Center

[0030] Address of the Preservation Institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0031] Preservation Date: October 30, 2024 Description of the Drawings

[0032] Figure 1 This is a line graph of the body weights and DAI scores of three groups of mice in Experimental Example 6 of the present invention; among them, Graph A is the line graph of the body weights of the mice in the model group and the normal feeding group, and Graph B is the line graph of the DAI scores of the mice in the model group and the normal feeding group.

[0033] Figure 2 This is a bar graph of the spleen indices of three groups of mice in Experimental Example 7 of the present invention.

[0034] Figure 3 This is a bar graph of the colon lengths of three groups of mice in Experimental Example 8 of the present invention.

[0035] Figure 4 This is a bar graph of 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.

[0036] Figure 5 This is a bar graph of the ACE index, Chao1 index, Shannon index, and Simpson index of three groups of mice in Experimental Example 10 of the present invention.

[0037] Figure 6 This is a bar graph of the relative abundances at the genus level of the cecal contents of three groups of mice in Experimental Example 10 of the present invention.

[0038] Figures 1 - 6 The meanings of the labels are listed as follows: NC is the mice in the normal group, DSS is the mice in the model group, and BLa79 is the mice in the treatment group with Bifidobacterium animalis subsp. lactis. Detailed Embodiments

[0039] The following further describes the detailed content of the present invention in combination with specific embodiments and experimental examples, but does not limit the protection scope of the present invention hereby.

[0040] I. Source of Biological Material

[0041] 1. The Caco-2 cells used in Experimental Example 3 were purchased from the Culture Collection Center of Wuhan University.

[0042] 2. Escherichia coli ATCC25922, Salmonella enteritidis ATCC14028, Staphylococcus aureus ATCC25923, Lactobacillus rhamnosus GG, and Clostridium perfringens used in Experimental Example 4 and Experimental Example 5 were purchased from the Guangdong Provincial Culture Collection Center of Microorganisms.

[0043] 3. Streptococcus pasteurianus 12758, Streptococcus anginosus strain 4997, and Streptococcus paris 21178 used in Experimental Example 4 have all been registered in NCBI. The accession number of Streptococcus pasteurianus 12758 is MW445225, the accession number of Streptococcus anginosus 4997 is MT459365, and the sequence number of Streptococcus paris 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 stored in the applicant's laboratory, and the applicant undertakes to distribute them to the public within 20 years from the filing date of this invention for verifying the technical effects of this invention.

[0044] 4. The BALB / c male mice used in Experimental Examples 6 - 10 were purchased from Beijing Speywood Biotechnology Co., Ltd.

[0045] II. Culture Media and Bacterial Strain Activation

[0046] 1. MRSc Medium (1 L): Peptone 10 g, Beef Extract Powder 5 g, Yeast Extract Powder 4 g, K 2 HPO 4 2 g, Ammonium Citrate 2 g, Sodium Acetate 5 g, Glucose 20 g, Tween 80 1 mL, MgSO 4 ·7H 2 O 0.58 g, MnSO 4 ·4H 2 O 0.25 g, L-Cysteine Hydrochloride 1 g, Distilled Water 1000 mL; pH value 6.2 ± 0.2. The preparation method of MRS medium refers to GB4789.35 - 2016, and 1.5% agar is added to the solid medium.

[0047] 2. 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, Mg SO 4 ·7H 2 O 0.2 g, MnSO 4 ·4H 2 O 0.2 g, Tween 80 1 ml, 1000 ml distilled water.

[0048] MRS Broth Medium

[0049] 3. MRS medium (1 L): Peptone 10 g, Beef extract powder 10 g, Yeast extract powder 5 g, Glucose 20 g, Diammonium citrate 2 g, Sodium acetate anhydrous 5 g, Dipotassium hydrogen phosphate 2 g, Magnesium sulfate 0.2 g, Manganese sulfate monohydrate 0.19 g, Tween-80 1 g. When preparing solid medium, add 2% agar powder and sterilize at 115°C for 20 min.

[0050] 4. LB medium (1 L): Tryptone 10 g, Yeast extract 5 g, Sodium chloride 10 g, Sterilize at 115°C for 20 min

[0051] 5. Strain activation: The test strain Bifidobacterium animalis subsp. lactis BLa79 was activated for three generations: The glycerol stock strain was inoculated into MRSc medium at 2% (v / v) and cultured at 37°C for 14 h; transferred to the second generation at an inoculation amount of 5% and cultured at 37°C for 8 h; transferred to the third generation at an inoculation amount of 2% and cultured at 37°C for 14 h to obtain the activated strain.

[0052] Group 1 Examples, Bifidobacterium animalis subsp. lactis BLa79 of the present invention

[0053] This group of examples provides a Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ) BLa79 with the efficacy of alleviating ulcerative colitis and broad-spectrum antibacterial, and its preservation number is CGMCC NO.32426.

[0054] Any act of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, reconstructing, selling, or offering to sell a strain of Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO.32426, and / or, the act of combining a strain of Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO.32426 with other probiotics, and / or, the antagonism of a strain of Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO.32426 against various pathogenic bacteria, and / or, the preparation of antibacterial products, and / or, the preparation of products for alleviating ulcerative colitis, and / or, the preparation of functional foods for regulating intestinal flora all fall within the protection scope of the present invention.

[0055] The other probiotics include, but are not limited to: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces cerevisiae, Torulaspora delbrueckii, Candida spp., Wickerhamomyces spp., Pichia spp., Saccharomyces boulardii, Trichosporonoides spp., Schwanniomyces occidentalis, Rhodotorula rubra, Schizosaccharomyces pombe, Pichia kudriavzevii, Bacillus thuringiensis, Brevibacillus laterosporus, Bacillus megaterium, Bacillus mucilaginosus, Bacillus azotofixans, Bacillus sphaericus, Clostridium butyricum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium infantis (i.e., Bifidobacterium longum subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis subsp. lactis), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium thermophilum, and Bifidobacterium thermophilum acidophilus.

[0056] Those skilled in the art can, according to actual production needs, combine conventional technical means or common knowledge in the production process of the pharmaceutical field (such as "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Inoculant Technology", etc.) to make routine selections or adjustments of pharmaceutical excipients, and then prepare products of different dosage forms, different storage conditions, and different shelf lives from a strain of Bifidobacterium animalis subsp. BLa79 with the preservation number CGMCC NO. 32426. This poses no technical obstacle to those skilled in the art and can be easily achieved.

[0057] Group 2 Examples, Uses of Bifidobacterium animalis subsp. BLa79 of the Present Invention

[0058] This group of examples provides the uses of Bifidobacterium animalis subsp. BLa79 with the preservation number CGMCC NO. 32426 in the preparation of bacteriostatic agents, functional foods for regulating intestinal flora, functional foods for enhancing immune function, products for relieving ulcerative colitis, or in vitro bacteriostasis for non-therapeutic purposes.

[0059] In some examples, the bacteriostatic spectrum of the bacteriostasis includes: Escherichia coli, Salmonella spp., Salmonella enteritidis, Staphylococcus aureus, Clostridium perfringens, Streptococcus paris, Streptococcus anginosus, Streptococcus pasteurianus;

[0060] Preferably, the regulation of intestinal flora includes: increasing the intestinal microbial diversity index, increasing the abundance of intestinal beneficial bacteria, and decreasing the abundance of intestinal harmful bacteria;

[0061] Preferably, the intestinal microbial diversity indices include: ACE index, Chao1 index, shannon index, and simpson index;

[0062] Preferably, the beneficial intestinal bacteria include: Ruminococcus ( Ruminococcus ), Eubacterium rectale ( Blautia ), Oscillibacter ( Oscillibacter ), Bifidobacterium ( Bifidobacterium ), and Butyricicoccus ;

[0063] Preferably, the harmful intestinal bacteria include: Helicobacter , Candidatus_Saccharimonas and Erysipelatoclostridium ;

[0064] Preferably, the immune enhancement includes: reducing the level of pro-inflammatory factors and promoting SIgA secretion;

[0065] Preferably, the remission 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;

[0066] Preferably, the pro-inflammatory factors include: TNF-α, IL-6, MPO.

[0067] Group 3 Examples, the bacteriostatic agent of the present invention

[0068] This group of examples provides a bacteriostatic agent. All examples in this group have the following common characteristics: The bacteriostatic agent includes: bacteriostatic active ingredients; The bacteriostatic active ingredients include: Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO. 32426.

[0069] In a further embodiment, the bacteriostatic agent further includes: excipients.

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

[0071] According to the content of the present invention, for different requirements in actual production applications, combined with the conventional technical means in the field of preparation (such as "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Bacterial Agents", etc.), those skilled in the art can select and formulate the above excipients, and make Bifidobacterium animalis subsp. lactis BLa79 with the preservation number CGMCC NO. 32426 into different dosage forms, such as powder, tablet, injection, oral liquid, suppository, gel, patch, spray, lotion, granule, etc.

[0072] In a specific embodiment, the dosage form of the bacteriostatic agent is selected from one or more of: powder, tablet, liquid, capsule.

[0073] Group 4 Examples, Functional Food for Regulating Intestinal Flora of the Present Invention

[0074] This group of examples provides a functional food for regulating intestinal flora. All examples in this group have the following common characteristics: The functional food for regulating intestinal flora includes: active ingredients; the active ingredients include: Bifidobacterium animalis subsp. lactis BLa79 with the preservation number CGMCC NO. 32426.

[0075] In a further embodiment, the functional food for regulating intestinal flora further includes: excipients.

[0076] In a more specific embodiment, the excipients are selected from: solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, glidant, flavoring agent, preservative, suspending agent, coating material, fragrance, antiadhesive, chelating agent, penetration enhancer, pH regulator, buffer, plasticizer, surfactant, foaming agent, defoaming agent, thickening agent, clathrating agent, humectant, absorbent, diluent, flocculant, deflocculant, filter aid, release retardant, etc.

[0077] According to the content of the present invention, for different requirements in actual production applications, combined with the conventional technical means in the field of food preparation (such as "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Bacterial Agents", etc.), those skilled in the art can select and formulate the above excipients, and make Bifidobacterium animalis subsp. lactis BLa79 with the preservation number CGMCC NO. 32426 into different functional food dosage forms, such as solid beverage, milk beverage, powder, tablet, injection, oral liquid, suppository, gel, patch, spray, lotion, granule, etc.

[0078] In a specific embodiment, the dosage form of the functional food for regulating intestinal flora is selected from one or more of the following: solid beverage, milk beverage, tablet, liquid preparation, capsule.

[0079] Group 5 embodiments, the immune-enhancing functional food of the present invention

[0080] This group of embodiments provides an immune-enhancing functional food. All embodiments in this group have the following common features: The immune-enhancing functional food includes: an active ingredient; the active ingredient includes: Bifidobacterium animalis subsp. lactis BLa79 with the preservation number CGMCC NO.32426.

[0081] In a further embodiment, the immune-enhancing functional food is characterized in that it further includes: excipients.

[0082] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0083] According to the content of the present invention, for different requirements in actual production applications, combined with conventional technical means in the field of food preparation (for example, "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Bacterial Agents", etc.), those skilled in the art can select and formulate the above excipients, and make Bifidobacterium animalis subsp. lactis BLa79 with the preservation number 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.

[0084] In a specific embodiment, the dosage form of the immune-enhancing functional food is selected from one or more of the following: solid beverage, milk beverage, tablet, liquid preparation, capsule.

[0085] Group 6 embodiments, the product for relieving ulcerative colitis of the present invention

[0086] 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 includes: an active ingredient; the active ingredient includes: Bifidobacterium animalis subsp. lactis BLa79 with the preservation number CGMCC NO.32426.

[0087] In a further embodiment, the product for relieving ulcerative colitis further comprises: excipients.

[0088] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0089] According to the content of the present invention, for different requirements in actual production applications, combined with conventional technical means in the field of preparation (such as "Encyclopedia of Pharmaceutical Technology", "Pharmaceutical Preparation Technology", "Research and Application of Microbial Bacterial Agents", etc.), those skilled in the art can select and formulate the above excipients, and make Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO.32426 into different dosage forms, such as powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, lotions, granules, etc.

[0090] In a specific embodiment, the dosage form of the product is selected from one or more of powders, tablets, liquids, and capsules.

[0091] Group 7 embodiments, the in vitro antibacterial method of the present invention

[0092] This group of embodiments provides an in vitro antibacterial method. All embodiments in this group have the following common features: using Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO.32426 for antibacterial.

[0093] The behavior of using a strain of Bifidobacterium animalis subsp. lactis BLa79 with the preservation number of CGMCC NO.32426 in combination with other probiotics for in vitro antibacterial falls within the protection scope of the present invention.

[0094] 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.

[0095] Experimental Example 1: Isolation, screening and identification of animal Bifidobacterium lactis subsp.

[0096] (1) Isolation and screening of strains

[0097] 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.

[0098] (2) Molecular biological identification

[0099] 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 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 2O 34.5 μL. 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. After detecting the PCR products by gel electrophoresis, they were sent to Wuhan Kingcare Bioengineering Co., Ltd. for sequencing. The identified gene sequences were submitted to the NCBI database (www.ncbi.nlm.nih.gov) for BLAST analysis and comparison. According to the identification results of molecular biology, this strain was determined to be Bifidobacterium animalis subsp. lactis, and it was named Bifidobacterium animalis subsp. lactis BLa79. This strain was sent for preservation, and its preservation information is as follows:

[0100] Preservation number: CGMCC NO. 32426

[0101] Taxonomic name: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis

[0102] Preservation unit: China General Microbiological Culture Collection Center

[0103] Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0104] Preservation date: October 30, 2024

[0105] Experimental Example 2. Evaluation of gastrointestinal tolerance

[0106] Simulated artificial gastric juice: Prepare 0.5% NaCl solution, add 0.3% pepsin, adjust the pH value to 2.5 with 1 mol / L HCl, and then filter and sterilize it with a 0.22 μm microporous membrane after complete dissolution for standby.

[0107] Bile salt solution: Add 0.2% (w / v) sodium thioglycolate to MRS liquid medium, then add 0.3% bile salt, filter and sterilize it with a 0.22 μm microporous membrane after complete dissolution for standby.

[0108] BLa79 was activated and cultured for 3 generations under anaerobic conditions, and the cells were collected after centrifugation. It was inoculated into the prepared simulated artificial gastric juice with pH 2.5 and 0.3% bile salt solution at a final concentration of 10 8 CFU / mL, mixed evenly, digested at 37°C. At the same time, the viable cell counts of the digestive juices at 0 h and 3 h were detected respectively, and the survival rates of the strain in artificial gastric juice and bile salt were calculated according to the following calculation formula:

[0109]

[0110] Among them, N1 represents the viable cell count in the strain system after treatment, that is, the viable cell count measured at 3 h, lgCFU / mL; N0 represents the initial viable cell count in the strain system, that is, the viable cell count measured at 0 h, lgCFU / mL.

[0111] Table 1. Tolerance of Bifidobacterium animalis subsp. lactis BLa79 to gastric juice and bile salts

[0112]

[0113] The results showed that after 3 h of digestion with artificial gastric juice or bile salts, the survival rates of Bifidobacterium animalis subsp. lactis BLa79 were 100.40% and 93.67% respectively, indicating that the strain Bifidobacterium animalis subsp. lactis BLa79 has strong resistance to gastric acid and bile salts and can successfully pass through the gastrointestinal tract to exert its probiotic function.

[0114] Example 3. Adhesion ability of Bifidobacterium animalis subsp. lactis BLa79 to Caco-2 cells

[0115] Preparation of bacterial suspension: Centrifuge the activated strain fermentation broth to collect the bacterial cells, wash them 3 times with PBS, and then resuspend the bacterial cells in DMEM culture medium without double antibiotics, and adjust the concentration of the bacterial suspension to 10 8 CFU / mL.

[0116] Culture of Caco-2 cells: Caco-2 cells are 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 in an incubator.

[0117] Cell adhesion experiment: ① Liquid culture of experimental strains: Activate the bacterial liquid for 2 generations, inoculate at 2% inoculum size, 9 mL of medium per 15 mL centrifuge tube, and incubate statically at 37 °C for 15 h. ② Preparation of single cell layer: Inoculate Caco-2 cells into DMEM medium supplemented with 20% (v / v) fetal bovine serum, transfer to a 12-well cell culture plate, and add 1 mL of 10 5 cell / ml cells per well, incubate at 5% CO 2 at 37 °C, change the medium every other day until a single cell layer is obtained for standby; ③ Preparation of bacterial suspension: At the same time, take the cultured target strain bacterial liquid, centrifuge at 10000 r / min at room temperature for 1 min to collect the bacterial cells, wash them twice with sterile PBS, resuspend with DMEM medium, and adjust the bacterial liquid concentration to 10 8 CFU / mL; ④ Co-culture: For the prepared single cell layer of Caco-2 cells, aspirate the medium, add PBS buffer to rinse 2 times, aspirate the buffer, then add 1 mL of the prepared bacterial suspension per well, mix well, and incubate at 5% CO 2, Incubate at 37°C for 2 h; ⑤ Carefully remove the culture supernatant, and add sterile PBS to rinse 5 times to remove the non - adherent bacteria; ⑥ Add 0.2 mL / well of trypsin cell digestive solution and digest for 5 min to elute the cells from the culture plate wells, and the collected solution is the sample; ⑦ Gradient - dilute and perform viable cell counting on the collected sample.

[0118] The adhesion ability is calculated according to the following formula: Adhesion ability (CFU / cell) = Number of bacteria adhering to cells (CFU) / Number of cells in the well (cells).

[0119] Table 2. Adhesion ability of Bifidobacterium animalis subsp. lactis BLa79 to Caco - 2 cells (CFU / cell)

[0120]

[0121] The results are shown in Table 2. The adhesion ability of Bifidobacterium animalis subsp. lactis BLa79 to Caco - 2 cells is 7.50 CFU / cell, which is better than that of the control strain LGG. It shows that BLa79 has good adhesion ability to Caco - 2 cells and has the potential to colonize in the intestine.

[0122] Example 4. Experiment on the antagonism of Bifidobacterium animalis subsp. lactis BLa79 against pathogenic bacteria

[0123] Preparation of pathogenic bacteria suspension:

[0124] Escherichia coli ATCC25922, Salmonella enteritidis ATCC14028, and Staphylococcus aureus ATCC25923 were inoculated into LB liquid medium at 2% (v / v), and after culturing at 37°C for 16 - 20 h, the bacterial suspension concentration was adjusted to 10 8 CFU / mL.

[0125] Streptococcus paris, Streptococcus pasteurianus, and Streptococcus anginosus were inoculated into the medium of BHI + 5% sheep blood at 5% (v / v), and after culturing at 37°C for 16 - 20 h, the bacterial suspension concentration was adjusted to 10 8 CFU / mL.

[0126] Experiment on antagonizing pathogenic bacteria:

[0127] (1) Cool the MRS broth medium containing 1.5% (w / v) agar to about 55°C, mix it with the suspension of indicator bacteria (Escherichia coli, Salmonella enteritidis, Staphylococcus aureus) in a certain proportion, so that the viable cell count of the indicator bacteria is 10 6At the order of magnitude of CFU / mL, and then quickly pour it into the plate pre-placed with Oxford cups. After the medium cools and solidifies, take out the Oxford cups, inject 200 μL of the fermentation broth of Bifidobacterium animalis subsp. lactis BLa79 into each well, and after overnight incubation at 37 °C, measure the diameter of the inhibition zone.

[0128] (2) Cool the BHI broth medium (containing 5% sheep blood) containing 1.5% (w / v) agar to about 55 °C, mix it with the suspension of indicator bacteria (Streptococcus paris, Streptococcus anginosus, Streptococcus pasteurianus) in a certain proportion, so that the viable count of the indicator bacteria is at 10 6 At the order of magnitude of CFU / mL, and then quickly pour it into the plate pre-placed with Oxford cups. After the medium cools and solidifies, take out the Oxford cups, inject 200 μL of the fermentation broth of Bifidobacterium animalis subsp. lactis BLa79 into each well, and after overnight incubation at 37 °C, measure the diameter of the inhibition zone.

[0129] Table 3. Inhibition zones (mm) of Bifidobacterium animalis subsp. lactis BLa79 against different pathogenic bacteria

[0130]

[0131] As can be seen from Table 3, the inhibition zones of Bifidobacterium animalis subsp. lactis BLa79 against Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus paris, Streptococcus anginosus 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 Bifidobacterium animalis subsp. lactis BLa79 can inhibit the growth of different pathogenic bacteria and has good antibacterial ability.

[0132] Example 5. Antibacterial experiment of co-culturing Bifidobacterium animalis subsp. lactis BLa79 and intestinal pathogenic bacteria

[0133] Activate Escherichia coli, Salmonella enteritidis and Clostridium perfringens in the corresponding media respectively, and adjust the bacterial amount to 10 8 CFU / ml. Escherichia coli and Salmonella enteritidis are inoculated into the modified MRS liquid medium at an inoculation amount of 2% respectively, and Clostridium perfringens is inoculated into the BHI liquid medium at an inoculation amount of 2%. Then, the same amount of BLa79 is inoculated into the media inoculated with pathogenic bacteria and mixed for culture. At the same time, Escherichia coli, Salmonella enteritidis and Clostridium perfringens are inoculated into the corresponding liquid media at the same inoculation amount as a control culture. Incubate statically at 37 °C for 24 h, and observe the inhibitory effect of BLa79 on pathogenic bacteria after co-culturing BLa79 and pathogenic bacteria. The results are shown in Table 4.

[0134] Table 4. Co-culture experiment of BLa79 and intestinal pathogenic bacteria

[0135]

[0136] The results are shown in Table 4. After co - culturing with pathogenic bacteria, BLa79 has a strong inhibitory effect on three intestinal pathogenic bacteria. The inhibition rates against Escherichia coli, Salmonella enteritidis, and Clostridium perfringens reach 94.13%, 99.78%, and 97.69% respectively, indicating that BLa79 has broad - spectrum antibacterial properties, can resist the invasion of multiple pathogenic bacteria to the intestine, and protect the intestinal barrier function.

[0137] Example 6. Effects of Bifidobacterium animalis subsp. lactis BLa79 on the body weight and DAI score of colitis mice

[0138] Establishment of colitis model:

[0139] The animal model was induced by DSS. Thirty SPF BALB / c male mice (19 - 20 g) were used in the experiment. The room temperature for animal breeding was 23 ± 2 °C, the humidity was 50% ± 10%, with artificial light for 12 h / d, and free access to food and water. All mice were divided into 3 groups, with 10 mice in each group, namely the normal group (NC), the model group (DSS), and the Bifidobacterium animalis subsp. lactis treatment group (BLa79). The specific experimental procedure was as follows: After one - week of adaptive feeding, in the second week, except for the normal group, all mice freely drank 3.5% DSS solution for 7 days, and the DSS solution was changed every 2 days to ensure the concentration and quality of the DSS solution. At the same time, 0.2 ml of the corresponding solution was intragastrically administered to the experimental mice at a fixed time every day. The normal group (NC) and the model group (DSS) were intragastrically administered PBS, and the Bifidobacterium animalis subsp. lactis treatment group (BLa79) was intragastrically administered a suspension of Bifidobacterium animalis subsp. lactis BLa79 at a concentration of 5×10 9 CFU / ml. The model group (DSS) and the Bifidobacterium animalis subsp. lactis treatment group (BLa79) freely drank 3.5% DSS solution for 7 d, and then changed to normal drinking water. Intragastric administration was performed once a day at a fixed time for 14 d, and the intragastric administration volume each time was 0.2 ml; the normal group and the model group were intragastrically administered an equal volume of PBS every day. After the experiment, the mice were fasted for 12 h, sacrificed, and dissected, and the samples were collected for subsequent index determination and analysis.

[0140] Changes in DAI score of mice: During the experiment, the mice were weighed and recorded every day to statistically analyze the body weight changes. During the modeling period, the feces of the mice were collected every day, the fecal traits were observed, and the fecal occult blood of the mice was detected with an occult blood test paper to calculate the DAI score. The specific scoring criteria are shown in Table 5.

[0141] Table 5. DAI scoring criteria

[0142]

[0143] By Figure 1As can be seen from A, the body weight of mice decreased after DSS induction, while the body weight of mice in the normal group gradually increased. At the end of the experiment, compared with before modeling, the body weight of mice in the model group decreased by 17.42%, and the body weight of mice in the Lactobacillus animalis subsp. lactis treatment group only decreased by 2.96%. This indicates that BLa79 intervention can significantly inhibit the decrease in body weight of mice ( p < 0.001). The change trend of DAI was opposite to that of body weight. Figure 1 As shown in B, during the modeling period, the DAI score of the model group continued to increase. At the end of the experiment, the DAI score of mice in the model group was significantly higher than that of the normal group ( p < 0.0001), while the DAI score of the Lactobacillus animalis subsp. lactis treatment group was significantly lower than that of the model group ( p < 0.001), indicating that Lactobacillus animalis subsp. lactis BLa79 effectively inhibited the occurrence of colitis and reduced the toxic effect of DSS on mice.

[0144] Example 7. Effect of Lactobacillus animalis subsp. lactis BLa79 on the spleen index of mice

[0145] The grouping and modeling of mice were the same as in Example 6. After dissecting the mice, the spleens were collected, weighed, and the spleen index was calculated. As Figure 2 can be seen, DSS induced splenomegaly in mice. Compared with the normal group, the spleen index of the model group increased extremely significantly ( p < 0.001), while the spleen index of the Lactobacillus animalis subsp. lactis treatment group was significantly lower than that of the model group ( p < 0.001), indicating that the intake of Lactobacillus animalis subsp. lactis BLa79 can reduce the spleen injury of mice with DSS-induced colitis.

[0146] Example 8. Effect of Lactobacillus animalis subsp. lactis BLa79 on the colon length

[0147] The grouping and modeling of mice were the same as in Example 6. After sacrificing the mice, the colon tissues of the mice were collected and the colon length was measured. As Figure 3 shown, the colon length of the model group was significantly lower than that of the normal group. After BLa79 intervention, the colon atrophy of mice was inhibited. Compared with the model group (4.83 cm), the colon length of the Lactobacillus animalis subsp. lactis treatment group (7.43 cm) increased significantly, about 1.54 times that of the model group. These results indicate that Lactobacillus animalis subsp. lactis BLa79 can effectively protect the colon of mice from damage caused by DSS.

[0148] Example 9. Effect of Lactobacillus animalis subsp. lactis BLa79 on the content of myeloperoxidase (MPO) and cytokines in the colon of mice with colitis

[0149] The grouping and model establishment of mice were the same as in Example 6. After the experiment ended, the mice were sacrificed and dissected. The colon was taken, placed in phosphate buffered saline solution and homogenized. According to the detection method in the ELISA kit manufacturer's instruction manual, the contents of TNF-α, IL-6, MPO and SIgA in the colon homogenate of each group of mice were measured. The results are shown in Figure 4 . Compared with the control group, the contents of pro-inflammatory factors TNF-α and IL-6 in the model group increased significantly ( p <0.05, p <0.01), and the content of MPO increased extremely significantly ( p <0.01). Compared with the model group, the concentrations of TNF-α, IL-6 and MPO in the colon of mice in the Lactobacillus animalis subsp. lactis treatment group decreased significantly ( p <0.05). In addition, the intake of Lactobacillus animalis subsp. lactis BLa79 could promote the secretion of SIgA in the colon. Compared with the model group, the concentration of SIgA in the Lactobacillus animalis subsp. lactis treatment group increased extremely significantly ( p <0.001), about 187.02% of that in the model group. Thus, it can be seen that Lactobacillus animalis subsp. lactis BLa79 reduces the levels of pro-inflammatory factors in the colon of DSS-induced colitis mice, and alleviates neutrophil aggregation. In addition, it also promotes the production of SIgA, improves the intestinal mucosal immune function, thereby relieving the intestinal inflammatory response and maintaining intestinal homeostasis.

[0150] Example 10. Effect of Lactobacillus animalis subsp. lactis BLa79 on the intestinal flora of mice

[0151] The grouping and model establishment of mice were the same as in Example 6. After the experiment ended, the mice were sacrificed and dissected, and the cecal contents were collected. The 16S rRNA sequencing and analysis of the cecal contents of mice were carried out. The results showed ( Figure 5 ), DSS caused the disorder of the intestinal flora of mice. Compared with the normal group, the ACE index, Chao1 index and Shannon index in the model group decreased significantly ( p < 0.01). Compared with the model group, the ACE index, Chao1 index, Shannon index and Simpson index in the α-diversity of intestinal microorganisms in the Lactobacillus animalis subsp. lactis treatment group increased, and among them, the ACE index, Chao1 index and Shannon index increased extremely significantly ( p < 0.01). The above results indicate that supplementing Lactobacillus animalis subsp. lactis BLa79 can improve the richness and community diversity of the intestinal microbial flora.

[0152] The changes in the relative abundances of the genus level of the cecal contents of mice among groups were analyzed. The results showed ( Figure 6 ), in the Lactobacillus animalis subsp. lactis treatment group, Ruminococcus ( Ruminococcus ), Eubacterium coprostanoligenes ( Blautia ), Oscillibacter (Oscillibacter ), Bifidobacterium ( Bifidobacterium ), and Butyricicoccus the relative abundance was significantly increased compared with the control group ( p < 0.05, p < 0.001). In the model group, Helicobacter ( Helicobacter ), Candidatus_Saccharimonas and Erysipelatoclostridium the relative abundance was significantly increased compared with the normal group ( p <0.05, p < 0.001), but BLa79 intervention could significantly reduce its relative abundance. Ruminococcus and Eubacterium mucigenes are considered key flora in ulcerative colitis and usually have a reduced abundance in UC patients. Ruminococcus is a genus of bacteria that produces propionic acid and butyric acid and can participate in food digestion and maintain intestinal barrier function. Bifidobacterium is generally considered a probiotic and has various functions such as regulating the intestinal flora structure, repairing the intestinal barrier, regulating immune responses, and antioxidant effects. This study found that BLa79 intervention could extremely significantly increase the relative abundance of Bifidobacterium in the mouse intestine, and the relative abundance of Bifidobacterium animalis subsp. lactis in the treatment group was about 22 times that of the control group or the model group. Eubacterium mucigenes, Oscillibacter, and Butyricicoccus can ferment carbohydrates to produce short-chain fatty acids, provide energy for host cells, and regulate immune responses. In addition, Eubacterium mucigenes can also produce bacteriocins to prevent pathogens from colonizing in the intestine. Helicobacter, Candidatus_Saccharimonas and Erysipelatoclostridium are considered intestinal pathogenic bacteria and have an adverse impact on host health. Erysipelatoclostridium is a potential pathogen associated with colon diseases, and its abundance is significantly increased in mice with IBD. The results showed that Bifidobacterium animalis subsp. lactis BLa79 could restore the intestinal flora structure, significantly increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, promote the restoration of intestinal flora homeostasis, and then 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. The use of animal Bifidobacterium lactis subspecies BLa79 with a deposit number of CGMCC NO.32426 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function or in vitro antibacterial for non-therapeutic purposes, characterized in that: The antibacterial spectrum of the antibacterial agent is: Escherichia coli, Salmonella, Salmonella enteritidis, Staphylococcus aureus, Clostridium perfringens, Streptococcus Parisi, Streptococcus pharyngitis and Streptococcus pasteurianus.

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 or in vitro antibacterial for non-therapeutic purposes, characterized in that: The intestinal flora regulation includes: improving the intestinal microbial diversity index, increasing the abundance of beneficial intestinal bacteria, and reducing the abundance of harmful intestinal bacteria.

4. The use of animal Bifidobacterium lactis subspecies BLa79 with a deposit number of CGMCC NO.32426 according to claim 3 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function or in vitro antibacterial for non-therapeutic purposes, characterized in that: The intestinal microbial diversity index includes: ACE index, Chao1 index, Shannon index and Simpson index.

5. The use of animal Bifidobacterium lactis subspecies BLa79 with a deposit number of CGMCC NO.32426 according to claim 3 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function or in vitro antibacterial for non-therapeutic purposes, characterized in that: The beneficial intestinal bacteria are: Ruminococcus ( Ruminococcus ), by Eubacterium mucosum ( Blautia ), Oscillibacterium spp. Oscillibacter ), Bifidobacterium spp. ( Bifidobacterium )and Butyricicoccus .

6. The use of animal Bifidobacterium lactis subspecies BLa79 with a deposit number of CGMCC NO.32426 according to claim 3 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function or in vitro antibacterial for non-therapeutic purposes, characterized in that: The harmful intestinal bacteria are: Helicobacter , Candidatus_Saccharimonas and Erysipelatoclostridium .

7. Use of animal Bifidobacterium lactis subsp. BLa79 with a deposit number of CGMCC NO.32426 according to any one of claims 2 to 6 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function or in vitro antibacterial for non-therapeutic purposes, characterized in that: The immunity enhancement includes: reducing the level of pro-inflammatory factors and promoting SIgA secretion.

8. The use of animal Bifidobacterium lactis subspecies BLa79 with a deposit number of CGMCC NO.32426 according to claim 7 in the preparation of antibacterial agents, functional foods for regulating intestinal flora, foods for enhancing immune function or in vitro antibacterial for non-therapeutic purposes, characterized in that: The pro-inflammatory factors include: TNF-α, IL-6, and MPO.

9. 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.

10. 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.

11. The functional food for regulating intestinal flora according to claim 10, characterized in that: Also includes: Auxiliary materials.

12. 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.

13. The food for enhancing immune function according to claim 12, characterized in that: Also includes: Auxiliary materials.

14. 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.

Images