Bifidobacterium bifidum and application of bifidobacterium bifidum in preparation of preparation for immunoregulation and intestinal barrier repair

Abstract

The invention relates to the field of detection of mucosal immune defense, in particular to bifidobacterium bifidum and application of the bifidobacterium bifidum in preparation of a preparation for immunoregulation and intestinal barrier repair. The bifidobacterium bifidum is a bifidobacterium bifidum BB16 of which the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.33423, and the bifidobacterium bifidum is a bifidobacterium bifidum BB16. The BB16 strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 15, 2025, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The BB16 strain has the function of directly promoting secretion of mucosal immune barrier SIgA.

Description

Technical Field

[0001] The present application relates to the field of detection of mucosal immune defense, and specifically to a strain of Bifidobacterium bifidum and its application in the preparation of preparations for immune regulation and intestinal barrier repair. Background Art

[0002] Within the human mucosal immune barrier, secretory immunoglobulin A (SIgA) serves as a core immune molecule on the mucosal surface, fulfilling key functions such as building the immune barrier, neutralizing toxins, and agglutinating pathogens. Its unique dimeric structure, combined with the secretory lamina, forms a dense protective layer on mucosal surfaces in the respiratory and digestive tracts, effectively preventing pathogens such as bacteria and viruses from adhering to epithelial cells. For example, in the intestines, SIgA can encapsulate Salmonella typhi, blocking its invasion of the intestinal mucosa; in the respiratory tract, SIgA neutralizes the hemagglutinin on the surface of the influenza virus, inhibiting viral infection. However, current drug treatments for mucosal immune-related diseases have significant limitations in promoting mucosal immune defenses and SIgA function.

[0003] Existing drug treatment options primarily include antibiotics, glucocorticoids, and immunomodulators, but these drugs struggle to precisely regulate mucosal immunity. While suppressing pathogens, antibiotics often disrupt the balance of intestinal flora, leading to a decrease in the number of beneficial bacteria such as Bifidobacteria and Lactobacilli, which in turn weakens the mucosal immune microenvironment. While glucocorticoids can quickly relieve inflammation, long-term use can inhibit the synthesis and secretion of SIgA, reducing mucosal immune barrier function. Immunomodulators such as interferon, while able to activate the immune system, lack the ability to specifically target the SIgA secretion pathway and are unable to effectively enhance local mucosal immune defenses. Furthermore, oral medications are susceptible to factors such as gastric acid and digestive enzymes during absorption through the digestive tract, making it difficult to maintain effective concentrations. This results in insufficient drug delivery to the mucosa and an inability to achieve sustained stimulation of SIgA secretion.

[0004] In contrast, biological preparations represented by probiotics have shown unique potential in assisting the improvement of mucosal immunity. Many studies have confirmed the positive effects of probiotics on mucosal immunity. According to a study published in the journal Gut (HartAL, et al. Gut. 2004 Nov;53 (11):1602-1609), researchers co-cultured human intestinal lamina propria mononuclear cells, whole blood or enriched blood dendritic cell populations with cell wall components of the probiotic preparation VSL#3 (containing four lactobacilli, three bifidobacteria and one streptococcal strain) and found that VSL#3 can effectively induce dendritic cells to produce IL-10. Dendritic cells play a key role in the activation of T lymphocytes and indirectly promote the secretion of SIgA. Summary of the Invention

[0005] The present application provides a strain of Bifidobacterium bifidum, which is Bifidobacterium bifidum BB16 with a deposit number of CGMCC NO. 33423. The BB16 strain was deposited on January 15, 2025, at the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The BB16 strain has the function of directly promoting the secretion of SIgA in the mucosal immune barrier.

[0006] The present application provides a composition comprising a mixture of at least one or more of dead cells, live cells and inactivated cells of Bifidobacterium bifidum BB16, or a lysate of one or more Bifidobacterium bifidum BB16 strains, or one or more metabolites of Bifidobacterium bifidum BB16, or one or more analogs of Bifidobacterium bifidum BB16, or one or more derivatives of Bifidobacterium bifidum BB16, or one or more fragments of Bifidobacterium bifidum BB16, or a combination thereof.

[0007] The present application also provides a preparation containing Bifidobacterium bifidum BB16 as an active ingredient and excipients for forming the preparation.

[0008] The present application also provides an auxiliary regulator of mucosal immune barrier function, which contains Bifidobacterium bifidum BB16 as an effective ingredient.

[0009] The present application also provides a preparation for repairing the intestinal barrier, which contains Bifidobacterium bifidum BB16 as an effective ingredient.

[0010] The present application also provides a fermented product containing Bifidobacterium bifidum BB16 as a bacterial species.

[0011] The present application also provides the use of a strain of Bifidobacterium bifidum in preparing a preparation. The preparation is selected from at least one of the following: an auxiliary regulator of mucosal immune barrier function; a preparation for repairing the intestinal barrier; a fermented product; a preparation for alleviating intestinal inflammation; a preparation for inhibiting the growth of harmful bacteria; a preparation for improving intestinal function; a preparation for regulating immune function; a preparation for regulating metabolism; and a prebiotic preparation.

[0012] According to the technical solutions provided in this application, the BB16 strain has the function of directly promoting the secretion of SIgA in the mucosal immune barrier. The BB16 strain can tolerate gastric acid and has extremely strong adhesion ability, and has the application prospect of being developed into a high-efficiency probiotic. The BB16 strain can increase the spleen index and thymus index of immunocompromised mice, promote the release of serum SIgA, IL-10, IL-17, and TNF-α, enhance the mucosal immune barrier function, and improve their immune capacity. The BB16 strain can repair ileal tissue in immunocompromised mice and reduce spleen tissue damage.

[0013] Furthermore, Bifidobacterium bifidum BB16 increased the relative abundance of intestinal Lactobacillus, Lactobacillus, unclassified Lachnospiraceae, Lachnospiraceae NK4A136 group, and Alternaria, while decreasing the relative abundance of Bacteroides, unclassified Desulfovibrio, and Helicobacter. This strain has potential applications as an adjuvant modulator of mucosal immune barrier function, a preparation for repairing the intestinal barrier, a fermented product, a preparation for alleviating intestinal inflammation, a preparation for inhibiting the growth of harmful bacteria, a preparation for improving intestinal function, a preparation for regulating immune function, a preparation for metabolic regulation, and a prebiotic. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The weight line graph of mice in the NC, MC, and BB16 groups provided in the experimental examples.

[0015] Figure 2 Bar graphs showing the spleen index (left) and thymus index (right) of mice in the NC, MC, and BB16 groups provided for experimental examples.

[0016] Figure 3 The serum SIgA bar graph (A), TNF-α bar graph (B), IL-17 bar graph (C), and IL-10 bar graph (D) of mice in the NC group, MC group, and BB16 group provided in the experimental examples.

[0017] Figure 4 H&E staining images of ileum tissues of mice in the NC, MC, and BB16 groups provided as experimental examples.

[0018] Figure 5 H&E staining images of spleen tissues of mice in the NC group, MC group, and BB16 group provided for experimental examples.

[0019] Figure 6 The results of α-diversity analysis of intestinal flora in the NC group, MC group, and BB16 group of mice provided as experimental examples; in the figure, (A) is the ACE index graph, (B) is the Chao1 index graph, (C) is the Simpson index graph, and (D) is the Shannon index graph.

[0020] Figure 7The results of β-diversity analysis of intestinal flora in the NC group, MC group and BB16 group of mice provided as experimental examples.

[0021] Figure 8 The OTUs cluster analysis results of the intestinal flora of mice in the NC group, MC group and BB16 group provided for the experimental example.

[0022] Figure 9 A bar chart showing the phylum-level species diversity analysis results of the intestinal flora of mice in the NC, MC, and BB16 groups provided as experimental examples.

[0023] Figure 10 The bar graph of the genus-level species diversity analysis results of the intestinal flora of the NC, MC, and BB16 groups of mice provided for the experimental examples includes, in order, Lactobacillus, Lactobacillus, unclassified Lachnospiraceae, Helicobacter NK4A136 group, Alternaria, unclassified Desulfovibrioaceae, Bacteroides, and Helicobacter. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0025] Although Hart AL, et al. "Gut. 2004 Nov;53 (11):1602 - 1609" co-cultured the probiotic preparation VSL#3, which contains cell wall components of eight bacterial strains including four lactobacilli, three bifidobacteria and one streptococcal strain, with human intestinal lamina propria mononuclear cells, whole blood or enriched blood dendritic cell populations, it was found that VSL#3 can indirectly promote the secretion of SIgA.

[0026] However, the present application still provides a strain of Bifidobacterium bifidum, with a deposit number of CGMCC No. 33423, Bifidobacterium bifidum BB16. The BB16 strain was deposited on January 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The BB16 strain can directly promote the secretion of SIgA in the mucosal immune barrier. This single strain can directly act on immunocompromised animals, promoting SIgA secretion and thereby enhancing the mucosal immune barrier function.

[0027] In some embodiments, the Bifidobacterium bifidum strain according to the present application may be an isolated bacterial strain.

[0028] The present application discloses Bifidobacterium bifidum BB16, including the following pure culture colonies of Bifidobacterium bifidum deposited in the General Microbiological Center of China Culture Collection Administration Committee or their analogs, fragments, lysates, or combinations thereof.

[0029] The present application provides a composition comprising a mixture of at least one or more of dead, live, and inactivated cells of Bifidobacterium bifidum BB16, or a lysate of one or more Bifidobacterium bifidum BB16 strains, or one or more metabolites of Bifidobacterium bifidum BB16, or one or more analogs of Bifidobacterium bifidum BB16, or one or more derivatives of Bifidobacterium bifidum BB16, or one or more fragments of Bifidobacterium bifidum BB16, or a combination thereof. The composition can promote and / or enhance serum SIgA secretion, thereby enhancing mucosal immune barrier function.

[0030] In the compositions provided in some embodiments, the concentration of the mixture of at least one or more of the dead bacteria, live bacteria and inactivated bacteria is 10 3 to 10 17 In the range of colony forming units (CFU), for example, 10 5 -10 17 CFU range, for example, 10 6 -10 17 CFU range, for example, 10 7 -10 17 CFU range, for example, 10 8 -10 17 CFU range, for example, 10 9 -10 17 CFU range, for example, 1010 -10 17 CFU range, for example, 10 11 -10 16 CFU range, for example, 10 12 -10 16 CFU range, for example, 10 13 -10 16 CFU range, for example, 10 7 -10 16 CFU range, for example, 10 8 -10 15 CFU range, for example, 10 9 -10 15 CFU range, for example, 10 10 -10 15 CFU range, for example, 10 11 -10 15 CFU range, for example, 10 12 -10 15 CFU range.

[0031] The present application also provides a preparation containing Bifidobacterium bifidum BB16 as an active ingredient and excipients for forming the preparation.

[0032] In some embodiments, the preparations provided herein include Bifidobacterium bifidum BB16 as an active ingredient, comprising a mixture of at least one or more of dead, live, and inactivated bacteria, or a lysate of one or more Bifidobacterium bifidum BB16 strains, or one or more metabolites of Bifidobacterium bifidum BB16, or one or more analogs of Bifidobacterium bifidum BB16, or one or more derivatives of Bifidobacterium bifidum BB16, or one or more fragments of Bifidobacterium bifidum BB16, or a combination thereof. The concentration of the active ingredient is 0.0001% (w / w) to 99% (w / w).

[0033] In the context of this application, the Bifidobacterium bifidum BB16 defined herein can be provided in the composition according to this application in the form of a mixture of at least one or more of dead, live, and inactivated cells. Live cells refer to live Lactobacillus reuteri bacteria with an intact cell structure, capable of normal metabolism and reproduction. For example, these cells are cultured in a culture medium (e.g., MRS culture medium), centrifuged and washed to retain live cells, and are typically stored in a freeze-dried form (e.g., freeze-dried bacterial powder). Dead cells refer to cells that have died naturally or lost their activity through physical or chemical treatment (e.g., high temperature, ultraviolet light). Their cell structure may be intact or partially destroyed. Inactivated cells specifically refer to cells that have been killed through controlled methods (e.g., heat inactivation, formaldehyde treatment, autoclaving), but retain their cell surface structures (e.g., cell wall, capsule). Inactivated cells emphasize "structure preservation," while dead cells may have their structure destroyed due to treatment.

[0034] As used herein, the term "lysate" or "extract" specifically refers to a solution or suspension of components of a microbial cell according to the present application in an aqueous medium, and comprises, for example, macromolecules (e.g., DNA, RNA, proteins, peptides, lipids, carbohydrates, etc.) and cell debris. The lysate preferably comprises cell walls or cell wall components, including binding receptors. Methods for producing lysates are well known to those skilled in the art and include, for example, the use of a "French press" or enzymatic lysis, a ball mill with glass beads or iron beads. Cells can be broken by enzymatic, physical, or chemical methods. Examples of enzymatic cell lysis can include individual enzymes and enzyme mixtures, such as proteases, proteinase K, lipases, glycosidases; chemical lysis can be induced by ionophores, detergents (e.g., SDS), acids, or bases; physical methods can also be implemented by using, for example, high pressure, osmotic pressure, temperature changes, or alternating hot and cold, such as a French press. In addition, chemical, physical, and enzymatic methods can of course be combined.

[0035] As used herein, the term "metabolites" refers to various substances produced by the microbial cells of the present application during their growth, reproduction, and metabolism. These metabolites are closely related to their physiological functions, probiotic properties, and applications, such as organic acids, bacteriocins, exopolysaccharides, amino acids and their derivatives, vitamins and coenzymes, secondary metabolites, and signaling molecules.

[0036] As used herein, the term "derivative" refers to a substance with a specific structure or function produced by chemical modification, biotransformation or other treatment methods using the microbial cells of this application as raw materials. It may be a modified product or metabolic intermediate of a cell component, such as a derivative of peptidoglycan, a component of the bacterial cell wall.

[0037] As used herein, the term "analog" refers to a substance that has a structure similar to, but not identical to, a certain type of component in the dead cells of the microorganisms according to the present application. It may be artificially synthesized or obtained from other sources and can simulate the function of the original component. For example, based on the structure of the active component in the cell, a compound with a similar structure (such as a peptide segment that simulates a bacterial surface antigen) is designed and synthesized, for example, a substance with a structure similar to that of a bacterial component is extracted from other organisms (such as a polysaccharide analogue from a plant).

[0038] Herein, the term "fragment" refers to a partial structure or component fragment of a microbial cell according to the present application, which is usually obtained by physical cutting, enzymatic hydrolysis or genetic engineering, such as cell wall fragments, protein fragments, nucleic acid fragments, etc.

[0039] Some embodiments provide a formulation that is a topical, gastric, or enteral formulation.

[0040] Some embodiments provide formulations comprising pharmaceutically, nutraceutically, or food-acceptable carriers or excipients. In some embodiments, the formulations can be provided in solid form, liquid form, viscous form, emulsion, or as a dry form.

[0041] The formulations provided in some embodiments can be preferably formulated into pastes, soft gelatin capsules, hard gelatin capsules, powders, talc, granules, beads, pastilles, effervescent tablets, lozenges, buccal tablets, chewable tablets, sublingual tablets, oils, liquids, solutions, tinctures, emulsions, juices, concentrates, syrups, sprays, mists, drinkable ampoules, gels, gels, tablets or coated pills.

[0042] In some embodiments, the formulation can be a topical formulation for human or animal skin or mucous membranes. Formulations for topical application can preferably be formulated as a paste; talcum powder; lotion; custard; foam; cream; or ointment.

[0043] In some embodiments, the topical formulation may be a powder composition comprising hydrated magnesium silicate (talc) and at least one Bifidobacterium bifidum BB16 of the present application.

[0044] In a further embodiment according to the present application, a powder composition for topical administration comprises hydrated magnesium silicate, at least one carbohydrate and at least one composition of the present application's Bifidobacterium bifidum BB16.

[0045] In a preferred embodiment, the topical composition may be formulated as a lotion; a custard; a foam; a cream; or an ointment, oil, or emulsion.

[0046] Some embodiments provide formulations that are powders, tablets, ointments, emulsions, oils, suspensions, lotions, gels, pastes, foams, dairy products, gels, mists, or fermented formulations.

[0047] The preparations provided in some embodiments may contain one or more thickeners, and / or one or more sweeteners and / or one or more artificial sweeteners, wherein the thickener is preferably selected from cellulose ethers, polysaccharides, selected from the group comprising: xanthan gum, gelatin, highly dispersed silicon dioxide, starch, carrageenan, alginate, tragacanth gum, agar, gum arabic, pectin and polyvinyl ester, and the sweetener is selected from the group comprising: glucose, fructose, sucrose, glucose syrup, sorbitol, mannitol, xylitol, maltitol, steviol glycosides, saccharin, cyclamate, acesulfame K and / or aspartame.

[0048] Preferred food and nutritional supplements in the sense of the present application may include effervescent tablets, vitamin tablets, dietary supplements, mineral tablets, trace element tablets, beverage powders, beverages, juices, milk drinks, yogurt, mineral water, non-carbonated water, filled gummies, chewable tablets, juices or syrups, coated pills and lozenges, and aerosols.

[0049] Furthermore, the formulations may comprise builders, enzymes, electrolytes, pH regulators, thickeners, prebiotics, optical brighteners, graying inhibitors, dye transfer inhibitors, foam regulators and / or colorants.

[0050] Surprisingly, the Bifidobacterium bifidum BB16 and its composition provided in this application can not only promote and / or enhance serum SIgA secretion and enhance mucosal immune barrier function, but also repair the ileum structure of immunocompromised animals, reduce spleen tissue damage, and maintain intestinal microecological balance.

[0051] In the context of this application, a "prebiotic" is a non-digestible food ingredient that promotes the growth of specific microorganisms. A "synbiotic" is a composition containing at least one probiotic and at least one prebiotic. Such compositions are understood to promote the growth of probiotics. Thus, powerful synbiotics are based on the combination of specific strains of probiotics with carefully selected prebiotics. They can provide important health benefits to mammals.

[0052] According to another aspect of the present application, a probiotic composition comprising Bifidobacterium bifidum BB16 and at least one more active ingredient is provided.

[0053] Prebiotics are chemical products that induce the growth and / or activity of commensal microorganisms (e.g., bacteria and fungi) that contribute to the health of the host. Prebiotics are non-digestible carbohydrates that pass through the upper gastrointestinal tract undigested and stimulate the growth and / or activity of beneficial bacteria that colonize the intestinal or skin microbiome.

[0054] Some oligosaccharides used as prebiotics are fructooligosaccharides (FOS), xylooligosaccharides (XOS), polydextrose, pectin, galacto-oligosaccharides (GOS) or human milk oligosaccharides (HMO). In addition, disaccharides such as lactulose or some monosaccharides such as lactose or tagatose can also be used as prebiotics.

[0055] In one embodiment of the present application, at least one prebiotic compound may be included in the composition of the present application. In a very broad concept, prebiotics are all compounds that can be metabolized by probiotics.

[0056] Preferably, prebiotics are indigestible or difficult to digest for mammals. Therefore, after being taken in by mammals, indigestible prebiotics can pass through the small intestine and enter the large intestine to stimulate the growth of probiotics in this compartment. Therefore, prebiotics can be used as a food source for probiotics. It is believed that prebiotics (many of which are indigestible carbohydrates) promote the growth of probiotics. Prebiotics are naturally present in, for example, cabbage, onion, whole grains, banana, garlic, honey, leek, artichoke, fortified food and beverage and dietary supplements. Prebiotics are well known in the art, and when used for the present application, there is no particular limitation on prebiotics themselves.

[0057] In one embodiment, the at least one prebiotic product in the composition is selected from the following compounds and compositions: non-digestible carbohydrates, β-glucans, manno-oligosaccharides, inulin, fructooligosaccharides, human milk oligosaccharides (HMOs), galacto-oligosaccharides (GOS), lactulose, lactofructooligosaccharides, galactotriose, fructooligosaccharides (FOS), cellobiose, cellodextrins, cyclodextrins, maltitol, lactitol, glycosilsucrose, betaine, vitamin E or a variant thereof (wherein the variant is selected from α, β, γ, δ tocopherol, tocotrienols and tocopherols). Optionally, manno-oligosaccharides and / or inulin may be preferred. HMOs may include lacto-N-tetraose, lacto-N-fucopentaose, lacto-N-triose, 3'-sialyllactose, lacto-N-neofucopentaose, sialic acid, L-fucose, 2-fucosyllactose, 6'-sialyllactose, lacto-N-neotetraose, and 3-fucosyllactose.

[0058] Prebiotics may also be used in the topical compositions of the present application.

[0059] In one embodiment, at least one of the following prebiotic compounds is used in the topical composition of the present application: lactose, β-glucan, manno-oligosaccharide, inulin, fructooligosaccharide, galacto-oligosaccharide (GOS), lactulose, lactofructooligosaccharide, galactotriose, fructooligosaccharide (FOS), cellobiose, cellodextrin, cyclodextrin, maltitol, lactitol, glucosylsucrose, betaine, vitamin E or a variant thereof (wherein the variant is selected from α, β, γ, δ tocopherol, tocotrienol and tocopherol), lacto-N-tetraose, lacto-N-fucosylpentaose, lacto-N-triose, 3'-sialyllactose, lacto-N-neofucopentaose, sialic acid, 2-fucosyllactose, 6'-sialyllactose, lacto-N-neotetraose and 3-fucosyllactose. Optionally, lactose and / or manno-oligosaccharide and / or inulin may be preferred.

[0060] D- and L-fucose strengthen the natural defenses of the skin or mucous membranes, stimulate epidermal immune defenses and / or prevent and / or treat skin autoimmune diseases. In one embodiment of the present application, the composition comprises D- or L-fucose.

[0061] In one embodiment of the present application, the composition further comprises L-fucose at a concentration of 10 mM to 500 mM in the composition.

[0062] In the context of the present application, acceptable additives and / or excipients for pharmaceutical or food applications include auxiliary substances known to those skilled in the art for the preparation of solid, semi-solid or liquid forms, for example, diluents, solvents (including water, glycerol, ethanol), solubilizers, acidulants, thickeners, sweeteners, flavoring agents, colorants, sweeteners, lubricants, surfactants, preservatives, pH stability buffers and mixtures thereof.

[0063] In various embodiments of the present application, the composition of the present application comprising Bifidobacterium bifidum BB16 may be a dietary supplement, a food (or a novel food or a functional food), or a composition for a dietary supplement or a food.

[0064] The gut microbiome is closely linked to intestinal inflammation, intestinal function, intestinal mucosal immunity, intestinal metabolism, and cardiovascular health. Obese individuals experience an imbalance in their gut microbiome, accompanied by a significant decrease in gut microbial diversity. Numerous experimental and clinical studies have confirmed the promising potential of probiotic-based adjunctive therapies. However, existing probiotic preparations or products may not always match the specific characteristics of a patient's gut microbiome, necessitating further screening for appropriate, personalized probiotics.

[0065] The Bifidobacterium bifidum BB16 provided in the present application can promote the relative abundance of intestinal Lactobacillus, Lactobacillus, unclassified Lachnospiraceae, Lachnospiraceae NK4A136 group, and other mycobacteria, and reduce the relative abundance of Bacteroides, unclassified Desulfovibrioaceae, and Helicobacter.

[0066] It can be seen that the Bifidobacterium bifidum BB16 provided in this application has application prospects for alleviating intestinal inflammation. The increase of beneficial bacteria such as Lactobacillus helps maintain the homeostasis of intestinal flora, prevent harmful substances from entering the body, maintain the integrity of the intestinal barrier, and alleviate intestinal inflammation. For example, Lactobacillus plantarum can alleviate the symptoms of inflammatory bowel disease by regulating the activity of intestinal immune cells and reducing the secretion of proinflammatory cytokines. Certain species of Bacteroides are associated with inflammatory responses, and their reduced abundance may help alleviate intestinal inflammation.

[0067] Thus, the present application shows that Bifidobacterium bifidum BB16 has the potential to inhibit the growth of harmful bacteria. By combining with Lactobacillus and other species, Bifidobacterium bifidum BB16 can inhibit the growth and reproduction of harmful bacteria such as Bacteroides, Desulfovibrio, and Helicobacter by competing for nutrients and producing antimicrobial substances such as bacteriocins, thereby reducing the risk of intestinal infections and preventing intestinal diseases such as diarrhea and enteritis.

[0068] This suggests that the Bifidobacterium bifidum BB16 provided in this application has promising applications in improving intestinal function. Unclassified bacteria such as the Lachnospiraceae family and the Helicobacter NK4A136 group participate in the fermentation of dietary fiber in the intestine, producing short-chain fatty acids such as butyrate. Butyrate is an important energy source for intestinal epithelial cells, helping to maintain normal intestinal cell metabolism and function, promoting intestinal motility, improving digestion and absorption, and alleviating constipation.

[0069] Thus, the Bifidobacterium bifidum BB16 provided in this application has application prospects for regulating immune function. The intestinal flora is closely related to the immune system. Increasing the abundance of beneficial bacteria can stimulate the development and maturation of the intestinal immune system, enhance the activity of immune cells, improve the body's immunity, help the body resist the invasion of foreign pathogens, and prevent infectious diseases. It also helps regulate immune balance and reduce the risk of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

[0070] It can be seen that the Bifidobacterium bifidum BB16 provided by the present application has application prospects for metabolic regulation. Beneficial bacteria such as Lactobacillus can participate in the metabolic conversion of various substances during the metabolic process. For example, they help break down complex components in food and promote the absorption of nutrients; they may also participate in the regulation of fat metabolism, reduce fat accumulation in the body, and play a certain role in preventing metabolic diseases such as obesity and hyperlipidemia. Certain bacteria in the Desulfovibrio family are related to the sulfate reduction process, and their overgrowth may be related to some metabolic disorders. Reducing their abundance helps maintain a normal metabolic state.

[0071] Based on this, another aspect of the present application also provides the use of Bifidobacterium bifidum BB16 as a probiotic preparation. Based on the above-mentioned beneficial regulatory effects on intestinal flora, Bifidobacterium bifidum BB16 can be developed into a probiotic product as a preparation that promotes the growth of these beneficial bacteria or contains these beneficial bacteria, thereby enhancing or regulating the mucosal immune barrier function, inhibiting the growth of harmful bacteria, alleviating intestinal inflammation, improving intestinal function, regulating metabolism, and promoting repair.

[0072] To help understand the mechanism of action of Bifidobacterium bifidum BB16 on the mucosa of animals, the effects of in vivo administration of Bifidobacterium bifidum BB16 on serum SIgA, ileum tissue, spleen tissue and intestinal flora of different mice were studied in a mouse model.

[0073] 1. Strain Isolation

[0074] The collected infant fecal samples were placed in sterile sampling tubes and transported in ice boxes. They were diluted with 0.85% saline under sterile conditions, spread on LMRS agar plates supplemented with 5% (V / V) mupirocin lithium salt, and incubated under anaerobic conditions at 37°C for 48-72 hours. Suspected single colonies were selected by visual observation of their colony morphology, observed under a microscope, and initially screened and purified. After purification, they were incubated in MRS liquid anaerobic tubes containing 0.05% L-cysteine ​​hydrochloride at 37°C for 12-16 hours, centrifuged, and resuspended in sterile 30% glycerol aqueous solution for storage in the strain bank of Wuhan Weikang Probiotics Research Institute.

[0075] 2. Strain identification

[0076] The selected target strain was cultured in liquid, the cells were collected, genomic DNA was extracted, and PCR amplification was performed. The content and purity of the PCR amplification products were tested. After passing the test, the strain was sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. Based on the sequencing results and relevant molecular biological identification, the Latin name of the strain is Bifidobacterium bifidum, and the strain was confirmed to be Bifidobacterium bifidum. The strain was named Bifidobacterium bifidum BB16 and deposited. Its deposit information is as follows:

[0077] Accession number: CGMCC NO. 33423

[0078] Classification name: Bifidobacterium bifidum BB16

[0079] Deposit date: January 15, 2025

[0080] Depository: General Microbiology Center of China Culture Collection Administration

[0081] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0082] 3. Gastric juice tolerance test

[0083] A 0.5% NaCl solution was prepared, 0.3% pepsin was added, and the pH was adjusted to 3.0 with 1 mol / L HCl. The solution was then fully dissolved and sterilized by filtration with a 0.22 μm microporous filter membrane to simulate artificial gastric fluid. One mL of activated Bifidobacterium bifidum BB16 culture was centrifuged at 10,000 rpm for 1 minute. The supernatant was discarded, and the cells were collected and added to 1 mL of simulated gastric fluid at pH 3.0, mixed thoroughly, and digested at 37°C. The viable cell counts were measured at 0 and 3 hours, and the survival rate was calculated. The survival rate (%) of the strain was calculated as Nt / N0 × 100%, where N0 represents the viable cell count (CFU / mL) of the strain at 0 hours, and Nt represents the viable cell count (CFU / mL) of the strain at 3 hours. The results showed that the gastric fluid tolerance of Bifidobacterium bifidum was 96.99%.

[0084] 4. Adhesion experiment of Bifidobacterium bifidum BB16 to Caco-2 cells

[0085] (1) Caco-2 cell culture

[0086] Caco-2 cells were cultured in DMEM high-glucose medium supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin and streptomycin in a 37°C, 90% humidity, 5% CO2 incubator.

[0087] (2) Adhesion test steps

[0088] ① Liquid culture of experimental strains: transfer the second generation according to the inoculum volume of 2%, culture at 37℃ for 14-16 hours.

[0089] ② Preparation of monolayer: Caco-2 cells were cultured in DMEM medium containing 20% ​​(v / v) fetal bovine serum, and then transferred to a 12-well cell culture plate. 1 mL of 2.4×10 5Cells were cultured at 5% CO2 and 37°C, with the medium changed every other day until a monolayer was obtained.

[0090] ③ Preparation of bacterial suspension: Take the cultured target strain bacterial suspension, centrifuge at 10000 r / min at room temperature for 1 min to collect the bacteria, wash twice with sterile PBS, and resuspend in DMEM medium to adjust the bacterial suspension concentration to 1×10 8 CFU / mL;

[0091] ④ Co-culture: Remove the culture medium from the prepared Caco-2 cell monolayer, wash twice with PBS buffer, remove the buffer, and add 1 mL / well of the prepared bacterial suspension. Mix well and incubate at 5% CO2 and 37°C for 2 h.

[0092] ⑤ Carefully remove the culture supernatant and rinse 5 times with sterile PBS to remove unadhered bacteria;

[0093] 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;

[0094] ⑦ Perform gradient dilution and live bacteria count on the collected samples.

[0095] Adhesion capacity was calculated using the following formula: Adhesion capacity (CFU / cell) = number of bacteria adhering to cells (CFU) / number of cells in the well (cell)

[0096] The results showed that the adhesion ability of Bifidobacterium bifidum BB16 to Caco-2 cells was 70.94±2.78 CFU / cell, indicating that Bifidobacterium bifidum BB16 has good colonization ability on intestinal epithelial cells.

[0097] 5. Establishment of immunocompromised mouse model and group intervention

[0098] Specific pathogen-free BALB / c male mice (18–20 g) were purchased from Beijing Sibeifu Biotechnology Co., Ltd. and housed in the animal room of the Hubei Provincial Center for Food and Drug Safety Evaluation. The room temperature was maintained at 22 ± 2°C, humidity at 50 ± 5%, and a 12-h light-dark cycle. They were allowed free access to water and food. Animal experiments were conducted in accordance with the guidelines of the Ethics Committee of the Hubei Provincial Center for Disease Control and Prevention (Ethics Committee No. 202410263).

[0099] Before the experiment, the mice were adaptively fed for one week and then randomly divided into three groups: normal control group (NC), immunosuppression group (MC), and Bifidobacterium bifidum BB16 group (BB16).

[0100] During the whole experiment, mice in the NC and MC groups were gavaged with 0.2 mL of sterile saline every day, and mice in the BB16 group were gavaged with an equal amount of Bifidobacterium bifidum BB16 suspension (viable count of 10 9 CFU / mL). On days 7 to 9 of the experiment, mice in the MC and BB16 groups were injected with cyclophosphamide (CTX, 80 mg / kg / day) for 3 consecutive days to establish an immunosuppressive mouse model. The NC group was injected with an equal volume of sterile saline. Mice were sacrificed on day 15, and samples were collected for subsequent analysis. Details of the animal experiments are shown in Table 1.

[0101] Table 1 In vivo experimental design and grouping

[0102] Group Name Group Description NC group The patients were gavaged with normal saline for 14 days, and 0.2 mL of normal saline was injected intraperitoneally on the 7th, 8th, and 9th days. MC Group The patients were gavaged with normal saline for 14 consecutive days, and 0.2 mL of cyclophosphamide (80 mg / kg) was injected intraperitoneally on days 7, 8, and 9. BB16 group <![CDATA[Continuous intragastric administration of live cells of Bifidobacterium bifidum BB16 (viable cell count is 10 9 CFU / mL, 0.2 mL / day / animal) for 14 days, and intraperitoneal injection of 0.2 mL of cyclophosphamide (80 mg / kg) on the 7th, 8th, and 9th days]]>

[0103] (1) Thymus index and spleen index

[0104] After one week of adaptive feeding, the mice were randomly divided into three groups. The body weights of the experimental mice were measured on days 1, 4, 7, 10, and 14. During autopsy, the spleen and thymus of the mice were collected, and the thymus index and spleen index of the mice were calculated.

[0105] The calculation formula of immune organ index is as follows:

[0106] Immune organ index = immune organ mass (mg) / body weight (g)

[0107] like Figure 1 As shown in the results, compared with the MC group, oral administration of BB16 could significantly increase the body weight of mice, thereby effectively alleviating the damage of cyclophosphamide to the body.

[0108] like Figure 2 As shown, immune organ indices decreased in the MC group, indicating that cyclophosphamide injection caused atrophy of the mice's immune organs and a decline in their immune function. Following oral administration of BB16, spleen and thymus indices increased by 169.4% and 101.9%, respectively, demonstrating that oral administration of BB16 can enhance the immune capacity of immunocompromised mice.

[0109] (2) Effects of Bifidobacterium bifidum BB16 on serum SIgA, IL-10, IL-17, and TNF-α in mice

[0110] The cells were centrifuged and the serum of the mice was collected. The SIgA content in the serum of each group of mice was detected by ELISA, the IL-10 content in the serum of each group of mice was detected by ELISA, the IL-17 content in the serum of each group of mice was detected by ELISA, and the TNF-α content in the serum of each group of mice was detected by ELISA.

[0111] like Figure 3As shown in the results, after cyclophosphamide injection, the levels of SIgA, IL-10, IL-17, and TNF-α in the serum of mice in the MC group were significantly lower than those in the NC group, while the levels of SIgA, IL-10, IL-17, and TNF-α in the serum of mice in the BB16 group increased and were significantly higher than those in the MC group. This indicates that the Bifidobacterium bifidum BB16 provided by the present invention can not only increase the serum inflammatory factor levels of immunocompromised model mice in vivo, but also promote SIgA expression and secretion, thereby increasing their serum SIgA levels.

[0112] (3) Effects of Bifidobacterium bifidum BB16 on mouse ileum tissue

[0113] After the mice were sacrificed, approximately 0.5 cm of ileum tissue was collected, fixed with 4% paraformaldehyde, and then embedded in paraffin. 4 μm thick sections were prepared using a pathology slicer and then stained with hematoxylin-eosin (H&E). Structural changes in ileum tissue were observed under a microscope. Figure 4 As shown in the figure, compared with the NC group, the villi in the small intestine of the MC group were shortened and their structure was damaged. After treatment with Bifidobacterium bifidum BB16, the small intestinal structure was improved, with the villi arranged neatly and the structure intact, indicating that BB16 can repair the ileum tissue of cyclophosphamide-induced immunocompromised mice.

[0114] (4) Effects of Bifidobacterium bifidum BB16 on mouse spleen tissue

[0115] After the mice were sacrificed, the spleen tissues were collected, fixed with 4% paraformaldehyde, and then embedded in paraffin. 4 μm thick sections were made using a pathology slicer and then stained with hematoxylin-eosin (H&E). The structural changes of the spleen tissue were observed under a microscope. Figure 5 As shown, in the NC group, the white pulp lymphocytes were densely distributed, with a clear boundary between them and the red pulp. In the MC group, the white pulp lymphocytes were reduced in number, dispersed in structure, and had a poorly defined boundary between them and the red pulp. In the BB16 group, the boundary between the white and red pulps was clear, and the number of lymphocytes increased. This suggests that Bifidobacterium bifidum BB16 can reduce splenic tissue damage.

[0116] (5) Effects of Bifidobacterium bifidum BB16 on the intestinal flora of mice

[0117] DNA was extracted from mouse cecal contents, and the V3-V4 region of bacterial 16S rRNA was amplified using PCR. Paired-end sequencing was performed on the Illumina Novaseq platform. Reads were spliced, filtered, clustered, or denoised, and species annotation and abundance analysis were performed to reveal the species composition of the samples. Alpha-diversity, beta-diversity, and species diversity analyses were further performed to uncover differences between samples.

[0118] 1) α-diversity analysis, β-diversity analysis

[0119] α-diversity reflects the species richness and species diversity of a single sample. There are many measurement indicators: Chao1, ACE, Shannon, Simpson, etc. Chao1 and ACE indexes measure species richness, that is, the number of species. Shannon and Simpson indices are used to measure species diversity. The larger the value, the higher the species diversity of the sample. Figure 6 As shown in the figure, Chao1 index, ACE index, Shannon index, and Simpson index were significantly reduced in the MC group. After treatment with Bifidobacterium bifidum BB16, Chao1 index, ACE index, Shannon index, and Simpson index were significantly increased, indicating that oral administration of BB16 can improve the richness and diversity of the intestinal flora in mice.

[0120] β-diversity analysis is used to compare the similarity of species diversity among different samples. Figure 7 As shown in the figure, the BB16 group was clearly separated from the MC group and adjacent to the NC group, indicating that the composition of the intestinal flora of mice in the BB16 group was closer to that of the NC group.

[0121] 2) OTUs cluster analysis

[0122] The results are as follows Figure 8 As shown in the figure, the number of shared OTUs between the MC group and the NC group was 516, and the number of shared OTUs between the BB16 group and the NC group was 553, indicating that oral administration of BB16 can increase the shared bacterial flora in the intestines of immunosuppressed mice and normally fed mice.

[0123] 3) Phylum-level species diversity analysis

[0124] The experimental results are as follows Figure 9 As shown in the figure, the relative abundance of Firmicutes and Bacteroidetes accounted for approximately 80% of the mouse intestinal microbiota. Compared with the NC group, the relative abundance of Firmicutes in the MC group was significantly reduced, while the relative abundance of Bacteroidetes was significantly increased. After oral administration of BB16, the relative abundance of Firmicutes in the mouse intestinal microbiota increased, while the relative abundance of Bacteroidetes decreased.

[0125] 4) Genus-level species diversity analysis

[0126] The experimental results are as follows Figure 10As shown, compared with the NC group, the relative abundance of unclassified Lachnospiraceae, Helicobacter NK4A136, and Alternaria species decreased in the MC group, while the relative abundance of Helicobacter, Bacteroides, and unclassified Desulfovibrio species increased. Compared with the MC group, the relative abundance of Lactobacillus, Lactobacillus, unclassified Lachnospiraceae, Helicobacter NK4A136, and Alternaria species increased in the Bifidobacterium bifidum BB16 group, while the relative abundance of Bacteroides, unclassified Desulfovibrio species, and Helicobacter decreased. The abundance of Helicobacter NK4A136 is associated with overall health and is a potential butyrate producer. Butyrate is a short-chain fatty acid (SCFA) that plays multiple regulatory roles in the innate and adaptive immune systems. Furthermore, Lactobacillus species can interact with immune receptors, leading to the production of immunomodulatory cytokines to defend against invading pathogens. The experimental results show that BB16 can increase the relative abundance of beneficial bacteria in the host intestine and reduce the relative abundance of harmful bacteria, which is beneficial to maintaining the homeostasis of the intestinal flora.

[0127] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. A strain of Bifidobacterium bifidum, characterized in that It is Bifidobacterium bifidum BB16 with a preservation number of CGMCCNO. 33423.

2. A composition, characterized in that A mixture comprising at least one or more of dead cells, live cells and inactivated cells of Bifidobacterium bifidum BB16 as claimed in claim 1, or a lysate of one or more Bifidobacterium bifidum BB16 strains, or one or more metabolites of Bifidobacterium bifidum BB16, or one or more analogs of Bifidobacterium bifidum BB16, or one or more derivatives of Bifidobacterium bifidum BB16, or one or more fragments of Bifidobacterium bifidum BB16, or a combination thereof.

3. The composition according to claim 2, characterized in that The concentration of the mixture of at least one or more of the dead cells, live cells and inactivated cells of Bifidobacterium bifidum BB16 is 10 3 to 10 17 within the range of colony-forming units. A preparation comprising the Bifidobacterium bifidum BB16 according to claim 1 as an active ingredient and auxiliary materials for forming the preparation.

5. The preparation according to claim 4, characterized in that The Bifidobacterium bifidum BB16 uses a mixture of at least one or more of dead bacteria, live bacteria and inactivated bacteria, or a lysate of one or more Bifidobacterium bifidum BB16 strains, or one or more metabolites of Bifidobacterium bifidum BB16, or one or more analogs of Bifidobacterium bifidum BB16, or one or more derivatives of Bifidobacterium bifidum BB16, or one or more fragments of Bifidobacterium bifidum BB16, or a combination thereof as an active ingredient.

6. An auxiliary regulator of mucosal immune barrier function, comprising the Bifidobacterium bifidum BB16 according to claim 1 as an active ingredient. 7 . A preparation for repairing the intestinal barrier, comprising the Bifidobacterium bifidum BB16 according to claim 1 as an active ingredient.

8. A fermented product comprising the Bifidobacterium bifidum BB16 according to claim 1 as a bacterial species.

9. Use of the Bifidobacterium bifidum according to claim 1 in preparing a preparation.

10. The use according to claim 7, characterized in that The preparation is selected from at least one of the following: Auxiliary regulator of mucosal immune barrier function; Preparations to repair the intestinal barrier; fermented products; Preparations to relieve intestinal inflammation; Preparations that inhibit the growth of harmful bacteria; Preparations that improve intestinal function; Preparations that modulate immune function; Metabolic regulation agents; Probiotic preparations.

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