Bifidobacterium breve with enhanced immunomodulatory function and application thereof

Abstract

The application relates to the technical field of immune enhancement, in particular to a Bifidobacterium breve BBr53 capable of enhancing immune regulation function and application thereof. The Bifidobacterium breve is the BBr53 with a preservation number of CGMCC NO.34559. The BBr53 strain is preserved in the China General Microbiological Culture Collection Center on May 15, 2025, and the address is No.3, Xibei Road, Beichen, Chaoyang District, Beijing. The BBr53 strain can directly inhibit the proliferation of Edwardsiella tarda and has the function of enhancing immune regulation.

Description

Technical Field

[0001] This application relates to the technical field of immune enhancement, specifically to a short Bifidobacterium that enhances immune regulation and its applications. Background Technology

[0002] The immune system plays a crucial role in protecting the host from external viruses, pathogens, parasites, fungi, yeast, and internal abnormal cells such as tumor cells. The interaction between host immunity and the gut microbiota has been discovered. Many immune disorders, such as inflammation and autoimmune diseases, are associated with gut dysbiosis. Enhancing immune function primarily involves strengthening the intestinal mucosal barrier, regulating immune cells, and modulating anti-inflammatory effects. For example, maintaining intestinal pH can inhibit pathogen colonization and enhance intestinal mucosal barrier function. Another example is activating the activity of immune cells such as macrophages and natural killer cells (NK cells), promoting Th1 immune responses (such as IFN-γ secretion), balancing the Th1 / Th2 ratio, and alleviating allergic reactions. Yet another example is downregulating pro-inflammatory factors (such as IL-6 and TNF-α) and upregulating anti-inflammatory factors (such as IL-10) to suppress respiratory and intestinal inflammation.

[0003] Cyclophosphamide (CTX) is an alkylating agent widely used in chemotherapy for cancers such as leukemia, breast cancer, lymphoma, and autoimmune diseases. Its primary mechanism of action is through DNA alkylation, producing an anti-mitotic effect that leads to cell death. However, CTX's cytotoxic effect on tumor cells is not specific. CTX also has myelosuppressive and immunosuppressive effects. Furthermore, CTX causes serious intestinal problems, such as gut microbiota dysbiosis, mucosal damage, and disruption of the skin barrier. High-dose CTX can reduce body weight, relative spleen and thymus weight, leukocyte and NK cell activity, and the absolute number of B cells and T cells. Therefore, patients using cyclophosphamide for related diseases may face various risks of infection and complications, thus delaying diagnosis and treatment outcomes. To reduce, alleviate, suppress, or improve the side effects of cyclophosphamide and enhance the immune function of patients using cyclophosphamide, it is necessary to find safe immunomodulators and functional modulators with immunomodulatory properties. Summary of the Invention

[0004] This application provides a strain of Bifidobacterium breve, specifically Bifidobacterium breve BBr53 with accession number CGMCC NO.34559. This BBr53 strain was deposited on May 15, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] This application provides a composition comprising at least one or more of the following: dead, live, and inactivated cells of Bifidobacterium breve BBr53; lysates of one or more strains of Bifidobacterium breve BBr53; metabolites of one or more Bifidobacterium breve BBr53; analogues of one or more Bifidobacterium breve BBr53; derivatives of one or more Bifidobacterium breve BBr53; fragments of one or more Bifidobacterium breve BBr53; or combinations thereof.

[0006] This application also provides a formulation containing Bifidobacterium breve BBr53 as an active ingredient, and excipients for forming the formulation.

[0007] This application also provides an immunomodulatory preparation containing Bifidobacterium breve BBr53 as an active ingredient.

[0008] This application also provides an antibacterial preparation containing Bifidobacterium breve BBr53 as an active ingredient.

[0009] This application also provides a probiotic composition comprising Bifidobacterium breve BBr53 and at least one other active ingredient.

[0010] This application also provides a culture containing Bifidobacterium breve BBr53 or its progeny.

[0011] This application also provides the use of a strain of Bifidobacterium breve (BBr53) in the preparation of formulations. The formulation is selected from at least one of the following: formulations that improve the immunity of a subject; formulations that enhance immunomodulatory function; antibacterial formulations; probiotic compositions; formulations that promote the growth of aquatic animals; feed additives; gastric probiotics; intestinal probiotics; and metabolic regulators.

[0012] The technical effects of the technical solution provided in this application will be described in detail in the specific implementation, and will not be elaborated further here. Attached Figure Description

[0013] Figure 1 Plate inhibition zone diagrams of Edwardsiella tarda (A) and Aeromonas hydrophila (B) provided for the test examples.

[0014] Figure 2 The curves showing the change in body weight of mice in the NC, MC, and BBr53 groups provided for the experimental cases.

[0015] Figure 3 Statistical bar charts of spleen index (A) and thymus index (B) for mice in the NC, MC and BBr53 groups provided for the experimental cases.

[0016] Figure 4 Statistical bar charts of SIgA (A), TNF-α (B), IL-10 (C) and IL-17 (D) in the serum of mice in the NC, MC and BBr53 groups provided for the experimental cases.

[0017] Figure 5 H&E staining images of ileal tissue from mice in the NC, MC, and BBr53 groups provided for the experimental cases.

[0018] Figure 6 H&E staining images of spleen tissue from mice in the NC, MC, and BBr53 groups provided for the experimental cases.

[0019] Figure 7 Statistical graphs of ACE index (A), Chao1 index (B), Simpson index (C), and Shannon index (D) for α-diversity in mice of the NC group, MC group, and BBr53 group provided for the experimental case.

[0020] Figure 8 Statistical charts showing the β-diversity analysis of mice in the NC, MC, and BBr53 groups provided for the experimental cases.

[0021] Figure 9 Cluster analysis statistics of OTUs in mice from the NC, MC and BBr53 groups provided for the experimental cases.

[0022] Figure 10 Statistical charts showing the species diversity analysis at the phylum level for mice in the NC, MC, and BBr53 groups provided for the experimental cases.

[0023] Figure 11 Statistical bar charts showing the relative abundance of Firmicutes (A), Bacteroidetes (B), and the decrease in the Firmicutes / Bacteroidetes relative abundance ratio (C) in mice from the NC, MC, and BBr53 groups provided for the experimental cases.

[0024] Figure 12 Heatmaps showing species diversity at the phylum level in the NC, MC, and BBr53 mouse groups provided for the experimental cases.

[0025] Figure 13 The relative abundance of unclassified rodentaceae (A), Lactobacillus (B), Lactobacillus (C), unclassified spirulina (D), spirulina NK4A136 (E), Bacteroides (F), Bifidobacterium (G), and Desulfovibrioceae (H) in the cecal contents of mice in the NC, MC, and BBr53 groups provided for the experimental cases is shown in the bar chart. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0027] This application provides a strain of *Bifidobacterium breve*, with accession number CGMCC NO. 34559, namely *Bifidobacterium breve* BBr53. This strain BBr53 was deposited on May 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0028] In some embodiments, the Bifidobacterium breve strain according to this application may be an isolated bacterial strain.

[0029] This application discloses Bifidobacterium breve BBr53, including pure cultures of Bifidobacterium breve deposited at the China General Microbiological Culture Collection Center, or their analogues, fragments, lysates, or combinations thereof.

[0030] This application provides a composition comprising at least one or more of the following: dead, live, and inactivated cells of *Bifidobacterium breve* BBr53; lysates of one or more strains of *Bifidobacterium breve* BBr53; metabolites of one or more *Bifidobacterium breve* BBr53; analogues of one or more *Bifidobacterium breve* BBr53; derivatives of one or more *Bifidobacterium breve* BBr53; fragments of one or more *Bifidobacterium breve* BBr53; or combinations thereof. This composition can directly inhibit the proliferation of *Edwardsiella pneumoniae* and *Aeromonas hydrophila*.

[0031] In some embodiments of the provided compositions, the concentration of at least one or more of the dead bacterial cells, live bacterial cells, and inactivated bacterial cells is 10. 3 Up to 10 17 Within the range of colony-forming units per gram or milliliter (CFU / g or CFU / mL), for example, in 10 5 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 6 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 7-10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 11 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 12 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 13 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 7 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 11 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 12 -10 15 Within the range of CFU / g or CFU / mL.

[0032] This application also provides a formulation containing Bifidobacterium breve BBr53 as an active ingredient, and excipients for forming the formulation.

[0033] In some embodiments of the formulation, the *Bifidobacterium breve* BBr53 is used as an active ingredient in a mixture of at least one or more of dead, live, and inactivated bacterial cells, or lysates of one or more *Bifidobacterium breve* BBr53 strains, or one or more metabolites of *Bifidobacterium breve* BBr53, or one or more analogs of *Bifidobacterium breve* BBr53, or one or more derivatives of *Bifidobacterium breve* BBr53, or fragments of one or more *Bifidobacterium breve* BBr53, or combinations thereof. The concentration of the active ingredient is from 0.0001% (w / w) to 99% (w / w).

[0034] In the context of this application, *Bifidobacterium breve* BBr53 as defined herein may be provided in the composition or formulation according to this application in the form of dead cells, live cells, and inactivated cells, or a mixture of at least one or more of these. Live cells refer to live *Lactobacillus reuteri* bacteria with intact cell structure, capable of normal metabolism and reproduction, for example, cultured in a culture medium (such as MRS medium), centrifuged, washed, and then preserving the viability, typically in lyophilized form (such as lyophilized bacterial powder). Dead cells refer to cells that have died naturally or lost their activity through physical / chemical treatment (such as high temperature, ultraviolet light), and whose cell structure may be intact or partially destroyed. Inactivated cells specifically refer to cells that have been killed by controlled methods (such as heat inactivation, formaldehyde treatment, high pressure treatment) but retain their cell surface structures (such as cell walls, capsules). Inactivated cells emphasize "structural preservation," while dead cells may suffer structural damage due to the treatment method.

[0035] In this document, the terms "lysate" or "extract" specifically refer to a solution or suspension of components from microbial cells according to this application in an aqueous medium, and include, for example, macromolecules (e.g., DNA, RNA, proteins, peptides, lipids, carbohydrates, etc.) and cell debris. The lysate preferably includes the cell wall or cell wall components, including binding receptors. Methods for producing lysates are well known to those skilled in the art, including, for example, using a "French press" or enzymatic lysis, or a ball mill with glass or iron beads. Cells can be lysed by enzymatic, physical, or chemical methods. Examples of enzymatic cell lysis include individual enzymes and mixtures of enzymes, such as proteases, proteinase K, lipases, and glycosidases; chemical lysis can be induced by ionophores, detergents (e.g., SDS), acids, or bases; physical methods can also be implemented using, for example, high pressure, osmotic pressure, temperature variations, or alternating hot and cold treatments during a French press. Furthermore, chemical, physical, and enzymatic methods can, of course, be combined.

[0036] In this document, the term "metabolite" refers to various substances produced by the microbial cells according to this 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, extracellular polysaccharides, amino acids and their derivatives, vitamins and coenzymes, secondary metabolites and signaling molecules.

[0037] In this document, the term "derivative" refers to a substance with a specific structure or function generated from microbial cells as raw materials through chemical modification, biotransformation or other treatments. It may be a modified product of cell components or a metabolic intermediate, such as a derivative of peptidoglycan, a component of bacterial cell walls.

[0038] In this document, the term "analyte" refers to a substance whose structure is similar to, but not identical to, a component in the dead cells of a microorganism according to this application, and which may be artificially synthesized or obtained from other sources and can mimic 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 is designed and synthesized (such as a peptide that mimics a bacterial surface antigen), or a substance extracted from other organisms that has a structure similar to that of a bacterial component (such as a polysaccharide analog from a plant).

[0039] In this document, the term "fragment" refers to a partial structural or component fragment of a microbial cell according to this application, which is usually obtained by physical cutting, enzymatic digestion or genetic engineering, such as cell wall fragments, protein fragments, nucleic acid fragments, etc.

[0040] Some embodiments provide formulations containing pharmaceutically, health-promoting, or food-grade carriers or excipients. In some embodiments, the formulation may be provided in solid, liquid, viscous, emulsion, or dry form.

[0041] Some of the formulations provided in the embodiments can preferably be formulated as pastes, soft gelatin capsules, hard gelatin capsules, powders, talc, granules, beads, tablets, effervescent tablets, lozenges, chewable tablets, sublingual tablets, oils, liquids, solutions, tinctures, emulsions, fruit juices, concentrates, syrups, sprays, mists, drinking ampoules, gels, tablets, or coated pills.

[0042] Some of the embodiments provide formulations that are powders, tablets, ointments, emulsions, oils, suspensions, lotions, gels, pastes, foams, dairy products, gels, mists, or fermented preparations.

[0043] Some embodiments provide formulations that 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, and selected from the group consisting of xanthan gum, gelatin, highly dispersed silica, starch, carrageenan, alginate, astragalus gum, agar, gum arabic, pectin and polyvinyl ester, and the sweetener is selected from the group consisting of glucose, fructose, sucrose, glucose syrup, sorbitol, mannitol, xylitol, maltitol, steviol glycosides, saccharin, cyclamate, acesulfame K and / or aspartame.

[0044] Preferred foods and nutritional supplements in the sense of this application may include effervescent tablets, vitamin tablets, dietary supplements, mineral tablets, trace element tablets, beverage powders, beverages, fruit juices, dairy beverages, yogurt, mineral water, non-carbonated water, filled gummies, chewable tablets, fruit juices or syrups, coated pills and tablets, and aerosols.

[0045] In addition, the formulation may also contain detergents, enzymes, electrolytes, pH adjusters, thickeners, prebiotics, fluorescent whitening agents, ashing inhibitors, dye transfer inhibitors, foam modifiers and / or colorants.

[0046] In some embodiments, the formulation is an immunomodulatory enhancer. Treatment of cyclophosphamide-immunized mice with a formulation containing *Bifidobacterium breve* BBr53 as an active ingredient enhances the mice's immunomodulatory function, strengthens the intestinal mucosal barrier, and reduces ileal tissue damage. Furthermore, this formulation or *Bifidobacterium breve* BBr53 alleviates cyclophosphamide-induced thymus and spleen atrophy in mice. Moreover, this formulation or *Bifidobacterium breve* BBr53 upregulates the expression of immunoglobulins and cytokines in cyclophosphamide-immunized mice. Additionally, this formulation or *Bifidobacterium breve* BBr53 reduces splenic damage induced by cyclophosphamide (CTX).

[0047] In some implementations, the formulation is a gut probiotic. Treatment of immunocompromised mice with a formulation containing *Bifidobacterium breve* BBr53 as the active ingredient with cyclophosphamide resulted in a gut-promoting effect in these mice. For example, the formulation or *Bifidobacterium breve* BBr53 promoted an increase in the relative abundance of unclassified rodentaceae, *Lactobacillus*, *Lactobacillus*, unclassified *Trichophyton*, *Trichophyton NK4A136*, and *Bifidobacterium*, while decreasing the relative abundance of *Bacteroides* and *Desulfovibrio*.

[0048] In some embodiments, the formulation can be a growth promoter for aquatic animals or a feed additive. For example, a concentration of 10... 8 A bacterial suspension of *Bifidobacterium breve* BBr53 (cfu / mL) is mixed with fish feed at a weight ratio of 1:4 to obtain fish feed containing *Bifidobacterium breve* BBr5. This fish feed may have an ameliorative effect on fish mortality or disease symptoms caused by *Edwards tarda* and *Aeromonas hydrophila*.

[0049] Surprisingly, the *Bifidobacterium breve* BBr53 and its composition provided in this application can directly inhibit the proliferation of *Edwardsiella tarda* and *Aeromonas hydrophila*, enhance the immunomodulatory function of cyclophosphamide-induced immunosuppressed mice, strengthen the intestinal mucosal barrier, and reduce ileal tissue damage. Furthermore, *Bifidobacterium breve* BBr53 and its composition can also improve the intestinal flora of immunosuppressed mice, increasing its richness and diversity, and maintaining intestinal flora homeostasis. In particular, *Bifidobacterium breve* BBr53 and its composition can reduce the relative abundance of *Bacteroides* and *Desulfovibrio* species in the intestine, showing promise for development into a highly effective intestinal probiotic.

[0050] According to another aspect of this application, a probiotic composition comprising Bifidobacterium breve BBr53 and at least one other active ingredient is provided.

[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 comprising at least one probiotic and at least one prebiotic. Such compositions are understood to promote the growth of probiotics. Therefore, a powerful synbiotic is based on a combination of specific strains of probiotics and carefully selected prebiotics. These can provide important health benefits to mammals.

[0052] Prebiotics are chemical products that induce the growth and / or activity of symbiotic microorganisms (such as bacteria and fungi) that contribute to the health of the host. Prebiotics are indigestible carbohydrates that pass through the upper gastrointestinal tract undigested and stimulate the growth and / or activity of beneficial bacteria that colonize the gut or skin microbiota.

[0053] Some oligosaccharides used as prebiotics are fructooligosaccharides (FOS), xylooligosaccharides (XOS), polydextrose, pectin, galactooligosaccharides (GOS), or human milk oligosaccharides (HMOs). In addition, disaccharides such as lactulose or some monosaccharides such as lactose or tagatose can also be used as prebiotics.

[0054] In one embodiment of this application, at least one prebiotic compound may be included in the composition or formulation of this application. In a very broad concept, prebiotics are all compounds that can be metabolized by probiotics.

[0055] Preferably, prebiotics are indigestible or poorly digestible by mammals. Therefore, after being ingested by mammals, indigestible prebiotics can pass through the small intestine and enter the large intestine to stimulate the growth of probiotics in that compartment. Thus, prebiotics can serve as a food source for probiotics. It is believed that prebiotics (many of which are poorly digestible carbohydrates) promote the growth of probiotics. Prebiotics are naturally found in, for example, cabbage, onions, whole grains, bananas, garlic, honey, leeks, artichokes, fortified foods and beverages, and dietary supplements. Prebiotics are well known in the art, and there are no particular limitations on prebiotics themselves when used in this application.

[0056] In one embodiment, at least one prebiotic product in the composition is selected from the following compounds and compositions: indigestible carbohydrates, β-glucan, mannooligosaccharides, inulin, fructooligosaccharides, human milk oligosaccharides (HMO), galactooligosaccharides (GOS), lactulose, lactulose oligosaccharides, galactotriose, fructooligosaccharides (FOS), cellobiose, cellodextrin, cyclodextrin, maltitol, lactitol, glycosilsucrose, betaine, vitamin E, or variants thereof (wherein variants are selected from α, β, γ, δ tocopherols, tocotrienols, and tocomonenophenols). Optionally, mannooligosaccharides and / or inulin may be preferred. HMOs may include lact-N-tetrasaccharide, lact-N-fucopentose, lact-N-triose, 3'-sialyllactose, lact-N-neofucopentose, sialic acid, L-fucose, 2-fucosyllactose, 6'-sialyllactose, lact-N-neotetrasaccharide, and 3-fucosyllactose.

[0057] Prebiotics can also be used in the topical compositions of this application.

[0058] In one embodiment, at least one of the following prebiotic compounds is used in the topical composition of this application: lactose, β-glucan, mannooligosaccharide, inulin, fructooligosaccharide, galactooligosaccharide (GOS), lactulose, lactulose oligosaccharide, galactotriose, fructooligosaccharide (FOS), cellobiose, cellodextrin, cyclodextrin, maltitol, lactitol, glucosylsucrose, betaine, vitamin E or variants thereof (wherein the variants are selected from α, β, γ, δ tocopherols, tocotrienols and tocomonenophenols), lact-N-tetrasaccharide, lact-N-fucopentose, lact-N-triose, 3'-sialyllactose, lact-N-neofucopentose, sialic acid, 2-fucosyllactose, 6'-sialyllactose, lact-N-neotetrasaccharide, and 3-fucosyllactose. Optionally, lactose and / or mannooligosaccharide and / or inulin may be preferred.

[0059] D- and L-fucose enhance the natural defenses of the skin or mucous membranes, stimulate epidermal immune defenses, and / or prevent and / or treat autoimmune diseases of the skin. In one embodiment of this application, the composition comprises D- or L-fucose.

[0060] In one embodiment of this application, the composition further comprises L-fucose at a concentration of 10 mM to 500 mM in the composition.

[0061] In the context of this application, acceptable additives and / or excipients for pharmaceutical or food applications include adjuvant substances known to those skilled in the art for preparing solid, semi-solid, or liquid forms, such as diluents, solvents (including water, glycerol, and ethanol), solubilizers, acidifiers, thickeners, sweeteners, flavor enhancers, colorants, lubricants, surfactants, preservatives, pH-stabilizing buffers, and mixtures thereof.

[0062] In various embodiments of this application, the composition or formulation of this application containing Bifidobacterium breve BBr53 may be a dietary supplement, a food (or a novel food or functional food), or a composition for use as a dietary supplement or food.

[0063] According to one aspect of the invention, a culture comprising Bifidobacterium breve BBr53 or its progeny is provided.

[0064] As used herein, the term "progeny" refers to daughter cells produced by a microorganism through growth (e.g., growth in a culture medium). It is readily understood that during the growth and culture of microorganisms, particularly bacteria, genetic material may undergo changes (e.g., mutations of one or more bases). These changes can occur spontaneously or as a result of mutagenesis induced by chemical and / or physical agents (e.g., mutagens) and / or recombinant DNA techniques known in the art. Therefore, in this document, the term "progeny of Bifidobacterium breve" is intended to encompass both progeny whose genetic material has not changed and those whose genetic material has changed compared to the Bifidobacterium breve of the present invention. Of course, said progeny still retain the functions of the strain from which they are derived (e.g., the ability to enhance immunity, inhibit the proliferation of Edwardsiella tarda and Aeromonas hydrophila, etc.).

[0065] In some embodiments, the culture also includes components that provide nutrition (e.g., solid or liquid culture medium, feeding cell layer).

[0066] In some embodiments, the nutrient-providing ingredients are selected from prebiotics, proteins (e.g., enzymes), carbohydrates, lipids (e.g., fats), probiotics, vitamins, immunomodulators, milk substitutes, minerals, amino acids, or any combination thereof.

[0067] In some embodiments, the culture also comprises a cell-free culture filtrate of Lactobacillus paracasei or its progeny.

[0068] In some embodiments, the culture also contains Lactobacillus paracasei or derivatives of its progeny.

[0069] In some embodiments, the derivative is selected from metabolites, enzymes, cellular structural components (e.g., cell walls or components thereof), extracellular polysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

[0070] In some embodiments, the microorganism can be in any form, for example, alive or dead, as a lysate or extract, or as a bacterial product, or as a supernatant.

[0071] Furthermore, it was found that the Bifidobacterium breve BBr53 provided in this application can promote an increase in the relative abundance of unclassified rodentaceae, Lactobacillus spp., Lactobacillus spp., unclassified spirochetesceae, spirochetes NK4A136 group and Bifidobacterium spp. in the gut, while reducing the relative abundance of Bacteroides spp. and Desulfovibrioceae.

[0072] Furthermore, it was found that the *Bifidobacterium breve* BBr53 provided in this application shows promise in alleviating intestinal inflammation. An increase in beneficial bacteria such as *Lactobacillus* helps maintain intestinal flora homeostasis, prevents harmful substances from entering the body, maintains the integrity of the intestinal barrier, and reduces intestinal inflammation. For example, *Lactobacillus plantarum* can alleviate symptoms of inflammatory bowel disease by regulating the activity of intestinal immune cells and reducing the secretion of pro-inflammatory cytokines. Meanwhile, certain species of *Bacteroides* are associated with inflammatory responses, and a decrease in their abundance may help alleviate intestinal inflammation.

[0073] Furthermore, it was found that the Bifidobacterium breve BBr53 provided in this application has the potential to inhibit the growth of harmful bacteria. By competing for nutrients and producing antibacterial substances such as bacteriocins, Bifidobacterium breve BBr53 can inhibit the growth and reproduction of harmful bacteria such as Bacteroides and Desulfovibrioceae, thereby reducing the risk of intestinal infection and preventing intestinal diseases such as diarrhea and enteritis.

[0074] Furthermore, it was found that the *Bifidobacterium breve* BBr53 provided in this application has the potential to improve intestinal function. Unclassified bacteria such as those belonging to the *Helicobacter* family and *Helicobacter breve* NK4A136 group participate in the fermentation of dietary fiber in the intestine, producing short-chain fatty acids such as butyric acid. Butyric acid is an important energy source for intestinal epithelial cells, helping to maintain normal metabolism and function of intestinal cells, promoting intestinal peristalsis, improving digestion and absorption, and relieving constipation.

[0075] Furthermore, it was discovered that the *Bifidobacterium breve* BBr53 provided in this application has promising applications in metabolic regulation. Beneficial bacteria such as *Lactobacillus* can participate in the metabolic transformation of various substances during metabolism. For example, they help break down complex components in food, promoting the absorption of nutrients; they may also participate in the regulation of fat metabolism, reducing fat accumulation in the body, and playing a certain role in preventing metabolic diseases such as obesity and hyperlipidemia. Meanwhile, some bacteria of the *Desulfovibrioceae* family are involved in the sulfate reduction process, and their excessive growth may be associated with some metabolic disorders; reducing their abundance helps maintain a normal metabolic state.

[0076] Based on this, another aspect of this application also provides the application of Bifidobacterium breve BBr53 as a probiotic preparation. Based on the aforementioned beneficial regulatory effects on the intestinal flora, Bifidobacterium breve BBr53 can be developed into a probiotic product as a preparation that promotes the growth of these beneficial bacteria or contains these beneficial bacteria itself, 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.

[0077] To help understand Bifidobacterium breve BBr53 and its applications, a detailed explanation is provided below in conjunction with relevant experiments.

[0078] 1. Strains Isolation

[0079] Infant fecal samples were transported in sterile sampling tubes in ice packs and serially diluted with 0.85% physiological saline under sterile conditions. The samples were then spread onto LMRS agar plates supplemented with 5% (v / v) mupirocin lithium salt and incubated anaerobically at 37°C for 48–72 h. Suspected single colonies were picked by visual observation of colony morphology, examined under a microscope, and subjected to preliminary screening and purification. After purification, the samples were incubated in MRS liquid anaerobic tubes containing 0.05% L-cysteine ​​hydrochloride at 37°C for 12–16 h. After centrifugation to remove the supernatant, the samples were resuspended in sterile 30% glycerol aqueous solution and stored in the strain bank of Wuhan Weikang Probiotics Research Institute.

[0080] 2. Strain identification

[0081] The selected target strain was cultured in liquid medium, the bacterial cells were collected, genomic DNA was extracted, and PCR amplification was performed. The content and purity of the PCR amplification products were tested, and those that passed the test were sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. Based on the sequencing results and relevant molecular biological identification, the Latin name of the strain was determined to be *Bifidobacterium breve*, confirming it as *Bifidobacterium breve*. This strain was named *Bifidobacterium breve* BBr53 and deposited for preservation. Its preservation information is as follows:

[0082] Accession number: CGMCC NO.34559

[0083] Classification and nomenclature: Bifidobacterium breveBBr53

[0084] Deposit date: May 15, 2025

[0085] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0086] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0087] 3. Bifidobacterium breve BBr53 inhibits the growth of Edwardsiella tarda and Aeromonas hydrophila.

[0088] Bifidobacterium breve BBr53 strain was inoculated into liquid MRS medium at a 2% inoculum and incubated at 37°C for 24 h. The bacterial suspension was then set aside. Edwardsiella tarda and Aeromonas hydrophila were inoculated into LB medium at a 1% inoculum and incubated at 37°C for 16 h. The bacterial suspensions were then set aside. The cultured Edwardsiella tarda and Aeromonas hydrophila were centrifuged, and the bacterial cells were collected. The cells were washed twice with PBS buffer, then resuspended in PBS and adjusted to an OD600 value between 0.3 and 0.4. The suspensions were thoroughly mixed by vortexing. LB solid medium containing 1.5% agar was cooled to approximately 55°C and mixed separately with the Edwardsiella tarda and Aeromonas hydrophila suspensions at specific ratios to ensure a viable count of at least 10⁻⁶ for the indicator bacteria. 6 The concentration was on the order of CFU / mL. The culture was then rapidly poured into plates pre-filled with Oxford cups. After the medium cooled and solidified, the Oxford cups were removed, and 200 μL of the bacterial fermentation broth (with a viable count of 10⁻⁶) was injected into each well. 8 (on the order of CFU / mL), after overnight incubation at 37°C, the diameter of the inhibition zone was measured.

[0089] like Figure 1 As shown, inhibition zones appeared on both Edwardsiella tarda and Aeromonas hydrophila plates, with an average diameter of 16.5 mm for Edwardsiella tarda and 14.2 mm for Aeromonas hydrophila. This indicates that the Bifidobacterium breve BBr53 provided by this invention can inhibit the growth of Edwardsiella tarda and Aeromonas hydrophila.

[0090] 4. Establishment and grouping of an in vivo immunodeficiency mouse model

[0091] Male BALB / c mice (18-20g) without specific pathogens were purchased from Beijing Spefol Biotechnology Co., Ltd., and housed in the animal room of the Hubei Provincial Center for Food and Drug Safety Evaluation. The temperature was maintained at 22±2℃, humidity at 50±5%, with a 12-hour light-dark cycle, and free access to water and food was provided. Animal experiments were conducted in accordance with the guidelines of the Ethics Committee of the Hubei Provincial Center for Disease Control and Prevention (No.: Safety Evaluation Center Animal (Fujian) No. 202410263).

[0092] Before the experiment, the mice were acclimatized for one week and then randomly divided into three groups: normal control group (NC), immunosuppressed group (MC), and Bifidobacterium breve BBr53 group (BBr53).

[0093] Throughout the experiment, mice in the NC and MC groups were administered 0.2 mL of sterile saline by gavage daily, while mice in the BBr53 group were administered an equal volume of Bifidobacterium breve BBr53 bacterial suspension (with a viable count of 10^6). 9(CFU / mL). From day 7 to day 9 of the experiment, mice in the MC and BBr53 groups were continuously injected with cyclophosphamide (CTX, 80 mg / kg / d) for 3 days to establish an immunosuppressed mouse model, while 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. Detailed animal experiments are shown in Table 1.

[0094] Table 1. In vivo experimental design grouping

[0095] NC Group The patient was given normal saline by gavage for 14 consecutive days, with 0.2 mL of normal saline injected intraperitoneally on days 7, 8, and 9. MC Group The patient was given normal saline by gavage for 14 consecutive days, followed by intraperitoneal injection of 0.2 mL of cyclophosphamide (80 mg / kg) on ​​days 7, 8, and 9. BBr53 group <![CDATA[Continuous intragastric administration of live Bifidobacterium breve BBr53 cells (the viable count is 10 9 CFU / mL, 0.2 mL / day / animal) for 14 days; 0.2 mL of cyclophosphamide (80 mg / kg) was intraperitoneally injected on the 7th, 8th and 9th days]]>

[0096] (1) Thymus index and spleen index

[0097] After one week of acclimatization, the mice were randomly divided into three groups. The weight of the mice was measured on days 1, 4, 7, 10 and 14. During dissection, the spleen and thymus of the mice were collected, and the thymus index and spleen index of the mice were calculated.

[0098] The formula for calculating the immune organ index is as follows:

[0099] Immune organ index = Immune organ mass (mg) / Body weight (g)

[0100] like Figure 2 As shown, compared with the MC group, gavage with BBr53 significantly increased the body weight of mice, thereby effectively alleviating the damage of cyclophosphamide to the body.

[0101] like Figure 3 As shown, the immune organ indices in the MC group all decreased, indicating that immune organ atrophy occurred in mice after cyclophosphamide injection. The spleen and thymus indices of mice administered BBr53 by gavage were significantly higher than those in the MC group, with increases of 112.3% and 88.7% respectively. This indicates that BBr53 can alleviate cyclophosphamide-induced thymus and spleen atrophy in mice, suggesting that intervention with Bifidobacterium breve (BBr53) can effectively alleviate the damage to immune organs caused by CTX.

[0102] (2) Effects of Bifidobacterium breve BBr53 on serum SIgA, IL-10, IL-17 and TNF-α in mice

[0103] Mouse serum was collected by centrifugation. The levels of SIgA, IL-10, IL-17, and TNF-α in the serum of each group of mice were detected by ELISA.

[0104] like Figure 4As shown, BBr53 treatment significantly increased the levels of SIgA, TNF-α, IL-10, and IL-17 in mouse serum, increasing by 58.3%, 90.1%, 75.6%, and 109.4%, respectively, compared to the MC group. These results indicate that BBr53 can upregulate the expression of serum immunoglobulins and cytokines in CTX-treated mice, suggesting that the *Bifidobacterium breve* BBr53 provided by this invention enhances the immune system of mice.

[0105] (3) Effects of Bifidobacterium breve BBr53 on mouse ileum tissue

[0106] After the mice were sacrificed, approximately 0.5 cm of ileal tissue was collected, fixed with 4% paraformaldehyde, and then embedded in paraffin. Sections with a thickness of 4 μm were prepared using a pathological microtome, and then stained with hematoxylin and eosin (H&E). The structural changes of the ileal tissue were observed under a microscope.

[0107] like Figure 5 As shown, the small intestinal tissue structure in the NC group was normal, with tightly packed villi and intact intestinal epithelial cells. Conversely, the small intestinal tissue in the MC group showed damage, with epithelial cells sloughing off from the lamina propria. These lesions were improved in the Bifidobacterium breve BBr53 group. This indicates that the Bifidobacterium breve BBr53 provided by this invention can enhance the intestinal mucosal barrier and reduce ileal tissue damage.

[0108] (4) Effects of Bifidobacterium breve BBr53 on mouse spleen tissue

[0109] After the mice were sacrificed, their spleen tissue was collected, fixed with 4% paraformaldehyde, and then embedded in paraffin. Sections with a thickness of 4 μm were prepared using a pathological microtome, and then stained with hematoxylin and eosin (H&E). The structural changes of the spleen tissue were observed under a microscope.

[0110] like Figure 6 As shown, in the NC group, the white pulp lymphocytes in the spleen tissue were densely distributed and clearly demarcated from the red pulp. In the MC group, the number of lymphocytes in the white pulp was reduced, the structure was dispersed, and the boundary with the red pulp was unclear. After intervention with Bifidobacterium breve BBr53, the boundary between the white pulp and the red pulp became clear, the number of lymphocytes increased, and Bifidobacterium breve BBr53 alleviated spleen damage induced by CTX.

[0111] (5) Effects of Bifidobacterium breve BBr53 on the intestinal flora of mice

[0112] Cecal contents were collected from mice in each group, and DNA was extracted. The V3-V4 region of bacterial 16S rRNA was amplified by PCR. Small fragment libraries were constructed and sequenced using paired-end sequencing on the Illumina Novaseq platform. Read splicing, filtering, clustering, or noise reduction, followed by species annotation and abundance analysis, revealed the species composition of the samples. Further α-diversity analysis, β-diversity analysis, and species diversity analysis were performed to explore differences between samples.

[0113] 1) Alpha-diversity analysis, β-diversity analysis

[0114] Alpha diversity reflects the species richness and diversity of a single sample, and there are several metrics for this, including Chao1, ACE, Shannon, and Simpson. The Chao1 and ACE indices measure species richness, i.e., the number of species. The Shannon and Simpson indices measure species diversity; higher values ​​indicate greater species diversity in the sample. The results of mouse gut alpha diversity analysis are shown below. Figure 7 As shown, compared with the NC group, the Chao1 index, ACE index, Shannon index, and Simpson index were significantly decreased in the MC group, indicating that the richness and diversity of the intestinal flora in mice decreased after injection of cyclophosphamide. However, after gavage administration of the *Bifidobacterium breve* BBr53 provided by this invention, the Chao1 index, ACE index, Shannon index, and Simpson index all significantly increased, indicating that BBr53 treatment improved the richness and diversity of the intestinal flora in mice, which is beneficial for maintaining intestinal flora homeostasis.

[0115] β-diversity analysis was used to compare the similarity in species diversity among different samples. Results are as follows: Figure 8 As shown, the MC group and the NC group were clearly separated, indicating that the microbial composition of the MC group was significantly different from that of the NC group. The BBr53 group was adjacent to the NC group, indicating that gavage administration of BBr53 brought the composition of the intestinal microbiota in immunocompromised mice closer to that of the normal group.

[0116] 2) OTUs cluster analysis

[0117] OTUs cluster analysis, such as Figure 9 The results showed that the MC group and the NC group had a total of 127 OTUs, and the BBr53 group and the NC group had a total of 220 OTUs, indicating that oral administration of Bifidobacterium breve BBr53 can increase the common flora in the intestines of immunosuppressed mice and normally fed mice.

[0118] 3) Phylum-level species diversity analysis

[0119] Phylum-level species diversity analysis, such as Figure 10As shown, at the phylum level, each group mainly consists of Bacteroidetes, Firmicutes, Proteobacteria, Iron-depleting Bacteria, Actinobacteria, Cyanobacteria, and Fusobacteria. Among them, Firmicutes and Bacteroidetes are the two dominant phyla.

[0120] like Figure 11 As shown, compared with the NC group, the relative abundance of Firmicutes was significantly decreased and the relative abundance of Bacteroidetes was significantly increased in the MC group, resulting in a decrease in the Firmicutes / Bacteroidetes ratio. After BBr53 treatment, the relative abundance of Firmicutes significantly increased and the relative abundance of Bacteroidetes significantly decreased, ultimately increasing the Firmicutes / Bacteroidetes ratio, indicating that gavage administration of BBr53 can improve the intestinal flora structure of mice.

[0121] like Figure 12 As shown, compared with the NC group, the relative abundance of Firmicutes was significantly decreased and the relative abundance of Bacteroidetes was significantly increased in the MC group, resulting in a decrease in the Firmicutes / Bacteroidetes ratio. After BBr53 treatment, the relative abundance of Firmicutes significantly increased and the relative abundance of Bacteroidetes significantly decreased, ultimately increasing the Firmicutes / Bacteroidetes ratio, indicating that gavage administration of BBr53 can improve the intestinal flora structure of mice.

[0122] 4) Genus-level species diversity analysis

[0123] Figure 13 The relative abundance of genera in the cecal contents of mice in each group is shown. The relative abundance of unclassified Muribaculaceae, Ligilactobacillus, Lactobacillus, unclassified Lachnospiraceae, Lachnospiraceae NK4A136 group, Bacteroides, Bifidobacterium, and Desulfovibrionaceae is analyzed.

[0124] like Figure 13 As shown, compared with the NC group, the relative abundance of unclassified *Rhizoctoniaceae*, *Lactobacillus*, *Lactobacillus*, unclassified *Trichophyceae*, and *Trichophyceae* NK4A136 was significantly decreased in the MC group, while the relative abundance of *Bacteroides* and *Desulfovibrioceae* increased. Compared with the MC group, the relative abundance of unclassified *Rhizoctoniaceae*, *Lactobacillus*, *Lactobacillus*, unclassified *Trichophyceae*, *Trichophyceae* NK4A136, and *Bifidobacterium* increased in the *Bifidobacterium breve* BBr53 group. Furthermore, compared with the MC group, the relative abundance of *Bacteroides* and *Desulfovibrioceae* was decreased in the *Bifidobacterium breve* BBr53 group.

[0125] The abundance of unclassified rodentaceae was closely correlated with propionate concentration. The abundance of *Helicobacter tumefaciens* NK4A136 group was associated with host health and is a potential butyrate producer. Both propionate and butyrate belong to the category of short-chain fatty acids (SCFAs), which play various regulatory roles in the innate and adaptive immune systems. Furthermore, *Bifidobacterium* and *Lactobacillus* can interact with immune receptors, leading to the production of immunomodulatory cytokines to defend against invading pathogens. The results indicate that *Bifidobacterium breve* BBr53 can increase the abundance of beneficial bacteria (unclassified rodentaceae, *Lactobacillus*, *Lactobacillus*, unclassified *Helicobacter*, *Helicobacter tumefaciens* NK4A136 group, and *Bifidobacterium*) in the host gut, while decreasing the abundance of harmful bacteria (Bacteroides and Desulfovibrioceae), maintaining gut microbiota homeostasis, improving the intestinal barrier, and playing an immunomodulatory role.

[0126] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A strain of Bifidobacterium breve ( Bifidobacterium breve ), characterized in that, It is Bifidobacterium breve BBr53 with accession number CGMCC NO.34559.

2. A composition for inhibiting the proliferation of Edwardsiella tarda and Aeromonas hydrophila, characterized in that, The included Bifidobacterium breve BBr53 is the Bifidobacterium breve with accession number CGMCC NO.34559. Bifidobacterium breve ) BBr53 strain.

3. A gut probiotic composition, characterized in that, It contains Bifidobacterium breve BBr53, wherein Bifidobacterium breve BBr533 is the Bifidobacterium breve with accession number CGMCC NO.34559. Bifidobacterium breve The concentration of *Bifidobacterium breve* strain BBr53 was 10... 3 Up to 10 17 Within the range of colony-forming units per gram or milliliter.

4. A preparation for enhancing immune regulation, characterized in that, The formulation contains *Bifidobacterium breve* BBr53 as the active ingredient and excipients for forming the formulation, wherein *Bifidobacterium breve* BBr53 is *Bifidobacterium breve* with accession number CGMCC NO.34559. Bifidobacterium breve ) BBr53 strain.

5. A preparation for inhibiting the proliferation of Edwardsiella tarda and Aeromonas hydrophila, characterized in that, The formulation contains *Bifidobacterium breve* BBr53 as the active ingredient and excipients for forming the formulation, wherein *Bifidobacterium breve* BBr53 is *Bifidobacterium breve* with accession number CGMCC NO.34559. Bifidobacterium breve ) BBr53 strain.

6. A culture, characterized in that, The culture medium contains Bifidobacterium breve BBr53 as described in claim 1, and the culture system is an anaerobic MRS liquid medium supplemented with 0.05% L-cysteine ​​hydrochloride.

7. The Bifidobacterium breve as described in claim 1 ( Bifidobacterium breve Its application in the preparation of formulations, characterized in that, The formulation is selected from at least one of the following: Preparations that enhance immune regulation; Antibacterial agents that inhibit the growth of Edwardsiella tarda and Aeromonas hydrophila; The intestinal probiotic preparation increases the relative abundance of unclassified rodentaceae, Lactobacillus spp., Lactobacillus spp., unclassified spirochetaceae, spirochetaceae NK4A136 group and Bifidobacterium spp. in the intestine, and decreases the relative abundance of Bacteroidetes spp. and Desulfovibrioceae. The intestinal probiotic preparation also increases the relative abundance of Firmicutes phylum in the intestine, and decreases the relative abundance of Bacteroidetes phylum.

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