Bifidobacterium animalis subsp. Lactis microbial inoculum, application and product thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a bifidobacterium animalis subsp. Lactis microbial agent, application and a product thereof. The bifidobacterium animalis subsp. Lactis provided by the invention is bifidobacterium animalis subsp. Lactis BL-5M, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.33449. By means of intervention of the animal bifidobacterium subsp. Lactis BL-5M, the skin itch degree can be reduced, and the scratching behavior is reduced; skin spongy edema, abnormal keratosis and spinous hyperplasia phenomena are relieved; the diseased skin tissue structure repair is promoted; skin dermal superficial layer inflammatory cell infiltration is relieved, and the anti-inflammatory capacity of the body is improved; the serum IgE level is obviously reduced, and the anti-allergy or desensitization capability of the body is improved; the symptoms such as erythema, edema and pruritus of people with easily-contacted allergic constitution are effectively reduced, and the uncomfortable body feeling is reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bifidobacterium animalis subsp. lactis, its inoculum, applications, and products. Background Technology

[0002] Allergic contact dermatitis (ACD) is a common inflammatory skin condition characterized by persistent itching and pain after local skin contact with reactive chemicals.

[0003] The pathogenesis of Acute Crohn's disease (ACD) may be related to primary irritants, allergic reactions, genetics, and autoimmunity. The main clinical manifestations include itching, skin erythema, and fever. Western medicine often uses a combination of glucocorticoids and antihistamines to treat this disease, which helps alleviate clinical symptoms. However, long-term treatment can easily lead to dependence, resulting in relapse after discontinuation. Traditional Chinese medicine classifies ACD under the category of "damp sores." Baihu Tang (White Tiger Decoction) is a common Chinese herbal decoction, known as the "ancestor of cold remedies," primarily treating symptoms such as high fever, thirst, irritability, and a floating and slippery pulse. It has shown significant clinical efficacy in treating ACD. However, there are currently few clinical reports on the efficacy of Baihu Tang combined with Western medicine in treating this disease.

[0004] Relieving allergic reactions through microorganisms is not only sustainable—that is, the live bacteria colonize and multiply in the intestines, and their own and their metabolites continue to increase, thus exerting a probiotic effect—but also, because the number of beneficial microorganisms is large, their reproduction rate is fast, and they live in the human intestines and coexist with the human body, they will not cause any damage to the body or any toxic side effects. They can gently and effectively relieve allergic reactions from the inside out, and the effect is even better with long-term consumption.

[0005] Relevant patent documents retrieved:

[0006] This document, published in China (CN120158394A, June 17, 2025), discloses a *Bifidobacterium longum* subspecies FN103 with accession number GDMCC No. 65836. It effectively utilizes the extracellular release of monosaccharides or disaccharides by *Bifidobacterium longum* to achieve high-efficiency proliferation and produce large amounts of short-chain fatty acids. Furthermore, the proliferation and short-chain fatty acid production capabilities of this *Bifidobacterium longum* subspecies FN103 are stronger than those of two other strains belonging to the same subspecies. In addition, the combined use of *Bifidobacterium longum* subspecies FN103 with human milk oligosaccharides and *Bifidobacterium longum* can inhibit the expression of inflammatory factors, improve symptoms such as skin redness and desquamation, and exert a therapeutic effect on atopic dermatitis. Therefore, this *Bifidobacterium longum* subspecies has high application value in allergic diseases.

[0007] Providing a Bifidobacterium lactis subsp. animalis that improves allergic contact dermatitis is of great significance for breaking through the current bottleneck of ACD treatment, expanding the pathway of microbial intervention in allergic diseases, and meeting the health needs for safe and long-term effects. Summary of the Invention

[0008] The purpose of this invention is to provide: Bifidobacterium animalis subsp. lactis, its agents, applications and products, and related technologies to address and provide effective solutions to technical problems such as contact dermatitis, or combinations thereof.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] The definition of standard chemical terms can be found in the reference "Principles and Identification Techniques of Bacterial and Archaea Systematic Taxonomy, Higher Education Press, Chief Editors Li Wenjun, Liu Lan, Jiao Jianyu, and Fang Baozhu, 2025-01".

[0012] Unless otherwise stated, conventional methods within the scope of the art, such as growth curves, in vitro antioxidant capacity, toxicity tests, antibacterial capacity, resistance to artificial gastrointestinal fluids, and adhesion methods, shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms "optional / arbitrary" or "optionally / arbitrarily" mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: "The bacterial agent comprises any one or more of Bifidobacterium animalis subsp. lactis cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder" means that the bacterial agent may contain Bifidobacterium animalis subsp. lactis cells, or the bacterial agent may contain Bifidobacterium animalis subsp. lactis fermentation broth, or the bacterial agent may contain Bifidobacterium animalis subsp. lactis cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder.

[0015] The term "Bifidobacterium lactis subsp. animalis" as used in this article refers to: Bifidobacterium genus ( Bifidobacterium Animal Bifidobacterium species ( B. animalis It is a subspecies of probiotics and is one of the most widely used and studied strains in the field of probiotics. It has clear probiotic characteristics and safety properties, and is widely found in the human gut, breast milk and fermented dairy products. It is also a core functional strain in food, health food and microecological preparations.

[0016] The term "culture medium" as used in this article refers to an artificially prepared nutrient substrate suitable for the growth, reproduction, or accumulation of metabolic products by microorganisms (such as bacteria, fungi, and viruses). Its components typically include carbon sources, nitrogen sources, inorganic salts, vitamins, and water, and the formulation can be adjusted according to the needs of the cultured organism (e.g., solid culture media require the addition of agar, and anaerobic microbial culture requires a special anaerobic environment). Culture media are indispensable basic materials in experiments and industrial production involving microbial isolation, identification, cultivation, and fermentation.

[0017] The term "contact dermatitis" used in this article refers to an inflammatory skin disease that occurs at and around the contact site after the skin or mucous membranes come into contact with a specific external substance. Its core characteristics include symptoms such as erythema, papules, vesicles, and itching at the contact site. In severe cases, it may be accompanied by erosion, exudation, or even systemic discomfort. It is one of the most common allergic / irritant skin diseases in clinical practice and can be divided into two main types based on its pathogenesis: irritant and allergic.

[0018] On the one hand, the present invention provides a subspecies of Bifidobacterium lactis (Bifidobacterium animalis) Bifidobacterium animalis subsp.lactis The Bifidobacterium animalis subsp. lactis mentioned is Bifidobacterium animalis subsp. lactis BL-5M, with accession number CGMCC No. 33449.

[0019] On the other hand, the present invention provides a microbial agent comprising the above-mentioned Bifidobacterium animalis subsp. lactis.

[0020] Specifically, the bacterial agent includes one or more of the following: Bifidobacterium animalis subsp. lactis cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder.

[0021] Preferably, the bacterial cells are a liquid suspension or a solid bacterial powder.

[0022] Specifically, the microbial agent also includes nutritionally acceptable nutrient additives.

[0023] Preferably, the nutritional additives include any one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0024] In another aspect, the present invention provides a composition comprising the above-mentioned Bifidobacterium lactis subsp. or bacterial agent.

[0025] Specifically, the composition has the function of improving contact dermatitis.

[0026] Preferably, the composition has any one or more of the following functions: (1) Reduce the degree of skin itching caused by contact dermatitis and reduce scratching behavior; (2) Reduces spongy edema, abnormal keratinization and acanthosis of the skin caused by contact dermatitis; (3) Promotes the repair of the damaged skin tissue structure caused by contact dermatitis; (4) Reduces superficial dermal inflammatory cell infiltration caused by contact dermatitis and improves the body's anti-inflammatory ability; (5) Reduce the level of IgE immunoglobulin in the body caused by contact dermatitis and improve the body's anti-allergy and desensitization ability; (6) Reduce symptoms such as erythema, edema and itching in people with easily contact allergies, and reduce discomfort.

[0027] More preferably, the composition has any one or more of the following functions: (1) It is used to relieve the skin itching symptoms of patients with contact dermatitis, reduce repeated scratching behavior caused by itching, and improve the patient's daily comfort and sleep quality; (2) Used to improve skin pathological damage caused by contact dermatitis and reduce histological lesions such as spongiform edema, abnormal keratinization and acanthosis of the skin; (3) It is used to promote the recovery of tissue structure of skin damaged by contact dermatitis, accelerate the healing of skin lesions, and maintain the integrity of the skin barrier; (4) It is used to reduce the local inflammatory response of contact dermatitis, reduce the degree of inflammatory cell infiltration in the superficial dermis, and improve the overall anti-inflammatory level of the body; (5) It is used to downregulate the abnormally elevated IgE associated with contact dermatitis, enhance the body's anti-allergy and desensitization ability, and reduce the recurrence and aggravation of allergic reactions; (6) It is used to improve the skin reaction of people with allergies after contact with allergens, reduce discomfort symptoms such as erythema, edema, and itching, and improve daily life tolerance.

[0028] Specifically, the composition contains at least 1×10 8 CFU of Bifidobacterium animalis subsp. lactis BL-5M.

[0029] Preferably, the composition contains 1×10 8 -1×10 12 CFU of Bifidobacterium animalis subsp. lactis BL-5M.

[0030] More preferably, the composition contains 1×10 9 CFU of Bifidobacterium animalis subsp. lactis BL-5M.

[0031] Specifically, the composition also includes excipients.

[0032] Preferably, the excipients include any one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, and buffer.

[0033] In another aspect, the present invention provides the use of the above-mentioned Bifidobacterium lactis subsp. or bacterial agent in the preparation of compositions having the function of improving contact dermatitis.

[0034] The beneficial effects of this invention are as follows: The *Bifidobacterium lactis* subsp. BL-5M provided by this invention can enhance the ability to improve contact allergies. Intervention with *Bifidobacterium lactis* subsp. BL-5M of this invention can reduce the severity of skin itching and scratching; alleviate spongiform edema, abnormal keratinization, and acanthosis; promote the repair of diseased skin tissue structure; reduce inflammatory cell infiltration in the superficial dermis, improving the body's anti-inflammatory capacity; significantly reduce serum IgE levels, enhancing the body's anti-allergic or desensitization ability; and effectively reduce symptoms such as erythema, edema, and itching in people with easily contact allergies, reducing discomfort.

[0035] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for improving contact dermatitis, the method comprising using the above-mentioned Bifidobacterium animalis subsp. lactis, bacterial agent, or composition.

[0036] Specifically, the method includes administering an effective amount of Bifidobacterium animalis subsp. lactis, a bacterial agent, or a composition to the subject.

[0037] Among them, the technical characteristics of the subjects included mammals.

[0038] Among them, the preferred subjects for technical characteristics are human beings.

[0039] Preservation Instructions Preserved strain: Bifidobacterium animalis subsp. lactis BL-5M; Classification and nomenclature: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp.lactis ; Accession number: CGMCC No. 33449; Preservation period: January 17, 2025; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Abbreviation of depositary institution: CGMCC; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0040] Figure 1 The colony is Bifidobacterium lactis subsp. BL-5M from MRS plate.

[0041] Figure 2 Gram staining for Bifidobacterium lactis subsp. BL-5M.

[0042] Figure 3 The colony is *Bifidobacterium lactis* subsp. BL-5M from the Columbia blood plate.

[0043] Figure 4 The growth curve of Bifidobacterium lactis subsp. BL-5M.

[0044] Figure 5 Mice were subjected to gavage for 4 days to observe scratching behavior; (see image) This represents p < 0.05; This means p < 0.01. This means p < 0.0001.

[0045] Figure 6 Scratching behavior in mice was observed after 6 days of gavage; ns in the figure represents no significant difference. This means p < 0.0001.

[0046] Figure 7 Scratching behavior in mice was observed after 8 days of gavage; ns in the figure represents no significant difference. This means p < 0.0001.

[0047] Figure 8 The figure shows scratching behavior in mice; ns in the figure represents no significant difference. This represents p < 0.05; This means p < 0.01. This means p < 0.001. This means p < 0.0001.

[0048] Figure 9 The appearance of the skin at the sensitized site in mice.

[0049] Figure 10 HE staining of skin tissue from sensitized sites in mice.

[0050] Figure 11 The figure shows the epidermal thickness of the skin at the sensitization site in mice; ns in the figure represents no significant difference. This means p < 0.0001.

[0051] Figure 12 These are inflammatory cells in the dermal layer of the skin at the sensitization site in mice; ns in the figure represents no significant difference. This means p < 0.05. This means p < 0.0001.

[0052] Figure 13 The value represents the IgE concentration in mouse serum; ns in the figure indicates no significant difference. This means p < 0.0001.

[0053] Figure 14 The figure shows the score of contact sensitization symptoms in susceptible individuals treated with bacterial strains; This means p < 0.001. Detailed Implementation

[0054] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0055] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0056] Example 1: Isolation and Identification of Bifidobacterium animalis subsp. lactis BL-5M The *Bifidobacterium lactis* subspecies BL-5M of this invention was isolated from healthy breast milk. The 16S rRNA sequencing results are shown in SEQ ID NO.1:

[0057] Strain Preservation: Bifidobacterium animalis subsp. lactis BL-5M was deposited on January 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33449, and classified as Bifidobacterium animalis subsp. lactis. Bifidobacterium animalis subsp.lactis .

[0058] Example 2: Detection of physicochemical characteristics of Bifidobacterium animalis subsp. lactis BL-5M strain 2.1 Morphological and colony observation, hemolytic characteristics Pure bacterial culture was evenly spread onto a glass slide and fixed in the outer flame of an alcohol lamp. After Gram staining, the slide was slowly rinsed with deionized water to remove excess staining solution. After the slide dried, it was observed and photographed under an oil immersion microscope. One loopful of bacterial culture was streaked onto MRS agar medium and incubated at 37°C for 48 hours. The colony morphology was then observed.

[0059] The microbiological characteristics of Bifidobacterium animalis subsp. lactis BL-5M are as follows: (1) Cultured on MRS agar medium, such as Figure 1 As shown, the colonies are white, opaque, round, with a smooth and moist surface, neat edges, and a raised center.

[0060] (2) Gram staining morphology: such as Figure 2 As shown, the bacteria are irregular rod-shaped, straight or curved, forked, and arranged singly, in pairs or in a V-shape. They are Gram-positive, do not produce spores, and are non-motile.

[0061] (3) Columbia blood plate culture, such as Figure 3 As shown, there was no hemolysis around the colony, indicating a negative hemolysis reaction.

[0062] 2.2 Utilization of different carbohydrates by *Bifidobacterium animalis* subsp. *lactam* BL-5M Preparation of colony plates: The strain is streaked on MRS agar plates. After colonies grow, single colonies are picked and streaked again until single colonies grow.

[0063] Preparation of bacterial test solution: Pick the colonies on the plate into 2 mL of physiological saline, shake and mix well, take an appropriate amount of bacterial solution (v) into 5 mL of physiological saline, measure the OD value, and then take (2v) from the original bacterial solution into 10 mL of API-matched culture medium and mix well.

[0064] Incubation reaction: Add sterile deionized water to the bottom plate of the incubation box to ensure a humid environment. Take out the test strips (0-19, 20-39, 40-49) from the packaging bag, separate them, place them in the bottom plate of the incubation box, and gently tilt them forward.

[0065] Using a pipette, draw 115 μL of bacterial culture, placing the pipette tip against the edge of the cup to add it, avoiding air bubbles. Fill only the top of the tube (cup) completely, and seal the top with sterile liquid paraffin to maintain an anaerobic environment. Incubate the tube at 37°C, observing the color changes of the reagent strips after 24 and 48 hours. The results are shown in Table 1. BL-5M can utilize 19 carbon sources.

[0066] Table 1. Identification results of Bifidobacterium animalis subsp. lactis BL-5M API-50

[0067] Note: + indicates that the color is different from the control color, and - indicates that the color is the same as the control color.

[0068] 2.3 Determination of growth curve of Bifidobacterium animalis subsp. lactis BL-5M After activating BL-5M for two generations at a 1% inoculum in 96-well plates, the third generation was inoculated at a 1% inoculum, and the growth curve was measured at regular intervals using a microplate reader.

[0069] Specific procedures: The test strain preserved in glycerol was inoculated into sterile MRS liquid medium at a 1% inoculum and anaerobically cultured at 37°C for 16 h. 1% of the culture was then transferred to a sterile 96-well plate containing 200 µl of MRS broth. After purging with nitrogen, the plate was cultured at 37°C for 8 h. 5% of the culture was then transferred to a new 96-well plate. Each strain was replicated in 3 wells. The plates were sealed and cultured at 37°C with shaking. OD600 was measured every 1 h for 30 h.

[0070] The results are as follows Figure 4 The results show that BL-5M enters the logarithmic growth phase at 4 hours and the stationary growth phase at 24 hours, with an OD600 value of approximately 1.5 at the stationary phase.

[0071] 2.4 In vitro antioxidant capacity of Bifidobacterium animalis subsp. lactis BL-5M After activation with BL-5M, the cells were cultured for three generations. Cell lysates and fermentation broth supernatants were prepared. The DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, and total antioxidant capacity of the cell lysates and fermentation broth supernatants were tested using the Nanjing Jiancheng FRTP kit (catalog number: A015-3-1). The experimental procedures were performed in accordance with the kit instructions. The results are shown in Table 2.

[0072] Table 2. Antioxidant capacity of Bifidobacterium animalis subsp. lactis BL-5M strain

[0073] 2.5 Virulence and safety testing of Bifidobacterium animalis subsp. lactis BL-5M Bifidobacterium animalis subsp. lactis BL-5M strain was inoculated into MRS liquid medium and cultured anaerobically at 37°C for three generations. The number of viable bacteria in the culture medium was counted and concentrated to 1×10⁻⁶. 10 CFU / mL, 20 mL / kg was administered orally to test mice for 3 consecutive days, and the mice were observed for 7 days. Results: Oral administration of BL-5M strain resulted in no statistically significant change in body weight in the test mice (p > 0.05), and no toxic reactions or deaths were observed. This meets the requirements of the European Food Safety Authority (EFSA) for evaluating antibiotic resistance in edible bacteria. Bifidobacterium animalis subsp. lactis BL-5M does not contain exogenous antibiotic resistance genes and is safe for consumption.

[0074] According to Appendix E of GB31615.2—2025 "National Food Safety Standard - Procedure for Safety Evaluation of Food-grade Microbial Strains" (Determination of Antimicrobial Resistance of Food-grade Bacterial Strains (Microbroth Dilution Method), the susceptibility of BL-5M antibiotics was evaluated, and the results are shown in Table 3.

[0075] Table 3. Antibiotic susceptibility of Bifidobacterium animalis subsp. lactis BL-5M strain

[0076] 2.6 Antibacterial activity of Bifidobacterium animalis subsp. lactis BL-5M The antibacterial activity of the strains was determined using the Oxford cup method.

[0077] The BL-5M strain was inoculated into sterile MRS liquid medium at a 1% inoculum and cultured at 37°C for 16 hours. Then, it was inoculated into MRS liquid medium at a 5% inoculum and cultured at 37°C for 8 hours. Finally, it was inoculated into MRS liquid medium at a 5% inoculum and cultured at 37°C for 48 hours.

[0078] Six common pathogenic bacteria were selected as indicator bacteria: *Escherichia coli* ATCC25922, *Staphylococcus aureus* ATCC25923, *Pseudomonas aeruginosa* ATCC27853, *Enterococcus faecalis* ATCC29212, *Shigella flexneri* CICC 21534, and *Cronobacter sakazakii* CICC 21560. A 1% inoculum was added to sterile BHI liquid medium and incubated overnight at 37°C for approximately 16 hours. The OD600 value was adjusted to 0.8, and the medium was then stored at 4°C for later use.

[0079] Preparation of agar plates (10 mL / plate): Add the pathogenic bacterial suspension at a 1% (v / v) ratio to BHI agar medium maintained at approximately 50°C. Mix well and pour onto the agar plate (5 mL / plate). After the upper layer of medium has solidified, place it into an Oxford cup using sterile forceps. Add 200 µL of BL-5M supernatant to the Oxford cup. Simultaneously, use autoclaved uninoculated MRS liquid medium as a negative control and MRS + lactic acid (pH=4.15) as a positive control. Incubate at 4°C for 24 h for diffusion, then incubate statically at 37°C for 24 h. Observe the inhibition zone; measure the diameter of the outer inhibition zone with calipers. The results of the antibacterial ability are shown in Table 4.

[0080] Table 4. Antibacterial activity of Bifidobacterium animalis subsp. lactis BL-5M strain against pathogens.

[0081] The results showed that Bifidobacterium animalis subsp. lactis BL-5M had an inhibitory effect on all six pathogenic bacteria, and the antibacterial effect was relatively good.

[0082] 2.7 Resistance of Bifidobacterium animalis subsp. lactis BL-5M to artificial gastrointestinal fluid Preparation of artificial gastric fluid: Measure 0.2 mL of hydrochloric acid, add 0.8 mL of water and mix well to obtain dilute hydrochloric acid. Weigh 0.2 g of NaCl and 0.35 g of pepsin, add 100 mL of water and dissolve completely. Adjust the pH to 2.5 with dilute hydrochloric acid, mix well, and filter under sterile conditions using a 0.22 μm filter membrane.

[0083] Preparation of artificial intestinal fluid: Dissolve 0.68g of potassium dihydrogen phosphate in 50mL of water, adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution, and dissolve 1g of pancreatic enzyme in an appropriate amount of water. Mix the two solutions, dilute with water to 100mL, mix well, and filter under sterile conditions using a 0.22μm filter membrane.

[0084] Activation of the strain: 2% of the strain cryopreservation solution was inoculated into 1.5 mL of MRS broth and incubated overnight at 37°C. The next day, 5% of the bacterial solution was inoculated into 1.5 mL of MRS broth and incubated at 37°C for 7 hours. 2% of the activated second-generation bacterial solution was inoculated into 15 mL of MRS broth and incubated overnight at 37°C.

[0085] Bacterial culture treatment: Take 10 mL of culture medium, centrifuge at 6000 g for 10 min, discard the supernatant, wash the bacteria twice with sterile physiological saline, resuspend the bacterial sludge in 3 mL of artificial gastric fluid, mix well, and then bring the volume to 10 mL with artificial gastric fluid. Take 100 μL and perform 10-fold serial dilutions, and use MRS plate counting to determine the initial viable count. After anaerobic incubation at 37℃ for 3 h, take 100 μL and perform serial dilutions, and use MRS plate counting to determine the viable count after 3 h of gastric fluid treatment. Artificial intestinal fluid treatment of bacterial culture: The treatment method is the same as that of artificial gastric fluid treatment, only the artificial gastric fluid in the system is replaced with artificial intestinal fluid. The results are shown in Table 5 below.

[0086] Table 5. Statistical analysis of viable bacterial counts of Bifidobacterium lactis subsp. BL-5M in artificial gastric and intestinal fluids.

[0087] 2.8 Determination of the adhesion ability of Bifidobacterium animalis subsp. lactis BL-5M Strain preparation: Activate BL-5M strain, culture for three generations, centrifuge at 10000g for 5 min in 0.01M pBS (pH 7.2-7.4), wash twice, and adjust the cell concentration to approximately 5 × 10⁻⁶ cells / mL in DMEM medium. 7 CFU / mL.

[0088] Cell preparation: Resuscitate and culture Caco-2 cells, digest the cells and plate them into 24-well cell culture plates. The Caco-2 cells should form a monolayer with a confluence of 80-100%. Wash the cells with 0.01M pBS (pH 7.2-7.4), 1 mL / well, twice. The washing process should be gentle to avoid cell detachment.

[0089] Adhesion experiment: After cell washing, add 1 mL of solution containing 5 × 10⁶ cells of the bacterial strain. 7 Incubate at CFU / mL in a 37℃, 5% CO2 incubator for 2 hours. Use the washed bacterial suspension as a reference for co-incubation. After incubation, discard the cell culture supernatant containing unattached bacterial strains. Wash cells with 0.01M PBS (pH 7.2-7.4), 1 mL / well, 3 times. Digest cells with Trypsin-EDTA (0.25%) for 15 min, 0.5 mL / well. Count the cells in each well of a 24-well cell culture plate. Dilute the digested bacterial suspension with physiological saline to a final volume of 10. -3 10 -4 10 -5 The bacteria were spread on MRS plates and incubated in an anaerobic environment at 37°C for 2 days. The number of colonies formed by the strains on the MRS plates before and after adhesion was recorded, and the adhesion index and adhesion rate were calculated.

[0090] Adhesion index = Number of viable bacteria after adhesion / Number of cells in negative control well; Adhesion rate (%) = Number of viable bacteria after adhesion / Number of viable bacteria before adhesion.

[0091] The adhesion experiment results of BL-5M strain are shown in Table 6.

[0092] Table 6 Adhesion ability of Bifidobacterium animalis subsp. lactis BL-5M strain

[0093] Example 3: Freeze-drying process and preparation of Bifidobacterium animalis subsp. lactis BL-5M powder Bifidobacterium animalis subsp. lactis BL-5M was subcultured and activated three times at 37℃ using MRS medium. The bacterial culture was then inoculated into MRS liquid medium for large-scale culture (the culture volume depends on the actual needs). The culture conditions were: static culture at 37℃ for 16 hours to obtain the culture medium of the strain. Centrifuge 6000g of the cultured bacterial solution for 5 minutes, discard the supernatant, mix the bacterial sludge and skim milk at a mass ratio of 1:1, mix well, and then put it into a freeze dryer for vacuum freeze drying. After vacuum freeze drying for 48 hours, the bacterial powder is obtained.

[0094] Example 4: Animal Model of Contact Dermatitis Induced by Intervention with Bifidobacterium lactis subsp. BL-5M Experimental animals: 6-week-old SPF-grade C57 BL / 6 female mice.

[0095] Main reagent: 2,4-Dinitrofluorobenzene (DNFB), purchased from Sigma-Aldrich, CAS No.: 70-34-8. Solvent: Acetone-olive oil (4:1) mixture; Abdominal sensitization reagent was DNFB diluted with solvent at a concentration of 0.5%; Neck and back sensitization reagent was DNFB diluted with solvent at a concentration of 0.2%.

[0096] Animal intervention: Mice were continuously administered Lactobacillus paracasei LP33, Bifidobacterium animalis subsp. lactis I88-01-001, or Bifidobacterium animalis subsp. lactis BL-5M by gavage for 15 days, with the concentration adjusted to 1×10⁻⁶. 10 CFU / mL, gavage dose 100 μL / mouse / day. On days 9 and 10 of the experiment, mice were sensitized by abdominal administration at a dose of 50 μL / mouse; on days 16, 18, 20 and 22 of the experiment, mice were sensitized by neck and back administration at a dose of 50 μL / mouse. Information on animal gavage and sensitization reagents is provided in Table 7.

[0097] Results observation: On days 19, 21, and 23 of the experiment, i.e., days 4, 6, and 8 after gavage, the scratching behavior of mice was recorded by VR; skin from the back of the neck was collected, stained with HE, and the pathological changes, changes in epidermal thickness, and degree of inflammatory cell infiltration of the skin tissue were observed; mouse serum was collected to test the IgE concentration.

[0098] Table 7 Information on animal gavage and sensitizing agents

[0099] Bacterial culture preparation: The bacterial strain was activated and cultured for three generations. The bacterial concentration was adjusted to 1×10⁻⁶ by centrifugation (12000 rpm, 5 min). 10 CFU / mL. The system included a blank group, a solvent group, and a model group; the bacterial suspension was used as a negative control (MRS); the commercially available anti-allergy strain LP33 was purchased from Jingyue Biotechnology Co., Ltd., model: GM-080, *Lactobacillus paracasei*, and served as the animal control strain; strain I88-01-001 was isolated from healthy breast milk, *Bifidobacterium lactis*, and served as the animal control strain; strain BL-5M was isolated from healthy breast milk, *Bifidobacterium lactis*, and served as the animal experimental strain; strain information and gavage dosage are shown in Table 8.

[0100] Table 8. Strains and Gavage Dosage

[0101] 4.1 Scratching behavior in mice Scratching behavior: A scratching action was recorded when a mouse raised its forepaw or hind paw to scratch the sensitized area behind its ear or on the back of its neck, and then returned to the ground or its mouth. Scratching behavior was recorded in a VR (Vibration Therapy) system on days 4, 6, and 8 after gavage. Before VR recording, mice were allowed 10 minutes to acclimatize to the environment. The plastic box was covered with transparent plastic wrap, and ventilation holes were provided. Spontaneous scratching behavior was recorded for 60 minutes. Researchers were not allowed to enter or leave the mouse enclosure during VR recording.

[0102] Figures 5-8 The results show the total scratching behavior statistics at 4, 6, and 8 days after gavage, and Table 9 shows the scratching behavior statistics of mice after bacterial strain intervention.

[0103] The results showed that the total number of scratching behaviors in mice treated by gavage with LP33 and BL-5M strains was significantly lower than that in the blank group, solvent group, model group, and I88-01-001 strain (P < 0.0001), indicating that the mouse model of contact dermatitis was successfully established. The number of scratching behaviors in LP33 and BL-5M strains was significantly lower than that in the model group (P < 0.01), indicating that LP33 and BL-5M strains effectively reduced scratching behaviors caused by contact dermatitis, and the total number of scratching behaviors in mice treated with BL-5M strains was the lowest.

[0104] Table 9. Statistics on the scratching behavior of mice after intervention by bacterial strains

[0105] Note: The total number of scratching behaviors on days 4, 6, and 8 after gavage were compared between each group and the model group. The number of scratching behaviors in each group was lower than that in the model group. This means P < 0.05. This means P < 0.01. This means P < 0.001. This means P < 0.0001.

[0106] 4.2 Histopathological study of skin tissue at the sensitization site in mice with contact dermatitis 4.2.1 Skin tissue appearance and HE staining Skin samples were taken from the sensitized sites of mice and stained with hematoxylin-eosin (HE). Changes in skin morphology and structure, as well as inflammatory cell infiltration, were observed. Changes in epidermal thickness and the number of inflammatory cells were measured and statistically analyzed. The appearance of the sensitized skin sites in mice was referenced. Figure 9 HE staining results of skin tissue at the sensitized site were referenced. Figure 10 .

[0107] Microscopic examination of the skin tissue at the sensitized site on the neck and back of mice in the blank control and solvent groups showed normal structure. Microscopic examination of the skin tissue at the sensitized site in the model group, MRS group, and I88-01-001 group showed atopic dermatitis-like changes such as stratum corneum shedding, acanthosis, spongiosis, and superficial dermal inflammatory cell infiltration. Except for the blank control and solvent groups, the stratum corneum in the LP33 and BL-5M groups was shed, the thickness of the non-keratinized epidermis was reduced, and the skin appendages and dermis atrophied simultaneously. Compared to the model group, the LP33 and BL-5M groups showed reduced spongiosis, abnormal keratinization, and acanthosis, indicating that strains LP33 and BL-5M promote the repair or normalization of the diseased skin tissue structure.

[0108] 4.2.2 Epidermal thickness of skin tissue at sensitization sites in mice HE-stained slides of mouse skin tissue from sensitized sites were photographed under a microscope with specific parameters. Three regions were randomly selected from each slide. The epidermal thickness of the skin tissue from sensitized sites in mice under different intervention methods was statistically analyzed. The results are shown in [Table missing]. Figure 11 See Table 10.

[0109] The results showed that the epidermal thickness in the blank, solvent, LP33, I88-01-001, and BL-5M groups was significantly lower than that in the model group (P < 0.0001). This indicates that intervention with strains LP33, I88-01-001, and BL-5M significantly alleviated the thickening of the skin at the sensitized sites in mice caused by edema, inflammation, hyperkeratosis, and acanthosis, with strain BL-5M showing the best effect.

[0110] Table 10. Statistics on the thickness of inflammatory skin in mice after bacterial strain intervention.

[0111] Note: Skin thickness at the sensitization site in mice treated with the strain was statistically analyzed. Compared with the model group, the skin thickness in each group was lower in the former group than in the latter. This means P < 0.05. This means P < 0.01. This means P < 0.001. This means P < 0.0001.

[0112] 4.2.3 Inflammatory cells in the skin tissue of mice at the sensitization site HE-stained slides of mouse skin tissue from sensitized sites were photographed under a microscope with specific parameters. Three regions were randomly selected from each slide. The number of inflammatory cells in the skin tissue of mice sensitized by different intervention methods was counted. The results of the number of inflammatory cells in the dermis of the sensitized sites of mice are shown in [the table below]. Figure 12 See Table 11.

[0113] The results showed that when mice were treated with the strains via gavage, the number of inflammatory cells at the sensitized site and the skin thickness in the blank group and the solvent group were lower than those in the model group (P < 0.0001). The skin thickness in the LP33 and I88-01-001 groups was also lower than that in the model group (P < 0.05); the skin thickness in the BL-5M group was also lower than that in the model group (P < 0.0001). This indicates that intervention with strains LP33, I88-01-001, and BL-5M in mice reduced the infiltration of superficial dermal inflammatory cells at the sensitized site and improved the body's anti-inflammatory capacity, with strain BL-5M exhibiting the strongest anti-inflammatory effect.

[0114] Table 11. Statistical analysis of inflammatory cells at the sensitization site in mice treated with bacterial strains.

[0115] Note: Statistics on inflammatory cells at the sensitization sites in mice treated with the strain. Compared with the model group, the number of inflammatory cells in each group was lower. This means P < 0.05. This means P < 0.01. This means P < 0.001. This means P < 0.0001.

[0116] 4.3 Serum IgE Levels in Mice Blood was collected from the eyeballs of experimental animals, incubated at room temperature for 1 hour, and then in tilted blood collection tubes at 4°C for 1 hour. The tubes were then centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected and stored at -80°C for later use. A Mouse IgE ELISA Kit was purchased from Solarbio (catalog number: SEKM-0345). The procedure was performed according to the kit instructions. Samples were diluted 4-fold. The kit's detection range was 33-5000 pg / mL. The OD values ​​of each well were measured at 450 nm. Statistical analysis was then performed. The results of IgE detection in the serum of mice in each group are shown below. Figure 13 And Table 12.

[0117] The results showed that compared with the model group, the BL-5M group significantly reduced serum IgE levels (p<0.0001), while the LP33 group and I88-01-001 group showed no difference from the model group, indicating that strain BL-5M has excellent ability to enhance the body's anti-allergy or desensitization capabilities.

[0118] Table 12. Statistics of serum IgE in mice treated with bacterial strains

[0119] Note: Statistical analysis of serum IgE concentration in mice treated with this strain showed that, compared to the model group, the IgE concentration was lower in the latter group. This means P < 0.05. This means P < 0.01. This means P < 0.001. This means P < 0.0001.

[0120] 4.4 Comprehensive Evaluation of Animal Experiment Detection Indicators Animal experiments were conducted with blank, solvent, and model groups. Different bacterial strains were used to intervene in mice, with commercially available anti-allergy strains LP33 and I88-01-001 serving as control strains. Four indicators were measured after strain intervention in the animals: scratching behavior, number of inflammatory cells in the mouse dermis, epidermal thickness, and serum IgE concentration. Strain BL-5M showed the best performance, effectively reducing inflammation in the contact dermatitis model mice. Specifically, after strain intervention, scratching behavior was significantly reduced; the repair of diseased skin tissue structure was promoted, and spongiform edema, abnormal keratinization, and acanthosis were alleviated; superficial dermal inflammatory cell infiltration was reduced; serum IgE levels were significantly decreased, indicating strong anti-allergic ability.

[0121] Example 5: Study on the intervention of bacterial strains in people with contact allergies Strain preparation: Bifidobacterium animalis subsp. lactis BL-5M was fermented, and the bacterial cells were collected by centrifugation. After two washings, 10% skim milk powder was added as a preservative, and the mixture was freeze-dried under vacuum to prepare lyophilized bacterial powder. Maltodextrin and lyophilized bacterial powder were mixed in a certain proportion to prepare a strain preparation with a theoretical viable count of 2 × 10⁻⁶. 10 CFU / g, fluorescent staining and flow cytometry showed a dead bacterial count of 10-15%, dispensed into 0.5g bags, and stored at 2-8℃.

[0122] Volunteer Recruitment: Individuals with allergies, including those allergic to metals such as jewelry, watches, and eyeglass frames, as well as those allergic to chemicals in daily necessities.

[0123] Intervention Method: Twenty volunteers were randomly divided into two groups, one receiving a preparation of Bifidobacterium animalis subsp. lactis BL-5M strain, and the other receiving a placebo of maltodextrin. Each group consisted of 10 volunteers. One sachet was administered twice daily, morning and evening, for 30 consecutive days. The strain was dissolved in warm or ice water. Allergic symptoms were observed for 30 days after the intervention. The objective scoring system for contact sensitization symptoms was used (see Table 13), and the subjective scoring system was used (see Table 14). Volunteer scores were then analyzed. Figure 14 .

[0124] Table 13 Objective Scoring System for Contact Allergy Symptoms

[0125] Table 14 Subjective Scoring System for Contact Allergy Symptoms

[0126] The results showed that after 30 days of intervention with Bifidobacterium animalis subsp. lactis BL-5M in people with a predisposition to allergic reactions, the objective and subjective scores of allergic symptoms were lower than those in the placebo group (P < 0.0001). This indicates that Bifidobacterium animalis subsp. lactis BL-5M can effectively reduce symptoms such as erythema, edema, and itching in people with a predisposition to allergies, and reduce their discomfort.

[0127] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A subspecies of Bifidobacterium lactis ( Bifidobacterium animalis subsp.lactis ), characterized in that, The aforementioned Bifidobacterium animalis subsp. lactis is Bifidobacterium animalis subsp. lactis BL-5M, with accession number CGMCCNo.33449.

2. A microbial agent, characterized in that, The bacterial agent comprises Bifidobacterium lactis subsp. animalis as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent comprises any one or more of the following: Bifidobacterium animalis subsp. lactis cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder.

4. A composition comprising Bifidobacterium animalis subsp. lactis as described in claim 1 or the bacterial agent as described in any one of claims 2-3.

5. The composition according to claim 4, characterized in that, The composition described herein has the function of improving contact dermatitis.

6. The composition according to claim 5, characterized in that, The composition has any one or more of the following functions: (1) Reduce the degree of skin itching caused by contact dermatitis and reduce scratching behavior; (2) Reduces spongy edema, abnormal keratinization and acanthosis of the skin caused by contact dermatitis; (3) Promotes the repair of the damaged skin tissue structure caused by contact dermatitis; (4) Reduces superficial dermal inflammatory cell infiltration caused by contact dermatitis and improves the body's anti-inflammatory ability; (5) Reduce the level of IgE immunoglobulin in the body caused by contact dermatitis and improve the body's anti-allergy and desensitization ability; (6) Reduce symptoms such as erythema, edema and itching in people with easily contact allergies, and reduce discomfort.

7. The composition according to claim 6, characterized in that, The composition has any one or more of the following functions: (1) It is used to relieve the skin itching symptoms of patients with contact dermatitis, reduce repeated scratching behavior caused by itching, and improve the patient's daily comfort and sleep quality; (2) Used to improve skin pathological damage caused by contact dermatitis and reduce histological lesions such as spongiform edema, abnormal keratinization and acanthosis of the skin; (3) It is used to promote the recovery of tissue structure of skin damaged by contact dermatitis, accelerate the healing of skin lesions, and maintain the integrity of the skin barrier; (4) It is used to reduce the local inflammatory response of contact dermatitis, reduce the degree of inflammatory cell infiltration in the superficial dermis, and improve the overall anti-inflammatory level of the body; (5) It is used to downregulate the abnormally elevated IgE associated with contact dermatitis, enhance the body's anti-allergy and desensitization ability, and reduce the recurrence and aggravation of allergic reactions; (6) It is used to improve the skin reaction of people with allergies after contact with allergens, reduce discomfort symptoms such as erythema, edema, and itching, and improve daily life tolerance.

8. The composition according to claim 4, characterized in that, The composition contains at least 1×10 8 CFU of Bifidobacterium animalis subsp. lactis BL-5M.

9. The composition according to claim 8, characterized in that, The composition further includes excipients, which include any one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, and buffers.

10. The use of the Bifidobacterium lactis subsp. animalis according to claim 1 or the bacterial agent according to any one of claims 2-3 in the preparation of a composition having the function of improving contact dermatitis.

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