Application of bifidobacterium bifidum NKUFB3-12 and composition thereof in preparation of product for relieving allergic diseases and / or regulating intestinal flora caused by allergy

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to application of bifidobacterium bifidum NKUFB3-12 and a composition thereof in preparation of products for relieving allergic diseases and / or regulating intestinal flora caused by allergy. The preservation number of the bifidobacterium bifidum NKUFB3-12 is GDMCC (China General Microbiological Culture Collection Center) No. 64013. The bifidobacterium bifidum NKUFB3-12 disclosed by the invention has excellent functional characteristics of relieving allergic diseases and regulating allergic intestinal flora, and also has excellent probiotic characteristics of acid resistance, cholate resistance and oxidation resistance, and the strain is safe and has no adverse side effect after being taken. The invention provides a composition. The composition comprises bifidobacterium bifidum NKUFB3-12 and 2 '-fucosyllactose (2'-FL), and the NKUFB3-12 is compounded with the 2 '-FL to show an effective inhibition effect on the imbalance of allergic immune response.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Bifidobacterium bifidum NKUFB3-12 and its composition in the preparation of products for relieving allergic diseases and / or regulating intestinal flora caused by allergies. Background Technology

[0002] In recent years, the global incidence of allergic diseases (such as allergic rhinitis, asthma, and atopic dermatitis) has increased significantly, seriously affecting patients' quality of life and imposing a heavy burden on individuals, families, and society. Their pathogenesis is closely related to immune system imbalance and gut microbiota dysbiosis. Studies have found that the core mechanism of allergic reactions involves an imbalance in the Th1 / Th2 immune response, with overexpression of Th2 cytokines (such as IL-4, IL-5, and IL-13) leading to abnormally high levels of IgE antibodies and the release of inflammatory mediators, thereby causing tissue damage. Meanwhile, the gut, as the largest immune organ in the human body, plays a crucial role in regulating systemic immunity through its barrier function and gut microbiota homeostasis. If the barrier is damaged due to dysbiosis or pathogen invasion, intestinal permeability increases (i.e., "leaky gut"), allowing undigested antigens (such as food proteins) to enter the bloodstream, activating the immune system and exacerbating allergic reactions. Modern dietary structures, antibiotic overuse, and environmental stress have led to a decline in gut microbiota diversity, a decrease in the abundance of beneficial bacteria such as Bifidobacteria, and an overgrowth of opportunistic pathogens, further exacerbating inflammation and immune dysbiosis. Therefore, there is an urgent need to solve the technical problem of simultaneously alleviating allergic diseases and targeting and regulating the gut microbiota and its metabolic functions to restore immune homeostasis. Summary of the Invention

[0003] The purpose of this invention is to provide an application of Bifidobacterium bifidum NKUFB3-12 and its composition in the preparation of products for relieving allergic diseases and / or regulating intestinal flora caused by allergies. The Bifidobacterium bifidum NKUFB3-12 has excellent functional characteristics for relieving allergic diseases and regulating allergic intestinal flora. It also has excellent probiotic characteristics such as acid and bile salt resistance and antioxidant properties. Moreover, the strain is safe and has no adverse side effects after consumption.

[0004] This invention provides the application of Bifidobacterium bifidum NKUFB3-12 in the preparation of products for relieving allergic diseases and / or regulating intestinal flora caused by allergies, wherein the preservation number of Bifidobacterium bifidum NKUFB3-12 is GDMCCNo.64013.

[0005] As a preferred embodiment, the viable count of the Bifidobacterium bifidum NKUFB3-12 is ≥1×10⁻⁶. 9 CFU / mL or ≥1×10 9 CFU / g.

[0006] As a preferred option, the symptoms of the allergy include one or more of the following: body swelling, scratching, slow movement, and mild diarrhea.

[0007] As a preferred embodiment, the product includes at least one of the following effects: significantly reduced spleen index and / or serum allergy-related biochemical indicators; significantly alleviated intestinal barrier dysfunction and inflammatory damage; effectively regulated intestinal flora imbalance caused by allergies; and significantly increased the content of short-chain fatty acids in the intestine.

[0008] As a preferred embodiment, the product includes: pharmaceuticals and / or functional foods;

[0009] When the product is a pharmaceutical product, the application is in the preparation of pharmaceutical products for relieving allergic diseases and / or regulating intestinal flora caused by allergies;

[0010] When the product is a functional food, the application is in the preparation of functional foods that regulate intestinal flora caused by allergies.

[0011] The present invention provides a composition comprising: Bifidobacterium bifidum NKUFB3-12 and 2'-fucosylated lactose, wherein the preservation number of Bifidobacterium bifidum NKUFB3-12 is GDMCC No. 64013.

[0012] As a preferred embodiment, the viable count of the Bifidobacterium bifidum NKUFB3-12 in the composition is ≥1×10⁻⁶. 9 CFU / mL or ≥1×10 9 CFU / g.

[0013] As a preferred embodiment, the concentration of 2'-fucosylated lactose in the composition is 0.2–8 mg / mL or 0.2–8 mg / g.

[0014] This invention provides the use of the above composition in the preparation of products for relieving allergic diseases and / or regulating intestinal flora caused by allergies.

[0015] As a preferred embodiment, the product includes: pharmaceuticals and / or functional foods;

[0016] When the product is a pharmaceutical product, the application is in the preparation of pharmaceutical products for relieving allergic diseases and / or regulating intestinal flora caused by allergies;

[0017] When the product is a functional food, the application is in the preparation of functional foods that regulate intestinal flora caused by allergies.

[0018] Beneficial Effects: This invention provides the application of *Bifidobacterium bifidum* NKUFB3-12 in the preparation of products for alleviating allergic diseases and / or regulating intestinal flora caused by allergies. The preservation number of *Bifidobacterium bifidum* NKUFB3-12 is GDMCC No. 64013. This invention verifies in multiple aspects that *Bifidobacterium bifidum* NKUFB3-12 has the functional characteristics of alleviating allergic diseases and regulating intestinal flora caused by allergies. Using *Bifidobacterium bifidum* NKUFB3-12 intervention, allergic symptoms such as body edema, scratching, slow movement, and mild diarrhea in allergic mice were effectively alleviated; spleen index and serum allergy-related biochemical indicators were significantly reduced; intestinal barrier dysfunction and inflammatory damage in mice were significantly alleviated; the imbalance of intestinal flora structure caused by allergies in mice was effectively regulated, and the abundance of probiotic *Lactobacillus* was increased; the content of short-chain fatty acids propionic acid and butyric acid in the mouse intestine was significantly increased. Simultaneously, *Bifidobacterium bifidum* NKUFB3-12 has excellent acid and bile salt resistance, antioxidant probiotic properties, and the strain is safe, with no adverse side effects after administration.

[0019] This invention provides a composition comprising: *Bifidobacterium bifidum* NKUFB3-12 and 2'-fucosylated lactose, wherein the preservation number of *Bifidobacterium bifidum* NKUFB3-12 is GDMCC No. 64013. The gene of *Bifidobacterium bifidum* NKUFB3-12 contains GH95 encoding fucosidase, meaning it can use 2'-fucosylated lactose (2'-FL) as a fermentation substrate for its own proliferation. This effectively enhances the adhesion ability of NKUFB3-12 to intestinal cells, enabling it to exert a long-term effect in the intestine. In vitro cell experiments demonstrate that NKUFB3-12 combined with 2'-FL exhibits an effective inhibitory effect on the imbalance of allergic immune responses, effectively regulating the expression of Th1 / Th2 cell-related immune factors, restoring the Th1 / Th2 cell differentiation imbalance, and showing potential for alleviating allergic diseases.

[0020] This invention provides the application of the above composition in the preparation of products for alleviating allergic diseases and / or regulating intestinal flora caused by allergies. 2'-Fucose-based lactose (2'-FL), as a major component of HMOs (human milk oligosaccharides), can selectively stimulate the growth of Bifidobacteria and enhance their metabolic activity through the "bifidus factor" effect. Bifidobacteria can effectively utilize human milk oligosaccharides for growth and reproduction, and by competitively binding to intestinal epithelial cells, prevent harmful pathogens from adhering and colonizing, thus strengthening the intestinal barrier function. After 2'-FL interacts with Bifidobacteria, it can regulate immune cell activity, affect cytokine secretion, and transmit regulatory signals to the immune system, thereby helping to correct abnormal immune responses and reduce the risk of allergies, showing great application potential in the prevention and treatment of allergic diseases and the maintenance of intestinal health. Attached Figure Description

[0021] Figure 1 A complete genome circle of Bifidobacterium bifidum NKUFB3-12;

[0022] Figure 2 The growth curves of Bifidobacterium bifidum NKUFB3-12 with different oligosaccharides as carbon sources are shown.

[0023] Figure 3 The graph shows the weight results of mice, including the weight changes of mice in different groups over time and the final weight gain of mice in different groups;

[0024] Figure 4 Spleen weight and spleen index of mice in different groups;

[0025] Figure 5 Scoring of allergic symptoms in different groups of mice;

[0026] Figure 6 The levels of allergy-related biochemical indicators in the serum of mice from different groups were measured, including the levels of OVA-specific IgE (OVA-sIgE), IgG1, IgG2a, IgG1 / IgG2a, mast cell protease 1 (Mcpt-1), and histamine (HIS).

[0027] Figure 7 H&E staining and histological scoring of colon tissue sections from different groups of mice;

[0028] Figure 8 PAS staining and goblet cell count of colon tissue sections from different groups of mice;

[0029] Figure 9 The relative expression levels of mouse colonic inflammatory factor mRNA;

[0030] Figure 10 To analyze the α-diversity of gut microbiota species in different groups of mice, including differences in Simpson index and ACE index;

[0031] Figure 11 Bar chart showing the species richness of gut microbiota in different groups of mice (phylum level);

[0032] Figure 12 Plots showing the relative abundance of Firmicutes and Bacteroidetes in mice from different groups;

[0033] Figure 13 Bar chart showing the species richness of gut microbiota in mice from different groups (genus level);

[0034] Figure 14 The content of short-chain fatty acids in the feces of mice in different groups;

[0035] Figure 15 The relative expression levels of G-protein-coupled receptor mRNA in the colon of mice from different groups;

[0036] Figure 16 To observe the adhesion ability of A. muciniphila after different interventions using an inverted microscope;

[0037] Figure 17 To investigate the effect of different dosage ratios of intervention groups on the relative expression level of IFN-γ mRNA in spleen cells after OVA stimulation;

[0038] Figure 18 To investigate the effect of different dosage ratios of intervention groups on the relative expression level of IL-4 mRNA in spleen cells after OVA stimulation;

[0039] In this context, the same letter indicates that the difference is not significant, while different letters indicate that the difference is significant. Detailed Implementation

[0040] This invention provides the application of Bifidobacterium bifidum NKUFB3-12 in the preparation of products for relieving allergic diseases and / or regulating intestinal flora caused by allergies, wherein the preservation number of Bifidobacterium bifidum NKUFB3-12 is GDMCCNo.64013.

[0041] Information on the strain of *Bifidobacterium bifidum* NKUFB3-12 described in this invention can be found in patent publication CN 118773037A, entitled "A type of *Bifidobacterium bifidum* and its application." The viable count of *Bifidobacterium bifidum* NKUFB3-12 described in this invention can be ≥1×10⁻⁶. 9 Any value within the range of CFU / mL; or ≥1×10 9 Any value within the CFU / g range. Symptoms of the allergy described in this invention may include one or more of the following: body edema, scratching, slow movement, and mild diarrhea. Allergies described in this invention may include OVA-induced allergies.

[0042] The products of this invention include pharmaceuticals and / or functional foods. The products of this invention may include at least one of the following effects: significant reduction in spleen index and / or serum allergy-related biochemical indicators; significant relief of intestinal barrier dysfunction and inflammatory damage; effective regulation of intestinal flora imbalance caused by allergies; and significant increase in the content of short-chain fatty acids in the intestine. The serum allergy-related biochemical indicators of this invention may include one or more of the following: OVA-specific IgE (OVA-sIgE), IgG1, IgG2a, mast cell protease 1 (Mcpt-1), and histamine (HIS). The inflammatory factors causing the inflammatory damage of this invention may include: IL-6 and IL-1β. The effective regulation of intestinal flora imbalance caused by allergies of this invention may include: increasing the abundance of the probiotic Lactobacillus. The medium- and short-chain fatty acids of this invention may include: propionic acid and / or butyric acid. When the product of this invention is a pharmaceutical, the application is in the preparation of pharmaceuticals for relieving allergic diseases and / or regulating intestinal flora caused by allergies; when the product is a functional food, the application is in the preparation of functional foods for regulating intestinal flora caused by allergies.

[0043] This invention provides a composition comprising: *Bifidobacterium bifidum* NKUFB3-12 and 2'-fucosylated lactose, wherein the preservation number of *Bifidobacterium bifidum* NKUFB3-12 is GDMCC No. 64013. 2'-fucosylated lactose (2'-FL), as a major component of HMOs (human milk oligosaccharides), can selectively stimulate the growth of Bifidobacteria and enhance their metabolic activity through the "bifidus factor" effect. Studies have shown that Bifidobacteria can effectively utilize human milk oligosaccharides for growth and reproduction, and by competitively binding to intestinal epithelial cells, prevent harmful pathogens from adhering and colonizing, thus strengthening the intestinal barrier function. Simultaneously, the interaction between 2'-FL and other human milk oligosaccharides and *Bifidobacterium bifidum* can regulate immune cell activity, affect cytokine secretion, and transmit regulatory signals to the immune system, thereby helping to correct abnormal immune responses and reduce the risk of allergies, demonstrating significant application potential in the prevention and treatment of allergic diseases and the maintenance of intestinal health.

[0044] The viable count of Bifidobacterium bifidum NKUFB3-12 in the composition of this invention is ≥1×10⁻⁶. 9 CFU / mL or ≥1×10 9CFU / g. When the composition is a liquid formulation, the concentration of 2'-fucosylated lactose in the composition can be any value within the range of 0.2 to 8 mg / mL, for example, 0.2, 0.5, 1, 3, 5, or 8 mg / mL; or when the composition is a solid formulation, the concentration of 2'-fucosylated lactose in the composition can be any value within the range of 0.2 to 8 mg / g, for example, 0.2, 0.5, 1, 3, 5, or 8 mg / g. In cell experiments, Bifidobacterium bifidum NKUFB3-12 combined with 2'-FL showed an effective inhibitory effect on the imbalance of allergic immune response. In specific embodiments of this application, the composition used in cell experiments, based on the total amount of the composition, may include any one of the following: 1×10 7 CFU / mL Bifidobacterium bifidum NKUFB3-12 and 0.5 mg / mL 2'-fucosylated lactose; 1×10 8 CFU / mL Bifidobacterium bifidum NKUFB3-12 and 0.5 mg / mL 2'-fucosylated lactose; 1×10 7 CFU / mL Bifidobacterium bifidum NKUFB3-12 and 5 mg / mL 2'-fucosylated lactose; 1×10 7 CFU / mL of Bifidobacterium bifidum NKUFB3-12 and 5 mg / mL of 2'-fucosylated lactose were used in cell experiments. Lower concentrations were used primarily because the in vitro cell culture environment is relatively simple and controllable, cells directly benefit from the probiotics, and the experimental period is shorter. Changes in cell activity and inflammatory factors can be observed at low concentrations, while avoiding the potential cytotoxicity of high concentrations. In animal (in vivo) experiments, however, probiotics need to undergo a complex physiological environment. The International Society for the Study of Probiotics and Prebiotics (ISAPP) consensus statement on the scope and rational use of the term "probiotics" recommends that for non-strain-specific probiotic preparations (including Bifidobacterium bifidum) in food and dietary supplements, the minimum dose should be 1 × 10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL (Reference: Hill C, Guarner F, Reid G, et al. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the termprobiotic[J]. Nat Rev Gastro Hepat, 2014, 11(8):506-14.).

[0045] This invention provides the use of the above-described composition in the preparation of products for relieving allergic diseases and / or regulating intestinal flora caused by allergies. When the product of this invention is a pharmaceutical, the application is in the preparation of a pharmaceutical for relieving allergic diseases and / or regulating intestinal flora caused by allergies; when the product is a functional food, the application is in the preparation of a functional food for regulating intestinal flora caused by allergies.

[0046] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, illustrates the application of Bifidobacterium bifidum NKUFB3-12 and its composition provided by the present invention in the preparation of products for alleviating allergic diseases and / or regulating intestinal flora caused by allergies. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1: The extraction method of Bifidobacterium bifidum NKUFB3-12 is as follows:

[0048] (I) Isolation and screening of Bifidobacterium bifidum NKUFB3-12:

[0049] (1) The collected infant fecal samples were serially diluted with sterile physiological saline. The serially diluted bacterial solutions were plated on solid screening medium and anaerobically cultured at 37°C for 24-48 hours in a normal incubator until colonies grew. Single colonies with Bifidobacterium colony morphology were picked and examined under a microscope by Gram staining to observe the morphological characteristics of the bacteria. Gram-positive bacteria were selected as the target strain.

[0050] The solid screening medium was prepared as follows: 50 mL of filtered horse serum, 0.05% L-cysteine ​​hydrochloride and 0.05 mg / mL mupirocin were added per liter to the MRS medium. The L-cysteine ​​hydrochloride was sterilized by filtration through a sterile filter membrane and then autoclaved. After the medium cooled to 40-50°C, mupirocin was added.

[0051] (2) Purification of strains: The strains that still have the morphology of Bifidobacterium colonies after repeated streaking purification on solid screening medium are used as target strains. The colonies and bacterial states under the microscope are recorded. Single colonies are picked and cultured in liquid modified MRS liquid medium (MRSC) to prepare for subsequent molecular biological identification.

[0052] The modified MRS liquid medium (MRSC) is prepared as follows: 0.05% (w / v) of L-cysteine ​​hydrochloride is added to the liquid MRS medium.

[0053] The colony morphology of the purified Bifidobacterium bifidum NKUFB3-12 strain was studied. The results showed that the colonies of this strain were small, smooth, milky white, with neat and raised edges and a soft texture. The strain also exhibited V-shaped, Y-shaped, or club-shaped morphologies after initial division or subculture.

[0054] (3) Identification of the strain: The strain was cultured in an anaerobic environment at 37℃ to mid-log phase. After washing several times with PBS, a sufficient amount of bacterial cells was collected. Genomic DNA was extracted using a bacterial DNA extraction kit, and the quality of the extracted DNA was tested. Genome sequencing and bioinformatics analysis were then performed. By comparing with a local database, the genomes of 19 strains most closely related to Bifidobacterium at the species level were selected for comparison based on the 16S rRNA sequence. According to the analysis results, NKUFB3-12 was identified as Bifidobacterium bifidum. Further whole-genome sequencing analysis of NKUFB3-12 was performed, and the results are as follows: Figure 1 As shown.

[0055] Sequencing results showed that the chromosomal genome of Bifidobacterium bifidum NKUFB3-12 was circular and lacked plasmids. The genome length was 2245486 bp; the GC content was 62.75; it contained 1821 DNA coding sequences (CDS), with a total gene length of 2245486 bp, accounting for 85.87% of the total genome length; the genome contained 53 tRNAs and 6 rRNAs.

[0056] Example 2: Growth characterization of Bifidobacterium bifidum NKUFB3-12 using different oligosaccharides as carbon sources:

[0057] Bifidobacterium strain NKUFB3-12 was activated using MRSC liquid medium and cultured to the third generation stable phase. The cultured Bifidobacterium was then centrifuged at 4000×g for 10 minutes, washed three times with PBS, and finally resuspended in sugar-free liquid medium for later use. Liquid MRS medium without added carbohydrates served as a control (Neg. control). Different carbohydrates were added to the MRS medium at 1% (w / v) to obtain different carbohydrate media. These carbohydrates were 2'-fucosylated lactose (2'-FL), 3-fucosylated lactose (3-FL), fructooligosaccharides (FOS), galactooligosaccharides (GOS), lactose (Lac), and glucose (Glc). The resuspended Bifidobacterium was inoculated into different media at a 1.5% inoculum and anaerobically cultured in Hengette anaerobic glass tubes at 37°C in a standard incubator. The optical density (OD) was manually measured every 4 hours. 600 The growth curves of Bifidobacterium bifidum NKUFB3-12 in different carbohydrate media were obtained, with 60 h of culture as the final growth period, as shown in Table 1 and 2. Figure 2 .

[0058] Table 1. Final growth of NKUFB3-12 with different carbon sources

[0059] intervention Neg.control 3-FL 2'-FL FOS GOS Glc Lac <![CDATA[End OD 600 > 0.232589 0.906907 1.191307 0.237163 1.170946 0.741803 1.147093

[0060] like Figure 2 As shown, *Bifidobacterium bifidum* NKUFB3-12 can grow in various carbon source media and has a relatively rich carbohydrate glycolytic capacity. Specifically, the final OD of NKUFB3-12 in a medium with 2'-FL as the sole carbon source is [not specified]. 600 The value was the highest and greater than that of glucose medium, indicating that NKUFB3-12 can utilize 2'-FL well, providing a solid theoretical basis for the combined intervention of Bifidobacterium bifidum NKUFB3-12 and 2'-FL.

[0061] Example 3: Test on the allergic reaction of Bifidobacterium bifidum NKUFB3-12 in mice:

[0062] (1) Set up a blank group (abbreviated as CON group, denoted as CON), a model group (abbreviated as OVA group, denoted as OVA) constructed using conventional sensitizing protein ovalbumin, and an experimental group (abbreviated as FB3-12 group, denoted as FB3-12) to compare the results. Each group has 6 mice.

[0063] The construction of a mouse food allergy model is divided into two stages: sensitization and challenge.

[0064] Sensitization: Mice in the OVA group and FB3-12 group were sensitized by intraperitoneal injection of OVA on days 0, 7, and 14. The first sensitization used OVA (purchased from Merck Sigma). (Made in America) with complete Freund's adjuvant (purchased from Merck Sigma) OVA (origin: America) was administered at a final concentration of 0.25 μg / mL, with a total volume of 200 mL, designated as day 0. The second sensitization and third booster immunization were performed using OVA combined with incomplete Freund's adjuvant (each OVA final concentration was 0.25 μg / mL, total volume 200 mL). The blank control group (COM group, denoted as CON) mice underwent three sensitizations using 0.9% sterile saline instead. Sensitization was completed 14 days after the first OVA injection. Starting from day 15, FB3-12 group mice were continuously immunized with Bifidobacterium bifidum FB3-12 (1×10⁻⁶) for three weeks. 9 CFU / mouse / day was administered orally, while the CON and OVA groups were treated with 200 μL of 0.9% sterile saline instead.

[0065] Challenge: On day 32 of the sensitization test, all mice in the OVA and FB3-12 groups underwent initial challenge (OVA concentration 0.25 μg / mL, volume 200 μL) to enhance the immune response. Four days later, the mice underwent a final challenge (OVA concentration 2.5 μg / mL, volume 200 μL), ending the experiment. Mice in the CON group were given an equal volume of 0.9% sterile saline. After the experiment, mice were fasted for 12 hours, anesthetized with ether, blood was collected from the posterior orbital venous plexus, and the mice were euthanized by cervical dislocation. Serum, spleen, and colon samples were collected. All animal experimental procedures strictly adhered to the relevant provisions of the Tianjin Municipal Regulations on the Management of Experimental Animals. The body weight of the three groups of mice was recorded weekly throughout the intervention period. After the intervention, the weight gain was calculated using the following formula, and the results are as follows: Figure 3 As shown. After the intervention, the spleen weight of the sacrificed mice was measured, and the spleen index was calculated using the following formula. The results are shown below. Figure 3 As shown.

[0066] Weight gain = Final body weight of mouse - Body weight of mouse before first OVA injection

[0067] Spleen index = spleen weight / final body weight of mouse.

[0068] like Figure 3 and Figure 4 As shown, the body weight of mice in the CON group and FB3-12 group differed significantly from that in the OVA group starting from day 21 and day 28 of the sensitization test, respectively. After the intervention, the body weight gain and spleen weight after mouse sacrifice in the OVA group were significantly higher than those in the other two groups throughout the intervention period. The spleen index of mice in the OVA group was significantly increased, suggesting an enhanced immune response in the sensitized mice.

[0069] Within 30 minutes after the last challenge to the three groups of mice, the allergic symptoms of the mice in different groups were observed and evaluated according to the evaluation criteria in Table 2. The evaluation results are shown in Table 2. Figure 5 OVA-induced allergies caused physiological and behavioral changes in the OVA-treated mice, including body edema, scratching, slow movement, and mild diarrhea, but FB3-12 intervention effectively improved these allergy symptoms (see [link to article]). Figure 5 These results preliminarily demonstrate the significant allergic effect of Bifidobacterium bifidum NKUFB3-12 on allergic diseases.

[0070] Table 2 Scoring criteria for allergic symptoms in mice

[0071] Fraction Allergy symptoms 0 No allergic symptoms 1 bristling hair, repeatedly scratching mouth and nose 2 Reduced activity, swelling around the eyes and mouth, shortness of breath, diarrhea 3 Prolonged inactivity, shortness of breath, and rashes around the mouth or tail. 4 No response to stimulation, muscle spasms, loose stools 5 shock or death

[0072] Example 4: The sample obtained in Example 3 was subjected to the following tests:

[0073] (1) Effects of Bifidobacterium bifidum NKUFB3-12 intervention on allergy-related immune markers in mice:

[0074] Serum from mice in the CON, FB3-12, and OVA groups in Example 3 was collected after sacrifice. Following the manufacturer's instructions, an ELISA kit (purchased from Nanjing Jiancheng Biotechnology Institute, Nanjing, China) was used to determine the levels of allergy-related markers in the serum of the three groups of mice, including OVA-specific IgE (OVA-sIgE), IgG1, IgG2a, mast cell protease 1 (Mcpt-1), and histamine (HIS). The results are shown in [Figure number missing]. Figure 6 And Table 3.

[0075] Table 3 Serum allergy-related biochemical indicators

[0076] Indicator (Average) CON OVA FB3-12 OVA-sIgE (ng / mL) <![CDATA[188.8 b ]]> <![CDATA[274.6 a ]]> <![CDATA[148.2 c ]]> Mcpt-1 (ng / mL) <![CDATA[0.7760 b ]]> <![CDATA[1.379 a ]]> <![CDATA[0.8214 b ]]> HIS (ng / mL) <![CDATA[73.21 b ]]> <![CDATA[92.8 a ]]> <![CDATA[73.45 b ]]> IgG2a (mg / mL) <![CDATA[3.498 a ]]> <![CDATA[3.450 a ]]> <![CDATA[3.874 a ]]> IgG1 (mg / mL) <![CDATA[6.921 b ]]> <![CDATA[11.94 a ]]> <![CDATA[7.444 b ]]> IgG1 / IgG2a <![CDATA[1.923 b ]]> <![CDATA[3.454 a ]]> <![CDATA[1.908 b ]]>

[0077] Note: In the table, the same letter indicates that the difference is not significant, and different letters indicate that the difference is significant.

[0078] like Figure 6 As shown in Table 3, compared to the unstimulated CON group, the significantly elevated levels of OVA-sIgE, Mcpt-1, and HIS in the serum of mice in the OVA group may have been the cause of the itching. Additionally, the serum levels of Th2 cell-related IgG1 and the IgG1 / IgG2a ratio in the OVA group were significantly higher than in the other two groups. However, after three weeks of FB3-12 intervention, these allergy-related indicators in the mouse serum returned to normal levels. These results indicate that FB3-12 intervention improved relevant biochemical indicators in allergic mice and may exert its allergy-relieving effect primarily by influencing the Th2 immune response.

[0079] (2) Bifidobacterium bifidum NKUFB3-12 can alleviate intestinal barrier dysfunction and inflammation in mice:

[0080] Impaired intestinal barrier function makes the submucosal immune system more susceptible to allergen stimulation. To assess the impact of NKUFB3-12 on the intestinal barrier, H&E staining and PAS staining were used to visualize histopathological features. The distal colon of mice was collected and fixed in 4% paraformaldehyde for 24 h, followed by graded ethanol dehydration, paraffin embedding, and sectioning. Hematoxylin-eosin (H&E) and periodic acid-Schiffstain (PAS) staining were then used for histomorphological observation and pathological evaluation. Results are shown in [Figure number missing]. Figure 7 and Figure 8Six colonic slices from each group were randomly numbered and interpreted in a blinded manner. Each slice was individually scored by an independent researcher unaware of the treatment type according to a given scoring criterion. The scores for each mouse were then decoded, and the regrouped values ​​were statistically analyzed. The scoring criteria are shown in Table 4.

[0081] Table 4. Scoring criteria for colonic histopathology

[0082] score Colon histology score 0 No inflammation, no epithelial / crypt damage 1 Mild inflammation, damage to the basal third of the epithelium / crypts 2 Moderate inflammation, lesions of the basal two-thirds epithelium / crypts 3 Accumulated inflammation in the mucosa and submucosa, resulting in crypt loss. 4 Accumulated inflammation throughout the entire layer, destruction of crypt and surface epithelium.

[0083] like Figure 7 As shown, FB3-12 intervention significantly improved the aggregated lymphocytes and crypt structure changes in the colon of allergic mice, and resulted in lower histological scores. Figure 8 As shown, PAS-stained goblet cell mucin was significantly reduced in the colon of mice in the OVA group, but intervention with FB3-12 significantly increased the number of goblet cells in the crypts.

[0084] In addition, the relative mRNA expression levels of inflammatory factors in the mouse colon were determined. mRNA was extracted from the mouse colon using TriQuick reagent and reverse transcribed into cDNA according to the manufacturer's instructions. The cDNA was then used for real-time quantitative PCR (RT-qPCR) to determine the expression levels of related genes. Specific primer sequences are shown in Table 5. The final results were obtained using the 2-ΔΔCt method, with β-actin as an internal control for normalization. The relative mRNA expression levels of other groups were calculated using the CON group as the standard. The results are shown in Table 5. Figure 9 See Table 6.

[0085] Table 5 Primer sequences of related genes

[0086]

[0087] like Figure 9 As shown in Table 6, after three weeks of intervention with FB3-12, the levels of intestinal inflammatory factors IL-6 and IL-1β in allergic mice were significantly improved. In summary, NKUFB3-12 has a significant effect on improving colonic barrier function and inhibiting overall inflammation in allergic mice.

[0088] Table 6. Relative expression levels of mouse intestinal inflammatory factors mRNA

[0089] Indicator (Average) CON OVA FB3-12 IL-1β <![CDATA[1 b ]]> <![CDATA[2.780 a ]]> <![CDATA[1.403 b ]]> IL-6 <![CDATA[1 c ]]> <![CDATA[4.541 a ]]> <![CDATA[2.808 b ]]> TNF-α <![CDATA[1 a ]]> <![CDATA[0.9891 a ]]> <![CDATA[0.8053 a ]]>

[0090] Note: In the table, the same letter indicates that the difference is not significant, and different letters indicate that the difference is significant.

[0091] (3) Bifidobacterium bifidum NKUFB3-12 regulates the gut microbiota structure in allergic mice:

[0092] After extracting microbial DNA from mouse fecal samples, the bacterial V3-V4 region sequence was amplified using the universal 16S rRNA gene primers 338F (5'-ACTCCTACGGGAGGCAGCA-3', as shown in SEQ ID NO.7) and 806R (5'-GGACTACHVGGGTWTCTAAT-3', as shown in SEQ ID NO.8). Species clustering, diversity analysis, and principal coordinates analysis (PCoA) were performed on the NovoMagic cloud platform (Beijing Novogene Technology Co., Ltd.). The results are shown in [Figure number missing]. Figures 10-13 .

[0093] from Figure 10 As can be seen, NKUFB3-12 intervention reversed the decline in the Simpson index, which represents α diversity, caused by OVA sensitization, and to some extent restored the structural shift in the gut microbiota of allergic mice.

[0094] To better clarify the composition of the microbial community, a relative abundance bar chart was used to show the top 10 annotated species at the phylum level (see [link to relevant data]). Figure 11 The two major phyla, Firmicutes and Bacteroides, showed significant differences in the intestines of the three groups of mice. FB3-12 intervention significantly increased the abundance of Firmicutes and the F / B ratio (see...). Figure 12 In addition, at the genus level, Akkermansia was significantly enriched in the OVA group, while Lactobacillus and Coridextribacter were enriched in the intestines of FB3-12-treated mice. We also found that NKUFB3-12 promoted the abundance of Oscillospiraceaea and Ruminococcaceae and inhibited the abundance of Enterorhabdus (see [link to article]). Figure 13 ).

[0095] Overall, the data indicate that intervention with Bifidobacterium bifidum NKUFB3-12 remodeled the gut microbiota ecology of allergic mice.

[0096] (4) Effects of Bifidobacterium bifidum NKUFB3-12 on short-chain fatty acid metabolism in the intestines of allergic mice:

[0097] On the day the intervention ended, fresh mouse feces were collected using an ice box. The supernatant obtained after centrifugation with 10% sulfuric acid was extracted with ether and the concentrations of short-chain fatty acids (SCFAs) were determined by high-performance gas chromatography. The results are shown below. Figure 14 See Table 7.

[0098] Table 7. Short-chain fatty acid content in mouse intestines

[0099] Short-chain fatty acids (average value) CON OVA FB3-12 Acetic acid (nmol / mg) <![CDATA[7.660 ab ]]> <![CDATA[6.855 b ]]> <![CDATA[9.800 a ]]> Propionic acid (nmol / mg) <![CDATA[1.470 b ]]> <![CDATA[1.615 b ]]> <![CDATA[2.756 a ]]> Butyric acid (nmol / mg) <![CDATA[1.484 b ]]> <![CDATA[1.542 b ]]> <![CDATA[3.589 a ]]> Valeric acid (nmol / mg) <![CDATA[0.5251 ab ]]> <![CDATA[0.4409 b ]]> <![CDATA[0.7647 a ]]>

[0100] Note: In the table, the same letter indicates that the difference is not significant, and different letters indicate that the difference is significant.

[0101] like Figure 14 As shown in Table 7, compared with the other two groups, intervention with NKUFB3-12 significantly increased the content of propionic acid and butyric acid in the mouse intestine.

[0102] In addition, mRNA was extracted from mouse colon using TriQuick reagent and reverse transcribed into cDNA according to the manufacturer's instructions for real-time quantitative PCR (RT-qPCR) to determine the expression level of G protein-coupled receptors (GPRs). Specific primer sequences are shown in Table 8. OVA stimulation resulted in a decrease in the relative expression levels of SCFA-related receptors GPR41 and GPR43 mRNA in the colon of allergic mice, but this was significantly alleviated by three consecutive weeks of FB3-12 intervention, while the GPR109A content did not change significantly among different treatments (see Table 8). Figure 15 Therefore, FB3-12 not only reprogrammed the ecological niche of the gut microbiota in allergic mice, but also altered the metabolic function of the microbiota, promoting the production of SCFAs.

[0103] Table 8 Primer sequences of related genes

[0104]

[0105] Example 5: Bifidobacterium bifidum NKUFB3-12 combined with 2'-FL modulates allergy-related immune responses:

[0106] Given that Bifidobacterium bifidum NKUFB3-12 has been shown to have significant benefits in relieving allergy symptoms, and that NKUFB3-12 can fully hydrolyze and utilize 2'-FL as a fermentation substrate, the role of Bifidobacterium bifidum NKUFB3-12 in combination with 2'-FL in regulating allergic diseases was further explored.

[0107] (1) Pretreatment of NKUFB3-12 adherent intestinal cells with different carbon sources

[0108] Intestinal cell pretreatment: HT-29MTX cells were removed from liquid nitrogen, revived, passaged, and plated to construct an HT-29MTX intestinal barrier model. After 21 days of differentiation, the cells were washed three times with PBS, and then incubated in a cell culture incubator for 30 min with sterile PBS containing 10 μM CMFDA fluorescent dye to label the cells. After labeling, the cells were washed three times with sterile PBS to remove excess fluorescent dye and were ready for use.

[0109] Pretreatment of NKUFB3-12: NKUFB3-12 cells were activated two days in advance and passaged to a stable third generation at a 2% inoculum rate. Cells were washed twice with sterile saline and resuspended. The resuspended Bifidobacteria were then inoculated at a 2% inoculum rate into liquid medium containing 1% different carbohydrates (2'-fucosylated lactose 2'-FL, galactooligosaccharides GOS, and glucose Glu) and cultured for 24 h. After washing three times with sterile PBS, the cells were resuspended in sterile PBS containing 10 μM CellTrackerRed CMTPX fluorescent dye and then incubated statically at 37°C for 30 min to label the cells. After labeling, the cells were washed three times with sterile PBS to remove excess fluorescent dye and resuspended in antibiotic-free DMEM medium, adjusting the bacterial concentration to 10. 8 Approximately CFU / mL, for later use.

[0110] Bacterial adhesion: 10 8 One mL of NKUFB3-12 bacterial suspension (CFU / mL) was inoculated into an HT-29MTX cell intestinal model and co-cultured in a cell culture incubator for 2 hours. The bacterial suspension was then aspirated, and the cells were washed three times with sterile PBS to remove bacteria that failed to adhere to the intestinal barrier cells (HT-29MTX cells). Subsequently, the adhesion ability of NKUFB3-12 cells after different interventions was observed using a Nikon Eclipse Ti2 inverted microscope. The results are shown in the figure below. Figure 16 .

[0111] like Figure 16 As shown, compared with the Glu and GOS intervention groups, NKUFB3-12 cells cultured in 2'-fucosylated lactose medium showed significantly enhanced adhesion to intestinal cells. The results indicate that 2'-FL intervention can improve the adhesion of NKUFB3-12 cells to HT-29MTX cells and exert a long-term effect in the intestine.

[0112] (2) NKUFB3-12 combined with 2'-fucosylated lactose (2'-FL) regulates T cell immune response.

[0113] Two days in advance, FB3-12 cells were activated and passaged to a stable third generation at a 2% inoculation rate. The cells were then washed twice with sterile saline and the bacterial concentration was adjusted to 10-1. 9 CFU / mL was diluted for subsequent experiments. Additionally, a 500 ng / mL stock solution of 2'-FL (100×) was prepared and diluted for subsequent experiments.

[0114] After euthanizing Balb / c mice, the spleen was quickly removed and placed in a 70 μm cell filter. A single-cell suspension of the mouse spleen was prepared using a grinding method and erythrocyte lysis buffer, and the cell concentration was adjusted to 102.6 Cells were seeded at a concentration of 1 mL / well in 24-well cell culture plates and incubated at 37°C with 5% CO2 for 2 hours to allow for slight cell adhesion. A 100 μg / mL OVA solution was prepared using RPMI 1640 medium. Except for the blank control group, the OVA group (positive control) and the four intervention groups each received 1 mL of OVA solution to achieve a final concentration of 50 μg / mL in the cell culture medium, and continued culturing to stimulate cells. After 24 hours, the four intervention groups were supplemented with NKUFB3-12 and 2'-FL at the concentrations corresponding to groups A through D, and incubated at 37°C with 5% CO2 for 8 hours to treat mouse spleen single cells stimulated by OVA. The four intervention groups used Bifidobacterium bifidum NKUFB3-12 at a concentration of 1 × 10⁻⁶ in the composition. 7 CFU / mL and 1×10 8 Four combinations of CFU / mL and 2'-FL were prepared at concentrations of 0.5 mg / mL or 5 mg / mL, including Group A: 0.5 mg / mL 2'-FL and 1×10⁻⁶ CFU / mL. 7 Group B: CFU / mL FB3-12; Group B: 0.5 mg / mL 2'-FL and 1×10 8 CFU / mL FB3-12; Group C: 5 mg / mL 2'-FL and 1×10 7 Group D: CFU / mL FB3-12; Group D: 5 mg / mL 2'-FL and 1×10 8 CFU / mLFB3-12.

[0115] After culturing at 37℃ and 5% CO2 for 8 hours, cells and their culture supernatant were collected for detecting the relative mRNA expression levels of Th1 / 2-related cytokines IFN-γ and IL-4, to explore their alleviating effect on allergy-related immune response dysregulation. Cultured cells were collected in 1.5 mL centrifuge tubes, and mRNA was extracted from the cells using TriQuick reagent. Following the manufacturer's instructions, this mRNA was reverse transcribed into cDNA using a template for real-time quantitative PCR (RT-qPCR) to determine the expression levels of related genes. Specific primer sequences are shown in Table 9. The final results were obtained using the 2-ΔΔCt method, with β-actin as an internal control for normalization. The relative mRNA expression levels of other groups were calculated using the CON group as the standard. The results are shown in Table 9. Figure 17 and Figure 18 .

[0116] Table 9 Primer sequences of related genes

[0117]

[0118] like Figure 17As shown, all four intervention combinations significantly activated IFN-γ expression in Th1 cells, with group C exhibiting the highest average expression level. Figure 18 As shown, spleen immune cells stimulated by OVA highly expressed the Th2-related cytokine IL-4, while the expression level of this indicator was significantly reduced after intervention in groups A and C. In summary, in cell experiments, Bifidobacterium bifidum NKUFB3-12 combined with 2'-FL effectively inhibited the imbalance of the allergic immune response induced by OVA. Group C, consisting of 5 mg / mL 2'-FL and 10 mg / mL IL-4, showed the most significant effect. 7 The intervention condition with CFU / mLFB3-12 showed the best effect.

[0119] Therefore, the Bifidobacterium bifidum NKUFB3-12 exhibits excellent functional properties in alleviating allergic diseases and regulating allergic intestinal flora. It also possesses excellent acid and bile salt resistance, antioxidant properties, and is a safe strain with no adverse side effects after administration. The combination of NKUFB3-12 and 2'-FL demonstrates an effective inhibitory effect on allergic immune response imbalances.

[0120] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of Bifidobacterium bifidum NKUFB3-12 in the preparation of products for relieving allergic diseases and / or regulating intestinal flora caused by allergies, characterized in that, The preservation number of the Bifidobacterium bifidum NKUFB3-12 is GDMCC No. 64013.

2. The application according to claim 1, characterized in that, The viable count of the Bifidobacterium bifidum NKUFB3-12 is ≥1×10⁻⁶. 9 CFU / mL or ≥1×10 9 CFU / g.

3. The application according to claim 1, characterized in that, The symptoms of the allergy include one or more of the following: swelling, scratching, slow movement, and mild diarrhea.

4. The application according to claim 1, characterized in that, The product includes at least one of the following effects: significantly reduced spleen index and / or serum allergy-related biochemical indicators; significantly alleviated intestinal barrier dysfunction and inflammatory damage; effectively regulated intestinal flora imbalance caused by allergies; and significantly increased the content of short-chain fatty acids in the intestine.

5. The application according to claim 1 or 4, characterized in that, The products include: pharmaceuticals and / or functional foods; When the product is a pharmaceutical product, the application is in the preparation of pharmaceutical products for relieving allergic diseases and / or regulating intestinal flora caused by allergies; When the product is a functional food, the application is in the preparation of functional foods that regulate intestinal flora caused by allergies.

6. A composition, characterized in that, The composition comprises: Bifidobacterium bifidum NKUFB3-12 and 2'-fucosylated lactose, wherein the preservation number of Bifidobacterium bifidum NKUFB3-12 is GDMCCNo.64013.

7. The composition according to claim 6, characterized in that, The viable count of the Bifidobacterium bifidum NKUFB3-12 in the composition is ≥1×10⁻⁶. 9 CFU / mL or ≥1×10 9 CFU / g.

8. The composition according to claim 6, characterized in that, The concentration of 2'-fucosylated lactose in the composition is 0.2–8 mg / mL or 0.2–8 mg / g.

9. The use of the composition according to any one of claims 6 to 8 in the preparation of products for relieving allergic diseases and / or regulating intestinal flora caused by allergies.

10. The application according to claim 9, characterized in that, The products include: pharmaceuticals and / or functional foods; When the product is a pharmaceutical product, the application is in the preparation of pharmaceutical products for relieving allergic diseases and / or regulating intestinal flora caused by allergies; When the product is a functional food, the application is in the preparation of functional foods that regulate intestinal flora caused by allergies.

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