Use of bifidobacterium longum subsp. infantis in modulating antibiotic-induced immune and immunoglobulin dysregulation

Abstract

The application discloses application of Bifidobacterium longum subsp. infantis in regulating antibiotic-induced immune and immunoglobulin disorders, and belongs to the technical field of microorganisms. The application finds that Bifidobacterium longum subsp. infantis CCFM1269 can effectively relieve immune disorders caused by antibiotic exposure in early life or during pregnancy, and has great application prospect. The relieving effect of the strain includes: for a newborn individual, regulating intestinal flora; improving colon IFN-gamma, IgG2a, IgM, IgA and fecal IgA levels; reducing colon IL-4 and IgE levels; and improving spleen CD4+ T cell levels. For a pregnant individual, the SIgA level of colostrum and transitional milk of a pregnant rat exposed to antibiotics is improved, the IgG level of mature milk is improved, the expression levels of pIgR, J chain and chemokine CCL28 mRNA in mammary gland tissue are up-regulated, the IgA + Recruitment and distribution of plasma cells.

Description

Technical Field

[0001] This invention relates to the application of Bifidobacterium longum subsp. infantis in regulating antibiotic-induced immune and immunoglobulin imbalances, and belongs to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Early life is a critical window for the colonization of the gut microbiota and the development and maturation of the immune system. The gut microbiota and the host immune system have a close bidirectional symbiotic relationship, jointly maintaining gut barrier homeostasis and immune balance. A healthy gut microbiota can stimulate the development of immune organs, promote the formation of immune tolerance, and maintain the dynamic balance of Th1 / Th2 immune responses. However, dysbiosis during this stage will significantly increase the risk of allergic diseases and autoimmune diseases in childhood and adulthood.

[0003] Breast milk is a core link in maintaining the gut microbiota and immune homeostasis in early life, and its role is reflected in two key dimensions: First, it provides sufficient maternal SIgA to infants and young children with extremely weak endogenous secretory immunoglobulin A (SIgA) synthesis capacity, which blocks the invasion of pathogenic bacteria through immune exclusion mechanisms, regulates the intestinal flora, induces immune tolerance, and significantly reduces the incidence of infection and necrotizing enterocolitis (NEC); Second, the human milk oligosaccharides (HMOs) it contains specifically nourish the core dominant pioneer bacterium of infants' gut, Bifidobacterium longum subsp. infantum. The short-chain fatty acids (SCFAs) produced by the metabolism of this bacterium can strengthen immune regulation and intestinal barrier function, promote the development of infants' own mucosal immunity and endogenous SIgA synthesis.

[0004] The widespread use of perinatal antibiotics has become the most significant risk factor for disrupting the aforementioned protective system, with two independent but overlapping exposure pathways: First, direct antibiotic administration to newborns: Early exposure to antibiotics, while effectively controlling pathogen infection, also indiscriminately disrupts the fragile gut microbiota of infants, leading to reduced gut microbiota diversity and a decrease in the abundance of beneficial bacteria. This early gut microbiota imbalance caused by antibiotics severely interferes with the normal developmental trajectory of the host's immune system, directly inhibiting the infant's own endogenous SIgA synthesis and causing immune dysregulation. Second, antibiotic use by pregnant women during the perinatal period: Approximately 20%-25% of pregnant women use antibiotics during pregnancy. Prenatal antibiotic exposure in late pregnancy significantly reduces the level and activity of SIgA in breast milk through gut-mammary axis disruption, resulting in infants facing insufficient maternal passive immunity after birth. If both of the above conditions occur simultaneously, a dual immunodeficiency of "insufficient maternal SIgA supply + impaired endogenous SIgA synthesis" will be formed, which will significantly increase the incidence of NEC, recurrent respiratory and gastrointestinal infections in premature infants, and significantly increase the long-term risk of developing allergic diseases, autoimmune diseases and inflammatory bowel disease in childhood and adulthood.

[0005] Current interventions for this clinical problem have significant limitations and cannot simultaneously address the two core issues mentioned above: commercially available formula milk powder cannot simulate the structure and function of SIgA in breast milk; the strains used in conventional microecological interventions are mostly exogenous species, lacking early intestinal niche adaptation, and have limited effectiveness in addressing specific immune dysregulation and endogenous SIgA synthesis disorders caused by neonatal antibiotics. Crucially, the regulation of breast milk SIgA by probiotics exhibits high strain specificity: *Lactobacillus reuteri* ATCC55730 can regulate maternal immunity but has no effect on breast milk SIgA; *Lactobacillus rhamnosus* HN001 and *Bifidobacterium animalis* subsp. *lactotrichum* HN019 can only slightly increase the SIgA detection rate, but cannot significantly increase its absolute concentration, confirming that improving maternal immunity and regulating breast milk SIgA are independent technical effects. Furthermore, prebiotics and nutritional factors lack clear intervention mechanisms and stable clinical effects, and the translation of traditional Chinese medicine compound formulas is difficult.

[0006] Therefore, existing technologies cannot simultaneously address the two core issues of decreased SIgA levels in breast milk due to antibiotic exposure during the perinatal period and insufficient endogenous SIgA synthesis in newborns due to direct antibiotic exposure. There is an urgent need to develop a novel *Bifidobacterium longum* infant subspecies strain that possesses both highly efficient intestinal colonization capabilities in early life and can act on both mother and child generations. This strain could both enhance breast milk SIgA levels by regulating the maternal gut-mammary axis and repair damaged intestinal flora in newborns, promoting their own endogenous SIgA synthesis, thereby providing a safe and efficient microecological solution for the prevention and treatment of related immune disorders. Summary of the Invention

[0007] [Technical Issues] The technical problem to be solved by this invention is to provide a strain of *Bifidobacterium longum* infantile subsp. that has the effect of regulating immune dysregulation caused by early life antibiotic exposure and alleviating the dysregulation of SIgA and major immunoglobulins in breast milk caused by late pregnancy antibiotic exposure. Bifidobacterium longum subsp. infantis ).

[0008] [Technical Solution] To solve the technical problem of this invention, this invention provides a strain of Bifidobacterium longum infantis subsp. ( Bifidobacterium longum subsp. infantis The use of CCFM1269 in the preparation of medicines for the prevention and / or treatment of immune dysregulation caused by early life antibiotic exposure and dysregulation of SIgA and major immunoglobulins in breast milk caused by late pregnancy antibiotic exposure.

[0009] In one embodiment of the present invention, the *Bifidobacterium longum* subsp. infantis CCFM1269 was isolated from a breast milk sample from Wuxi City, Jiangsu Province. After isolation and screening, the strain was identified as *Bifidobacterium longum* subsp. infantis using steps including bacterial genomic DNA extraction, 16S rDNA-specific primer PCR amplification, amplification product purification, DNA sequencing, and sequence alignment. It was named *Bifidobacterium longum* subsp. infantis CCFM1269, with accession number GDMCC No:62839, and has been published in patent CN115820488B.

[0010] In one embodiment of the present invention, the colonies of Bifidobacterium longum subsp. infantis CCFM1269 on MRS medium are convex to cushion-shaped, with intact edges, soft, moist, white and glossy.

[0011] In one embodiment of the present invention, the viable count of the above-mentioned Bifidobacterium longum subsp. infantis CCFM1269 in the medicine is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0012] In one embodiment of the invention, the early-life antibiotic exposure-induced immune dysregulation is caused by the broad-spectrum antibiotic ampicillin. The dysregulation of immunoglobulins in breast milk caused by antibiotic exposure during pregnancy is caused by a combination of the antibiotics ampicillin, neomycin, and erythromycin.

[0013] In one embodiment of the present invention, the drug contains the Bifidobacterium longum subsp. infantis CCFM1269, a drug carrier, and / or pharmaceutical excipients.

[0014] In one embodiment of the present invention, the pharmaceutical product further comprises conventional pharmaceutical carriers and / or pharmaceutical excipients.

[0015] In one embodiment of the present invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0016] In one embodiment of the present invention, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

[0017] In one embodiment of the present invention, the dosage form of the medicine includes powder, granules, capsules, tablets, pills, or oral liquid.

[0018] This invention provides *Bifidobacterium longum* subsp. *infantitidis* (B. *longum* subsp. *infantitidis*). Bifidobacterium longum subsp. infantisThe application of CCFM1269 in the preparation of health products that help regulate intestinal flora, wherein the preservation number of Bifidobacterium longum subsp. infantis CCFM1269 is GDMCC No:62839.

[0019] In one embodiment of the present invention, the regulation of intestinal flora includes targeting and increasing the abundance of Bifidobacterium and Alternaria.

[0020] [Beneficial Effects] 1. This invention screened out a strain of Bifidobacterium longum infantis subspecies (… Bifidobacterium longum subsp. infantis CCFM1269, the infant subspecies of Bifidobacterium longum, has a mitigating effect on immune dysregulation caused by antibiotic exposure in early life or during pregnancy, specifically in the following ways: (1) Increase the level of IFN-γ in the colon of antibiotic-exposed mice; (2) Reduced IL-4 levels in the colon of antibiotic-exposed mice; (3) Increase the level of IgG2a in the colon of antibiotic-exposed mice; (4) Reduce colonic IgE levels in antibiotic-exposed mice; (5) Increase the colonic IgM level in antibiotic-exposed mice; (6) Increase colonic IgA levels in antibiotic-exposed mice; (7) Increase fecal IgA levels in antibiotic-exposed mice; (8) Increase the level of CD4+ T cells in the spleen of antibiotic-exposed mice; (9) Regulate the gut microbiota of antibiotic-exposed mice and increase the abundance of Bifidobacterium and Alternaria. (10) Increase SIgA levels in breast milk of individuals exposed to antibiotics during pregnancy; (11) Increase the number of IgA+ plasma cells in the breast of individuals exposed to antibiotics during pregnancy; (12) Increase the level of pIgRNA in the breast of individuals exposed to antibiotics during pregnancy; (13) Increase the level of CCL28 RNA in the breast of individuals exposed to antibiotics during pregnancy; (14) Increase the level of J chain RNA in the breast of individuals exposed to antibiotics during pregnancy.

[0021] Therefore, Bifidobacterium longum infantis subsp. CCFM1269 has great potential for application in the preparation of products (such as food or pharmaceuticals) for the prevention and / or treatment of immune dysregulation caused by early life antibiotic exposure and dysregulation of SIgA and major immunoglobulins in breast milk caused by late pregnancy antibiotic exposure.

[0022] 2. Bifidobacterium longum subsp. infantis is a type of probiotic and has been included in the "List of Bacterial Strains that Can Be Used in Food" issued by the Ministry of Health. Therefore, the Bifidobacterium longum subsp. infantis CCFM1269 screened by this invention will not pose any potential safety risks to patients with immune dysregulation caused by early life antibiotic exposure or dysregulation of SIgA and major immunoglobulins in breast milk caused by late pregnancy antibiotic exposure.

[0023] 3. The cultivation process of Bifidobacterium longum infantis subsp. only requires culture medium and some control of culture conditions, and the cost is relatively low. It will not impose too much economic burden on patients with immune disorders caused by early life antibiotic exposure or disorders of SIgA and major immunoglobulins in breast milk caused by late pregnancy antibiotic exposure. Attached Figure Description

[0024] Figure 1 Comparison of colonic IFN-γ levels in mice exposed to different antibiotics.

[0025] Figure 2 Comparison of IL-4 levels in the colon of mice exposed to different antibiotics.

[0026] Figure 3 Comparison of colonic IgG2a levels in mice exposed to antibiotics in different groups.

[0027] Figure 4 Comparison of colonic IgE levels in mice exposed to antibiotics in different groups.

[0028] Figure 5 Comparison of colonic IgM levels in mice exposed to antibiotics in different groups.

[0029] Figure 6 Comparison of colonic IgA levels in mice exposed to antibiotics in different groups.

[0030] Figure 7 Comparison of fecal IgA levels in mice exposed to antibiotics in different groups.

[0031] Figure 8 Comparison of CD4+ T cell levels in the spleen of mice exposed to antibiotics in different groups.

[0032] Figure 9 Comparison of Chao1 index levels in fecal microbiota of mice in different groups exposed to antibiotics.

[0033] Figure 10 β-diversity of fecal microbiota in mice exposed to antibiotics in different groups.

[0034] Figure 11 Comparison of Bifidobacterium levels in the gut microbiota of mice exposed to antibiotics in different groups.

[0035] Figure 12 Comparison of the levels of Alternaria species in the gut microbiota of mice exposed to antibiotics in different groups.

[0036] Figure 13 Comparison of SIgA levels in the milk of rats exposed to antibiotics during pregnancy in different groups.

[0037] Figure 14 Comparison of IgG levels in the milk of pregnant rats exposed to antibiotics during pregnancy in different groups.

[0038] Figure 15 Comparison of IgM levels in the milk of pregnant rats exposed to antibiotics during pregnancy in different groups.

[0039] Figure 16 IgA+ plasma cell staining results in mammary glands of rats exposed to antibiotics during pregnancy in different groups.

[0040] Figure 17 Quantitative results of IgA+ plasma cells in the mammary glands of rats exposed to antibiotics during pregnancy in different groups.

[0041] Figure 18 Comparison of pIgR mRNA levels in mammary glands of rats exposed to antibiotics during pregnancy in different groups.

[0042] Figure 19 Comparison of J-chain mRNA levels in mammary glands of rats exposed to antibiotics during pregnancy in different groups.

[0043] Figure 20 Comparison of CCL28 mRNA levels in mammary glands of rats exposed to antibiotics during pregnancy in different groups. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0045] The C57BL / 6 mice used in the following examples were purchased from Zhejiang Vital River Pharmaceutical Co., Ltd.; the Bifidobacterium longum subsp. infantis used in the following examples ( Bifidobacterium longum subsp. infantis CCFM1269 was isolated by the Biotechnology Center of the School of Food Science and Technology, Jiangnan University, and has been disclosed in patent CN115820488B, with accession number GDMCC No:62839; the ELISA kits used in the following examples were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., and the fecal genomic DNA extraction kits were purchased from MP Biopharmaceuticals, Inc., USA.

[0046] The culture media involved in the following examples are as follows: MRS solid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, agar 15 g / L, cysteine ​​1 g / L.

[0047] MRS liquid culture medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, cysteine ​​1 g / L.

[0048] Example 1: Preparation of a suspension of Bifidobacterium longum subsp. infantis CCFM1269 The preparation methods of Bifidobacterium longum infantis subsp. in Examples 1-9 are as follows: Streaking of *Bifidobacterium longum* subsp. infantis culture onto MRS solid medium and incubating at 37°C for 48 h yielded single colonies. These single colonies were then inoculated into MRS liquid medium and incubated at 37°C for 24 h to obtain an activated culture. This activated culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and incubated at 37°C for 24 h to obtain a primary seed culture. The primary seed culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and incubated at 37°C for 24 h to obtain a secondary seed culture. The secondary seed culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and incubated at 37°C for 24 h to obtain a bacterial culture. The bacterial culture was centrifuged at 6000 g for 15 min, and the precipitate was collected. The precipitate was washed twice with physiological saline buffer and then centrifuged again at 6000 g for 10 min to obtain bacterial cells. The *Bifidobacterium longum* subsp. infantis bacterial cells were resuspended in physiological saline to a cell concentration of 1. 10 9 CFU / mL was used to obtain a culture of Bifidobacterium longum subsp. infantis.

[0049] Example 2: Effects of Bifidobacterium longum infantis subsp. CCFM1269 on colonic cytokine dysregulation induced by early-life antibiotic exposure The specific steps are as follows: Eight SPF-grade, 6-week-old male, pathogen-free C57BL / 6 female mice and four male mice were used as experimental animals. They were housed at a room temperature of 22–24°C and humidity of 40–60%, with a 12-hour / 12-hour day-night cycle and free access to food and water for one week. Afterward, they were housed together at a female-to-male ratio of 2:1. Once the females became pregnant, the males were removed. The gestation period was 3 weeks. Newborn pups were nursed by their mothers until weaning at 3 weeks of age, and then separated and housed until 4 weeks of age. After birth, the pups were randomly divided into a control group, a model group, and an intervention group (n=8 in each group). Mice in the control group received 10 μL of physiological saline by gavage from 5 to 10 days as a control, and 100 μL of physiological saline by gavage from 11 to 28 days as a control. Mice in the model group were administered ampicillin antibiotics by gavage (10 μL, 100 mg / kg, once daily) from day 5 to 10, and saline by gavage from day 11 to 28 as a control. Mice in the intervention group were administered ampicillin antibiotics by gavage (10 μL, 100 mg / kg, once daily) from day 5 to 10, and Bifidobacterium longum subsp. infantis 10 from day 11 to 28. 9 CFU / Tianhuo bacteria solution was administered by gavage. Young mice were euthanized at four weeks of age, and colon tissue was collected. The levels of cytokines IFN-γ and IL-4 were measured using an enzyme-linked immunosorbent assay (ELISA) kit (results are shown below). Figure 1-2 ).

[0050] like Figure 1 As shown, compared with the model group (0.077 ng / mg), *Bifidobacterium longum* subsp. infantis CCFM1269 significantly increased the IFN-γ level in mouse colon tissue (0.128 ng / mg) (p<0.05). Figure 2 As shown, compared with the model group (0.161 ng / mg), *Bifidobacterium longum* subsp. infantis CCFM1269 significantly reduced IL-4 levels in mouse colon tissue (0.135 ng / mg) (p<0.05). Therefore, *Bifidobacterium longum* subsp. infantis CCFM1269 inhibits the dysregulation of Th1 / Th2 cytokine levels in the gut under early-life antibiotic exposure, thereby helping to prevent the risk of allergic diseases caused by abnormal Th2 immune responses.

[0051] Example 3: Effects of Bifidobacterium longum infantis subsp. CCFM1269 on colonic immunoglobulin dysregulation induced by early life antibiotic exposure For specific experimental methods, refer to Example 2. When the young mice reached four weeks of age, blood was collected and the mice were euthanized. Then, colon tissue was collected, and the levels of immunoglobulins IgG2a and IgE were measured using an enzyme-linked immunosorbent assay (ELISA) kit (results are shown below). Figure 3-4 ).

[0052] like Figure 3As shown, compared with the model group (4.181 μg / mg), *Bifidobacterium longum* subsp. infantis CCFM1269 significantly increased the IgG2a level in mouse colon tissue (5.531 μg / mg) (p<0.05). Figure 4 As shown, compared with the model group (7.728 μg / mg), *Bifidobacterium longum* subsp. infantis CCFM1269 significantly reduced IgE levels in mouse colon tissue (6.393 μg / mg) (p<0.05). Therefore, *Bifidobacterium longum* subsp. infantis CCFM1269 inhibits the dysregulation of Th1 / Th2 immunoglobulin levels in the gut under early-life antibiotic exposure, thereby helping to prevent the risk of allergic diseases caused by abnormal Th2-type immune responses.

[0053] Example 4: Effects of Bifidobacterium longum infantis subsp. CCFM1269 on colonic IgM and IgA levels in mice exposed to antibiotics in early life For specific experimental methods, refer to Example 2. When the young mice reached four weeks of age, blood was collected and the mice were euthanized. Then, colon tissue was collected, and the levels of immunoglobulins IgM and IgA were measured using an enzyme-linked immunosorbent assay (ELISA) kit (results are shown in Figure 2). Figure 5-6 ).

[0054] like Figure 5 As shown, compared with the model group (1.947 ng / mg), *Bifidobacterium longum* subsp. infantis CCFM1269 significantly increased the level of IgM, the precursor of IgA after B-cell class switching, in mouse colon tissue (3.830 ng / mg) (p<0.05). Figure 6 As shown, compared with the model group (7.961 ng / mg), *Bifidobacterium longum* subsp. infantis CCFM1269 significantly increased IgA levels in mouse colon tissue (14.696 ng / mg) (p<0.05). Therefore, *Bifidobacterium longum* subsp. infantis CCFM1269 enhances the first line of immune defense of the intestinal epithelium composed of IgA in early life antibiotic exposure, thereby helping to protect the intestinal epithelium from intestinal toxins and pathogens.

[0055] Example 5: Effect of Bifidobacterium longum infantis subsp. CCFM1269 on fecal IgA levels in mice exposed to antibiotics in early life For specific experimental methods, refer to Example 2. When the young mice were four weeks old, blood was collected and the mice were euthanized. Then, mouse feces were collected, and the level of immunoglobulin IgA was measured using an enzyme-linked immunosorbent assay (ELISA) kit (results are shown in Figure 2). Figure 7 ).

[0056] like Figure 7As shown, compared with the model group (4.048 μg / g feces), *Bifidobacterium longum* subsp. infantis CCFM1269 significantly increased the level of IgA in the feces of early-life antibiotic-exposed mice (7.531 μg / g feces) (p<0.05). This indicates that *Bifidobacterium longum* subsp. infantis CCFM1269 enhances the ability of the intestines of early-life antibiotic-exposed mice to secrete IgA into the intestinal lumen, thereby facilitating the binding of intestinal toxins and pathogens and their clearance from the intestinal lumen.

[0057] Example 6: Effect of Bifidobacterium longum infantis subsp. CCFM1269 on CD4+ T cell levels in spleen of mice exposed to antibiotics in early life For specific experimental methods, refer to Example 2. Blood was collected from four-week-old mice, and the mice were euthanized. Single-cell suspensions were prepared from the spleen of the mice, and CD4+ T cells in the spleen were measured by flow cytometry. The results are shown in […]. Figure 8 .

[0058] CD4+ T cells are key immune cells that mediate the production of IgA in the gut via a T cell-dependent pathway. For example... Figure 8 As shown, the proportion of CD4+ T cells in the model group (10.35%) was significantly lower than that in the control group (14.17%) (p<0.05). After intervention with CCFM1269, the proportion of CD4+ T cells significantly increased (14.08%) (p<0.05), indicating that it may restore the level of IgA produced by the T cell-dependent pathway by restoring the decrease in CD4+ T cell level caused by antibiotic exposure in early life.

[0059] Example 7: Effects of Bifidobacterium longum infantis subsp. CCFM1269 on gut microbiota α-diversity in mice exposed to antibiotics in early life The specific experimental method is as described in Example 2. When the young mice reached four weeks of age, blood was collected and the mice were euthanized. Fecal samples were then collected, and genomic DNA was extracted from the feces. Specific PCR amplification of the V3-V4 region of the extracted genomic DNA was performed, followed by 16S rDNA sequencing. The Chao1 index in the α-diversity of the fecal microbiota was analyzed (results are shown in Example 2). Figure 9 ).

[0060] like Figure 9 As shown, compared with the model group (23.625), *Bifidobacterium longum* subsp. infantis CCFM1269 significantly reduced the Chao1 index decrease induced by early-life antibiotic exposure (40.375) (p<0.05). This indicates that *Bifidobacterium longum* subsp. infantis CCFM1269 enhances the diversity of gut microbiota in mice exposed to antibiotics in early life, which contributes to the healthy homeostasis of gut microbiota in early life and ensures the healthy development of microbiota and intestinal immunity in early life.

[0061] Example 8: Effects of Bifidobacterium longum infantis subsp. CCFM1269 on gut microbiota β-diversity in mice exposed to antibiotics in early life The specific experimental method is as described in Example 2. When the young mice reached four weeks of age, blood was collected and the mice were euthanized. Fecal samples were then collected, and genomic DNA was extracted from the feces. Specific PCR amplification of the V3-V4 region of the extracted genomic DNA was performed, followed by 16S rDNA sequencing. The β-diversity of the fecal microbiota was analyzed (results are shown in Figure 2). Figure 10 ).

[0062] like Figure 10 As shown, early-life antibiotic exposure led to significant segregation of the gut microbiota in mice. After intervention with *Bifidobacterium longum* subsp. infantis CCFM1269, the composition of the gut microbiota showed a significant reversion towards the control group. This indicates that *Bifidobacterium longum* subsp. infantis CCFM1269 enhanced the diversity of the gut microbiota in mice exposed to antibiotics early in life, which contributes to healthy gut microbiota homeostasis in early life and ensures the healthy development of the microbiota and intestinal immunity in early life.

[0063] Example 9: Effects of Bifidobacterium longum infant subspecies CCFM1269 on Bifidobacterium and Alternaria species in the gut of mice exposed to antibiotics in early life The specific experimental method is as described in Example 2. When the young mice reached four weeks of age, blood was collected and the mice were euthanized. Fecal samples were then collected, and genomic DNA was extracted from the feces. Specific PCR amplification of the V3-V4 region of the extracted genomic DNA was performed, followed by 16S rDNA sequencing. The levels of Bifidobacterium and Alternaria genera in the fecal microbiota were analyzed (results are shown in Example 2). Figure 11-12 ).

[0064] like Figure 11 and 12 As shown, intervention with *Bifidobacterium longum* subsp. infantis CCFM1269 significantly increased the levels of *Bifidobacterium* (0.038%) and *Alternaria* (2.095%) species related to tryptophan metabolism in the gut microbiota (p<0.05). This indicates that *Bifidobacterium longum* subsp. infantis CCFM1269 enhances the levels of bacteria related to tryptophan metabolism in the gut microbiota, which helps promote the enrichment of tryptophan metabolites in the gut during early life and modulates early life gut immune development by activating the aryl hydrocarbon receptor pathway.

[0065] Example 10: Effects of Bifidobacterium longum infantis subsp. CCFM1269 on SIgA and major immunoglobulins in breast milk under antibiotic exposure during pregnancy The specific steps are as follows: Thirty-six SPF-grade, 6-week-old male pathogen-free SD rats and 18 male rats were used as experimental animals. They were housed at a room temperature of 22–24°C and humidity of 40–60%, with a 12-hour / 12-hour day-night cycle and free access to food and water for one week. Afterward, the females were grouped with males at a 2:1 ratio. Once the females became pregnant, the males were removed. Gestation was induced by free access to water between 13.5 and 18.5 days. The pregnant rats were randomly divided into three groups: a control group (CN), an antibiotic group (ABXS), and an intervention group (ABXS+CCFM1269). The control group received sterile water, while the model and intervention groups received an antibiotic solution. The antibiotic solution consisted of ampicillin (2 g / L), neomycin (2 g / L), and erythromycin (0.2 g / L), dissolved in 500 mL of sterile drinking water. Simultaneously, from day 13.5 of gestation until the mothers were sacrificed, the control group mothers received 1 mL of physiological saline via gavage, while the model group mothers received 1 mL of physiological saline via gavage without intervention. The intervention group mothers were given 10 mL of Bifidobacterium infantis. 10 CFU / day live bacteria solution was administered by gavage (1 mL). Eighteen animals were sacrificed on day 10 of lactation, and milk was collected from these 18 animals on days 4 and 7 of gestation (400 uL each); another 18 animals were sacrificed on day 21 of lactation, and milk was collected from these 18 animals on days 14 and 21 of gestation (400 uL each).

[0066] like Figure 13The results of SIgA content detection in breast milk are shown. On day 4 of lactation, compared with the normal group (330.118 ng / mL) and the model group (197.546 ng / mL), the SIgA content decreased by 40.16% (P<0.001). The SIgA content in the Bifidobacterium infantis subsp. CCFM1269 intervention group (253.299 ng / mL) was significantly increased by 28.22% compared with the model group (P<0.01), reaching 76.73% of the normal group (P<0.001). On day 7 of lactation, compared with the normal group (317.905 ng / mL) and the model group (205.432 ng / mL), the SIgA content decreased by 35.38% (P<0.001). The SIgA content in the intervention group (261.707 ng / mL) was significantly increased. The intervention group (335.55 ng / mL) showed a significant increase of 27.39% compared to the model group (P < 0.01), reaching 82.32% of the normal group (P < 0.01). On day 14 of lactation, compared to the normal group (335.55 ng / mL), the model group (314.547 ng / mL) decreased by 6.26%, while the intervention group (373.458 ng / mL) showed an increase of 18.72% compared to the model group (P < 0.05), with no statistically significant difference compared to the normal group. On day 21 of lactation, the intervention group (328.94 ng / mL) was 99.37% of the model group (331.037 ng / mL) and 93.89% of the normal group (350.343 ng / mL), with no statistically significant difference between the groups.

[0067] like Figure 14 The results of the detection of IgG content in breast milk are shown. On day 4 of lactation, the level of Bifidobacterium longum subsp. infantis CCFM1269 in the intervention group (2.17876 mg / mL) was 84% ​​higher than that in the model group (1.18413 mg / mL), reaching 94.70% of that in the normal group (2.30068 mg / mL). On day 7 of lactation, the level of this strain in the intervention group (3.12849 mg / mL) was 105.81% higher than that in the model group (1.52008 mg / mL), reaching 92.21% of that in the normal group (3.39288 mg / mL). On day 14 of lactation, there was no statistically significant difference between the intervention group (1.98969 mg / mL), the model group (2.11633 mg / mL), and the normal group (2.2594 mg / mL). On day 21 of lactation, the intervention group (3.35996 mg / mL) was 50.10% higher than the model group (2.23855 mg / mL) (P<0.05) and reached 138.28% of the normal group (2.42798 mg / mL).

[0068] like Figure 15The results of IgM content detection in breast milk are shown. On day 4 of lactation, the IgM content in the Bifidobacterium longum subsp. infantis CCFM1269 intervention group (405.177 ug / mL) was 26.15% lower than that in the model group (548.666 ug / mL), reaching 124.72% of that in the normal group (324.87 ug / mL mg / mL). On day 7 of lactation, the IgM content in the intervention group (263.124 ug / mL) was not significantly different from that in the model group (265.665 ug / mL), reaching 61.06% of that in the normal group (430.965 ug / mL). On day 14 of lactation, the IgM content in the intervention group (198.014 ug / mL) was 31.95% lower than that in the model group (290.958 ug / mL), reaching 101.94% of that in the normal group (194.25 ug / mL). On day 21 of lactation, the intervention group (277.982 ug / mL) was 39.53% higher than the model group (199.226 ug / mL), reaching 155.87% of the normal group (178.339 ug / mL).

[0069] Therefore, Bifidobacterium longum infant subspecies CCFM1269 effectively improves the immune dysfunction of breast milk induced by antibiotic exposure during pregnancy, significantly upregulates the levels of key immune factors SIgA and IgG in breast milk, and repairs the damage to the breast milk immune barrier caused by antibiotic exposure, thereby helping to reduce the risk of immune abnormalities and related diseases in offspring caused by low breast milk immunity.

[0070] Example 11: Verification of the effect of Bifidobacterium longum infantis subsp. CCFM1269 on upregulating the expression of CCL28, J chain and pIgR in mammary glands and promoting SIgA synthesis in breast milk. The specific experimental method is as described in Example 10. Eighteen mice were sacrificed on the 10th day of lactation, and another 18 mice were sacrificed on the 21st day of lactation. Their mammary glands were then harvested, and the number of IgA+ plasma cells in the mammary glands of the mother mice was observed by immunohistochemistry. pIgR mRNA, J chain mRNA, and CCL28 mRNA were measured by real-time quantitative polymerase chain reaction.

[0071] like Figure 16 Figure 17As shown, on day 10 of lactation, the number of SIgA-producing plasma cells in the mammary tissue of mother mice in the intervention group (343 mm2) increased by 54.50% compared to the model group (222 mm2), and was 82.65% of that in the normal group (415 mm2). On day 21 of lactation, the number of SIgA-producing plasma cells in the intervention group (448.5 mm2) increased by 44.44% compared to the model group (310.5 mm2), and was 109.66% of that in the normal group (409 mm2). There was no significant difference in the number of SIgA-producing plasma cells in the mammary tissue of the three groups. There was no significant difference in the number of SIgA-producing plasma cells in the mammary tissue of mother mice in the intervention group (448.5 mm2).

[0072] like Figure 18 As shown, on day 10 of lactation, compared with the normal group (1.05257), the model group (0.439328) significantly reduced the polyimmunoglobulin receptor (pIgR) mRNA in mammary tissue of mammary rats by 58.26% (p < 0.05). Compared with the model group, Bifidobacterium longum infantis CCFM1269 (1.13984) significantly increased pIgR mRNA by 159.45% (p < 0.01); the intervention group was 108.29% of the normal group. On day 21 of lactation, compared with the normal group (1.01595), the model group (0.880094) was 86.63% of the normal group; compared with the model group, the intervention group (1.22574) increased pIgR mRNA by 39.27%, which was 120.65% of the normal group. There was no significant difference among the three groups on day 21 of lactation.

[0073] like Figure 19 As shown, on day 10 of lactation, compared with the normal group (1.02062), the model group (0.737892) had a 27.70% reduction in the mRNA of the key small molecule protein (J chain) mediating the formation of immunoglobulin multimers in breast tissue, but the difference was not statistically significant. Compared with the model group, Bifidobacterium longum infantis CCFM1269 (1.1438) significantly increased J chain mRNA in maternal mice on day 10 of lactation by 55.01% (p < 0.05); the intervention group was 112.07% of the normal group. On day 21 of lactation, compared with the normal group (1.01688), the model group (0.62927) significantly reduced J chain mRNA in mammary tissue by 38.12% (p < 0.05); while compared with the model group, Bifidobacterium longum infantis CCFM1269 (1.1737) significantly increased J chain mRNA on day 21 of lactation by 86.52% (p < 0.01); the intervention group was 115.42% of the normal group.

[0074] female mouse Figure 20 As shown, on day 10 of lactation, compared with the normal group (1.02436), the model group (0.477351) significantly reduced the core chemotactic signal CCL28 mRNA in mammary tissue of mammary rats by 53.40% (p < 0.001); compared with the model group, Bifidobacterium longum infantis subsp. CCFM1269 (1.0265) significantly increased the CCL28 mRNA in mammary tissue of mammary rats by 115.04% on day 10 of lactation (p < 0.001); the intervention group was 100.21% of the normal group; on day 21 of lactation, compared with the normal group (1.00702), the model group (1.00836) was 100.13% of it, the intervention group (1.22948) increased the CCL28 mRNA in mammary tissue of mammary rats by 21.93% of the model group, and the intervention group was 121.93% of the model group; the CCL28 levels in the three groups were 100.001% of the normal group (1.00702). There was no significant difference in mRNA levels.

[0075] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Bifidobacterium longum infantis subspecies ( Bifidobacterium longum subsp. infantis The use of CCFM1269 in the preparation of medicines for the prevention and / or treatment of immune dysregulation caused by maternal or neonatal antibiotic exposure during pregnancy, wherein the accession number of Bifidobacterium longum subsp. infantis CCFM1269 is GDMCC No:62839.

2. The application according to claim 1, characterized in that, In the aforementioned drug, the bacterial count of *Bifidobacterium longum* subsp. infantis CCFM1269 is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

3. The application according to claim 2, characterized in that, The antibiotic exposures include ampicillin exposure and combined exposure to ampicillin, neomycin, and erythromycin.

4. The application according to claim 3, characterized in that, The drug contains the Bifidobacterium longum infantis subspecies CCFM1269, a drug carrier, and / or pharmaceutical excipients.

5. The application according to claim 4, characterized in that, The drug also includes conventional pharmaceutical carriers and / or pharmaceutical excipients.

6. The application according to claim 5, characterized in that, The pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and / or liposomes.

7. The application according to claim 6, characterized in that, The pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

8. The application according to claim 7, characterized in that, The dosage forms of the medicine include powder, granules, capsules, tablets, pills, or oral liquid.

9. Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis The application of CCFM1269 in the preparation of health products that help regulate intestinal flora, wherein the preservation number of Bifidobacterium longum subsp. infantis CCFM1269 is GDMCC No:62839.

10. The application according to claim 9, characterized in that, The regulation of gut microbiota includes targeting and increasing the abundance of Bifidobacterium and Alternaria.

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