Bifidobacterium longum subsp. Infantis CCFM1271 capable of adjusting Th1 / Th2 balance and application thereof

Abstract

The invention discloses bifidobacterium longum subsp.infantis CCFM1271 capable of adjusting Th1 / Th2 balance and application of the bifidobacterium longum subsp.infantis CCFM1271, and belongs to the technical field of microorganisms. The bifidobacterium longum subsp. Infantis provided by the invention can adjust Th1 / Th2 balance and IgA content of young rats, and the specific effects are as follows: (1) the percentage contents of B cells and T cells in mesenteric lymph nodes of mammals in early life are improved; the content of IFN-gamma, the content of IgG2a, the content of IgA and the content of sIgA in the colon are increased, and the ratio of IgG2a to IgE is increased; (3) the content of IFN-gamma and the ratio of IgG2a / IgE in serum of mammals in the early stage of life are increased; (4) the content of IL-4 and IgE in colons of mammals in the early stage of life is reduced, and the expression level of T-bet mRNA in the colons is improved; and (5) improving the relative abundance of the IgA binding state Alistipes of the feces of the lactating male animals at the early life stage.

Description

[0001] This application is a divisional application of patent application number "2022114738059" filed on 2022-11-22. Technical Field

[0002] This invention relates to Bifidobacterium longum infantis subspecies CCFM1271, which can regulate the Th1 / Th2 balance, and belongs to the field of microbial technology. Background Art

[0003] With the rising incidence of various non-communicable diseases, there is a strong interest in discovering the causes of these diseases and a focus on early immune establishment to reduce the risk of later disease development. Especially during the highly malleable period of infancy, proper nutritional supplementation can minimize the occurrence of diseases during growth. After birth, infants exhibit a preference for T helper cell type 2 immunity due to factors such as hormones and progesterone, which is a major reason for the increased risk of allergies, asthma, and other diseases in childhood. Furthermore, gut microbiota imbalance is prevalent in modern society and may be a factor contributing to the increased incidence of immune-mediated diseases. Breastfed infants may be more resistant to diarrhea, allergies, asthma, and inflammatory bowel disease. Breast milk not only provides essential nutrition for infants but also promotes the establishment of gut microbiota. Therefore, it is necessary to explore the microbiota that can combat allergies, asthma, and other potential immune diseases and promote the immune system.

[0004] Bifidobacterium longum subsp. infantis is a major Bifidobacterium colonizer in the infant gut and has shown advantages in promoting early life health. Current research has identified the loss of early life Bifidobacteria as associated with an increased risk of several immune diseases and intestinal inflammation, but the mechanisms remain unclear. Early life microbiota exposure may offer an opportunity to prevent the risk of allergies caused by Th1 / Th2 imbalance. In breastfed infants given Bifidobacterium longum subsp. infantis EVC001, intestinal T helper cell 2 (Th2) and Th17 cytokines were silenced. Administration of EVC001 to adult mice has a therapeutic effect on allergic asthma by promoting Th1 and silencing Th2 immune responses. Furthermore, the introduction of Bifidobacterium longum subsp. infantis reduces intestinal inflammation by stably and sustainably remodeling the gut microbiota of breastfed infants.

[0005] In recent years, studies have shown that *Bifidobacterium longum* subsp. *infantii* (EVC001) can promote the differentiation of T0 cells into Th1 cells in infants and regulate the Th1 / Th2 balance. Therefore, there is an urgent need to find a probiotic strain that can regulate the Th1 / Th2 balance and promote IgA production. Summary of the Invention

[0006] This invention provides *Bifidobacterium longum subsp. infantis*, which is *Bifidobacterium longum subsp. infantis* CCFM1269, *Bifidobacterium longum subsp. infantis* CCFM1270, *Bifidobacterium longum subsp. infantis* CCFM1271 or *Bifidobacterium longum subsp. infantis* CCFM1272.

[0007] The aforementioned Bifidobacterium longum subsp. infantis CCFM1269, taxonomically named Bifidobacterium longum subsp. infantis, was deposited on September 26, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 62839, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The aforementioned Bifidobacterium longum subsp. infantis CCFM1270, taxonomically named Bifidobacterium longum subsp. infantis, was deposited on September 26, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 62840, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The aforementioned Bifidobacterium longum subsp. infantis CCFM1271, taxonomically named Bifidobacterium longum subsp. infantis, was deposited on September 26, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 62841, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The aforementioned *Bifidobacterium longum* subsp. *infantis*, taxonomically named *Bifidobacterium longum*, was deposited on September 26, 2022, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 62842), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0011] The present invention also provides a probiotic agent containing one or more of the following: Bifidobacterium longum infantis subspecies CCFM1269, CCFM1270, CCFM1271, and CCFM1272.

[0012] In one embodiment, the probiotic agent contains at least 1 × 10⁻⁶ live bacteria of *Bifidobacterium longum* subsp. *infantitidis*. 9 CFU / mL or 1×10 9 CFU / g.

[0013] In one embodiment, the probiotic agent is a bacterial suspension of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

[0014] The present invention also provides a fermentation agent containing the said Bifidobacterium longum infant subspecies CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

[0015] In one embodiment, the fermentation agent is prepared by inoculating Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 into a culture medium and culturing at 37°C for 24-48 hours to obtain a culture solution; centrifuging the culture solution to obtain bacterial cells; and resuspending the bacterial cells in physiological saline to obtain the fermentation agent.

[0016] In one embodiment, the culture medium is MRS medium.

[0017] The present invention also provides a drug containing at least one strain of Bifidobacterium longum infantis subsp. CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

[0018] In one embodiment, the viable count of *Bifidobacterium longum* subsp. infantis in the drug is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0019] In one embodiment, the drug contains the *Bifidobacterium longum* subsp. infantis and a pharmaceutically acceptable carrier.

[0020] In one embodiment, the drug is used to regulate the Th1 / Th2 balance and / or promote IgA synthesis.

[0021] The present invention also provides the use of the aforementioned Bifidobacterium longum infant subspecies CCFM1269, CCFM1270, CCFM1271 or CCFM1272 in the preparation of health products that help enhance immunity.

[0022] The present invention also provides the application of one or more of the following Bifidobacterium longum infant subspecies CCFM1269, CCFM1270, CCFM1271 or CCFM1272 in probiotic products for regulating Th1 / Th2 balance and IgA synthesis.

[0023] In one embodiment, the product is used to increase the levels of Th1 / Th2-related cytokines and immunoglobulins in the colon of early-life mammals.

[0024] In one embodiment, the product is used to regulate the gut microbiota of early-life mammals.

[0025] In one embodiment, the adjustment of Th1 / Th2 balance and IgA synthesis includes at least one of the following functions:

[0026] (1) Increase the percentage of B cells, T cells and Th cells in the mesenteric lymph nodes of mammals in early life;

[0027] (2) Increase the levels of IFN-γ, IgG2a, IgA, and sIgA in the colon of early-life mammals, and increase the IgG2a / IgE ratio.

[0028] (3) Increase the content of IFN-γ and the ratio of IgG2a / IgE in the serum of early-life mammals;

[0029] (4) Reduces the levels of IL-4 and IgE in the colon of male mammals in early life;

[0030] (5) Increase the expression level of T-bet mRNA in the colon of early-life mammals;

[0031] (6) Increase the relative abundance of IgA-bound Alistipes in the feces of male mammals in early life.

[0032] The present invention also provides the use of one or more of the following subspecies of Bifidobacterium longum, CCFM1269, CCFM1270, CCFM1271 or CCFM1272, in the preparation of health products that help regulate the intestinal flora.

[0033] Beneficial effects:

[0034] 1. This invention screened out three subsp. infantis of Bifidobacterium longum (CCFM1269, CCFM1270, CCFM1271, and CCFM1272) that have the function of regulating Th1 / Th2 balance and IgA secretion, specifically in the following ways:

[0035] (1) Increase the percentage of B cells, T cells and Th cells in the mesenteric lymph nodes of female and male young mice;

[0036] (2) Increase the content of IFN-γ, IgG2a, IgA, and sIgA in the colon of female and male juvenile mice, and increase the IgG2a / IgE ratio;

[0037] (3) Increase the content of IFN-γ and the ratio of IgG2a / IgE in the serum of female and male young mice;

[0038] (4) Reduces the levels of IL-4 and IgE in the colon of male juvenile rats;

[0039] (5) Increase the expression level of T-bet mRNA in the colon of female and male juvenile mice;

[0040] (6) Increase the relative abundance of IgA-bound Alistipes in the feces of male juvenile mice.

[0041] 2. The *Bifidobacterium longum* subsp. infantis subsp. CCFM1269, CCFM1270, CCFM1271, and CCFM1272 screened in this invention are food-safe strains that can be used to prepare products that regulate Th1 / Th2 balance and IgA content, and have great application prospects.

[0042] 3. The cultivation process of the Bifidobacterium longum infant subspecies of the present invention only requires the control of culture medium and some culture conditions, which is relatively low in cost and easy to realize industrial production.

[0043] Preservation of biological materials

[0044] Bifidobacterium longum subsp.infantis (CCFM1269), taxonomically named Bifidobacterium longum subsp.infantis, was deposited on September 26, 2022, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 62839), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0045] Bifidobacterium longum subsp.infantis, CCFM1270, taxonomically named Bifidobacterium longum subsp.infantis, was deposited on September 26, 2022, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 62840), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0046] Bifidobacterium longum subsp.infantis, CCFM1271, taxonomically named Bifidobacterium longum subsp.infantis, was deposited on September 26, 2022, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 62841), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0047] Bifidobacterium longum subsp.infantis, CCFM1272, taxonomically named Bifidobacterium longum subsp.infantis, was deposited on September 26, 2022, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 62842), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Changes in B cells of mesenteric lymph nodes in different groups of female juvenile mice; in the figure, *: p<0.05.

[0049] Figure 2 Changes in T cells in the mesenteric lymph nodes of female juvenile mice in different groups; in the figure, *: p<0.05.

[0050] Figure 3 Changes in Th cells in the mesenteric lymph nodes of female juvenile mice in different groups; in the figure, *: p<0.05

[0051] Figure 4 Changes in colonic IFN-γ in different groups of female juvenile mice; in the figure, *: p<0.05.

[0052] Figure 5 Changes in colonic IgG2a in different groups of female juvenile mice; in the figure, *: p<0.05.

[0053] Figure 6 Changes in colonic IgA in different groups of female juvenile mice; in the figure, *: p<0.05.

[0054] Figure 7 Changes in colonic sIgA in different groups of female juvenile mice; in the figure, *: p<0.05.

[0055] Figure 8 Changes in colonic IgG2a / IgE ratio in different groups of female juvenile mice; in the figure, *: p<0.05.

[0056] Figure 9 Changes in serum IFN-γ in different groups of female juvenile mice; * in the figure: p<0.05.

[0057] Figure 10 Changes in serum IgG2a / IgE ratio in different groups of female juvenile mice; * in the figure: p<0.05.

[0058] Figure 11 Changes in T-bet mRNA in the colon of female juvenile mice in different groups; in the figure, *: p<0.05.

[0059] Figure 12 Changes in the percentage of mesenteric lymphocytes in different groups of male juvenile rats; * in the figure: p<0.05.

[0060] Figure 13 Changes in the percentage of mesenteric lymphocytes in different groups of male juvenile rats; * in the figure: p<0.05.

[0061] Figure 14 Changes in the percentage of Th cells in the mesenteric lymph nodes of male juvenile rats in different groups; in the figure, *: p<0.05.

[0062] Figure 15 Changes in colonic IgA in male juvenile mice in different groups; in the figure, *: p<0.05.

[0063] Figure 16 : Colonic IgE in male juvenile mice from different groups; In the figure, *: p<0.05.

[0064] Figure 17 IL-4 in the colon of male juvenile mice in different groups; in the figure, *: p<0.05.

[0065] Figure 18 Changes in colonic IgG / IgE ratio in different groups of male juvenile mice; * in the figure: p<0.05.

[0066] Figure 19 Changes in serum IgG2a in male juvenile mice in different groups; in the figure, *: p<0.05.

[0067] Figure 20 Changes in serum IgG2a / 2aIgE in different groups of male juvenile mice; in the figure, *: p<0.05.

[0068] Figure 21 Changes in T-bet mRNA in the colon of male juvenile mice in different groups; in the figure, *: p<0.05.

[0069] Figure 22 : Alistipes bound to sIgA in feces of male juvenile mice from different groups; in the figure, *: p<0.05. Detailed Implementation

[0070] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0071] The BALB / C mice used in the following examples were purchased from Zhejiang Vital River Co., Ltd.; the Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, and CCFM1272 used in the following examples were isolated from the Biotechnology Center of the School of Food Science and Technology, Jiangnan University.

[0072] The detection reagents involved in the following examples are as follows:

[0073] The ELISA kits for IFN-γ, IgG2a, IgE, IgA, sIgA, and IL-4 were purchased from Formex.

[0074] The culture media involved in the following examples are as follows:

[0075] 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 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, agar 15 g / L.

[0076] 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.

[0077] Example 1: Screening and identification of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, and CCFM1272

[0078] 1. Screening

[0079] Using breast milk samples from Wuxi City, Jiangsu Province, 0.5 mL of the sample, preserved in 30% (v / v) glycerol, was added aseptically to a 10 mL centrifuge tube containing 4.5 mL of physiological saline, yielding 10... -1 Diluent, repeat the above dilution steps to obtain 10 -2 10 -3 10 -4 10 -5 10 -6Diluents: 100 μL of each of the different gradient dilutions were spread onto MRS solid medium and incubated at 37°C for 72 h to obtain dilution plates. Typical colonies from the dilution plates were streaked onto MRS solid medium and incubated at 37°C for 48 h to obtain purified colonies. The purified colonies were inoculated into MRS liquid medium and incubated at 37°C for 48 h to obtain strains FJSWXI4MI, FJSWXI8TI, FJSWXI10TI, and BJSWXB6MNIM1, which were named CCFM1270, CCFM1271, CCFM1272, and CCFM1269, respectively.

[0080] 2. Identification

[0081] The genomes of CCFM1269, CCFM1270, CCFM1271, and CCFM1272 were extracted and amplified using 16S rDNA. The 16S rDNA amplification conditions were: 95℃ for 5 min; 35 cycles (95℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min); 72℃ for 10 min. Amplification primers: 27F: (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R: (5'-TACGGCTACCTTGTTACGACTT-3'). The purification and sequence alignment of the amplification products were performed according to the method described in the literature (Turroni F et al. Exploring the Diversity of the Bifidobacterial Population in the Human Intestinal Tract[J]. Appl Environ Microb. 2009; 75(6):1534–45). The 16S rDNA of CCFM1269, CCFM1270, CCFM1271, and CCFM1272 was amplified and sequenced (by Suzhou Genewiz Biotechnology Co., Ltd.). The 16S rDNA sequences of CCFM1269, CCFM1270, CCFM1271, and CCFM1272 obtained from the sequencing analysis were compared with GenBank. The results showed that this strain is Bifidobacterium longum subsp. infantis, and named it Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, and CCFM1272.

[0082] Example 2: Preparation of Bifidobacterium longum subsp. infantile suspension

[0083] The bacterial suspensions of *Bifidobacterium longum* subsp. *infantii* CCFM1269, CCFM1270, CCFM1271, and CCFM1272 were prepared according to the following method:

[0084] Streaking of Bifidobacterium longum subsp. infantis culture onto MRS solid medium and incubating at 37°C for 48 h yielded single colonies.

[0085] Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 24 hours to obtain an activated solution. The activated solution was then inoculated into MRS liquid medium at an inoculation rate of 1% (v / v) and cultured at 37°C for 24 hours to obtain a primary seed culture.

[0086] The primary seed culture was inoculated into MRS liquid medium at an inoculation rate of 1% (v / v) and cultured at 37°C for 24 h to obtain the secondary seed culture.

[0087] The secondary seed culture was inoculated into MRS liquid medium at an inoculation rate of 1% (v / v) and cultured at 37°C for 24 h to obtain bacterial culture. The bacterial culture was centrifuged at 6000g for 15 min, and the precipitate was collected. The precipitate was washed twice with physiological saline buffer, and then centrifuged again at 6000g for 10 min to obtain bacterial cells. The lactic acid bacteria were resuspended in physiological saline to a cell concentration of 1×10⁻⁶ cells / mL. 9 CFU / mL was used to obtain a culture of Bifidobacterium longum subsp. infantis.

[0088] Example 3: Effects of Bifidobacterium longum infantis subsp. CCFM1269, CCFM1270, and CCFM1272 on the lymphocyte content of mesenteric lymph nodes in female baby rats

[0089] Eight male SPF (Spectrum Free) BALB / C mice and four male mice, aged 6 weeks, were housed at a room temperature of 22–24°C and a humidity of 40–60%, with 12h / 12h day-night cycles and free access to food and water for one week. Afterward, the females were grouped together at a 2:1 female-to-male ratio. Once the females became pregnant, the males were removed. The gestation period was 3 weeks. The pups were given oral gavage starting at 1 week of age and divided into three groups: Control group (gavage with physiological saline) and CCFM1269, CCFM1270, and CCFM1272 (gavage with Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1272, respectively).

[0090] Animals were acclimatized for one week before the experiment began, which lasted for eight weeks. Specific treatments were as follows:

[0091] Control group: 200 μL of normal saline was administered by gavage starting 1 week after birth;

[0092] Bifidobacterium longum infantis subspecies CCFM1269, CCFM1270, and CCFM1272 groups: Starting one week after birth, 10 μL of the corresponding bacterial suspension was administered via gavage, bringing the gavage dose to 1 × 10⁻⁶.9 CFU / only / day.

[0093] Mice were administered the drug by gavage until 3 weeks of age. At the end of the experiment, mesenteric lymph nodes from female juvenile mice were collected and placed in D-PBS to prepare a single-cell suspension. Flow cytometry was used to analyze the T, B, and Th cells in the mesenteric lymph nodes. The results are shown below. Figure 1-3 .

[0094] The number of T cells, B cells, and Th cells in mesenteric lymph nodes can generally reflect whether a bacterial strain can stimulate an individual to produce adaptive immunity. Figure 1 The results showed that the B cell content in the mesenteric lymph nodes of the normal group was 13.16%, the CCFM1270 group was 22.6%, the CCFM1272 group was 19.51%, and the CCFM1269 group was 18.3%. The percentage of B cells in the mesenteric lymph nodes of female juvenile mice in the CCFM1270, CCFM1272, and CCFM1269 groups was significantly higher than that in the normal group (p<0.05).

[0095] Depend on Figure 2 It was found that the T cell content in the mesenteric lymph nodes of the normal group was 60.44%, that of the CCFM1270 group was 68.73%, and that of the CCFM1272 group was 70.61%. The percentage of T cells in the mesenteric lymph nodes of female juvenile mice in the CCFM1270 and CCFM1272 groups was significantly higher than that in the normal group (p<0.05, p<0.05).

[0096] Depend on Figure 3 It was found that the Th cell content in the mesenteric lymph nodes of the normal group was 68.7%, while that in the CCFM1269 group was 71.86%. The percentage of Th cells in the mesenteric lymph nodes of female juvenile mice in the CCFM1269 group was significantly higher than that in the normal group (p<0.05).

[0097] Example 4: Effects of Bifidobacterium longum infant subspecies CCFM1269, CCFM1270, and CCFM1272 on Th1 / Th2-related cytokines and immunoglobulins in female infant mice.

[0098] Colons of female juvenile mice were collected, homogenized, and analyzed using an ELISA kit. The colon homogenates from each group were then used to measure Th1 / Th2-related cytokines and immunoglobulins in the colon. Results are shown below. Figures 4-8 .

[0099] IFN-γ and IgG2a levels are the main cytokines and immunoglobulins of the Th1 immune type, respectively. IgA and sIgA levels are mainly immunoglobulins of mucosal immunity. Figure 4It was found that the IFN-γ content in the colon of female juvenile mice in the CCFM1269 group was significantly higher than that in the normal group (p<0.05). The IFN-γ concentration in the colon homogenate of the normal group was 2.60 ng / mg protein, while that in the CCFM1269 group was 3.56 ng / mg protein.

[0100] Depend on Figure 5 It was found that the IgG2a content in the colon of female juvenile mice in the CCFM1269 group was significantly higher than that in the normal group (p<0.05). The IgG2a concentration in the colon homogenate of the normal group was 1.89 μg / mg protein, while that in the CCFM1269 group was 2.70 μg / mg protein.

[0101] Depend on Figure 6 It was found that the IgA content in the colon of female juvenile mice in the CCFM1269, CCFM1270, and CCFM1272 groups was significantly higher than that in the normal group (p<0.05). The IgA concentration in the colon homogenate of the normal group was 4.29 μg / mg protein, the CCFM1269 group was 7.66 μg / mg protein, the CCFM1270 group was 6.21 μg / mg protein, and the CCFM1272 group was 6.98 μg / mg protein.

[0102] Depend on Figure 7 It was found that the sIgA content in the colon of female juvenile mice in the CCFM1269 and CCFM1272 groups was significantly higher than that in the normal group (p<0.05). The sIgA concentration in the colon homogenate of the normal group was 0.43 μg / mg protein, that of CCFM1269 was 0.84 μg / mg protein, and that of CCFM1272 group was 0.68 μg / mg protein.

[0103] An increased IgG2a / IgE ratio indicates a transition in an individual's immune type from Th2 to Th1, gradually reaching equilibrium. Figure 8 It was found that the IgG2a / IgE ratio in the colon of female juvenile mice in the CCFM1269 group was significantly higher than that in the normal group (p<0.05). The ratio of IgG2a to IgE in the colon homogenate of the normal group was 0.89 μg / mg protein, while that in the CCFM1269 group was 1.42 μg / mg protein.

[0104] Example 5: Effects of Bifidobacterium longum infant subspecies CCFM1269, CCFM1270, and CCFM1272 on serum Th1 / Th2-related cytokines and immunoglobulins in female infant mice.

[0105] The grouping and treatment of mice were the same as in Example 3. After the experiment, blood was collected and the mice were euthanized. The serum levels of Th1 / Th2-related cytokines and immunoglobulins in each group of mice were measured using an ELISA kit. The results are shown in [Figure 3]. Figure 9-10 .

[0106] like Figure 9As shown, the serum IFN-γ content in female juvenile mice in the CCFM1269 group was significantly higher than that in the normal group (p<0.05). The serum IFN-γ concentration in the normal group was 0.74 ng / mL, while that in the CCFM1269 group was 0.92 ng / mL.

[0107] like Figure 10 As shown, the serum IgG2a / IgE ratio in female juvenile mice in the CCFM1269 group was significantly higher than that in the normal group (p<0.05). The serum IgG2a to IgE ratio in the normal group was 0.60, while that in the CCFM1269 group was 0.81.

[0108] Example 6: Effects of Bifidobacterium longum infant subsp. CCFM1269, CCFM1270, and CCFM1272 on T-bet expression levels in the colon of female baby mice

[0109] The grouping and modeling of mice were the same as in Example 3. After the experiment, blood was collected and mice were sacrificed. RNA was extracted from the mouse colons and reverse transcribed using a kit. The expression level of T-bet in the colons of each group of mice was measured, and the results are shown in [see attached table]. Figure 11 .

[0110] Depend on Figure 11 It was found that CCFM1269 significantly increased the relative expression level of T-bet in the colon of female juvenile mice (p<0.01), with the CCFM1269 group showing a 1.45-fold increase compared to the normal group.

[0111] Example 7: Effects of Bifidobacterium longum infant subsp. CCFM1269, CCFM1270, and CCFM1271 on the lymphocyte content of mesenteric lymph nodes in male baby rats

[0112] Eight male SPF (Spectrum Free) BALB / c mice and four male mice, aged 6 weeks, were housed at a room temperature of 22–24°C and a humidity of 40–60%, with 12h / 12h day-night alternation, and free access to food and water for one week. After one week, the females were grouped 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. The pups were given oral gavage starting at 1 week of age and were divided into a Control group (gavage with physiological saline) and a CCFM1269, CCFM1270, and CCFM1271 group (gavage with suspensions of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271, respectively).

[0113] Animals were acclimatized for one week before the experiment began, which lasted for eight weeks. Specific treatments were as follows:

[0114] Control group: 200 μL of normal saline was administered by gavage starting 1 week after birth as a control;

[0115] Bifidobacterium longum infantile subspecies CCFM1269, CCFM1270, and CCFM1271: Starting one week after birth, administer 10 μL of bacterial suspension via gavage, bringing the gavage dose to 1 × 10⁻⁶. 9 CFU / only / day.

[0116] Mice were administered the drug by gavage until 3 weeks of age. At the end of the experiment, mesenteric lymph nodes from female juvenile mice were collected and placed in D-PBS to prepare a single-cell suspension. Flow cytometry was used to analyze the T and B cells in the mesenteric lymph nodes. The results are shown below. Figures 12-14 .

[0117] Depend on Figure 12 It was found that the percentage of B cells in the mesenteric lymph nodes of male young mice in the CCFM1270 and CCFM1271 groups was significantly higher than that in the normal group (p<0.05). The B cell content in the mesenteric lymph nodes of the normal group was 13.50%, that of the CCFM1270 group was 20.20%, and that of the CCFM1271 group was 18.27%.

[0118] Depend on Figure 13 It was found that the percentage of T cells in the mesenteric lymph nodes of female juvenile mice in the CCFM1270, CCFM1271, and CCFM1269 groups was significantly higher than that in the normal group (p<0.05, p<0.05). The T cell content in the mesenteric lymph nodes of the normal group was 41.9%, that of the CCFM1270 group was 61.2%, that of the CCFM1271 group was 66.8%, and that of the CCFM1269 group was 67.3%.

[0119] Depend on Figure 14 It was found that the percentage of Th cells in the mesenteric lymph nodes of female juvenile mice in the CCFM1269 group was significantly higher than that in the normal group (p<0.05). The Th cell content in the mesenteric lymph nodes of the normal group was 63.24%, that of CCFM1270 was 72.21%, and that of CCFM1269 group was 70.11%.

[0120] Example 8: Effects of Bifidobacterium longum infant subspecies CCFM1269, CCFM1270, and CCFM1271 on Th1 / Th2 balance and IgA content in the colon of male juvenile rats

[0121] The mice were grouped as in Example 3. After the experiment, mouse colons were placed in phosphate-buffered saline solution and homogenized. The colon homogenates of each group were measured using an ELISA kit. The results are shown in [Figure 3]. Figure 15-18 .

[0122] like Figure 15As shown, the CCFM1269, CCFM1270, and CCFM1271 groups significantly increased the IgA content in the colon of male juvenile rats (p<0.01). The IgA concentration in the colon homogenate of the normal group was 1.03 μg / mg protein, the CCFM1269 group was 1.78 μg / mg protein, the CCFM1270 group was 1.90 μg / mg protein, and the CCFM1271 group was 2.03 μg / mg protein.

[0123] like Figure 16 As shown, CCFM1269 and CCFM1271 significantly reduced the IgE content in the colon of male juvenile rats (p<0.05). The IgE concentration in the colon homogenate of the normal group was 2.43 μg / mg protein, the CCFM1269 group was 1.81 μg / mg protein, and the CCFM1271 group was 1.78 μg / mg protein.

[0124] like Figure 17 As shown, the CCFM1270 group significantly reduced the IL-4 content in the colon of male juvenile rats (p<0.05). The IL-4 content in the colon of the normal group was 390.56 pg / mg protein, while that in the CCFM1270 group was 309.58 pg / mg protein.

[0125] like Figure 18 As shown, CCFM1269, CCFM1270, and CCFM1271 significantly increased the IgG2a / IgE ratio in male juvenile mice (p<0.05). The IgG2a / IgE ratio in the colon homogenate of the normal group was 0.76, while that of the CCFM1269 group was 1.07, the CCFM1270 group was 1.07, and the CCFM1271 group was 0.98.

[0126] Example 9: Effects of Bifidobacterium longum infant subspecies CCFM1269, CCFM1270, and CCFM1271 on serum Th1 / Th2 balance in male baby mice

[0127] The grouping and treatment of mice were the same as in Example 3. After the experiment, blood was collected and the mice were euthanized. The serum levels of Th1 / Th2-related cytokines in each group of mice were measured using an ELISA kit. The results are shown below. Figures 19-20 .

[0128] like Figure 19 As shown, the CCFM1269 group significantly increased the serum IgG2a content in male juvenile mice (p<0.05), while the serum IgG2a concentration in the normal group was 0.58 ng / mL and that in the CCFM1269 group was 0.72 ng / mL.

[0129] like Figure 20As shown, the CCFM1269 group significantly increased the serum IgG2a / IgE ratio (p<0.05), while the ratio of serum IgG2a to IgE in the normal group was 0.60, and that in the CCFM1269 group it was 0.79.

[0130] Example 10: Effects of Bifidobacterium longum infant subspecies CCFM1269, CCFM1270, and CCFM1271 on T-bet expression levels in the colon of male baby rats

[0131] The grouping and modeling of mice were the same as in Example 3. After the experiment, blood was collected and mice were sacrificed. RNA was extracted from the mouse colons and reverse transcribed using a kit. The expression level of T-bet in the colons of each group of mice was measured, and the results are shown in [see attached table]. Figure 21 .

[0132] Transcription factor T-bet is a key factor that specifically regulates Th0 differentiation and acts as a Th1 / Th2 switching switch. T-bet transcription only occurs in Th1 cell lines. Therefore, it is believed that T-bet is selectively expressed in Th1 cells. As a Th1-specific transcription factor, T-bet is selectively expressed in Th1 cells and plays an important role in Th1 cell development by initiating the Th1 genetic program, while inhibiting the synthesis of Th2 cytokines.

[0133] Depend on Figure 21 It was found that CCFM1269 significantly increased the relative expression level of T-bet in the colon of male juvenile mice (p<0.05), with the CCFM1269 group showing a 1.41-fold increase compared to the normal group.

[0134] Example 11: Effects of Bifidobacterium longum infant subspecies CCFM1269, CCFM1270, and CCFM1271 on the composition of IgA-binding bacteria in the feces of male juvenile mice

[0135] The grouping and modeling of mice were the same as in Example 3. After the experiment, mouse feces were collected, fecal IgA-binding bacteria were enriched, and genomic DNA was extracted from the feces using a FastDNA Spin Kit (MP Biomedical, USA). Specific PCR amplification of the V3-V4 region of the extracted genomic DNA was performed, followed by 16S rDNA sequencing. Changes in fecal microbiota were analyzed, and the results are shown below. Figure 22 .

[0136] Depend on Figure 22 It was found that the relative abundance of IgA binding to Alistipes in the male juvenile control group was 0.018, in the CCFM1269 group it was 0.11, in the CCFM1270 group it was 0.039, and in the CCFM1271 group it was 0.052, which significantly increased the relative abundance of IgA binding to Alistipes.

[0137] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone 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 subsp. infantis CCFM1271, taxonomically named Bifidobacterium longum subsp. infantis, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with the collection number GDMCC No: 62841, and the collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A probiotic containing Bifidobacterium longum subspecies infantis CCFM1271 according to claim 1.

3. The probiotic according to claim 2, characterized in that The number of viable bacteria of Bifidobacterium longum infantis subspecies CCFM1271 in the probiotic is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

4. A starter containing the Bifidobacterium longum subspecies infantis CCFM1271 according to claim 1.

5. A medicine containing the Bifidobacterium longum subspecies infantis CCFM1271 according to claim 1.

6. Use of the Bifidobacterium longum subspecies infantis CCFM1271 described in claim 1 in the preparation of a health product that helps regulate intestinal flora.

7. Use of Bifidobacterium longum subspecies infantis CCFM1271 described in claim 1 in the preparation of a probiotic product for regulating Th1 / Th2 balance and IgA synthesis.

8. The use according to claim 7, characterized in that: The product is used to increase the levels of Th1 / Th2 related cytokines and immunoglobulins in the colon of early life mammals.

9. The use according to claim 8, characterized in that: Adjusting the Th1 / Th2 balance includes at least one of the following effects: (1) Increase the percentage of B cells, T cells, and Th cells in the mesenteric lymph nodes of early-life mammals; (2) Increase the levels of IFN-γ, IgG2a, IgA, sIgA, and the ratio of IgG2a / IgE in the colon of early-life mammals; (3) Increase the IFN-γ content and IgG2a / IgE ratio in the serum of mammals in early life; (4) reduce the levels of IL-4 and IgE in the colon of male mammals in early life; (5) increase the expression level of T-bet mRNA in the colon of early-life mammals; (6) Increase the relative abundance of IgA-bound Alistipes in the feces of male mammals in early life.

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