Bifidobacterium longum subsp. infantis capable of regulating Th1 / Th2 balance and IgA synthesis in young mice and its application

Abstract

The present invention discloses Bifidobacterium longum subsp. infantis capable of regulating the Th1 / Th2 balance and IgA synthesis in young mice and its application, belonging to the field of microbial technology. The Bifidobacterium longum subsp. infantis provided by the present invention can regulate the Th1 / Th2 balance and IgA content in young mice, specifically manifested in: (1) increasing the percentage contents of B cells, T cells, and Th cells in the mesenteric lymph nodes of early-life mammals; increasing the contents of IFN-γ, IgG2a, IgA, and sIgA in the colon, and the ratio of IgG2a / IgE; (3) increasing the content of IFN-γ in the serum of early-life mammals and the ratio of IgG2a / IgE; (4) decreasing the contents of IL-4 and IgE in the colon of early-life mammals and increasing the expression level of T-bet mRNA in the colon; (5) increasing the relative abundance of IgA-bound Alistipes in the feces of early-life male mammals.

Description

Technical Field

[0001] The present invention relates to Bifidobacterium longum subsp. infantis capable of regulating Th1 / Th2 balance and IgA synthesis in young mice and its application, belonging to the field of microbial technology. Background Art

[0002] With the rising incidence of various non-communicable diseases, people have shown great interest in discovering the causes of diseases and paying attention to early immune establishment to reduce the risk of late-onset diseases. Especially during the period when infants and young children are highly plastic, reasonable nutritional supplementation can minimize the occurrence of diseases during the growth process. After birth, infants show a preference for type 2 T helper cell immunity due to factors such as hormones and progesterone, which is the main cause of increased risks of allergies, asthma, and other diseases in childhood. In addition, the imbalance of the infant gut microbiota is common in modern society and may be a factor contributing to the increased incidence of immune-mediated diseases. Breastfed infants may be resistant to diarrhea, allergies, asthma, and inflammatory bowel disease. Breast milk not only provides basic nutrition for infants but also promotes the establishment of the gut microbiota. Therefore, it is necessary to explore the microbiota that can combat the occurrence of allergies, asthma, and other possible immune diseases and promote the immune system.

[0003] Bifidobacterium longum subsp. infantis is the main bifidobacterium colonizer in the infant gut and shows advantages in promoting early-life health. Current research has determined that the loss of bifidobacteria in early life is associated with an increased risk of several immune diseases and intestinal inflammation, but the mechanism is still unclear. Exposure to the early-life microbiota may provide an opportunity to prevent the allergy risk caused by Th1 / Th2 imbalance. In breastfed infants given Bifidobacterium longum subsp. infantis EVC001, gut T helper cell 2 (Th2) and Th17 cytokines are silenced. Administration of EVC001 to adult mice has a therapeutic effect on allergic asthma by promoting Th1 and silencing Th2 immune responses. In addition, the introduction of Bifidobacterium longum subsp. infantis reduces intestinal inflammation by stably and continuously remodeling the gut microbiota of breastfed infants.

[0004] In recent years, studies have shown that Bifidobacterium longum subsp. infantis EVC001 can promote the differentiation of T 0 cells into Th1 in infants and regulate the Th1 / Th2 balance in infants. Therefore, there is an urgent need to find a probiotic that can regulate the Th1 / Th2 balance and promote the production of IgA. Summary of the Invention

[0005] The present invention provides Bifidobacterium longum subsp. infantis, wherein the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. infantis CCFM1269, Bifidobacterium longum subsp. infantis CCFM1270, Bifidobacterium longum subsp. infantis CCFM1271 or Bifidobacterium longum subsp. infantis CCFM1272;

[0006] The Bifidobacterium longum subsp. infantis CCFM1269, taxonomically named Bifidobacterium longum subsp. infantis, was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with a deposit number of GDMCC No: 62839, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0007] The Bifidobacterium longum subsp. infantis CCFM1270, taxonomically named Bifidobacterium longum subsp. infantis, was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with a deposit number of GDMCC No: 62840, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0008] The Bifidobacterium longum subsp. infantis CCFM1271, taxonomically named Bifidobacterium longum subsp. infantis, was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with a deposit number of GDMCC No: 62841, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0009] The Bifidobacterium longum subsp. infantis CCFM1272, taxonomically named Bifidobacterium longumsubsp. infantis, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with the collection number GDMCC No: 62842, and the collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The present invention also provides a probiotic agent containing one or more of the Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 and CCFM1272.

[0011] In one embodiment, the number of viable bacteria of Bifidobacterium longum subspecies infantis in the probiotic is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0012] In one embodiment, the probiotic agent is a bacterial suspension of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

[0013] The present invention also provides a starter culture containing the Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

[0014] In one embodiment, the method for preparing the starter culture is as follows: inoculate Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 into a culture medium, culture at 37 °C for 24 - 48 h to obtain a culture solution; centrifuge the culture solution to obtain bacterial cells; resuspend the bacterial cells with physiological saline to obtain the starter culture.

[0015] In one embodiment, the culture medium is MRS medium.

[0016] The present invention also provides a drug containing at least one strain of the Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

[0017] In one embodiment, the viable count of Bifidobacterium longum subsp. infantis in the drug is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0018] In one embodiment, the drug contains the Bifidobacterium longum subsp. infantis and a pharmaceutically acceptable carrier.

[0019] In one embodiment, the drug is used to regulate the Th1 / Th2 balance and / or promote IgA synthesis.

[0020] The present invention also provides the application of the Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 in the preparation of a health product helpful for enhancing immunity.

[0021] The present invention also provides the application of one or more of the Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 in a probiotic product for regulating the Th1 / Th2 balance and IgA synthesis.

[0022] 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.

[0023] In one embodiment, the product is used to regulate the intestinal flora of early-life mammals.

[0024] In one embodiment, the regulation of Th1 / Th2 balance and IgA synthesis includes at least one of the following effects:

[0025] (1) Increasing the percentage contents of B cells, T cells, and Th cells in the mesenteric lymph nodes of early-life mammals;

[0026] (2) Increasing the contents of IFN-γ, IgG2a, IgA, and sIgA, and the ratio of IgG2a / IgE in the colon of early-life mammals;

[0027] (3) Increasing the content of IFN-γ and the ratio of IgG2a / IgE in the serum of early-life mammals;

[0028] (4) Decreasing the contents of IL-4 and IgE in the colon of early-life male mammals;

[0029] (5) Increasing the expression level of T-bet mRNA in the colon of early-life mammals;

[0030] (6) Increasing the relative abundance of IgA-bound Alistipes in the feces of early-life male mammals.

[0031] The present invention also provides the use of one or more of the Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, or CCFM1272 in the preparation of a health product helpful for regulating the intestinal flora.

[0032] Beneficial effects:

[0033] 1. The present invention has screened out Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, and CCFM1272 with the effect of regulating Th1 / Th2 balance and IgA secretion, specifically manifested as:

[0034] (1) Increasing the percentage contents of B cells, T cells, and Th cells in the mesenteric lymph nodes of female and male young mice;

[0035] (2) Increasing the contents of IFN-γ, IgG2a, IgA, and sIgA, and the ratio of IgG2a / IgE in the colon of female and male young mice;

[0036] (3) Increasing the content of IFN-γ and the ratio of IgG2a / IgE in the serum of female and male young mice;

[0037] (4) Reduced colonic IL-4 and IgE levels in male young mice;

[0038] (5) Increased T-bet mRNA expression levels in the colon of both male and female mice;

[0039] (6) Increase the relative abundance of IgA-bound Alistipes in the feces of male young mice.

[0040] 2. The Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 and CCFM1272 screened by the present invention are food safety strains and can be used to prepare products for regulating Th1 / Th2 balance and IgA content, and have great application prospects.

[0041] 3. The cultivation process of Bifidobacterium longum subspecies infantis of the present invention only requires the control of culture medium and some culture conditions, and the cost is relatively low, and it is easy to realize industrial production.

[0042] Biomaterial Deposit

[0043] Bifidobacterium longum subsp. infantis CCFM1269, taxonomically named Bifidobacterium longum subsp. infantis, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with the deposit number GDMCC No: 62839, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0044] Bifidobacterium longum subsp. infantis CCFM1270, taxonomically named Bifidobacterium longum subsp. infantis, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with the deposit number GDMCC No: 62840, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0045] 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 deposit number GDMCC No: 62841, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0046] Bifidobacterium longum subsp. infantis CCFM1272, taxonomically named Bifidobacterium longum subsp. infantis, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with the deposit number GDMCC No: 62842, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : Changes of B cells in mesenteric lymph nodes of female mice in different groups; In the figure, *: p<0.05.

[0048] Figure 2 : Changes of T cells in mesenteric lymph nodes of female mice in different groups; In the figure, *: p<0.05.

[0049] Figure 3 :Changes of Th cells in mesenteric lymph nodes of female mice in different groups; in the figure, *: p<0.05

[0050] Figure 4 : Changes of IFN-γ in the colon of female mice in different groups; In the figure, *: p<0.05.

[0051] Figure 5 : Changes in colon IgG2a in female mice of different groups; in the figure, *: p<0.05.

[0052] Figure 6 : Changes in colon IgA in female mice in different groups; In the figure, *: p<0.05.

[0053] Figure 7 : Changes in colon sIgA in female mice in different groups; In the figure, *: p<0.05.

[0054] Figure 8 : Changes in colon IgG2a / IgE in female mice of different groups; In the figure, *: p<0.05.

[0055] Figure 9 : Changes in serum IFN-γ of female mice in different groups; In the figure, *: p<0.05.

[0056] Figure 10 : Changes in serum IgG2a / IgE of female mice in different groups; In the figure, *: p<0.05.

[0057] Figure 11 : Changes of T-bet mRNA in the colon of female mice in different groups; In the figure, *: p<0.05.

[0058] Figure 12 : Changes in the percentage of mesenteric lymphatic B cells in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0059] Figure 13 : Changes in the percentage of mesenteric lymphatic T cells in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0060] Figure 14 : Changes in the percentage of mesenteric lymphatic Th cells in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0061] Figure 15 : Changes in colon IgA in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0062] Figure 16 : Colon IgE in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0063] Figure 17 : Colon IL-4 in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0064] Figure 18 : Changes in the ratio of colon IgG / IgE in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0065] Figure 19 : Changes in serum IgG2a in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0066] Figure 20 : Changes in the ratio of serum IgG2a / 2aIgE in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0067] Figure 21 : Changes in colon T-bet mRNA in male juvenile mice of different groups; in the figure, *: p < 0.05.

[0068] Figure 22 : sIgA-bound Alistipes in the feces of male juvenile mice of different groups; in the figure, *: p < 0.05. Detailed implementation manners

[0069] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings.

[0070] The BALB / C mice involved in the following examples were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.; the Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, and CCFM1272 involved in the following examples were isolated from the Biotechnology Center of the School of Food Science and Technology, Jiangnan University;

[0071] The detection reagents involved in the following examples are as follows:

[0072] The ELISA kits for IFN-γ, IgG2a, IgE, IgA, sIgA, and IL-4 were purchased from Fumentus.

[0073] The culture media involved in the following examples are as follows:

[0074] 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, K 2 PO 4 ·3H 2 O 2.6 g / L, MgSO 4 ·7H 2 O 0.1 g / L, MnSO 4 0.05 g / L, Tween 80 1 mL / L, Agar 15 g / L.

[0075] MRS liquid 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, K 2 PO 4 ·3H 2 O 2.6 g / L, MgSO 4 ·7H 2 O 0.1 g / L, MnSO 4 0.05 g / L, Tween 80 1 mL / L.

[0076] Example 1: Screening and strain identification of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, and CCFM1272

[0077] 1. Screening

[0078] Taking 0.5 mL of the breast milk sample from Wuxi City, Jiangsu Province, which was stored in 30% (v / v) glycerol, and adding it to a 10 mL centrifuge tube containing 4.5 mL of physiological saline in a sterile environment to obtain 10 -1 dilution, repeating the above dilution steps to obtain 10 -2, 10 -3 , 10 -4 , 10 -5 , 10 -6 Diluent; 100 μL of gradient diluents with different gradients were respectively taken and spread on MRS solid medium, and cultured at 37 °C for 72 h to obtain dilution-coated plates; typical colonies on the dilution-coated plates were picked and streaked on MRS solid medium respectively, and cultured at 37 °C for 48 h to obtain purified colonies; the purified colonies were picked and inoculated into MRS liquid medium, and cultured at 37 °C for 48 h to obtain strains FJSWXI4MI, FJSWXI8TI, FJSWXI10TI, BJSWXB6MNIM1, which were respectively named, CCFM1270, CCFM1271, CCFM1272, CCFM1269.

[0079] 2. Identification

[0080] The genomes of CCFM1269, CCFM1270, CCFM1271, and CCFM1272 were extracted for 16S amplification. The amplification conditions for 16S rDNA were: 95 °C for 5 min; 35 cycles (95 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min); 72 °C for 10 min. Amplification primers: 27F: (5’-AGAGTTTGATCCTGGCTCAG-3’), 1492R: (5’-TACGGCTACCTTGTTACGACTT-3’). The purification and sequence alignment processes of the amplification products were carried out according to the methods described in the literature (Turroni F et al. Exploring the Diversitym 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 by sequencing analysis were aligned in GenBank. The results showed that these strains were all Bifidobacterium longum subsp. infantis, named Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, CCFM1272.

[0081] Example 2: Preparation of Bifidobacterium longum subsp. infantis bacterial suspension

[0082] The bacterial suspensions of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, and CCFM1272 were prepared as follows:

[0083] Dip the bacterial suspension of Bifidobacterium longum subsp. infantis and streak it on MRS solid medium, and culture it at 37 °C for 48 h to obtain single colonies;

[0084] Pick the single colonies and inoculate them into MRS liquid medium, culture at 37 °C for 24 h to obtain an activation solution; inoculate the activation solution into MRS liquid medium at an inoculation amount of 1% (v / v), and culture at 37 °C for 24 h to obtain a primary seed solution;

[0085] Inoculate the primary seed solution into MRS liquid medium at an inoculation amount of 1% (v / v), and culture at 37 °C for 24 h to obtain a secondary seed solution;

[0086] Inoculate the secondary seed solution into MRS liquid medium at an inoculation amount of 1% (v / v), and culture at 37 °C for 24 h to obtain a bacterial suspension; centrifuge the bacterial suspension at 6000 g for 15 min, and collect the precipitate; wash the precipitate twice with physiological saline buffer, and then centrifuge at 6000 g for 10 min to obtain bacterial cells; resuspend the lactic acid bacteria cells with physiological saline to a cell concentration of 1×10 9 CFU / mL to obtain the bacterial suspension of Bifidobacterium longum subsp. infantis.

[0087] Example 3: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1272 on the content of mesenteric lymph node lymphocytes in female young mice

[0088] Eight 6-week-old female pathogen-free (SPF) BALB / C mice and four male mice were taken. After being raised for 1 week at a breeding room temperature of 22 - 24 °C, humidity of 40 - 60%, 12 h / 12 h day-night alternation, and free access to food and water, they were caged in a ratio of 2 females to 1 male. After the female mice became pregnant, the male mice were taken out. After a 3-week gestation period, the pups were gavaged starting from 1 week old. They were divided into a Control group (gavaged with physiological saline), CCFM1269, CCFM1270, and CCFM1272 (gavaged with Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1272 respectively).

[0089] The experiment started after 1 week of animal adaptive feeding and lasted for 8 weeks. The specific treatments were as follows:

[0090] Control group (Control): Starting from 1 week after birth, gavaged with 200 μL of physiological saline;

[0091] Groups of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1272: Starting from 1 week after birth, 10 μL of the corresponding bacterial suspension was intragastrically administered, with the intragastric dose being 1×10 9 CFU / rat / day.

[0092] The intragastric administration continued until the rats reached 3 weeks of age. After the experiment ended, mesenteric lymph nodes of female pups were collected, placed in D-PBS, and single-cell suspensions were prepared. The T cells, B cells, and Th cells in the mesenteric lymph nodes were measured by flow cytometry. The measurement results are shown in Figures 1-3 .

[0093] The numbers of T cells, B cells, and Th cells in the mesenteric lymph nodes can usually reflect whether the strain can stimulate an individual to produce adaptive immunity. As Figure 1 can be seen, the content of B cells in the mesenteric lymph nodes of the normal group was 13.16%, that of the CCFM1270 group was 22.6%, that of the CCFM1272 group was 19.51%, and that of the CCFM1269 group was 18.3%. The percentage content of B cells in the mesenteric lymph nodes of female pups in the CCFM1270, CCFM1272, and CCFM1269 groups was significantly higher than that of the normal group (p<0.05);

[0094] As Figure 2 can be seen, the content of T cells in the mesenteric lymph nodes of the normal group was 60.44%, that of the CCFM1270 group was 68.73%, that of the CCFM1272 group was 70.61%. The percentage content of T cells in the mesenteric lymph nodes of female pups in the CCFM1270 group and the CCFM1272 group was significantly higher than that of the normal group (p<0.05, p<0.05);

[0095] As Figure 3 can be seen, the content of Th cells in the mesenteric lymph nodes of the normal group was 68.7%, that of the CCFM1269 group was 71.86%. The percentage content of Th cells in the mesenteric lymph nodes of female pups in the CCFM1269 group was significantly higher than that of the normal group (p<0.05).

[0096] Example 4: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1272 on Th1 / Th2-related cytokines and immunoglobulins in female pups

[0097] The colon of female pups was collected and homogenized in phosphate buffered saline. The colon homogenates of each group of mice were measured by an ELISA kit to measure the Th1 / Th2-related cytokines and immunoglobulins in the colon. The measurement results are shown in Figures 4-8 .

[0098] The IFN-γ content and IgG2a content are the main cytokines and immunoglobulins of the Th1 immune type, respectively. The IgA content and sIgA content are mainly immunoglobulins of mucosal immunity. From Figure 4 It can be seen that the IFN-γ content in the colon of female young 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, and that in the CCFM1269 group was 3.56 ng / mg protein;

[0099] From Figure 5 It can be seen that the IgG2a content in the colon of female young 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, and that in the CCFM1269 group was 2.70 μg / mg protein;

[0100] From Figure 6 It can be seen that the IgA content in the colon of female young 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, that in the CCFM1269 group was 7.66 μg / mg protein, that in the CCFM1270 group was 6.21 μg / mg protein, and that in the CCFM1272 group was 6.98 μg / mg protein;

[0101] From Figure 7 It can be seen that the sIgA content in the colon of female young 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 in CCFM1269 was 0.84 μg / mg protein, and that in the CCFM1272 group was 0.68 μg / mg protein;

[0102] An increase in the IgG2a / IgE ratio indicates that the immune type of an individual transitions from the Th2 type to the Th1 type and gradually reaches equilibrium. From Figure 8 It can be seen that the IgG2a / IgE in the colon of female young 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, and that in the CCFM1269 group was 1.42 μg / mg protein.

[0103] Example 5: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1272 on Th1 / Th2-related cytokines and immunoglobulins in the serum of female young mice

[0104] The grouping and treatment of mice were the same as in Example 3. After the experiment, blood was collected and the mice were sacrificed. The contents of Th1 / Th2-related cytokines and immunoglobulins in the serum of mice in each group were measured using an ELISA kit. The detection results are shown in Figures 9-10 .

[0105] As Figure 9 shown, the IFN-γ content in the serum 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 normal group was 0.74 ng / mL, and that in the CCFM1269 group was 0.92 ng / mL;

[0106] As Figure 10 shown, the IgG2a / IgE in the serum of female juvenile mice in the CCFM1269 group was significantly higher than that in the normal group (p<0.05). The ratio of serum IgG2a to IgE in the normal group was 0.60, and that in the CCFM1269 group was 0.81.

[0107] Example 6: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1272 on the expression level of T-bet in the colon of female juvenile mice

[0108] The grouping and modeling of mice were the same as in Example 3. After the experiment, blood was collected and the mice were sacrificed. RNA was extracted from the colon of the mice, and reverse transcription was performed using a kit to measure the expression level of T-bet in the colon of mice in each group. The detection results are shown in Figure 11 .

[0109] It can be Figure 11 seen that CCFM1269 could significantly increase the relative expression of T-bet in the colon of female juvenile mice (p<0.01), and the CCFM1269 group was 1.45 times that of the normal group.

[0110] Example 7: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271 on the lymphocyte content in the mesenteric lymph nodes of male juvenile mice

[0111] Eight 6-week-old male specific pathogen-free (SPF) BALB / C female mice and four male mice were taken. After being raised for 1 week under the conditions of a breeding room temperature of 22-24 °C, a humidity of 40-60%, a 12h / 12h day-night cycle, and free access to food and water, they were caged in a male-female ratio of 2:1. After the female mice became pregnant, the male mice were taken out. The gestation period was 3 weeks. The juvenile mice started gavage at 1 week old and were divided into a Control group (gavaged with normal saline), CCFM1269, CCFM1270, and CCFM1271 (gavaged with suspensions of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271, respectively).

[0112] The experiment started after 1 week of adaptive feeding of the animals and lasted for 8 weeks. The specific treatments were as follows:

[0113] Control group: Starting from 1 week after birth, 200 μL of normal saline was intragastrically administered as a control.

[0114] Groups of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271: Starting from 1 week after birth, 10 μL of bacterial suspension was intragastrically administered, and the intragastric administration dose was 1×10 9 CFU / animal / day.

[0115] Intragastric administration was performed until 3 weeks of age. After the experiment ended, mesenteric lymph nodes of female young mice were taken, placed in D-PBS, and single-cell suspensions were prepared. Determination was carried out by flow cytometry. T cells and B cells in the mesenteric lymph nodes were measured, and the measurement results are shown in Figures 12-14 .

[0116] It can be seen from Figure 12 that the percentage content 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 content of B cells in the mesenteric lymph nodes of the normal group was 13.50%, that in the CCFM1270 group was 20.20%, and that in the CCFM1271 group was 18.27%.

[0117] It can be seen from Figure 13 that the percentage content of T cells in the mesenteric lymph nodes of female young mice in the CCFM1270, CCFM1271, and CCFM1269 groups was significantly higher than that in the normal group (p<0.05, p<0.05). The content of T cells in the mesenteric lymph nodes of the normal group was 41.9%, that in the CCFM1270 group was 61.2%, that in the CCFM1271 group was 66.8%, and that in the CCFM1269 group was 67.3%.

[0118] It can be seen from Figure 14 that the percentage content of Th cells in the mesenteric lymph nodes of female young mice in the CCFM1269 group was significantly higher than that in the normal group (p<0.05). The content of Th cells in the mesenteric lymph nodes of the normal group was 63.24%, that in the CCFM1270 was 72.21%, and that in the CCFM1269 group was 70.11%.

[0119] Example 8: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271 on the Th1 / Th2 balance and IgA content in the colon of male young mice

[0120] The grouping of the mice was the same as in Example 3. After the experiment ended, the colon of the mice was taken, placed in phosphate buffered saline and homogenized. Each group of mouse colon homogenates was measured by an ELISA kit, and the detection results are shown in Figures 15-18 .

[0121] As shown Figure 15 in the figure, the CCFM1269, CCFM1270, and CCFM1271 groups significantly increased the IgA content in the colon of male juvenile mice (p<0.01). The IgA concentration in the colon homogenate of the normal group was 1.03 μg / mg protein, that of the CCFM1269 group was 1.78 μg / mg protein, that of the CCFM1270 group was 1.90 μg / mg protein, and that of the CCFM1271 group was 2.03 μg / mg protein;

[0122] As shown Figure 16 in the figure, CCFM1269 and CCFM1271 significantly decreased the IgE content in the colon of male juvenile mice (p<0.05). The IgE concentration in the colon homogenate of the normal group was 2.43 μg / mg protein, that of the CCFM1269 group was 1.81 μg / mg protein, and that of the CCFM1271 group was 1.78 μg / mg protein;

[0123] As shown Figure 17 in the figure, the CCFM1270 group significantly decreased the IL-4 content in the colon of male juvenile mice (p<0.05). The content of IL-4 in the colon of the normal group was 390.56 pg / mg protein, and that of the CCFM1270 group was 309.58 pg / mg protein.

[0124] As shown Figure 18 in the figure, CCFM1269, CCFM1270, and CCFM1271 significantly increased IgG2a / IgE in male juvenile mice (p<0.05). The ratio of IgG2a to IgE in the colon homogenate of the normal group was 0.76, that of the CCFM1269 group was 1.07, that of the CCFM1270 group was 1.07, and that of the CCFM1271 group was 0.98.

[0125] Example 9: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271 on the Th1 / Th2 balance in the serum of male juvenile mice

[0126] The grouping and treatment of the mice were the same as in Example 3. After the experiment, blood was collected and the mice were sacrificed. The Th1 / Th2-related cytokines in the serum of each group of mice were measured by an ELISA kit, and the detection results are shown in Figures 19-20 .

[0127] As shown Figure 19 in the figure, the CCFM1269 group significantly increased the IgG2a content in the serum of male juvenile mice (p<0.05). The IgG2a concentration in the serum of the normal group was 0.58 ng / mL, and that of the CCFM1269 group was 0.72 ng / mL;

[0128] As shown Figure 20As shown, the CCFM1269 group significantly increased IgG2a / IgE in serum (p<0.05). The ratio of serum IgG2a to IgE in the normal group was 0.60, while that in the CCFM1269 group was 0.79.

[0129] Example 10: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271 on the expression level of T-bet in the colon of male young mice

[0130] The grouping and modeling of mice were the same as in Example 3. After the experiment, blood was collected and the mice were sacrificed. RNA was extracted from the colon of the mice, and reverse transcription was performed using a kit to measure the expression level of T-bet in the colon of each group of mice. The test results are shown in Figure 21 .

[0131] The transcription factor T-bet is a key factor that specifically regulates the differentiation of Th0 and plays a role as a Th1 / Th2 switch. T-bet transcription only appears in the Th1 cell line. Therefore, it is considered 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 the development of Th1 cells by initiating the Th1 genetic program and inhibiting the synthesis of Th2 cytokines.

[0132] As Figure 21 can be seen, CCFM1269 can significantly increase the relative expression level of T-bet in the colon of male young mice (p<0.05), and the CCFM1269 group is 1.41 times that of the normal group.

[0133] Example 11: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271 on the composition of fecal IgA-binding bacteria in male young mice

[0134] The grouping and modeling of mice were the same as in Example 3. After the experiment, mouse feces were collected, and fecal IgA-binding bacteria were enriched. Genomic DNA in the feces was extracted using the FastDNA Spin Kit (MP Biomedicals, USA). Specific PCR amplification was performed on the V3-V4 region of the extracted genomic DNA, and 16S rDNA sequencing was carried out to analyze the changes in the fecal flora. The analysis results are shown in Figure 22 .

[0135] As Figure 22 can be seen, the relative abundance of IgA-binding Alistipes in the Control group of male young mice was 0.018, that in the CCFM1269 group was 0.11, that in the CCFM1270 group was 0.039, and that in the CCFM1271 group was 0.052, significantly increasing the relative abundance of IgA-binding Alistipes.

[0136] Although the present invention has been disclosed above in its preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Bifidobacterium longum subsp. infantis CCFM1269, taxonomically named Bifidobacterium longum subsp. infantis, was deposited at the Guangdong Provincial Culture Collection of Microorganisms on September 26, 2022, with the deposit number GDMCC No: 62839.

2. A probiotic agent containing Bifidobacterium longum subsp. infantis CCFM1269 as claimed in claim 1.

3. The probiotic agent according to claim 2, wherein, it further contains one or more of Bifidobacterium longum subsp. infantis CCFM1270, Bifidobacterium longum subsp. infantis CCFM1271, and Bifidobacterium longum subsp. infantis CCFM1272; the Bifidobacterium longum subsp. infantis CCFM1270, taxonomically named Bifidobacterium longum subsp. infantis, was deposited at the Guangdong Provincial Culture Collection of Microorganisms on September 26, 2022, with the deposit number GDMCC No: 62840, and the deposit address is on the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou; the Bifidobacterium longum subsp. infantis CCFM1271, taxonomically named Bifidobacterium longum subsp. infantis, was deposited at the Guangdong Provincial Culture Collection of Microorganisms on September 26, 2022, with the deposit number GDMCC No: 62841, and the deposit address is on the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou; the Bifidobacterium longum subsp. infantis CCFM1272, taxonomically named Bifidobacterium longum subsp. infantis, was deposited at the Guangdong Provincial Culture Collection of Microorganisms on September 26, 2022, with the deposit number GDMCC No: 62842, and the deposit address is on the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

4. The probiotic agent according to claim 2 or 3, wherein, The viable count of *Bifidobacterium longum subsp. infantis* in the probiotic agent is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

5. A starter containing Bifidobacterium longum subsp. infantis CCFM1269 as claimed in claim 1.

6. Use of the Bifidobacterium longum subsp. infantis as claimed in claim 1 in the preparation of health products.

7. Use of the Bifidobacterium longum subsp. infantis CCFM1269 as claimed in claim 1 in the preparation of health products that help regulate the intestinal flora.

8. Use of the Bifidobacterium longum subsp. infantis CCFM1269 as claimed in claim 1 in the preparation of probiotic products for regulating the intestinal flora.

9. According to the use described in claim 8, wherein, the regulation of the intestinal flora is achieved by regulating the Th1 / Th2 balance and IgA synthesis.

10. According to the use described in claim 8, wherein, The probiotic product also contains one or more of Bifidobacterium longum subsp. infantis CCFM1270, Bifidobacterium longum subsp. infantis CCFM1271, and Bifidobacterium longum subsp. infantis CCFM1272; The Bifidobacterium longum subsp. infantis CCFM1270, taxonomically named Bifidobacterium longum subsp. infantis, was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on September 26, 2022, with the deposit number GDMCC No: 62840, and the deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou; The Bifidobacterium longum subsp. infantis CCFM1271, taxonomically named Bifidobacterium longum subsp. infantis, was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on September 26, 2022, with the deposit number GDMCC No: 62841, and the deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou; The Bifidobacterium longum subsp. infantis CCFM1272, taxonomically named Bifidobacterium longum subsp. infantis, was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on September 26, 2022, with the deposit number GDMCC No: 62842, and the deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

11. The application according to claim 9, wherein, the product is used to increase the levels of Th1 / Th2-related cytokines and immunoglobulins in the colon of early-life mammals.

12. The application according to claim 9, wherein, the regulation of Th1 / Th2 balance and IgA synthesis includes at least one of the following effects: (1) Increasing the percentage contents of B cells, T cells, and Th cells in the mesenteric lymph nodes of early-life mammals; (2) Increasing the contents of IFN-γ, IgG2a, IgA, and sIgA, and the IgG2a / IgE ratio in the colon of early-life mammals; (3) Increasing the content of IFN-γ and the IgG2a / IgE ratio in the serum of early-life mammals; (4) Decreasing the contents of IL-4 and IgE in the colon of early-life male mammals; (5) Increasing the expression level of T-bet mRNA in the colon of early-life mammals; (6) Increasing the relative abundance of IgA-bound Alistipes in the feces of early-life male mammals.

Images