Bifidobacterium longum subsp. Infantis CCFM1272 and application thereof in adjusting Th1 / Th2 balance and IgA synthesis

Abstract

The invention discloses bifidobacterium longum subsp. Infantis CCFM1272 and application thereof in adjusting Th1 / Th2 balance and IgA synthesis, 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, T cells and Th 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 the patent application with application number "2022114738059" and application date of 2022-11-22. Technical Field

[0002] The invention relates to Bifidobacterium longum subspecies infantis CCFM1272 and application thereof in regulating Th1 / Th2 balance and IgA synthesis, belonging to the technical field of microorganisms. Background Art

[0003] With the increasing incidence of various non-communicable diseases, people have a strong interest in discovering the causes of diseases and focusing on early immune establishment to reduce the risk of later diseases. Especially during the period when infants and young children are more plastic, reasonable nutritional supplementation can minimize the occurrence of diseases during growth. After birth, infants show a preference for T helper cell type 2 immunity due to factors such as hormones and progesterone, which is the main reason for the increased risk of allergies, asthma and other diseases in childhood. In addition, imbalance of infant intestinal flora is common in modern society and may be a factor in the increased incidence of immune-mediated diseases. Breastfed infants may resist diarrhea, allergies and asthma, as well as inflammatory bowel disease. Breast milk not only provides basic nutrition for infants, but also promotes the establishment of intestinal flora. Therefore, it is necessary to explore the microbiota that can fight the occurrence of allergies, asthma and other possible immune diseases and promote the immune system.

[0004] Bifidobacterium longum infantis is the main bifidobacterium colonizer in the infant gut and exhibits advantages in promoting health in early life. Current studies have identified that the loss of bifidobacteria in early life is associated with an increased risk of several immune diseases and intestinal inflammation, but the mechanisms are unclear. Early life microbiota exposure may provide an opportunity to prevent allergy risk caused by Th1 / Th2 imbalance. Intestinal T helper 2 (Th2) and Th17 cytokines were silenced in breastfed infants given Bifidobacterium longum infantis EVC001. 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 infantis reduced intestinal inflammation by stably and continuously reshaping the intestinal microbiota of breastfed infants.

[0005] In recent years, studies have shown that Bifidobacterium longum infantis subspecies EVC001 can promote the differentiation of T0 cells into Th1 cells in infants and regulate the Th1 / Th2 balance in infants. Therefore, it is urgent to find a probiotic that can regulate the Th1 / Th2 balance and promote the production of IgA. Summary of the invention

[0006] 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;

[0007] 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;

[0008] 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;

[0009] 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;

[0010] 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.

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

[0012] 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.

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

[0014] The present invention also provides a starter containing the Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

[0015] In one embodiment, the method for preparing the starter is: inoculating Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 into a culture medium, culturing at 37°C for 24 to 48 hours to obtain a culture solution; centrifuging the culture solution to obtain bacterial cells; and resuspending the bacterial cells with physiological saline to obtain a starter.

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

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

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

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

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

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

[0022] The present invention also provides the use of one or more of the Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 in a probiotic product 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 modulate the intestinal flora of an early life mammal.

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

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

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

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

[0029] (4) reduce 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 Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 in the preparation of a health product that helps regulate intestinal flora.

[0033] Beneficial effects:

[0034] 1. The present invention screened out Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, and CCFM1272, which have the functions of regulating Th1 / Th2 balance and IgA secretion, and are specifically embodied in:

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

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

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

[0038] (4) Reduced colonic IL-4 and IgE levels in male young mice;

[0039] (5) Increased T-bet mRNA expression levels in the colon of both male and female mice;

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

[0041] 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.

[0042] 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.

[0043] Biomaterial Deposit

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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

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

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

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

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

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

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

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

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

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

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

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

[0059] Fig.12 : Changes in the percentage of B cells in mesenteric lymphocytes of male young mice in different groups; In the figure, *: p<0.05.

[0060] Fig.13 : Changes in the percentage of mesenteric lymphocyte T cells in male young mice in different groups; In the figure, *: p<0.05.

[0061] Fig.14 : Changes in the percentage of Th cells in mesenteric lymphocytes of male young mice in different groups; In the figure, *: p<0.05.

[0062] Fig.15 : Changes in colon IgA in male young mice in different groups; In the figure, *: p<0.05.

[0063] Fig.16 : Colon IgE in male young mice in different groups; in the figure, *: p<0.05.

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

[0065] Fig.18 : Changes in colon IgG / IgE in male young mice in different groups; In the figure, *: p<0.05.

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

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

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

[0069] Fig. 22 : sIgA-bound Alistipes in feces of male young mice in different groups; in the figure, *: p<0.05. DETAILED DESCRIPTION

[0070] The present invention is further described below in conjunction with specific embodiments and drawings.

[0071] The BALB / C mice involved in the following examples were purchased from Zhejiang Weitong Lihua Company; 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 Engineering of Jiangnan University;

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

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

[0074] The culture medium involved in the following examples is as follows:

[0075] MRS solid culture medium: peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, agar 15g / L.

[0076] MRS liquid culture medium: peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L.

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

[0078] 1. Screening

[0079] From the breast milk sample from Wuxi City, Jiangsu Province, 0.5 mL of the sample stored in 30% (v / v) glycerol was added to a 10 mL centrifuge tube containing 4.5 mL of physiological saline under a sterile environment to obtain 10 -1 Dilution, repeat the above dilution steps to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6dilution; 100 μL of gradient dilutions of different gradients were respectively applied on MRS solid culture medium, and cultured at 37°C for 72 h to obtain dilution coating plates; typical colonies on the dilution coating plates were picked up and streaked on MRS solid culture medium, and cultured at 37°C for 48 h to obtain purified colonies; purified colonies were picked up and inoculated into MRS liquid culture medium, and cultured at 37°C for 48 h to obtain strains FJSWXI4MI, FJSWXI8TI, FJSWXI10TI, BJSWXB6MNIM1, which were named FJSWXI4MI, FJSWXI8TI, FJSWXI10TI, BJSWXB6MNIM1, and named as CCFM1270, CCFM1271, CCFM1272, and CCFM1269, respectively.

[0080] 2. Identification

[0081] The genomes of CCFM1269, CCFM1270, CCFM1271, and CCFM1272 were extracted and 16S amplified. The conditions for 16S rDNA amplification 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 of amplified products and sequence alignment were carried out according to the method 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 were amplified and sequenced (by Suzhou Jinweizhi Biotechnology Co., Ltd.), and the 16S rDNA sequences of CCFM1269, CCFM1270, CCFM1271, and CCFM1272 obtained by sequencing analysis were compared in GenBank. The results showed that these strains were all Bifidobacterium longum subsp. infantis, and were named Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, CCFM1271, and CCFM1272.

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

[0083] The bacterial liquids of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 and CCFM1272 were prepared as follows:

[0084] Dip the bacterial liquid of Bifidobacterium longum subsp. infantis on MRS solid medium and culture at 37°C for 48h to obtain a single colony;

[0085] Pick a single colony and inoculate it into MRS liquid culture medium, and culture it at 37°C for 24 hours to obtain an activation solution; inoculate the activation solution into MRS liquid culture medium at an inoculum volume of 1% (v / v), and culture it at 37°C for 24 hours to obtain a primary seed solution;

[0086] The first-level seed solution was inoculated into MRS liquid culture medium at a rate of 1% (v / v), and cultured at 37°C for 24 hours to obtain the second-level seed solution;

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

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

[0089] Eight 6-week-old male pathogen-free (SPF) BALB / C female mice and four male mice were taken. They were raised at a room temperature of 22-24°C, a humidity of 40-60%, a 12h / 12h day and night alternation, and free access to food and water for one week. Then, they were caged together in a ratio of 2:1 between male and female. After the female mice became pregnant, the male mice were taken out. The gestation period was 3 weeks. The pups were gavaged from the age of 1 week old and were divided into the Control group (gavage with normal saline), CCFM1269, CCFM1270, and CCFM1272 (gavage with Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1272, respectively).

[0090] The experiment started after the animals were adapted for one week and lasted for 8 weeks. The specific treatments were as follows:

[0091] Control group (Control): 200 μL normal saline was gavaged starting 1 week after birth;

[0092] Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1272 groups: 10 μL of the corresponding bacterial suspension was administered orally starting 1 week after birth, with the dose being 1×109 CFU / bird / day.

[0093] The mice were gavaged until 3 weeks old. After the experiment, the mesenteric lymph nodes of the female mice were taken and placed in D-PBS to prepare single cell suspension. The mesenteric lymph node T cells, B cells, and Th cells were measured by flow cytometry. The results are shown in the table below. Figure 1-3 .

[0094] The number of T cells, B cells, and Th cells in mesenteric lymph nodes can usually reflect whether the strain can stimulate the individual to produce adaptive immunity. Figure 1 It can be seen that 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 of B cells in the mesenteric lymph nodes of female young 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 can be seen that the content of T cells in the mesenteric lymph nodes of the normal group was 60.44%, the CCFM1270 group was 68.73%, and the CCFM1272 group was 70.61%. The percentage of T cells in the mesenteric lymph nodes of female young 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 can be seen that the content of Th cells in the mesenteric lymph nodes of the normal group was 68.7%, and that of the CCFM1269 group was 71.86%. The percentage 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).

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

[0098] The colon of female young mice was placed in phosphate buffered saline and homogenized. The colon homogenate of each group of mice was tested by ELISA kit to determine the colon Th1 / Th2 related cytokines and immunoglobulins. The test results are shown in Figures 4 to 8 .

[0099] IFN-γ content and IgG2a content are the main cytokines and immunoglobulins of Th1 immune type. IgA content and sIgA content are mainly immunoglobulins of mucosal immunity. Figure 4It 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, while that of the CCFM1269 group was 3.56 ng / mg protein.

[0100] Depend on 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 concentration of IgG2a in the colon homogenate of the normal group was 1.89μg / mg protein, while that of the CCFM1269 group was 2.70μg / mg protein.

[0101] Depend on Figure 6 It can be seen that the IgA content in the colon of female young mice in 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 4.29μg / mg protein, that of the CCFM1269 group was 7.66μg / mg protein, that of the CCFM1270 group was 6.21μg / mg protein, and that of the CCFM1272 group was 6.98μg / mg protein.

[0102] Depend on 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 0.43μg / mg protein, that of CCFM1269 was 0.84μg / mg protein, and that of CCFM1272 group was 0.68μg / mg protein.

[0103] The increase in the IgG2a / IgE ratio indicates that the individual's immune type is transitioning from Th2 to Th1 and gradually reaching a balance. Figure 8 It can be seen that the IgG2a / IgE in the colon of female 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 0.89μg / mg protein, while that of the CCFM1269 group was 1.42μg / mg protein.

[0104] Example 5: Effects of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, and CCFM1272 on Th1 / Th2-related cytokines and immunoglobulins in the serum of female young 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 killed. The levels of Th1 / Th2 related cytokines and immunoglobulins in the serum of each group of mice were determined by ELISA kit. The test results are shown in Figure 9-10 .

[0106] like Fig. 9As shown, the IFN-γ content in the serum of female 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 Fig.10 As shown, the IgG2a / IgE in the serum of female 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, while that in the CCFM1269 group was 0.81.

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

[0109] The grouping and modeling of mice were the same as in Example 3. After the experiment, blood was collected and the mice were killed. RNA was extracted from the colon of the mice and reverse transcribed 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 Fig.11 .

[0110] Depend on Fig.11 It can be seen that CCFM1269 can significantly increase the relative expression of T-bet in the colon of female young mice (p<0.01), and the CCFM1269 group is 1.45 times that of the normal group.

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

[0112] Eight 6-week-old male pathogen-free (SPF) BALB / C female mice and four male mice were taken. They were raised for one week at a room temperature of 22-24°C, a humidity of 40-60%, a 12h / 12h day and night alternation, and free access to food and water. Then, they were caged together in a ratio of 2:1 between male and female. After the female mice became pregnant, the male mice were taken out. The gestation period was 3 weeks. The pups were gavaged from the age of 1 week old and were divided into the Control group (gavage with normal saline), CCFM1269, CCFM1270, and CCFM1271 (gavage with bacterial suspensions of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271, respectively).

[0113] The experiment started after the animals were adapted for one week and lasted for 8 weeks. The specific treatments were as follows:

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

[0115] Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271 groups: 10 μL of bacterial suspension was administered orally starting 1 week after birth, with an intragastric dose of 1×10 9 CFU / bird / day.

[0116] The mice were gavaged until 3 weeks old. After the experiment, the mesenteric lymph nodes of the female mice were taken and placed in D-PBS to prepare single cell suspension. The mesenteric lymph node T cells and B cells were measured by flow cytometry. The results are shown in Table 1. Figures 12-14 .

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

[0118] Depend on Fig.13 It can be seen that the percentage of T cells in the mesenteric lymph nodes of female young mice in 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%, 61.2% in the CCFM1270 group, 66.8% in the CCFM1271 group, and 67.3% in the CCFM1269 group.

[0119] Depend on Fig.14 It can be seen that the percentage of Th cells in the mesenteric lymph nodes of female 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 subsp. infantis CCFM1269, CCFM1270, and CCFM1271 on Th1 / Th2 balance and IgA content in the colon of male young mice

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

[0122] like Fig.15As shown, CCFM1269, CCFM1270, and CCFM1271 groups significantly increased the IgA content in the colon of male young 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.

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

[0124] like Fig.17 As shown, the CCFM1270 group significantly reduced the IL-4 content in the colon of male young mice (p<0.05). The IL-4 content 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.

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

[0126] Example 9: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271 on serum Th1 / Th2 balance in male young 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 killed. The Th1 / Th2 related cytokines in the serum of each group of mice were determined by ELISA kit. The test results are shown in Figure 19-20 .

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

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

[0130] Example 10: Effects of Bifidobacterium longum subsp. infantis CCFM1269, CCFM1270, and CCFM1271 on T-bet expression levels in the colon of male young mice

[0131] The grouping and modeling of mice were the same as in Example 3. After the experiment, blood was collected and the mice were killed. RNA was extracted from the colon of the mice and reverse transcribed 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 Fig.21 .

[0132] The transcription factor T-bet is a key factor that specifically regulates Th0 differentiation and acts as a Th1 / Th2 switch. T-bet transcription only occurs in Th1 cell lines, so 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, plays an important role in the development of Th1 cells by initiating the Th1 genetic program, and inhibits the synthesis of Th2 cytokines.

[0133] Depend on Fig.21 It can be seen that CCFM1269 can significantly increase the relative expression 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.

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

[0135] The grouping and modeling of mice were the same as in Example 3. After the experiment, the feces of mice were collected, the fecal IgA-binding bacteria were enriched, and the genomic DNA in the feces was extracted using the FastDNA Spin Kit (MP Biopharmaceuticals, USA). The V3-V4 region of the extracted genomic DNA was specifically amplified by PCR, and 16S rDNA sequencing was performed to analyze the changes in fecal flora. The analysis results are shown in Fig. 22 .

[0136] Depend on Fig. 22 It can be seen that the relative abundance of IgA-bound Alistipes in the male young mice Control group was 0.018, the CCFM1269 group was 0.11, the CCFM1270 group was 0.039, and the CCFM1271 group was 0.052, which significantly increased the relative abundance of IgA-bound Alistipes.

[0137] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Bifidobacterium longum subsp. infantis CCFM1272, taxonomically named Bifidobacterium longum subsp. infantis, was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with the deposit number GDMCC No: 62840.

2. A probiotic containing Bifidobacterium longum subspecies infantis CCFM1272 according to claim 1.

3. The probiotic according to claim 2, characterized in that The number of viable bacteria of Bifidobacterium longum infantis subspecies CCFM1272 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 CCFM1272 according to claim 1.

5. Use of Bifidobacterium longum subspecies infantis CCFM1272 according to claim 1 in the preparation of health products that help enhance immunity.

6. Use of Bifidobacterium longum subspecies infantis CCFM1272 according to claim 1 in the preparation of a health product that helps regulate intestinal flora.

7. Use of Bifidobacterium longum subspecies infantis CCFM1272 according to 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: The regulation of Th1 / Th2 balance and IgA synthesis 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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