CN115820488A - Bifidobacterium longum subspecies neonatorum capable of adjusting Th1/Th2 balance and IgA synthesis of young mice and application thereof - Google Patents

Abstract

The invention discloses a bifidobacterium longum subspecies neonatorum capable of adjusting Th1/Th2 balance and IgA synthesis of a young mouse and application thereof, and belongs to the technical field of microorganisms. The bifidobacterium longum subspecies infantis provided by the invention can adjust the Th1/Th2 balance and IgA content of a young mouse, and is specifically embodied in that: (1) Improving the percentage content of B cells, T cells and Th cells in mesenteric lymph nodes of mammals at the early stage of life; improving the content of IFN-gamma, igG2a, igA and sIgA in the colon and the ratio of IgG2 a/IgE; (3) Increasing IFN-gamma content, igG2a/IgE ratio, in serum of mammal in early life; (4) Reducing the IL-4 and IgE content in the colon of mammals in early life, and increasing the expression level of T-beta mRNA in the colon; (5) Improving the relative abundance of the IgA associated state Alisipes in the feces of the mammal male in early life.

Description

Bifidobacterium longum subspecies neonatorum capable of adjusting Th1/Th2 balance and IgA synthesis of young mice and application thereof

Technical Field

The invention relates to a bifidobacterium longum subspecies infantis capable of adjusting Th1/Th2 balance and IgA synthesis of a young mouse and application thereof, belonging to the technical field of microorganisms.

Background

With the increasing incidence of various non-infectious diseases, there is a strong interest in finding the cause of the disease and concerns about early immune establishment to reduce the risk of later disease development. Especially in the period of stronger plasticity of infants, the nutrition is reasonably supplemented, and the occurrence of diseases in the growth process can be reduced to the greatest extent. Infants after birth show a preference for T helper type 2 immunotypes due to hormones and progesterone, among other factors, which are the major causes of increased risk of allergies, asthma and other diseases in childhood. Furthermore, an imbalance in infant gut flora is common in modern society and may be a factor in the increased incidence of immune-mediated diseases. Breast-fed infants may be resistant to diarrhea, allergies and asthma, as well as inflammatory bowel disease. Breast milk not only provides essential nutrition for the infant, but also promotes the establishment of intestinal flora. Therefore, there is a need to develop a microbiota that can fight the development of allergies, asthma and other possible immune diseases and boost the immune system.

Bifidobacterium longum subspecies infantis are the predominant colonizers of bifidobacteria in the infant's gut and exhibit advantages in promoting early life health. Studies have now established that the loss of bifidobacteria early in life is associated with several immune diseases and an increased risk of intestinal inflammation, but the mechanism is not yet clear. Early in life microbiota exposure may provide an opportunity to prevent allergic risk caused by Th1/Th2 imbalance. Intestinal T helper 2 (Th 2) and Th17 cytokines are silenced in breast-fed infants given bifidobacterium longum subspecies of infants EVC 001. Administration of EVC001 to adult mice has therapeutic effects on allergic asthma by promoting Th1 and silencing Th2 immune responses. Furthermore, the introduction of bifidobacterium longum subspecies infants reduces intestinal inflammation by stabilizing and continuously remodeling the intestinal microbiota of breast-fed infants.

In recent years, research shows that Bifidobacterium longum subspecies of infants EVC001 can promote T in infants ₀ The cells differentiate towards Th1, regulating the infant Th1/Th2 balance. Therefore, a probiotic strain capable of regulating Th1/Th2 balance and promoting IgA production is urgently needed.

Disclosure of Invention

The invention provides a Bifidobacterium longum subsp. infantis, wherein the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. CCFM1269, bifidobacterium longum subsp. CCFM1270, bifidobacterium longum subsp. CCFM1271 or Bifidobacterium longum subsp. CCFM1272;

the Bifidobacterium longum subsp. Infantum CCFM1269, which is taxonomically named as Bifidobacterium longum subsp. Infantis, has been deposited in Guangdong province collection of microorganisms in 26 th 9 th 2022, and has a deposit number of GDMCC No:62839 with preservation address of building 5 of large yard No. 59 of Pieli Zhonglu 100, guangzhou city;

the Bifidobacterium longum subsp. Infantum CCFM1270 is classified and named as Bifidobacterium longum subsp. Infantis, is deposited in Guangdong province collection center of microorganism strains in 26 th 9 th 2022, and has a deposit number of GDMCC No:62840 with preservation address of building 5 of large yard No. 59 of Pieli Zhonglu 100, guangzhou city;

the Bifidobacterium longum subsp infantis CCFM1271 is classified and named as Bifidobacterium longum subsp. Infantis, is deposited in 26 th 9 th 2022 in Guangdong province microorganism strain collection center, and the deposit number is GDMCC No:62841 with preservation address of building 5 of large yard No. 59 of Pieli Zhonglu 100, guangzhou city;

the Bifidobacterium longum subsp. Infantum CCFM1272 is classified and named as Bifidobacterium longum subsp. Infantis, has been deposited in Guangdong province collection center of microorganism strains in 26 months 9 and 2022, and has a deposit number of GDMCC No:62842, the preservation address is Guangzhou city's Renlie Dayu No. 100, no. 59, building 5.

The invention also provides a probiotic preparation containing one or more of the bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 and CCFM1272.

In one embodiment, the probiotic comprises a bifidobacterium longum subspecies infantis viable count of not less than 1 x 10 ⁹ CFU/mL or 1X 10 ⁹ CFU/g。

In one embodiment, the probiotic is a bacterial suspension of bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

The invention also provides a leavening agent containing the bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

In one embodiment, the preparation method of the leavening agent is as follows: inoculating bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 into a culture medium, and culturing at 37 ℃ for 24-48 h to obtain a culture solution; centrifuging the culture solution to obtain thalli; the cells were resuspended in physiological saline to obtain a starter culture.

In one embodiment, the medium is MRS medium.

The invention also provides a medicament containing at least one strain of the bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272.

In one embodiment, the medicament has a viable count of Bifidobacterium longum subspecies infantis of not less than 1X 10 ⁹ CFU/mL or 1X 10 ⁹ CFU/g。

In one embodiment, the medicament comprises said bifidobacterium longum subsp.

In one embodiment, the medicament is for modulating the regulation of the Th1/Th2 balance and/or promoting IgA synthesis.

The invention also provides application of the bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 in preparing health-care products beneficial to enhancing immunity.

The invention also provides application of one or more of bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 in probiotic products for regulating Th1/Th2 balance and IgA synthesis.

In one embodiment, the product is used to increase colonic Th1/Th2 associated cytokines and immunoglobulin levels in mammals early in life.

In one embodiment, the product is used for modulating the intestinal flora of mammals early in life.

In one embodiment, said modulating Th1/Th2 balance and IgA synthesis comprises at least one of the following effects:

(1) Improving the percentage content of B cells, T cells and Th cells in mesenteric lymph nodes of mammals at the early stage of life;

(2) Increasing IFN-gamma, igG2a, igA, sIgA content in colon of mammal in early life stage, and IgG2a/IgE ratio;

(3) Increasing IFN-gamma content, igG2a/IgE ratio, in serum of mammal in early life;

(4) Reducing the colon IL-4 and IgE content of the male mammal in the early life stage;

(5) Increasing the expression level of T-beta mRNA in the colon of a mammal early in life;

(6) Improving the relative abundance of IgA associated state Alisipes in male mammal feces in early life.

The invention also provides application of one or more of bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 in preparing health care products beneficial to regulating intestinal flora.

Has the advantages that:

1. the invention screens Bifidobacterium longum subspecies (Bifidobacterium longum subsp. Infantis) CCFM1269, CCFM1270, CCFM1271 and CCFM1272 with the functions of regulating Th1/Th2 balance and IgA secretion, which are specifically embodied in that:

(1) The percentage contents of B cells, T cells and Th cells in mesenteric lymph nodes of female and male young mice are improved;

(2) Improving the contents of IFN-gamma, igG2a, igA and sIgA in the colon of the female and male young mice and the ratio of IgG2 a/IgE;

(3) The content of IFN-gamma in the serum of the female and male young mice is improved, and the ratio of IgG2a/IgE is increased;

(4) Reducing the content of IL-4 and IgE in the colon of the male young mouse;

(5) Improving the expression level of T-beta mRNA in the colon of the female and male young mice;

(6) The relative abundance of male young mouse feces IgA binding state Alisipes is improved.

2. The bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 and CCFM1272 screened by the invention are food safe strains, can be used for preparing products for adjusting Th1/Th2 balance and IgA content, and have great application prospect.

3. The culture process of the bifidobacterium longum subspecies infantis only needs to control the culture medium and some culture conditions, has relatively low cost and is easy to realize industrial production.

Biological material preservation

Bifidobacterium longum subsp. infantis (Bifidobacterium longum subsp. Infantis) CCFM1269, which is taxonomically named Bifidobacterium longum subsp. Infantis and has been deposited at the guangdong province collection center at 26/9 of 2022 with the deposit number GDMCC No:62839, the storage address is No. 59 building 5 of large institute of Mieli Middy 100, guangzhou city.

Bifidobacterium longum subsp. infantis (Bifidobacterium longum subsp. Infantis) CCFM1270, which is taxonomically named as Bifidobacterium longum subsp. Infantis and has been deposited at the guangdong province collection center at 26/9/2022 with the deposit number GDMCC No:62840, the storage address is No. 59 building 5 of large institute of Mieli Middy 100, guangzhou city.

Bifidobacterium longum subsp. infantis (Bifidobacterium longum subsp. Infantis) CCFM1271, which is taxonomically named as Bifidobacterium longum subsp. Infantis and has been deposited at the guangdong province collection center at 26/9/2022 with the deposit number GDMCC No:62841, the storage address is No. 59 building 5 of large institute of Mieli Middy 100, guangzhou city.

Bifidobacterium longum subsp. infantis (Bifidobacterium longum subsp. Infantis) CCFM1272, which is taxonomically named as Bifidobacterium longum subsp. Infantis and has been deposited at the guangdong province collection center at 26/9/2022 with the deposit number GDMCC No:62842, the storage address is No. 59 building 5 of large institute of Mieli Middy 100, guangzhou city.

Drawings

FIG. 1: the change of mesenteric lymph node B cells of different groups of female mice; in the figure, x: p <0.05.

FIG. 2: change of T cells of mesenteric lymph nodes of different groups of female mice; in the figure, x: p <0.05.

FIG. 3: the change condition of the mesenteric lymph node Th cells of different groups of female mice; in the figure, x: p <0.05

FIG. 4: colon IFN-gamma changes in female mice of different groups; in the figure, x: p <0.05.

FIG. 5: colon IgG2a changes in different groups of female mice; in the figure, x: p <0.05.

FIG. 6: colon IgA changes in female mice of different groups; in the figure, x: p <0.05.

FIG. 7: colon sIgA changes in female mice of different groups; in the figure, x: p <0.05.

FIG. 8: colon IgG2a/IgE changes in different groups of female mice; in the figure, x: p <0.05.

FIG. 9: the change of IFN-gamma of the serum of female rats in different groups; in the figure, x: p <0.05.

FIG. 10: serum IgG2a/IgE changes in different groups of females; in the figure, x: p <0.05.

FIG. 11: colon T-beta mRNA changes in different groups of female mice; in the figure, x: p <0.05.

FIG. 12: percentage change of mesenteric lymphocyte B cells of different groups of male juveniles; in the figure, x: p <0.05.

FIG. 13: percentage change of mesenteric lymphocyte T cells of different groups of male young mice; in the figure, x: p <0.05.

FIG. 14: the percentage content change of the Th cells of mesenteric lymph of different groups of male young mice; in the figure, x: p <0.05.

FIG. 15: colon IgA changes in different groups of male young mice; in the figure, x: p <0.05.

FIG. 16: colon IgE of different groups of male pups; in the figure, x: p <0.05.

FIG. 17: colon IL-4 of male pups in different groups; in the figure, x: p <0.05.

FIG. 18: colon IgG/IgE changes in different groups of male young mice; in the figure, x: p <0.05.

FIG. 19: serum IgG2a changes of different groups of male young mice; in the figure, x: p <0.05.

FIG. 20: the serum IgG2a/2aIgE change condition of different groups of male young mice; in the figure, x: p <0.05.

FIG. 21: colon T-beta mRNA changes in different groups of male young mice; in the figure, x: p <0.05.

FIG. 22: different groups of male young mouse feces sIgA combined Alisipes; in the figure, x: p <0.05.

Detailed Description

The invention is further elucidated with reference to a specific embodiment and a drawing.

BALB/C mice referred to in the following examples were purchased from Wentonlihua, zhejiang; bifidobacterium longum subsp. Infarnatum (Bifidobacterium longum subsp. Infarnatum) CCFM1269, CCFM1270, CCFM1271 and CCFM1272, which are isolated from the biological technology center of the food institute of south Jiangnan university, are referred to in the examples below;

the detection reagents referred to in the following examples are as follows:

ELISA kits for IFN-. Gamma.IgG 2a, igE, igA, sIgA, IL-4 were purchased from Fomeis.

The media involved in the following examples are as follows:

MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate ₂ PO ₄ ·3H ₂ O 2.6g/L、MgSO ₄ ·7H ₂ O 0.1g/L、MnSO ₄ 0.05g/L, tween 80 mL/L and agar 15g/L.

MRS liquid culture medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate ₂ PO ₄ ·3H ₂ O 2.6g/L、MgSO ₄ ·7H ₂ O 0.1g/L、MnSO ₄ 0.05g/L and Tween 80 1mL/L.

Example 1: screening and strain identification of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 and CCFM1272

1. Screening

Taking 0.5mL of breast milk sample from Wuxi city of Jiangsu province, storing the sample in 30% (v/v) glycerol, adding the sample into a 10mL centrifuge tube filled with 4.5mL of physiological saline under the aseptic environment to obtain 10 ^-1 Diluting the solution, repeating the above dilution steps to obtain 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Diluting the solution; respectively sucking 100 mu L of gradient dilution liquid with different gradients, coating the gradient dilution liquid on an MRS solid culture medium, and culturing for 72h at 37 ℃ to obtain a diluted coating plate; selecting typical colonies on the diluted coating plate, streaking on an MRS solid culture medium, and culturing at 37 ℃ for 48h to obtain purified colonies; and (3) selecting a purified colony, inoculating the colony into an MRS liquid culture medium, and culturing at 37 ℃ for 48h to obtain strains FJSWXI4MI, FJSWXI8TI, FJSWXI10TI and BJSWXB6MNIM1 which are named as CCFM1270, CCFM1271, CCFM1272 and CCFM1269 respectively.

2. Identification

Extracting genomes of CCFM1269, CCFM1270, CCFM1271 and CCFM1272, and performing 16S amplification, wherein 16SrDNA amplification conditions are as follows: 5min at 95 ℃;35 cycles (95 ℃ 30s,55 ℃ 30s,72 ℃ 2 min); 10min at 72 ℃. An amplification primer: 27F: (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R: (5'-TACGGCTACCTTGTTACGACTT-3') the purification and sequence alignment of the amplified product was carried out according to the method described in the literature (Turroni F et al. Expanding the conversion of the bipolar amplification in the Human endogenous transform [ J ]. Appl Environ Microb.2009;75 (6): 1534-45). The 16S rDNA sequences of CCFM1269, CCFM1270, CCFM1271 and CCFM1272 were amplified and sequenced (Jin Weizhi Biotech, suzhou), and the 16S rDNA sequences of CCFM1269, CCFM1270, CCFM1271 and CCFM1272 obtained by sequencing analysis were aligned in GenBank, and the results showed that the strains were all Bifidobacterium longum subspecies, namely Bifidobacterium longum subspecies (Bifidobacterium longum subsp. Infantis) CCFM1269, CCFM1270, CCFM1271 and CCFM1272.

Example 2: preparation of Bifidobacterium longum infant sub-inoculum suspension

The bacterial liquid of bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 and CCFM1272 is prepared by the following method:

streaking the dipped bifidobacterium longum subspecies infantis liquid on an MRS solid culture medium, and culturing for 48h at 37 ℃ to obtain a single colony;

selecting a single colony, inoculating the single colony into an MRS liquid culture medium, and culturing for 24h at 37 ℃ to obtain an activating solution; inoculating the activated solution into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ for 24h to obtain a first-level seed solution;

inoculating the primary seed liquid into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ for 24h to obtain a secondary seed liquid;

inoculating the secondary seed liquid into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ for 24h to obtain a bacterial liquid; centrifuging 6000g of the bacterial liquid for 15min, and collecting precipitates; washing the precipitate with physiological saline buffer solution twice, and centrifuging again at 6000g for 10min to obtain thallus; resuspending the lactobacillus cells in physiological saline to a cell concentration of 1X 10 ⁹ CFU/mL to obtain Bifidobacterium longum subspecies infant liquid.

Example 3: effect of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270 and CCFM1272 on mesenteric lymph node lymphocyte content of female mouse

Taking 8 male pathogen-free (SPF) BALB/C female mice and 4 male mice of 6 weeks old, breeding for 1 week under the conditions of free feeding and drinking water at the room temperature of 22-24 ℃ and the humidity of 40-60 percent alternately day and night of 12h/12h, and then feeding according to the weight ratio of male to female 2:1, taking out male mice after female mice are pregnant, carrying out intragastric administration when the female mice are pregnant, wherein the gestation period is 3 weeks, and the young mice begin intragastric administration after the birth of 1 week, and are divided into a Control group (intragastric administration normal saline), a CCFM1269 group, a CCFM1270 group and a CCFM1272 group (respectively intragastric administration Bifidobacterium longum subsp.

The experiment was started 1 week after the animals were acclimatized for a total of 8 weeks. The specific treatment is as follows:

control group (Control): beginning gavage with 200 μ L of normal saline 1 week after birth;

bifidobacterium longum subspecies infantis CCFM1269 group, CCFM1270 group, CCFM1272 group: beginning to perform intragastric administration of 10 μ L of corresponding bacterial suspension 1 week after birth to make intragastric administration dosage be 1 × 10 ⁹ CFU/only/day.

And (3) perfusing the stomach to 3 weeks, after the experiment is finished, taking mesenteric lymph nodes of the female mice, placing the mesenteric lymph nodes in D-PBS to prepare single cell suspension, and measuring T cells, B cells and Th cells of the mesenteric lymph nodes by flow cytometry, wherein the measurement results are respectively shown in the figures 1-3.

The numbers of T cells, B cells and Th cells of mesenteric lymph nodes can generally reflect whether strains can stimulate individuals to generate adaptive immunity. As can be seen from FIG. 1, the mesenteric lymph node of the normal group has a B cell content of 13.16%, the CCFM1270 group has 22.6%, the CCFM1272 group has 19.51%, the CCFM1269 group has 18.3%, the female mouse mesenteric lymph nodes of the CCFM1270, CCFM1272 and CCFM1269 groups have a significantly higher percentage of B cells than the normal group (p < 0.05);

as can be seen from FIG. 2, the T cell content in mesenteric lymph nodes of the normal group is 60.44%, that in CCFM1270 is 68.73%, that in CCFM1272 is 70.61%, that in CCFM1270 and that in CCFM1272 female young mouse mesenteric lymph nodes are significantly higher (p <0.05 );

as can be seen from FIG. 3, the content of Th cells in mesenteric lymph nodes of the normal group was 68.7%, that of CCFM1269 group was 71.86%, and that of the female mouse mesenteric lymph nodes of CCFM1269 group was significantly higher than that of the normal group (p < 0.05).

Example 4: effect of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270 and CCFM1272 on female mouse Th1/Th2 associated cytokines and immunoglobulins

The colons of the female mice are taken and put into phosphate buffer saline solution and homogenized, the colons of each group of mice are measured by an ELISA kit, the colons Th1/Th2 related cytokines and immunoglobulins are measured, and the measurement results are respectively shown in figures 4-8.

IFN-gamma content, igG2a content, cytokines and immunoglobulins, respectively, that are the major Th1 immune types. IgA content and sIgA content are mainly immunoglobulins for mucosal immunity. As can be seen from FIG. 4, the content of IFN-gamma in the colon of female mice in CCFM1269 group is significantly higher than that in the normal group (p < 0.05), the concentration of IFN-gamma in colon homogenate of the normal group is 2.60ng/mg protein, and the concentration of IFN-gamma in CCFM1269 group is 3.56ng/mg protein;

as can be seen from FIG. 5, the content of IgG2a in the colon of female mice in CCFM1269 group is significantly higher than that in the normal group (p < 0.05), the concentration of IgG2a in colon homogenate of the normal group is 1.89 μ g/mg protein, and the concentration of IgG2a in CCFM1269 group is 2.70 μ g/mg protein;

as can be seen from FIG. 6, the IgA content in the colon of female mice in CCFM1269, CCFM1270 and CCFM1272 groups is significantly higher than that in the normal group (p < 0.05), the homogenized IgA concentration in the colon of the normal group is 4.29 μ g/mg protein, the IgA concentration in the colon of the CCFM1269 group is 7.66 μ g/mg protein, the IgA concentration in the colon of the CCFM1270 group is 6.21 μ g/mg protein, and the IgA concentration in the colon of the CCFM1272 group is 6.98 μ g/mg protein;

as can be seen from FIG. 7, the sIgA content in the colon of female mice in CCFM1269 and CCFM1272 groups is significantly higher than that in the normal group (p < 0.05), the colon homogenate sIgA concentration in the normal group is 0.43 μ g/mg protein, CCFM1269 is 0.84 μ g/mg protein, and CCFM1272 group is 0.68 μ g/mg protein;

an increased ratio of IgG2a/IgE indicates that the individual's immune type transitions from Th2 to Th1 and gradually reaches equilibrium. As can be seen from FIG. 8, the ratio of IgG2a/IgE in the colon of female mice in CCFM1269 group is 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 is 0.89. Mu.g/mg protein, and the ratio of IgG2a to IgE in CCFM1269 group is 1.42. Mu.g/mg protein.

Example 5: effect of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270 and CCFM1272 on female mouse serum Th1/Th2 associated cytokines and immunoglobulins

The mice were grouped and treated as in example 3. After the experiment is finished, blood is taken and mice are killed, the content of Th1/Th2 related cytokines and immunoglobulin in the serum of each group of mice is measured by an ELISA kit, and the detection result is shown in figures 9-10.

As shown in FIG. 9, the content of IFN-gamma in the serum of the female mouse in the CCFM1269 group is significantly higher than that in the normal group (p < 0.05), the concentration of IFN-gamma in the serum of the normal group is 0.74ng/mL, and the concentration of IFN-gamma in the serum of the CCFM1269 group is 0.92ng/mL;

as shown in fig. 10, the serum IgG2a/IgE of the female mice in CCFM1269 group was significantly higher than that in the normal group (p < 0.05), the ratio of IgG2a to IgE in the normal group serum was 0.60, and the ratio of IgG2a to IgE in CCFM1269 group was 0.81.

Example 6: effect of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1272 on Colon T-beta expression level in female mouse

The grouping and modeling of mice were the same as in example 3. After the experiment is finished, blood is taken and the mice are killed, the colon of each mouse is taken to extract RNA, reverse transcription is carried out by using the kit, the expression level of T-beta in the colon of each group of mice is measured, and the detection result is shown in figure 11.

As can be seen from FIG. 11, CCFM1269 can significantly increase the relative expression level of T-beta in colon of female mouse (p < 0.01), and CCFM1269 group is 1.45 times of that of normal group.

Example 7: effect of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 on mesenteric lymph node lymphocyte content in male young mice

8 male pathogen free (SPF) BALB/C female mice and 4 male mice of 6 weeks old are taken, and after being fed for 1 week under the conditions of free eating and drinking water at the room temperature of 22-24 ℃ and the humidity of 40-60 percent alternately for 12h/12h, the male mice are bred according to the ratio of male to female 2:1, taking out male mice after female mice are pregnant, carrying out intragastric administration when the female mice are pregnant, wherein the gestation period is 3 weeks, and the young mice begin intragastric administration after the birth of 1 week, and are divided into a Control group (intragastric administration normal saline), a CCFM1269 group, a CCFM1270 group and a CCFM1271 group (respectively intragastric administration Bifidobacterium longum subsp.

The experiment was started 1 week after the animals were acclimatized for a total of 8 weeks. The specific treatment is as follows:

control group: gavage of 200 μ L of saline was started as a control 1 week after birth;

bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 group: beginning to lavage 10 μ L bacterial suspension 1 week after birth to make the lavage dose be 1 × 10 ⁹ CFU/only/day.

And (3) irrigating the stomach to 3 weeks, after the experiment is finished, taking mesenteric lymph nodes of the female mice, placing the mesenteric lymph nodes in D-PBS to prepare single cell suspension, and measuring T cells and B cells of the mesenteric lymph nodes by flow cytometry, wherein the measurement results are respectively shown in figures 12-14.

As can be seen from FIG. 12, the percentage content of B cells in mesenteric lymph nodes of male young mice in the CCFM1270 and CCFM1271 groups is significantly higher than that in the normal group (p < 0.05), the content of B cells in mesenteric lymph nodes of the normal group is 13.50%, the content of B cells in the CCFM1270 group is 20.20%, and the content of B cells in the CCFM1271 group is 18.27%;

as can be seen from FIG. 13, the percentage of T cells in 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 T cell content in mesenteric lymph nodes of the normal group was 41.9%, that in the CCFM1270 group was 61.2% and that in the CCFM1271 group was 66.8%; CCFM1269 group 67.3%;

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

Example 8: effect of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270 and CCFM1271 on colonic Th1/Th2 balance and IgA content in male young mice

Groups of mice were as in example 3. After the experiment is finished, the colon of the mouse is taken and placed in phosphate buffer saline solution and homogenized, the colon homogenate of each group of mice is measured by an ELISA kit, and the detection result is shown in figures 15-18.

As shown in FIG. 15, the group CCFM1269, CCFM1270 and CCFM1271 significantly increased IgA content in the colon of male young mice (p < 0.01), the homogenized IgA concentration in the colon of the normal group was 1.03. Mu.g/mg protein, the group CCFM1269 was 1.78. Mu.g/mg protein, the group CCFM1270 was 1.90. Mu.g/mg protein, and the group CCFM1271 was 2.03. Mu.g/mg protein;

as shown in FIG. 16, CCFM1269 and CCFM1271 significantly reduced the IgE content in the colon of male young mice (p < 0.05), the homogenized IgE concentration in the colon of the normal group was 2.43. Mu.g/mg protein, that of CCFM1269 group was 1.81. Mu.g/mg protein, and that of CCFM1271 group was 1.78. Mu.g/mg protein;

as shown in FIG. 17, the content of IL-4 in the colon of the male pup was significantly reduced in the CCFM1270 group (p < 0.05), the content of IL-4 in the colon of the normal group was 390.56pg/mg protein, and the content of IL-4 in the colon of the CCFM1270 group was 309.58pg/mg protein.

As shown in fig. 18, CCFM1269, CCFM1270, CCFM1271 significantly increased IgG2a/IgE (p < 0.05) in the male pups, the ratio of homogenized IgG2a to IgE in the normal group was 0.76, that in ccfm1269 group was 1.07, that in ccfm1270 group was 1.07, and that in ccfm1271 group was 0.98.

Example 9: bifidobacterium longum subspecies infantis CCFM1269, CCFM1270 and CCFM1271 influence on male young mouse serum Th1/Th2 balance

The mice were grouped and treated as in example 3. After the experiment is finished, blood is taken and mice are killed, and the Th1/Th2 related cytokines in the serum of each group of mice are measured by an ELISA kit, and the detection results are shown in figures 19-20.

As shown in FIG. 19, the CCFM1269 group significantly increased the IgG2a content (p < 0.05) in the sera of the male pups, the normal group serum IgG2a concentration was 0.58ng/mL, and the CCFM1269 group was 0.72ng/mL;

as shown in fig. 20, the CCFM1269 group significantly increased IgG2a/IgE in serum (p < 0.05), the normal group serum IgG2a to IgE ratio was 0.60, and the ccfm1269 group was 0.79.

Example 10: effect of Bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 on Colon T-beta expression level in male young mouse

The grouping and modeling of mice were the same as in example 3. After the experiment is finished, blood is taken and mice are killed, the colon of the mice is taken to extract RNA, reverse transcription is carried out by using the kit, the expression level of T-beta in the colon of each group of mice is measured, and the detection result is shown in figure 21.

The transcription factor T-beta is a key factor which specifically regulates Th0 differentiation and plays a role of a Th1/Th2 switch. T-beta transcription occurs only in Th1 cell lines, and therefore, it is considered that T-beta is selectively expressed in Th1 cells, and T-beta is selectively expressed in Th1 cells as a Th 1-specific transcription factor, plays an important role in development of Th1 cells by initiating the Th1 genetic program, and inhibits synthesis of Th2 cytokines.

As can be seen from FIG. 21, CCFM1269 can significantly increase the relative expression of T-beta in colon of male young mouse (p < 0.05), and CCFM1269 group is 1.41 times of that of normal group.

Example 11: bifidobacterium longum subspecies infantis CCFM1269, CCFM1270 and CCFM1271 have influence on male young mouse fecal IgA binding bacteria

The mice were grouped and modeled as in example 3. After the experiment is finished, collecting mouse feces, enriching feces IgA binding bacteria, extracting genomic DNA in the feces by using FastDNA Spin Kit (American MP biological medicine company), carrying out specific PCR amplification on a V3-V4 region of the extracted genomic DNA, carrying out 16S rDNA sequencing, analyzing the change of the feces flora, and obtaining an analysis result shown in figure 22.

As can be seen from FIG. 22, the relative abundance of IgA binding Alisipes was 0.018 in the Control group, 0.11 in the CCFM1269 group, 0.039 in the CCFM1270 group, and 0.052 in the CCFM1271 group of male pups, which significantly increased the relative abundance of IgA binding Alisipes.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. Bifidobacterium longum subsp. Infantis, wherein said Bifidobacterium longum subsp. Infantis is Bifidobacterium longum subsp. Infantis CCFM1269, bifidobacterium longum subsp. Infantum CCFM1270, bifidobacterium longum subsp. Infantum CCFM1271 or Bifidobacterium longum subsp. Infantum CCFM1272;

the Bifidobacterium longum subsp. Infantum CCFM1269, which is taxonomically named as Bifidobacterium longum subsp. Infantis, has been deposited in Guangdong province collection of microorganisms in 26 th 9 th 2022, and has a deposit number of GDMCC No:62839 with preservation address of building 5 of large yard No. 59 of Pieli Zhonglu 100, guangzhou city;

the Bifidobacterium longum subsp. Infantum CCFM1270 is classified and named as Bifidobacterium longum subsp. Infantis, is deposited in Guangdong province collection center of microorganism strains in 26 th 9 th 2022, and has a deposit number of GDMCC No:62840 with preservation address of building 5 of large yard No. 59 of Pieli Zhonglu 100, guangzhou city;

the Bifidobacterium longum subsp. Infantum CCFM1271 is classified and named as Bifidobacterium longum subsp. Infantis, has been deposited in Guangdong province collection center of microorganism strains in 26 th 9 th 2022, and has a deposit number of GDMCC No:62841 with preservation address of building 5 of large yard No. 59 of Pieli Zhonglu 100, guangzhou city;

the Bifidobacterium longum subsp. Infantum CCFM1272 is classified and named as Bifidobacterium longum subsp. Infantis, has been deposited in Guangdong province collection center of microorganism strains in 26 months 9 and 2022, and has a deposit number of GDMCC No:62842, the preservation address is Guangzhou city's Renlie Dayu No. 100, no. 59, building 5.

2. A probiotic comprising one or more of bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271, CCFM1272 according to claim 1.

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

4. A starter culture comprising bifidobacterium longum subspecies infantis CCFM1269, CCFM1270, CCFM1271 or CCFM1272 according to claim 1.

5. A medicament comprising at least one strain of Bifidobacterium longum subsp.

6. Use of a bifidobacterium longum subsp.

7. Use of one or more of bifidobacterium longum subsp.

8. Use of one or more of bifidobacterium longum subsp.

9. The use according to claim 8, wherein the product is for increasing colonic Th1/Th2 associated cytokines and immunoglobulin levels in early life mammals.

10. The use of claim 8, wherein said modulation of Th1/Th2 balance and IgA synthesis comprises at least one of:

(1) Improving the percentage content of B cells, T cells and Th cells in mesenteric lymph nodes of mammals at the early stage of life;

(2) Increasing the IFN-gamma, igG2a, igA, sIgA content, igG2a/IgE ratio in the colon of the mammal in early life;

(3) Increasing IFN-gamma content, igG2a/IgE ratio, in serum of mammal in early life;

(4) Reducing the colon IL-4 and IgE content of the male mammal in the early life stage;

(5) Increasing the expression level of T-beta mRNA in the colon of a mammal early in life;

(6) Improving the relative abundance of IgA associated state Alisipes in male mammal feces in early life.

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