BIFIDOBACTERIUM BREVE 207-1 AND USE THEREOF

Abstract

The present application relates to a Bifidobacterium breve or progeny thereof. Specifically, the present application relates to Bifidobacterium breve 207-1 and a composition, culture, food product or dietary supplement containing the same. The present application also relates to the use of Bifidobacterium breve 207-1 and the composition, culture, food product or dietary supplement containing the same in medicine.

Claims

1-10. (canceled)

11. A Bifidobacterium breve or progeny thereof, in which the Bifidobacterium breve is deposited in Guangdong Microbial Culture Collection Center, and has an accession number of GDMCC No. 60962.

12. A composition, which comprises the Bifidobacterium breve or progeny thereof according to claim 11.

13. The composition according to claim 12, wherein the composition further comprises a microorganism selected from the group consisting of bacterium, fungus, or any combination thereof.

14. The composition according to claim 13, characterized by one or more of the following: (1) the microorganism is a probiotic; (2) the bacterium is selected from the group consisting of Lactobacillus spp., Bifidobacterium spp., Bacillus spp., Propionibacterium spp., Streptococcus spp., Lactococcus spp., Pediococcus spp., Enterococcus spp., Staphylococcus spp., or any combination thereof; (3) the fungus is a yeast; and (4) the fungus is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Kluyveromyces marxianus, or any combination thereof.

15. The composition according to claim 14, characterized by one or more of the following: (1) the bacterium of the Lactobacillus spp. is selected from the group consisting of: Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus jensenii, Lactobacillus iners, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, or any combination thereof; (2) the bacterium of the Bifidobacterium spp. is selected from the group consisting of: Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium adolescentis, or any combination thereof; (3) the bacterium of the Bacillus spp. is selected from the group consisting of: Bacillus subtilis, Bacillus coagulans, or any combination thereof; (4) the bacterium of the Propionibacterium spp. is selected from the group consisting of: Propionibacterium shermanii, Propionibacterium freudenreichii, Propionibacterium acidipropionici, or any combination thereof; (5) the bacterium of the Streptococcus spp. is selected from the group consisting of: Streptococcus thermophilus, Streptococcus salivarius, or any combination thereof; (6) the bacterium of the Lactococcus spp. is Lactococcus lactis; and (7) the bacterium of the Enterococcus spp. is selected from the group consisting of: Enterococcus faecalis, Enterococcus faecium, or any combination thereof.

16. The composition according to claim 12, characterized by one or more of the following: (1) the composition further comprises an additional additive; (2) the composition further comprises a nutrient; (3) the composition is used as a starter culture; (4) the Bifidobacterium breve in the composition is used as a starter culture to participate in a fermentation process; and (5) the Bifidobacterium breve is present in the composition in an amount of 10.sup.6 to 10.sup.12 CFU/dose.

17. The composition according to claim 16, characterized by one or more of the following: (1) the nutrient is selected from the group consisting of dietary fiber, prebiotic, protein, lipid, mineral, vitamin, plant ingredient, plant extract, plant polyphenol, or any combination thereof; (2) the starter culture is a starter culture for plant fermented product or a starter culture for dairy product; and (3) the Bifidobacterium breve is present in the composition in an amount of 10.sup.8 to 10.sup.12 CFU/dose.

18. A food product or dietary supplement, which comprises the Bifidobacterium breve or progeny thereof according to claim 11 or a composition comprising the Bifidobacterium breve or progeny thereof.

19. The food product or dietary supplement according to claim 18, characterized by one or more of the following: (1) the food product is selected from the group consisting of solid beverage, candy or fruit juice, or the food product is a dairy product; (2) the food product is selected from the group consisting of yogurt, flavored fermented milk, lactic acid bacteria beverage and cheese; (3) the dietary supplement is formulated for oral administration; (4) the dietary supplement is in the form of a pill, powder, capsule, tablet, granule powder, opercula, orally dissolving granule, sachet, dragee or liquid; (5) the Bifidobacterium breve is present in an amount of 10.sup.6 to 10.sup.12 CFU/dose in the food product or dietary supplement; and (6) the Bifidobacterium breve is present in an amount of 10.sup.8 to 10.sup.12 CFU/dose in the food product or dietary supplement.

20. A pharmaceutical composition, which comprises the Bifidobacterium breve or progeny thereof according to claim 11; and optionally, comprises a pharmaceutically acceptable carrier.

21. The pharmaceutical composition according to claim 20, characterized by one or more of the following: (1) the pharmaceutical composition comprises a formulation of the Bifidobacterium breve; (2) the pharmaceutical composition is formulated for oral administration; (3) the pharmaceutical composition is in the form of a pill, powder, capsule, tablet, granule powder, opercula, orally dissolving granule, sachet, dragee or liquid; (4) the Bifidobacterium breve is present in an amount of 10.sup.6 to 10.sup.12 CFU/dose in the pharmaceutical composition; and (5) the Bifidobacterium breve is present in an amount of 10.sup.8 to 10.sup.12 CFU/dose in the pharmaceutical composition.

22. A culture, which comprises the Bifidobacterium breve or progeny thereof according to claim 11; and optionally, comprises a nutrient-providing ingredient.

23. The culture according to claim 22, characterized by one or more of the following: (1) the culture further comprises a microorganism selected from the group consisting of bacterium, fungus, or any combination thereof; (2) the culture further comprises a yeast; (3) the nutrient-providing ingredient is selected from the group consisting of solid or liquid medium, feeder cell layer, or any combination thereof; (4) the nutrient-providing ingredient comprises protein, carbohydrate, fat, probiotic, enzyme, vitamin, immunomodulator, milk substitute, mineral, amino acid, or any combination thereof; (5) the culture further comprises a cell-free culture filtrate of the Bifidobacterium breve or progeny thereof; (6) the culture further comprises a derivative of the Bifidobacterium breve or progeny thereof; optionally, the derivative is selected from the group consisting of metabolite, enzyme, cellular structural component, extracellular polysaccharide, bacteriocin, compound containing immunogenic component, or any combination thereof; (7) the nutrient-providing ingredient further comprises cell wall or component thereof of the Bifidobacterium breve or progeny thereof; and (8) the microorganism is alive or dead, as a lysate or extract, or as a bacterial product, or as a supernatant.

24. Use of (1) the Bifidobacterium breve or progeny thereof according to claim 11 or (2) a composition comprising the Bifidobacterium breve or progeny thereof or (3) a pharmaceutical composition comprising the Bifidobacterium breve or progeny thereof or (4) a culture comprising the Bifidobacterium breve or progeny thereof in the manufacture of a medicament or dietary supplement or health care product, in which the medicament or dietary supplement or health care product is used for inhibiting inflammation or alleviating an inflammatory disease in a subject, or for improving an immunity of a subject, or for preventing a bacterial or viral infection or an autoimmune disease in a subject, or for improving or alleviating an allergic reaction or symptom thereof in a subject.

25. The use according to claim 24, characterized by one or more of the following: (1) the inflammatory disease is selected from the group consisting of a disease caused by retina inflammation, a disease caused by skin inflammation, a disease caused by respiratory tract inflammation, and a disease caused by digestive tract inflammation; (2) the bacterial or viral infection is selected from the group consisting of bacterial influenza, viral influenza, urinary tract infection, vaginitis and cervicitis; (3) the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, diabetes, rheumatoid arthritis and autoimmune encephalitis; (4) the allergic reaction or related symptom thereof is selected from the group consisting of eczema, atopic dermatitis, asthma and food allergy; (5) the medicament or dietary supplement or health care product can promote the activation of an immune cell, promote the production of a cytokine; (6) the medicament or dietary supplement or health care product can inhibit an IgE-mediated allergic reaction; (7) the medicament or dietary supplement or health care product is used alone or in combination with an additional antifungal agent, analgesic, anti-inflammatory drug, healing agent, or moisturizer; (8) the subject is a mammal; and (9) the subject is a human.

26. The use according to claim 25, characterized by one or more of the following: (1) the disease caused by retina inflammation is retinitis or keratitis; (2) the disease caused by skin inflammation is dermatitis or eczema; (3) the disease caused by respiratory tract inflammation is upper respiratory tract infection; (4) the disease caused by digestive tract inflammation is inflammatory bowel disease; (5) the cytokine is selected from the group consisting of IL-10, IL-6, IL-12, TNF-α, or any combination thereof; and (6) the cytokine is anti-inflammatory cytokines IL-10.

27. Use of the Bifidobacterium breve or progeny thereof according to claim 11 or a composition comprising the Bifidobacterium breve or progeny thereof or a culture comprising the Bifidobacterium breve or progeny thereof in the manufacture of a starter culture, in which the starter culture is used in the fermentation of a solid food or liquid food.

28. The use according to claim 27, characterized by one or two of the following: (1) the solid food is cheese; and (2) the liquid food is yogurt, flavored fermented milk, lactic acid bacteria drinks, or any combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1 shows the colony morphology of Bifidobacterium breve 207-1 cultured on the plate, wherein, FIG. 1A shows the colony morphology of Bifidobacterium breve 207-1 cultured on the BL plate, FIG. 1B shows the colony morphology of Bifidobacterium breve 207-1 cultured on the TOS plate.

[0096] FIG. 2 shows a microscopic view of Gram-stained Bifidobacterium breve 207-1.

[0097] FIG. 3 shows the serum IgE levels of OVA model mice that received or did not receive treatment with Bifidobacterium breve 207-1.

[0098] Notes on the Deposition of Biological Materials

[0099] The Bifidobacterium breve 207-1 has been deposited in the Guangdong Microbial Culture Collection Center (GDMCC) located on the 5th floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou. It has the accession number GDMCC No. 60962, and the deposit time is Jan. 15, 2020.

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

[0100] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than limiting the invention.

[0101] Unless otherwise specified, the experiments and methods described in the examples are basically performed according to conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in: Sambrook, Fritsch and Maniatis, “MOLECULAR CLONING: A LABORATORY MANUAL”, 2.sup.nd edition (1989); “CURRENT PROTOCOLS IN MOLECULAR BIOLOGY”, Edited by F. M. Ausubel et al., (1987); “METHODS IN ENZYMOLOGY” (series, Academic Publishing Company): “PCR 2: A PRACTICAL APPROACH”, Edited by M. J. MacPherson, B. D. Hames, and G. R. Taylor (1995); and ANIMAL CELL CULTURE, Edited by R. I. Frescheni (1987).

[0102] In addition, if the specific conditions were not specified in the examples, it should be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer thereof were all conventional products that could be purchased commercially. Those skilled in the art know that the embodiments describe the present invention by way of illustration, and are not intended to limit the protection scope claimed by the present invention. All publications and other references mentioned herein are incorporated into this application by reference in their entirety.

EXAMPLE 1

Isolation and Identification of Strains

[0103] In the present application, 265 strains were isolated from stool samples of normal term neonates born in West China Women's and Children's Hospital of Sichuan University, and one strain of Bifidobacterium breve was screened from them, and was named Bifidobacterium breve 207-1. Among them, the inclusion criteria for newborns were: living in five districts of Chengdu; gestational age of 37-42 weeks, birth weight between 2500-4000 g, and the babies had no congenital abnormalities or birth defects. The exclusion criteria were: mothers had used antibiotics within one month before delivery; parents had infectious diseases such as AIDS, tuberculosis, hepatitis B, and newborns were not suitable for collecting feces due to severe diseases such as neonatal pneumonia.

[0104] Fresh feces were collected from 1-4 months infants and placed in aseptic feces collection tubes. After sampling, it was temporarily stored at 4° C. and sent to the laboratory at low temperature by the sampling personnel for dilution and culture of the stool sample. If it could not be operated immediately, it was stored under anaerobic condition at 4° C. and cultured on the same day.

[0105] 0.5 g of feces was weighed, added with 4.5 mL of fecal diluent (4.5 g of KH.sub.2PO.sub.4, 6.0 g of Na.sub.2HPO.sub.4, 0.5 g of L-cysteine hydrochloride, 0.5 g of Tween-80, 1.0 g of agar were mixed with 1000 ml of distilled water, autoclaved for sterilization at 121° C. for 15 min for later use), shaken and mixed thoroughly, and diluted by 10 times in series. 100 μL of fecal mixture with suitable dilution degree was taken and spread on a plate with (5% horse blood) glucose, blood, liver culture medium (BL culture medium) by a L-shaped rod, and anaerobic culture was carried out at 37° C. for 48 h. The suspected Bifidobacterium colonies (those colonies which were brown or brownish red, had a smooth and moist surface, a round and raised shape and a neat edge, and had a diameter of about 3-5 mm) on the BL plate were picked up, subcultured on a plate with a Bifidobacterium selective medium, i.e. TOS propionate agar, and cultured under anaerobic condition at 37° C. for 48 hours. Then, the colonies were picked up from the TOS plate, inoculated on a MRS plate and cultured under anaerobic condition at 37° C. The growth of the colonies at the presence of oxygen was also observed. Gram staining and microscopic observation were performed on the strains.

[0106] The above experiments showed that the strain was a strictly aerobic strain, and was a gram-positive bacillus, with one or both ends swelled or bifurcated, and without spores. It is preliminarily determined that the strain isolated in the application is a Bifidobacterium (shown in FIGS. 1 and 2).

[0107] PCR method were used to identify the isolated strain. The bacterial DNA was extracted by a boiling method. Primers specifically for Bifidobacterium spp. and primers specifically for 8 Bifidobacterium strains (B. adolescentis group, B. angulatum, B. bifidum, B. breve, B. catenulatum group, B. dentium, B. infantis, B. longum) were used for PCR amplification of the bacterial DNA. The sequences of the primers could be found in a literature (Matsuki T, et al. 2004, Appl Environ Microbiol) and were shown in Table 1. The amplification system comprised 25 μL of 2× Taq MasterMix (Beijing Kangwei Century Biotechnology Co., Ltd.), 2 μL for each of upstream and downstream primers (10 μmol/L), 4 μL of template DNA, and 17 μL of ddH.sub.2O. The PCR amplification were carried out as follows: pre-denaturation at 94° C. for 2 min; denaturation at 94° C. for 30 s, annealing at 55° C. for 30 s, extension at 72° C. for 30 s (35 cycles); final extension at 72° C. for 2 min. PCR products were identified by 2% agarose gel electrophoresis (0.4 g agarose was dissolved in 20 mL 0.5× TBE buffer under heating, and 1 μL Goldview was added, and the loading volume was 5 μL, and electrophoresis was performed at 80V for 20 min), and ChemiDoc XRS+ System (a gel imaging system) was used to take photos for identification of Bifidobacterium.

[0108] The results of the PCR identification were shown in Table 2. A strong positive band was observed for the PCR products amplified from the Bifidobacterium spp. primers and the B. breve primers, and the remaining primers did not result in any PCR products. Therefore, the strain obtained in the application is Bifidobacterium breve.

[0109] Furthermore, the whole genome of Bifidobacterium breve 207-1 was determined using the PacBio Sequel2 sequencing platform, its genome length was 2,352,089 bp, the GC contents were 58.8% respectively (in which the GC contents referred to ratios of guanine and cytosine in the four bases of DNA), the chromosome genome contained 2,077 coding genes, 58 tRNA genes, 9 rRNA genes (including 5s rRNA, 16s rRNA and 23s rRNA), and 14 other types of RNA genes.

[0110] Based on the above-mentioned PCR identification and whole-genome sequencing experimental results, the present application obtained a new strain, i.e., Bifidobacterium breve 207-1, which was deposited on January 15, 2020.

TABLE-US-00001 TABLE 1 Sequences of primers SEQ primer Sequence ID NO. Bifidobacterium g-Bifid-F CTCCTGGAAAC  1 GGGTGG g-Bifid-R GGTGTTCTTCC  2 CGATATCTACA B. adolescentis BiADOg-1a CTCCAGTTGGA  3 group TGCATGTC BiADOg-1b TCCAGTTGACC  4 GCATGGT BiADO-2 CGAAGGCTTGC  5 TCCCAGT B. angulatum BiANG-1 CAGTCCATCGC  6 ATGGTGGT BiANG-2 GAAGGCTTGCT  7 CCCCAAC B. bifidum BiBIF-1 CCACATGATCG  8 CATGTGATTG BiBIF-2 CCGAAGGCTTG  9 CTCCCAAA B. breve BiBRE-1 CCGGATGCTCC 10 ATCACAC BiBRE-2 ACAAAGTGCCT 11 TGCTCCCT B. catenulatum BiCATg-1 CGGATGCTCCG 12 group ACTCCT BiCATg-2 CGAAGGCTTGC 13 TCCCGAT B. longum BiLON-1 TTCCAGTTGAT 14 CGCATGGTC BiLON-2 GGGAAGCCGTA 15 TCTCTACGA B. infantis BiINF-1 TTCCAGTTGAT 16 CGCATGGTC BiINF-2 GGAAACCCCAT 17 CTCTGGGAT B. dentium BiDEN-1 ATCCCGGGGGT 18 TCGCCT BiDEN-2 GAAGGGCTTGC 19 TCCCGA

TABLE-US-00002 TABLE 2 Identification results of Bifidobacterium name spp. Breve Infantis Bifidum catenulatum longum adolescentis dentium angulatum 207-1 ++ ++ − − − − − − − Note: ″++″ represents a strong positive band; ″+″ represents a positive band; ″±″ represents a weakly positive band; ″−″ represents negative.

EXAMPLE 2

Study on the Probiotic Characteristics of Bifidobacterium breve 207-1

[0111] 1. Experimental Screening for Acid and Bile Salt Tolerance of Bifidobacterium breve 207-1

[0112] 1.1 Preparation of Test Bacterial Suspension: Bifidobacterium breve 207-1 was resuscitated by TOS medium and subjected to passage twice for later use. After 48 hours of TOS culture, the bacteria were scraped into 2 mL of fecal diluent, vortexed and well mixed, and the concentration was roughly adjusted to 10.sup.8 to 10.sup.9 CFU/mL.

[0113] 1.2 Preparation of Simulated Gastric Acid and Bile Salt Culture Solutions

[0114] Preparation of simulated gastric acid culture solution: MRS broth culture solution was prepared, sterilized and then adjusted with 1 mol/L HCL to pH value of 3.0, thereby preparing the simulated gastric acid culture solution.

[0115] Preparation of simulated bile salt culture solution: MRS broth culture solution was prepared, sterilized and then added with bovine bile salt to a concentration of 0.1%, adjust with 1 mol/L HCL to pH of 8.0, and then filtered for sterilization with 0.22 μm microporous membrane, thereby preparing the bile salt culture solution.

[0116] 1.3 Gastric Acid and Bile Salt Tolerance Tests

[0117] Acid tolerance test of Bifidobacterium breve 207-1: 0.9 ml of the simulated gastric acid culture solution was placed into a EP tube, added with 0.1 ml of the prepared bacterial suspension, vortexed and well mixed, and then it was placed in an anaerobic incubator and cultured at 37° C. for 2 hours. Tests were carried out at 0 h and 2 h, and 3 parallel tests were set for the strain at each time point. The MRS broth (pH=7) without adjusting the pH was used as control, the growth of bacteria was observed, and the death of bacteria caused by other factors was excluded.

[0118] Bile salt tolerance test of Bifidobacterium breve 207-1: 0.9 ml of the bile salt culture solution was placed into a EP tube, added with 0.1 mL of the prepared bacterial suspension, vortexed and well mixed, and then it was placed in an anaerobic incubator and culture at 37° C. for 24 hours. Tests were carried out at 0 h and 24 h, and 3 parallel tests were set for the strain at each time point. The MRS broth (pH=7) without bile salt was used as control, the growth of bacteria was observed, and the death of bacteria caused by other factors was excluded.

[0119] After the above culture was completed, 10-fold gradient dilution was immediately carried out, the diluent with appropriate dilution degree was spot-inoculated to a TOS plate, cultured under anaerobic condition at 37° C. for 24-48 hours and subjected to counting. The counting result of the 0-hour dilution inoculation was used as the initial bacterial concentration. The results were compared with survival rates, which was calculated according to the following formula:

Survival rate (%)=concentration of viable bacteria after culture (CFU/mL)/0-hour concentration of viable bacteria (CFU/mL)×100

[0120] The experimental results showed that the survival rate of the Bifidobacterium breve 207-1 after 2 hours of digestion in the simulated gastric acid culture solution of pH=3 was 86.3%; the survival rate after 24 hours of culture in the bile salt culture solution was 73.6%. Therefore, it could be seen that the Bifidobacterium breve 207-1 had good tolerance to gastric acid and bile salts, and could effectively resist the extreme environment of the upper digestive tract, so as to reach the lower digestive tract, such as the colon, to perform healthy functions. The Bifidobacterium breve 207-1 therefore meets the basic requirements as a probiotic.

[0121] 2. Adhesion Ability of Bifidobacterium breve 207-1 to Intestinal Mucosa

[0122] 2.1 Adhesion test method: After resuscitating and subculturing Bifidobacterium breve 207-1 on the TOS plate for three generations, the OD600 absorbance of the bacterial suspension was adjusted to 1.051±0.005 (concentration of 1×10.sup.9 CFU/mL). The commercial strains Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus LGG were used as control strains for parallel experiments, and their bacterial suspensions were prepared according to the same method described above.

[0123] 500 μg/mL mucin solution was added to 96-well Maxisorp plate (Nunc), 100 μL per well, and incubated overnight in a refrigerator at 4° C. It was taken out and washed with PBS three times, 200 μL per well, then blocked with PBS containing 1% Tween 20 for 1 h, 100 μL per well. 100 μL of the prepared bacterial suspension was taken and added to microwell, and 3 parallels were set for each strain. At the same time, PBS was used instead of bacterial suspension to be added as a blank control, and 3 parallels were set. Incubation was carried out overnight in a refrigerator at 4° C. After the incubation, it was taken out and washed three times with PBS containing 0.05% Tween 20 to remove unadhered bacteria. Then, it was dried in an oven at 60° C. for 1 h. The dried microplate was added with 1% crystal violet solution, 100 μL per well, and stained for 45 min. Then it was washed with PBS for 6 times, added with absolute ethanol, and allowed to stand for 10 minutes to release the staining solution. Finally, the absorbance of each well at wavelength of 590 nm was measured by a microplate reader.

[0124] 2.2 Adhesion results of Bifidobacterium breve 207-1: The adhesion test results of Bifidobacterium breve 207-1 were shown in Table 3.

TABLE-US-00003 TABLE 3 Adhesion test results No. Strain No. Stain OD.sub.590 value (X ± S) 1 207-1 B. breve 0.648 ± 0.001 2 LGG L. rhamnosus 0.609 ± 0.018 3 Bb-12 B. animalis 0.696 ± 0.021 4 Blank control — 0.089 ± 0.003

[0125] Mucin is the main component of intestinal epithelial cells that produce a large amount of mucus. Mucus can protect intestinal mucosal cells from contact with pathogenic microorganisms, prevent pathogenic bacteria from invading epithelial cells, thereby protecting the normal function of epithelial cells. Studies have shown that probiotics can induce intestinal epithelial cells to secrete mucin that forms a biofilm on the surface of the intestinal mucosa by occupying the attachment points of the intestinal mucosa, preventing foreign bacteria from attaching to the intestinal mucosa. The oligosaccharide chain of mucin contains a large number of specific sites that bind to various probiotics, especially the epitope at the end of the saccharide chain can also screen and identify the flora of intestinal probiotics, and assist the colonization of intestinal probiotics.

[0126] The results of this experiment showed that Bifidobacterium breve 207-1 had similar mucin binding ability as compared with the commercial strains Bifidobacterium animalis BB12 and Lactobacillus rhamnosus LGG. Therefore, the applicant believes that Bifidobacterium breve 207-1 can effectively adhere to the intestinal mucosa and has excellent colonization ability. Based on this, it occupies the surface of the intestinal mucosa, forms a probiotic barrier, inhibits the colonization and invasion of harmful bacteria, thereby effectively regulating the intestinal flora and protecting the normal immune function of the intestinal immune cells.

EXAMPLE 3

Immune Properties of Bifidobacterium breve 207-1 by Cell Experiment

[0127] 1. Experimental Method

[0128] Mouse macrophages RAW264.7 (5×10.sup.5/mL) were cultured, the Bifidobacterium breve 207-1 suspension was adjusted to have a concentration of 10.sup.9 CFU/mL, diluted by 10 times and co-cultured with cells, and used as the experimental group; PGN was used as a positive control (PGN was peptidoglycan of gram-positive bacteria, and was the main substance in gram-positive bacteria that stimulated and induced inflammation of host immune cells, so it was used as a positive control for this experiment), Bb-12 and LGG were used as commercial control strains, RPMI1640 medium was used as a blank control, three parallel samples were set up for each group. After 24 hours of co-cultivation, the cell supernatant was collected, and the secretion levels of the cytokine expression proteins as shown in Table 4 were detected by the enzyme-linked immunosorbent assay (ELISA) method. The Bifidobacterium breve 207-1 was also used to stimulate human macrophages THP-1 by the same method, and its immunomodulatory effect on human cells was observed. 2. Experimental results and analysis of mouse macrophages RAW264.7

[0129] 2.1 Results and Analysis of Enzyme-Linked Immunosorbent Assay

[0130] The results of the ELISA experiment of co-culture of Bifidobacterium breve 207-1 and mouse macrophages RAW264.7 were shown in Table 4.

TABLE-US-00004 TABLE 4 ELISA test results (mean ± standard deviation) Cytokine expression level (pg/mL) Strain No. Strain IL-6 IL-10 TNF-α 207-1 B. breve 619.02 ± 13.83 1554.61 ± 305.37  4731.00 ± 173.31 Bb-12 B. animal 307.44 ± 65.03 931.44 ± 299.42 9887.97 ± 596.94 LGG L. rhamnosus  44.93 ± 39.48 92.07 ± 19.01  8097.62 ± 1006.08 PGN — 1005.75 ± 38.74  985.22 ± 130.50 7850.80 ± 467.76 Control group — 153.39 ± 20.75 450.99 ± 135.83 107.29 ± 58.44

[0131] The results showed that, compared with the control group, Bifidobacterium breve 207-1 could stimulate the increase of the expression levels of the genes of cytokines IL-6, IL-10 and TNF-α, and induced the activation of macrophages. In particular, the ELISA results showed that after stimulation with Bifidobacterium breve 207-1, the level of the anti-inflammatory factor IL-10 was significantly increased, especially significantly higher than that in the Bb-12, LGG, PGN group and the control group. The Bifidobacterium breve 207-1 could stimulate macrophages to secrete a large amount of anti-inflammatory cytokine IL-10, suggesting that this Bifidobacterium has a potential anti-inflammatory activity and can relieve inflammation and improve inflammatory diseases.

[0132] 3. Experimental Results and Analysis of Human Macrophages THP-1

[0133] The results of the ELISA experiment for co-culture of Bifidobacterium breve 207-1 and human macrophages THP-1 were shown in Table 5.

TABLE-US-00005 TABLE 5 ELISA results for THP-1 (mean ± standard deviation) Cytokine expression level (pg/mL) Strain No. Strain IL-6 IL-10 TNF-α 207-1 B. breve 247.24 ± 14.00 211.16 ± 11.46 1162.38 ± 102.36 PGN — 19.97 ± 0.67 14.77 ± 1.26 297.185 ± 24.65  Control —  0.34 ± 0.11  0.24 ± 0.33  6.57 ± 1.59 group

[0134] It could be seen from the results in Table 5 that Bifidobacterium breve 207-1 exhibited the same immunological effect on human macrophages as on animal cells, and its activation effect was significantly higher than that of the positive control PGN. It could be seen from the above two experiments that Bifidobacterium breve 207-1 has an important immune effect of stimulating the immune response of macrophages and causing them to secrete the anti-inflammatory cytokine IL-10.

Example 4

Immunomodulatory Ability Test of Bifidobacterium breve 207-1

[0135] 1. Experimental Method:

[0136] A total of 18 BALB/C mice, male, 6 weeks old, weighing 18-20 g, were selected. They were divided into 3 groups, including: 6 animals in the control group, 6 animals in the intervention control group of commercial probiotic strain LGG, which had been proved to have the most significant immunomodulatory ability in animal and clinical trials, and 6 animals in the intervention control group of Bifidobacterium breve 207-1. The animals of the control group were given 0.2 ml of normal saline per day, and the animals of the probiotic intervention groups were given 0.2 ml of bacterial solution per day (the bacterial content was 10.sup.9 cfu). The animals of each group were raised for 4 weeks and then sacrificed.

[0137] Sample collection: On the day before the start of the gavage experiment, all mice were weighed and their feces were collected. On the 14.sup.th day of the experiment, all mice were weighed and their feces were collected. On the 28.sup.th day of the experiment, all mice were weighed and their feces were collected, then they were sacrificed, and their organs (spleen, lung, thymus, liver) were collected and weighed.

[0138] Measurement of indicators: the organ coefficients of spleen and thymus were determined by use weighing method; the expression of cytokine genes in spleen tissue was determined by RT-PCR method.

[0139] 2. Experimental Results:

[0140] There was no significant difference in body weight in each group before and after the intervention, indicating that Bifidobacterium breve 207-1 had no adverse effect on the normal development of mice. There was no significant difference in spleen coefficient and thymus coefficient between the groups, indicating that the exertion of probiotics' immunomodulatory effect would not affect the normal function of the organs. As shown in table 6, the animal experiments showed that Bifidobacterium breve 207-1 activated macrophages and stimulated the secretion of various related cytokines. IL-10 was a multifunctional negative regulatory cytokine; the promoted secretion of a large amount of anti-inflammatory factor IL-10 when the probiotic activated pro-inflammatory responses (production of IL-6 and TNF-α) indicated that Bifidobacterium breve 207-1 could balance the inflammatory responses and maintain the homeostasis of cellular immunity. The ability of Bifidobacterium breve 207-1 to activate macrophages to secrete cytokines is higher than that of the commercial strain LGG (Table 6). The results of animal experiments were consistent with the results of cell experiments.

TABLE-US-00006 TABLE 6 Relative expression level of cytokine mRNA in spleen Group IL-10 IL-6 IL-12 TNF-a Control group 1.15 ± 0.28 1.19 ± 0.55 1.18 ± 0.62 0.99 ± 0.47 207-1 group 1.36 ± 0.49 1.23 ± 0.45 2.00 ± 0.88 2.12 ± 0.87 LGG group 0.47 ± 0.14 0.87 ± 0.3  0.35 ± 0.11 0.32 ± 0.11

EXAMPLE 5

Test of Bifidobacterium breve 207-1 Inhibiting Allergic Reaction

[0141] 1. Experimental Method:

[0142] In this experiment, ovalbumin (OVA) was used to establish a mouse serum IgE hypersensitivity model. The commercial probiotic strain LGG that had been shown to have a significant ability to inhibit allergic reactions in animal and clinical trials was used as a control to study the effect of Bifidobacterium breve 207-1 on the inhibition of allergic reactions.

[0143] Experimental grouping: a total of 60 male BALB/C mice, 6 weeks old, 18-20 g, were selected, and grouped as shown in Table 7:

TABLE-US-00007 TABLE 7 Grouping of experimental animals Group Number Gavage and intervention Control group 15 0.2 ml normal saline gavage + normal saline injection Model group 15 0.2 ml normal saline gavage + OVA intervention 207-1 test 15 0.2 ml 207-1 + OVA intervention group LGG group 15 0.2 ml LGG + OVA intervention

[0144] Intervention: The OVA reagent was 40 ug OVA+0.2 ml AL(OH).sub.3 adjuvant (absolute dose was 4 mg of AL(OH).sub.3). The absolute dose of the probiotic group was 10.sup.9 CFU, and the gavage volume was 0.2 ml/animal/day. After feeding for 42 days, 4 intraperitoneal OVA injection interventions were performed on the day 7, day 21, day 28, and day 35, respectively.

[0145] Sample collection and detection: The animals were sacrificed on the day 42, and the serum IgE contents and the expression of spleen cytokines were determined.

[0146] 2. Experimental Results:

[0147] IgE is the main mediator of type I hypersensitivity, and the type I hypersensitivity is the main physiological mechanism of most allergic diseases. The results showed that Bifidobacterium breve 207-1 could reduce the level of serum IgE in allergic mouse model, inhibit IgE-mediated allergic reactions, and alleviate allergic diseases (FIG. 3). This experiment also showed that the ability of Bifidobacterium breve 207-1 to reduce serum IgE was higher than that of the commercial strain LGG.

[0148] The detection results of spleen cytokines showed that Bifidobacterium breve 207-1 significantly increased the expression of anti-inflammatory factor IL-10 in spleen (Table 8). There are reports showing that IL-10 can participate in the regulation of TH1 and TH2. Based on the analysis of the above results, it is believed that the probiotics of the present application can induce the expression of IL-10, thereby inhibiting the activity of TH1 and TH2 cells, and down-regulating the body's immune response level, thereby achieving allergic reactions.

TABLE-US-00008 TABLE 8 Relative expression level of IL-10 cytokine mRNA in spleen Relative expression level of mRNA Group IL-10 Blank control group 1.69 ± 0.95 OVA model group 1.03 ± 0.32 207-1 + OVA group 4.65 ± 0.88 LGG + OVA group 1.84 ± 0.89