A type of Bifidobacterium longum capable of regulating gut microbiota abundance and gut health and its applications

Abstract

This invention discloses a *Bifidobacterium longum* strain capable of regulating intestinal flora abundance and intestinal health, and its applications, belonging to the field of microbial technology. This invention screened a *Bifidobacterium longum* strain CCFM5871, which significantly increased the relative abundance of *Rikenobacterium* spp. in the mouse intestine, promoted the growth of *Bacteroides davidii*, improved intestinal health, and increased the lifespan of nematodes. The *Bifidobacterium longum* CCFM5871 described in this invention, which significantly increases the abundance of *Rikenobacterium* and *Rikenobacterium* spp. in the intestine and promotes the growth of *Bacteroides davidii*, has broad application prospects in food and microecological preparations.

Description

Technical Field

[0001] This invention relates to a Bifidobacterium longum species capable of regulating intestinal flora abundance and intestinal health, and its applications, belonging to the field of microbial technology. Background Technology

[0002] *Rikenella* is a Gram-negative, obligate anaerobic bacterium. Rikenella can ferment propionate, producing energy in normal cells and promoting gluconeogenesis. It can also ferment carbohydrates (such as glucose) to promote growth. Rikenella is a potential probiotic, playing an important role in maintaining metabolic homeostasis, the immune system, and gut health. In recent years, numerous studies have shown a close correlation between the abundance of *Rikenella* and the host's immune system. For example, one patent indicates that the relative abundance of *Rikenella* can serve as a microbial biomarker for diagnosing food allergies (application publication number CN202210216093.6), and another study showed a significantly reduced relative abundance of *Rikenella* in food-allergic mice. Furthermore, research has shown an association between *Rikenella* and another autoimmune disease, rheumatoid arthritis, with significantly reduced *Rikenella* abundance in rheumatoid arthritis volunteers compared to normal volunteers. Another animal experiment showed a significantly reduced abundance of *Rikenella* in mice with autoimmune hepatitis (AIH). Therefore, regulating the abundance of *Rigigenella* in the gut can play a role in protecting health and alleviating disease.

[0003] Currently, some patents relate to health foods and their preparation methods that regulate gut microbiota. For example, patent CN108018248A discloses a *Lactobacillus casei* that can significantly restore antibiotic-induced changes in gut microbiota in mice; patent CN111004731A discloses a *Bacillus coagulans*, whose composition can significantly increase the abundance of *Allobaculum* in the gut. However, there are currently no reports on the design and use of *Bifidobacterium* to regulate the content of *Rikenella*. Furthermore, *Bacteroides* are important cornerstone bacteria in the gut and play a crucial role in maintaining gut microbiota homeostasis. *Bacteroides nordii* is an important species within the *Bacteroides* genus. Promoting the growth of *Bacteroides nordii* is beneficial for increasing its abundance in the gut, which is of great significance for maintaining gut microbiota homeostasis. Currently, there are no reports on *Bifidobacterium* promoting the growth of *Bacteroides nordii*.

[0004] Therefore, providing a solution that can upregulate the relative abundance of Rikenella in the gut and promote the growth of Bacteroides nordii, thereby improving gut microbiota dysbiosis, is a pressing technical problem that needs to be solved. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a strain of Bifidobacterium longum and a method for significantly increasing the relative abundance of Rikenella in the intestine, promoting the growth of Bacteroides davidii, regulating intestinal health, and increasing the lifespan of nematodes.

[0006] This invention provides a strain of Bifidobacterium longum CCFM5871, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection with accession number GDMCCNo:63537 and deposit date of June 7, 2023.

[0007] The *Bifidobacterium longum* CCFM5871 strain was isolated from a fecal sample of a 38-year-old male from Shuntouling Village, Shantang Town, Lianzhou City, Guangdong Province. Sequencing analysis revealed its 16S rDNA sequence as shown in SEQ ID NO.1. BLAST alignment of the sequenced data with *Bifidobacterium longum* was performed using NCBI, showing a 99.48% match, confirming the strain as *Bifidobacterium longum*. Therefore, it was named *Bifidobacterium longum* CCFM5871.

[0008] The growth characteristics of *Bifidobacterium longum* CCFM5871 on MRS medium were as follows: colonies were round on the front side. Figure 1 ), with a diameter between 1 and 2 mm, a convex lateral morphology, milky white, with a moist and smooth surface, and a Gram staining of blue-purple (). Figure 2 Gram-positive bacteria, non-spore-forming. Figure 1 ).

[0009] The growth characteristics of the Bifidobacterium longum CCFM5871 are as follows: it is an obligate anaerobic bacterium that is very sensitive to oxygen. It grows best at a temperature of 37–41°C and the optimal initial growth pH is 6.5–7.0.

[0010] The present invention also provides a microbial preparation containing the above-mentioned Bifidobacterium longum CCFM5871.

[0011] In one embodiment of the present invention, the viable count of Bifidobacterium longum CCFM5871 in the microbial preparation is not less than 1 × 10⁻⁶. 6CFU / mL or 1×10 6 CFU / g.

[0012] The present invention also provides a product containing the above-mentioned Bifidobacterium longum CCFM5871 or the above-mentioned microbial preparation.

[0013] In one embodiment of the present invention, the product includes food, medicine, or health products.

[0014] In one embodiment of the present invention, the viable count of Bifidobacterium longum CCFM5871 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0015] In one embodiment of the present invention, the food includes fermented fruits and vegetables, fermented milk, cheese, milk-containing beverages, milk powder, or other foods containing Bifidobacterium longum.

[0016] In one embodiment of the present invention, the drug contains the above-mentioned Bifidobacterium longum CCFM5871, a drug carrier, and / or pharmaceutical excipients.

[0017] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0018] In one embodiment of the present invention, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

[0019] The present invention also provides a method for promoting the proliferation of Bacteroides nordii, wherein the method comprises co-culturing Bifidobacterium longum CCFM5871 or the above-mentioned microbial preparation with Bacteroides nordii.

[0020] The present invention also provides a product that promotes the proliferation of Bacteroides nordii, the product containing the above-mentioned Bifidobacterium longum CCFM5871, or the above-mentioned microbial preparation.

[0021] In one embodiment of the present invention, the product includes food, medicine, or health products.

[0022] In one embodiment of the present invention, the viable count of Bifidobacterium longum CCFM5871 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0023] In one embodiment of the present invention, the food includes fermented fruits and vegetables, fermented milk, cheese, milk-containing beverages, milk powder, or other foods containing Bifidobacterium longum.

[0024] In one embodiment of the present invention, the drug contains the above-mentioned Bifidobacterium longum CCFM5871, a drug carrier, and / or pharmaceutical excipients.

[0025] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0026] In one embodiment of the present invention, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

[0027] The present invention also provides a method for increasing the abundance of Rikenella or Roseburia, characterized in that the method involves co-culturing in an environment containing Rikenella or Roseburia with the above-mentioned Bifidobacterium longum CCFM5871 or the above-mentioned microbial preparation.

[0028] In one embodiment of the present invention, the environment is an in vivo environment.

[0029] In one embodiment of the present invention, the environment is an in vitro environment.

[0030] The present invention also provides a product that enhances the levels of Rikenella or Roseburia, the product containing the aforementioned Bifidobacterium longum CCFM5871, or the aforementioned microbial preparation.

[0031] In one embodiment of the present invention, the product includes food, medicine, or health products.

[0032] In one embodiment of the present invention, the viable count of Bifidobacterium longum CCFM5871 in the microbial preparation is not less than 1 × 10⁻⁶. 6CFU / mL or 1×10 6 CFU / g.

[0033] In one embodiment of the present invention, the food includes fermented fruits and vegetables, fermented milk, cheese, milk-containing beverages, milk powder, or other foods containing Bifidobacterium longum.

[0034] In one embodiment of the present invention, the drug contains the above-mentioned Bifidobacterium longum CCFM5871, a drug carrier, and / or pharmaceutical excipients.

[0035] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0036] In one embodiment of the present invention, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

[0037] The present invention also provides the use of the above-mentioned Bifidobacterium longum CCFM5871, or the above-mentioned microbial preparation, in the preparation of products that promote the proliferation of Bacteroides davidii or increase the levels of Rikenella or Roseburia.

[0038] In one embodiment of the present invention, the product includes food, medicine, or health products.

[0039] In one embodiment of the present invention, the viable count of Bifidobacterium longum CCFM5871 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0040] In one embodiment of the present invention, the food includes fermented fruits and vegetables, fermented milk, cheese, milk-containing beverages, milk powder, or other foods containing Bifidobacterium longum.

[0041] In one embodiment of the present invention, the drug contains the above-mentioned Bifidobacterium longum CCFM5871, a drug carrier, and / or pharmaceutical excipients.

[0042] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0043] In one embodiment of the present invention, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

[0044] The present invention also provides a product containing the above-mentioned Bifidobacterium longum CCFM5871 and Bacteroides davidii.

[0045] In one embodiment of the present invention, the product includes food, medicine, or health products.

[0046] In one embodiment of the present invention, the viable count of Bifidobacterium longum CCFM5871 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0047] In one embodiment of the present invention, the food includes fermented fruits and vegetables, fermented milk, cheese, milk-containing beverages, milk powder, or other foods containing Bifidobacterium longum.

[0048] In one embodiment of the present invention, the drug contains the above-mentioned Bifidobacterium longum CCFM5871, a drug carrier, and / or pharmaceutical excipients.

[0049] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0050] In one embodiment of the present invention, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

[0051] The present invention also provides the application of the above-mentioned Bifidobacterium longum CCFM5871, or the above-mentioned microbial preparation, in the preparation of products that improve intestinal health, or in the preparation of probiotic health products, or in the preparation of probiotic foods.

[0052] In one embodiment of the present invention, the product includes food, medicine, or health products.

[0053] In one embodiment of the present invention, the viable count of Bifidobacterium longum CCFM5871 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0054] In one embodiment of the present invention, the food includes fermented fruits and vegetables, fermented milk, cheese, milk-containing beverages, milk powder, or other foods containing Bifidobacterium longum.

[0055] In one embodiment of the present invention, the drug contains the above-mentioned Bifidobacterium longum CCFM5871, a drug carrier, and / or pharmaceutical excipients.

[0056] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0057] In one embodiment of the present invention, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

[0058] Beneficial effects

[0059] 1. This invention screened out a strain of Bifidobacterium longum CCFM5871, which has the effects of regulating the abundance of Rickenella and Rochezoides, promoting the growth of Bacteroides davidii and increasing the lifespan of nematodes, increasing defecation frequency, fecal water content, fecal acetic acid content, small intestinal propulsion rate, and reducing intestinal transit time.

[0060] Therefore, Bifidobacterium longum CCFM5871 has great application potential in the preparation of products (such as food or medicine) that regulate the abundance of Rykenella spp., promote the growth of Bacteroides davidii, improve intestinal health, and thus prevent and / or treat related diseases.

[0061] 2. Bifidobacterium longum CCFM5871 is an edible probiotic. The present invention relates to Bifidobacterium longum CCFM5871 and its active ingredient.

[0062] Preservation of biological materials

[0063] A strain of Bifidobacterium longum, CCFM5871, taxonomically named Bifidobacterium longum, was deposited on June 7, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:63537, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0064] Figure 1 Results of dilution and plating of Bifidobacterium longum CCFM5871.

[0065] Figure 2 Gram microscopic examination results of Bifidobacterium longum CCFM5871.

[0066] Figure 3 Effect of Bifidobacterium longum CCFM5871 on the relative abundance of Riken bacteria in the mouse intestine.

[0067] Figure 4 The effect of Bifidobacterium longum CCFM5871 on the growth of Bacteroides davidii.

[0068] Figure 5 Effects of Bifidobacterium longum CCFM5871 on the lifespan of nematodes.

[0069] Figure 6 Effects of Bifidobacterium longum CCFM5871 on intestinal physiological indicators. Detailed Implementation

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

[0071] The MRS, BHI, and GMM culture media used in the following examples were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; the Fast DNA Spin Kit for Feces used in the following examples was purchased from MP Biomedicals.

[0072] Clinical trial probiotic powder: Both the probiotics and placebo products in the experimental products were food-grade and manufactured by Jiangsu Weikang Biotechnology Co., Ltd., commissioned by the Food Biotechnology Center of the School of Food Science and Technology, Jiangnan University. The production license for the probiotic powder is SC10632050900407. All products are powders with identical appearance and packaging. Each packet of probiotic powder weighs 2g and contains 5×10⁵ probiotics. 9 CFU live bacteria and maltodextrin, with a placebo of 2g maltodextrin. All experimental products were stored at 4°C during the trial.

[0073] The Balb / c mice used in the following examples were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; the Caenorhabditis elegans used in the following examples is the N2 wild-type Caenorhabditis elegans, which was kindly donated by the College of Food Science and Technology of Nanjing Agricultural University and is deposited at the Food Biotechnology Center of Jiangnan University.

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

[0075] Bifidobacterium selective medium (1L): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, distilled water: 1000mL; pH: 6.2-6.4; sterilize at 115℃ for 20min. When preparing the solid medium, add 15g agar, and before pouring the plates, add sterile mupirocin and sterile nystatin at 1‰ and 0.5‰ of the medium volume, respectively, resulting in final concentrations of 100μg / mL mupirocin and 25U / mL nystatin.

[0076] MRS liquid culture medium (1L): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, distilled water: 1000mL; pH: 6.2-6.4; sterilize at 115℃ for 20min.

[0077] MRS solid medium (1L): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, distilled water: 1000mL; pH: 6.2-6.4; sterilize at 115℃ for 20min. Add 15g agar when preparing the solid medium.

[0078] BHI liquid culture medium (1L): Dissolve 38.5g of brain heart extract culture medium powder in 1L of water, add vitamin K1 0.002g / L, heme chloride 0.01g / L, cysteine ​​hydrochloride 1g / L, and adjust the pH to 7.0.

[0079] S liquid culture medium: NaCl 5.85g, K2HPO4 1g, KH2PO4 6g, bring to a final volume of 1L, aseptically add 1mL of 5mg / mL cholesterol ethanol solution to prepare 1L of S basal culture medium; add 3mL each of 1mol / L CaCl2 solution and 1mol / L MgSO4 solution to the S basal culture medium; add 10mL of 1mol / L potassium citrate solution, pH 6.0 (citric acid·H2O 20g, potassium citrate·H2O 293.5g, bring to a final volume of 1L); add 10mL of trace metal solution (EDTA·2Na 1.86g, FeSO4·7H2O 0.69g, MnCl2·4H2O 0.2g, ZnSO4·7H2O 0.29g, CuSO4·5H2O 0.025g, bring to a final volume of 1L, autoclave, and store in the dark).

[0080] The preparation method of the Bifidobacterium longum suspension involved in the following examples is as follows:

[0081] Bifidobacterium longum was streaked onto MRS solid medium and cultured anaerobically at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h to activate the culture. The activation was repeated for two generations to obtain the activated solution.

[0082] The activation solution was inoculated into MRS liquid medium (inoculation amount 2%–4% (v / v)) and anaerobic cultured at 37°C for 18 h to obtain bacterial culture; the bacterial culture was centrifuged at 10000g for 5 min to obtain Bifidobacterium longum cells;

[0083] Bifidobacterium longum cells were washed with physiological saline and then resuspended in 30% glycerol solution (containing 1 g / L cysteine ​​hydrochloride) to achieve a bacterial concentration of 5 × 10⁻⁶. 9 CFU / mL, stored at -80℃.

[0084] The detection methods involved in the following embodiments:

[0085] Detection of abundance of Bacteroides northernis strains:

[0086] DNA was extracted from 1 mL of culture medium and qPCR was performed using specific primers. The qPCR mixture (total volume 20 μL) consisted of Tap enzyme Mix, 2 μL DNA template, and 0.2 μM of each primer. A standard curve was calculated using a 10-fold dilution series of DNA extracted from each strain to perform absolute quantification of the strain. The qPCR experimental procedure was as follows: Step 1: 94℃, 10 s; Step 2: 94℃, 20 s; Step 3: 55℃, 20 s; Step 4: 72℃, 50 s. Steps 2 to 4 were performed for 40 cycles. *B. longum* (F: 5′-TTCCAGTTGATCGCATGGTCTTCTA-3′, R: 5′-GGCTACCCGTCGAAGCCACG-3′), *B. nordii* (F: 5′-GCGGGGAACAGAATCAGACA-3′, R: 5′-ATTCCACCAAATGTAGGCGGGACGTTTAAT-3′)

[0087] Example 1: Isolation and screening of Bifidobacterium longum CCFM5871

[0088] 1. Sample collection

[0089] A fecal sample was collected from a 38-year-old male in Shuntouling Village, Shantang Town, Lianzhou City, Guangdong Province. The sample was placed in a sampling tube containing 30% glycerol and stored in an insulated box with ice packs. After being brought back to the laboratory, it was quickly placed in a -80℃ refrigerator for separation and screening.

[0090] 2. Isolation and purification of Bifidobacteria

[0091] (1) Dilution and plating: Take approximately 0.5g of fecal sample and add it to a 10mL centrifuge tube containing 4.5mL of physiological saline under sterile conditions to obtain 10 -1 Diluent, repeat the above dilution steps to obtain 10 -2 10 -3 10 -4 10 -5 10 -6 Diluent;

[0092] (2) Spread culture: Take 100 μL of the above 10 -4 10 -5 10 -6 Three serial dilutions were spread evenly on Bifidobacterium-specific selective culture medium and incubated at 37°C under anaerobic conditions for 48 hours.

[0093] (3) Primary purification culture: Take the diluted plating plates with a colony count in the range of 30 to 300. Randomly select 10 milky white, smooth, and neat single colonies from each sample and streak them onto the MRS solid medium. Incubate at 37°C under anaerobic conditions for 48 hours to obtain single colonies. Repeat the above steps to obtain the second streak single colonies.

[0094] (4) Secondary purification culture: Take a single colony from the second streak plate in step (3) and inoculate it into MRS liquid medium. Incubate at 37°C under anaerobic conditions for 12 hours to obtain the secondary purification culture.

[0095] 3. Preservation and Identification of Microbial Strains

[0096] (1) Preservation of microbial strains:

[0097] Shake the above-mentioned secondary purified culture medium to mix well. Take 700 μL of bacterial culture (cultured for 18-24 h) into a 2 mL clean bacterial culture preservation tube and make 6 parallel aliquots. Add 700 μL of 60% glycerol to 5 aliquots and resuspend them. After standing for 30 minutes, store them in a -80℃ refrigerator. Use 1 aliquot of bacterial culture for bacterial identification. Centrifuge at 8000 r / min for 3 min and discard the supernatant to obtain bacterial cells.

[0098] (2) Strain identification:

[0099] Step (1) Add 0.5 mL of sterile water to the preservation tube for bacterial identification, wash the bacterial cells by blowing, centrifuge at 10000 r / min for 1 min, discard the supernatant to obtain the bacterial cells, add 500 μL of sterile water to resuspend, and use it as a template for PCR strain identification system.

[0100] The primers and systems for 16S rDNA PCR are shown in Table 1 and Table 2, respectively.

[0101] Table 1: Primer Names

[0102] 16S rDNA PCR primer names sequence 27F 5'-AGAGTTTGATCCTGGCCTCA-3' 1492R 5'-GGTTACCTTGTTACGACTT-3'

[0103] Table 2: Bacterial strain identification 25μL 16S rDNA PCR reaction system

[0104] Components Dosage increase (μL) 27F 0.25 1492R 0.25 Taq enzyme Mix 12.5 template 1 Double distilled water 11

[0105] Conditions for 16S rDNA PCR: Step 1: 94℃, 10 min; Step 2: 94℃, 30 s; Step 3: 55℃, 30 s; Step 4: 72℃, 2 min; Step 5: 72℃, 10 min. Repeat steps 2 to 4 for 30 cycles.

[0106] After the PCR product was confirmed as a single bright band by nucleic acid electrophoresis, it was sent to BGI for sequencing. The 27F and 1492R spliced ​​sequences returned by sequencing were uploaded to NCBI's BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST) for species confirmation. The comparison results showed that the strain with the number CCFM5871 was a Bifidobacterium longum strain, and its 16S rDNA amplification sequence is shown in SEQ ID NO.1.

[0107] Example 2: Effects of Bifidobacterium longum CCFM5871 on the composition of intestinal flora in mice

[0108] The specific steps are as follows:

[0109] Twenty-four healthy male BALB / c mice aged 6-8 weeks and weighing 20-22g were randomly divided into four groups of six mice each: a blank control group, a model group, and an intervention group that was administered Bifidobacterium longum CCFM5871 and CCFM1114 (described in Chinese invention patent publication number CN113943682B) by gavage.

[0110] The experimental procedure is as follows:

[0111] Blank control group: 0.2 mL of normal saline was administered by gavage twice daily;

[0112] Model group (LOP): 0.2 mL loperamide solution (10 mg / kg bw) was administered by gavage, followed by 0.2 mL of normal saline by gavage 1 h later;

[0113] CCFM5871 group: 0.2 mL of loperamide solution (10 mg / kg bw) was administered by gavage daily, followed by 0.2 mL of a bacterial concentration of 5 × 10⁻⁶ cells / kg by gavage 1 hour later. 9 CFU / mL Bifidobacterium longum CCFM5871 bacterial suspension;

[0114] CCFM1114 group: 0.2 mL of loperamide solution (10 mg / kg bw) was administered by gavage daily, followed by 0.2 mL of a bacterial concentration of 5 × 10⁻⁶ cells / kg by gavage 1 hour later. 9 CFU / mL Bifidobacterium longum CCFM1114 bacterial suspension;

[0115] On day 14, mouse feces were collected. Bacterial genomes were extracted from fecal samples using the Fast DNA Spin Kit for Feces. The 16S V3-V4 region sequence was then amplified by PCR. Differences in gut microbiota composition in fecal samples were analyzed using next-generation sequencing. The relative abundance of major gut microbiota in each group of mice is shown in Table 3. Figure 3 .

[0116] Table 3: Relative abundance (%) of major gut microbiota in mice of each group

[0117] Fungi blank Loperamide CCFM5871 CCFM1114 g__Rikenella 0.9502 0.4605 5.9804 1.1959 g__Acinetobacter 0.0037 0.1766 0.0910 0.0029 g__Adlercreutzia 1.8517 1.7793 1.5720 1.4590 g__AF12 0.4783 0.1269 0.1758 0.7420 g__Bacteroides 11.3595 4.1614 36.8205 52.9094 g__Bifidobacterium 0.0000 0.0016 0.0145 0.0000 g__Coprococcus 0.1534 0.0205 0.2006 0.1559 g__Corynebacterium 0.0135 0.0262 0.0623 0.0083 g__Dorea 0.2015 0.0100 0.0738 0.0876 g__Enterococcus 1.1812 4.2490 0.9890 0.9028 g__Helicobacter 0.0856 0.0248 0.0923 0.1423 g__Mucispirillum 4.2623 1.6251 10.6890 1.7898 g__Oscillospira 1.1962 0.4542 0.6852 0.5927 g__other 2.4619 1.3905 2.2738 1.9034 g__Parabacteroides 0.3538 0.0927 0.0844 0.0102 g__Proteus 0.3867 0.0771 0.0087 0.0049 g__Roseburia 3.4547 0.3605 20.8645 7.0906 g__Ruminococcus 1.8232 0.8402 2.1574 2.5610 g__Staphylococcus 1.0174 2.6827 3.2503 0.2674 g__Streptococcus 0.1628 0.2691 0.1254 0.2294 g__Turicibacter 0.5101 1.0051 3.3741 3.2316

[0118] Rickenella plays an important role in maintaining intestinal health. The relative abundance of Rickenella in the intestines of mice in the blank control group was 0.95%, while that in the model group was 0.46%. The relative abundance of Rickenella in the intestines of mice treated with Bifidobacterium longum CCFM5871 was 5.98%, which was 12.99 times that of the model group. In contrast, the relative abundance of Rickenella in the intestines of mice treated with Bifidobacterium longum CCFM1114 was only 1.19%.

[0119] Reports indicate that *Roseburia* can increase butyrate levels in the gut, and a decrease in this genus is significantly associated with the development and progression of chronic kidney disease. *Roseburia* can regulate intestinal immune function by activating colonic TLR-5 receptors, thereby alleviating Crohn's disease. Furthermore, *Roseburia* also has the function of alleviating alcoholic fatty liver disease. The relative abundance of *Roseburia* in the gut of mice in the model group was 0.36%, while the relative abundance of *Roseburia* in the gut of mice treated with *Bifidobacterium longum* CCFM5871 was 20.86%, 57.9 times that of the model group, while the relative abundance of *Roseburia* in the gut of mice treated with *Bifidobacterium longum* CCFM1114 was only 7.09%.

[0120] It is evident that Bifidobacterium longum CCFM5871 can significantly improve the intestinal flora structure and increase the relative abundance of beneficial bacteria such as Rikenobacter and Rochezoides, while the other strain does not have this effect.

[0121] Example 3: Effect of Bifidobacterium longum CCFM5871 on the growth of Bacteroides davidii

[0122] The specific steps are as follows:

[0123] (1) Culture of Bacteroides davidii

[0124] Bacteroides davidii CCFM 1309 (GDMCC No. 63535) and Bacteroides davidii CCFM 1246 (deposited in the Biotechnology Center of the College of Food Science and Technology, Jiangnan University) were inoculated into BHI liquid medium at an inoculation rate of 2% (v / v) and cultured at 37°C for 18 h to prepare seed culture.

[0125] (2) Culture of Bifidobacterium longum

[0126] Bifidobacterium longum CCFM5871 and Bifidobacterium longum CCFM1114 were inoculated into MRS medium (with 0.05% [w / v] cysteine ​​added) at an inoculation rate of 2% (v / v) and activated by incubation at 37°C for 18 h. The activation was repeated for two generations to prepare seed culture of Bifidobacterium longum CCFM5871 and seed culture of Bifidobacterium longum CCFM1114, respectively.

[0127] (3) The two types of Bacteroides davidii seed cultures were inoculated into GMM medium (containing 0.5% arabinogalactan) to achieve a final cell concentration of 10. 5 CFU / mL, and simultaneously inoculated with two different Bifidobacterium longum seed cultures to achieve the same concentration (10). 5 CFU / mL), co-cultured at 37℃ for 24 h;

[0128] Data were collected periodically at 0h, 3h, 6h, 9h, 12h, 18h, and 24h for analysis of microbial growth (OD). 600 The results are shown in Table 4 and... Figure 4 As shown in the table, the data in the table are the growth OD values ​​of Bacteroides davidii.

[0129] Table 4: Effects of Bifidobacterium longum on the growth of Bacteroides cerevisiae

[0130]

[0131] The results show:

[0132] After co-culturing with Bifidobacterium longum CCFM5871, the OD values ​​of Bacteroides davidii CCFM1309 and Bacteroides davidii CCFM1246 were... 600 Both were improved, proving that Bifidobacterium longum CCFM5871 can promote the proliferation of Bacteroides davidii, while the growth of Bacteroides davidii was not significantly improved after co-culturing Bifidobacterium longum CCFM1114 with Bacteroides davidii.

[0133] Meanwhile, qPCR measurements of the abundance of *Bifidobacterium longum* and *Bacteroides davidii* during co-culture revealed results consistent with the above conclusions: *Bifidobacterium longum* CCFM5871 significantly promoted the growth of *Bacteroides davidii* CCFM1309 and CCFM1246, while *Bifidobacterium longum* CCFM1114 did not have this effect (see...). Figure 4 ).

[0134] Example 4: Effects of Bifidobacterium longum CCFM5871 on the intestines of healthy mice

[0135] The specific steps are as follows:

[0136] Fifteen healthy male BALB / c mice aged 6-8 weeks and weighing 20-22g were randomly divided into three groups of five mice each: a blank control group, a group treated with Bifidobacterium longum CCFM5871, and a group treated with Bifidobacterium longum CCFM1114.

[0137] The experimental procedure is as follows:

[0138] The control group received 0.2 mL of normal saline via gavage daily.

[0139] CCFM5871 group: 0.2 mL of bacterial strain was administered via gavage daily, with a concentration of 1×10⁻⁶. 9 CFU containing Bifidobacterium longum CCFM5871 bacterial culture;

[0140] CCFM1114 group: 0.2 mL of the bacterial strain was administered via gavage daily, with a concentration of 1×10⁻⁶. 9 CFU containing Bifidobacterium longum CCFM1114 bacterial culture.

[0141] Mice were continuously administered ink via gavage for 14 days until the last day of the experiment. All mice were fasted for 24 hours before being administered ink via gavage. The time of the first black stool excretion and the number of black stools excreted within 6 hours were recorded.

[0142] During the experimental period, the defecation frequency, intestinal transit time, small intestinal propulsion rate, fecal water content, and fecal acetic acid content of mice in each group were as follows: Figure 6 As shown.

[0143] The results showed that Bifidobacterium longum CCFM5871 significantly increased the acetic acid content, defecation frequency, fecal water content and small intestinal propulsion rate in healthy mice, and significantly reduced intestinal transit time, while another strain of Bifidobacterium longum CCFM1114 did not have this effect.

[0144] Example 5: Effect of Bifidobacterium longum CCFM5871 on the lifespan of Caenorhabditis elegans

[0145] The specific steps are as follows:

[0146] One hundred and twenty Caenorhabdit elegans N2 worms synchronized to stage L4 were randomly divided into four groups of 30 worms each: control group (fed with Escherichia coli OP50), CCFM5871 group (fed with Bifidobacterium longum CCFM5871), CCFM1232 group (fed with Bifidobacterium longum CCFM1232), and CCFM1114 group (fed with Bifidobacterium longum CCFM1114).

[0147] The experimental procedure is as follows:

[0148] (1) Inoculate Escherichia coli OP50 into 5 mL LB liquid medium and incubate at 37℃ with shaking at 150 rpm for 12 h. When OD 600 When the concentration is 1.0-1.2, after shaking and mixing, take 200 μL of bacterial solution and drop it onto an NGM plate and spread it evenly (about 60% of the entire plate). Incubate at 37°C for 12 h and store at 4°C for later use.

[0149] Bifidobacterium longum was inoculated into 5 mL of LB liquid medium and cultured at 37°C with shaking at 150 rpm for 12 h. When OD 600 When the concentration is 1.0-1.2, after shaking and mixing, take 200 μL of bacterial solution and drop it onto an NGM plate and spread it evenly (about 60% of the entire plate). Incubate at 37°C for 12 h and store at 4°C for later use.

[0150] Escherichia coli OP50 plates and long bifidobacteria plates were prepared separately.

[0151] (2) Place the frozen-thawed nematodes on NGM agar plates containing Escherichia coli OP50 and incubate at 15°C to revive them, obtaining a large number of oviposition-stage nematodes. Take 3.5 mL of sterile aqueous suspension containing nematodes and add it to a centrifuge tube. Add 0.5 mL of 5M sodium hydroxide solution and 1 mL of 5% sodium hypochlorite solution, mix thoroughly, wash repeatedly with M9 buffer 2-3 times, and resuspend in S agar medium. Incubate the S agar medium containing eggs at 20°C for 8-12 h, centrifuge at 1500×g for 3 min to collect L1 stage nematodes, transfer them to NGM agar plates containing nematode-eating Escherichia coli OP50, and incubate at 25°C for 48 h to obtain L4 stage nematodes.

[0152] (3) Place 180 L4 stage nematodes on plates containing Escherichia coli OP50 and different Bifidobacterium longum (30 nematodes per plate). Transfer the nematodes to new plates every day, record the number of nematode deaths and calculate the mortality rate (when observing, gently touch the nematode body with the end of the nematode pick; if there is no reaction, the nematode is considered dead).

[0153] Calculation formula: Survival rate (%) = (Number of surviving nematodes / Total number of nematodes) × 100%. Results are shown in Table 5 and... Figure 5 As shown.

[0154] Table 5: Effects of Bifidobacterium longum strains on nematode lifespan

[0155]

[0156] The results showed that Bifidobacterium longum CCFM5871 significantly increased the average lifespan of Caenorhabditis elegans by 18.3% and the longest lifespan by 21.6%. The experiment showed that Bifidobacterium longum CCFM5871 can significantly increase the lifespan of nematodes, while other strains did not have this effect.

[0157] Example 6: Bifidobacterium longum CCFM5871 used to prepare fermented milk

[0158] Bifidobacterium longum CCFM5871 can be used to prepare fermented milk. The specific preparation process for the milk is as follows:

[0159] (1) The secondary purified culture broth of Bifidobacterium longum CCFM5871 obtained in Example 1 was inoculated into the culture medium at an inoculation rate of 3% (v / v) and cultured at 37°C for 18 h to obtain bacterial suspension; the bacterial suspension was centrifuged to obtain bacterial sludge; the bacterial sludge was washed three times with phosphate buffer at pH 7.2 and then resuspended with a protectant to a concentration of 1×10⁻⁶. 10 The concentration of CFU / mL was used to obtain a suspension; the suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain the starter culture.

[0160] The culture medium is prepared by dissolving 10% enzyme hydrolysate, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0161] The ingredients of the protectant include: 100g / L skim milk powder, 30mL / L glycerin, 100g / L maltodextrin, 150g / L trehalose, and 10g / L L-monosodium glutamate;

[0162] (2) Sterilize the skim milk at 95℃ for 20 minutes and then cool it to 4℃ to obtain the raw material; add the starter culture prepared in step (1) to the raw material until the concentration is not less than 1×10⁻⁶. 6 CFU / mL was used to obtain milk (the milk should be refrigerated at 4°C).

[0163] Example 7: Bifidobacterium longum CCFM5871 used to prepare fermented soy milk

[0164] Bifidobacterium longum CCFM5871 can be used to prepare soy milk. The specific preparation process for soy milk is as follows:

[0165] (1) The secondary purified culture broth of Bifidobacterium longum CCFM5871 obtained in Example 1 was inoculated into the culture medium at an inoculation rate of 3% (v / v) and cultured at 37°C for 18 h to obtain bacterial suspension; the bacterial suspension was centrifuged to obtain bacterial sludge; the bacterial sludge was washed three times with phosphate buffer at pH 7.2 and then resuspended with a protectant to a concentration of 1×10⁻⁶. 10 The concentration of CFU / mL was used to obtain a suspension; the suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain the starter culture.

[0166] The culture medium is prepared by dissolving 10% skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0167] The ingredients of the protectant include: 100g / L skim milk powder, 30mL / L glycerin, 100g / L maltodextrin, 150g / L trehalose, and 10g / L L-monosodium glutamate;

[0168] (2) Soak soybeans at 80℃ for 2 hours, then remove the soybean skins to obtain peeled soybeans; drain the soaking water from the peeled soybeans, add boiling water and grind them into a paste to obtain soy milk; keep the soy milk at a temperature above 80℃ for 12 minutes to obtain cooked soy milk; filter the cooked soy milk through a 150-mesh sieve and centrifuge to obtain crude soy milk; heat the crude soy milk to 140-150℃ and quickly introduce it into a vacuum cooling chamber for vacuuming, so that the off-flavor substances in the crude soy milk are quickly expelled with water vapor to obtain cooked soy milk; cool the cooked soy milk to about 37℃ and add the starter culture obtained in step (1) to the cooked soy milk until the concentration is not less than 1×10 6 CFU / mL yields soy milk (soy milk must be refrigerated at 4℃).

[0169] Example 8: Bifidobacterium longum CCFM5871 used in the preparation of fruit and vegetable beverages

[0170] Bifidobacterium longum CCFM5871 can be used to prepare vegetable beverages. The specific preparation process for vegetable beverages is as follows:

[0171] (1) The secondary purified culture broth of Bifidobacterium longum CCFM5871 obtained in Example 1 was inoculated into the culture medium at an inoculation rate of 3% (v / v) and cultured at 37°C for 18 h to obtain bacterial suspension; the bacterial suspension was centrifuged to obtain bacterial sludge; the bacterial sludge was washed three times with phosphate buffer at pH 7.2 and then resuspended with a protectant to a concentration of 1×10⁻⁶. 10 The concentration of CFU / mL was used to obtain a suspension; the suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain the starter culture.

[0172] The culture medium is prepared by dissolving 10% enzyme hydrolysate, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0173] The ingredients of the protectant include: 100g / L skim milk powder, 30mL / L glycerin, 100g / L maltodextrin, 150g / L trehalose, and 10g / L L-monosodium glutamate;

[0174] (2) Wash the fresh vegetables and juice them to obtain vegetable juice; sterilize the vegetable juice at 140℃ for 2 seconds to obtain sterilized vegetable juice; cool the sterilized vegetable juice to about 37℃ and add the fermentation agent prepared in step (1) to the sterilized vegetable juice until the concentration is not less than 1×10 6 CFU / mL yields a vegetable beverage (which must be refrigerated at 4°C).

[0175] Example 9: Bifidobacterium longum CCFM5871 used in the preparation of fermented dairy products

[0176] Bifidobacterium longum CCFM5871 can be used to prepare fermented milk. The specific preparation process for fermented milk is as follows:

[0177] (1) The secondary purified culture broth of Bifidobacterium longum CCFM5871 obtained in Example 1 was inoculated into the culture medium at an inoculation rate of 3% (v / v) and cultured at 37°C for 18 h to obtain bacterial suspension; the bacterial suspension was centrifuged to obtain bacterial sludge; the bacterial sludge was washed three times with phosphate buffer at pH 7.2 and then resuspended with a protectant to a concentration of 1×10⁻⁶. 10 The concentration of CFU / mL was used to obtain a suspension; the suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain a freeze-dried powder with a bacterial cell concentration of 1×10⁻⁶. 9 CFU / g;

[0178] The culture medium is prepared by dissolving 10% enzyme hydrolysate, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0179] The ingredients of the protectant include: 100g / L skim milk powder, 30mL / L glycerin, 100g / L maltodextrin, 150g / L trehalose, and 10g / L L-monosodium glutamate;

[0180] (2) Mix the freeze-dried powder with the commercial dry powder starter Lactobacillus bulgaricus (cell concentration of 1×10⁻⁶). 9 CFU / g) and commercial dry powder starter culture Streptococcus thermophilus (cell concentration of 1×10⁻⁶) 9The starter culture was prepared by mixing CFU / g in a mass ratio of 1:1:1, with the concentration of Lactobacillus bulgaricus and Streptococcus thermophilus in the starter culture being 2%.

[0181] (3) Add sugar to fresh milk to a concentration of 5% to obtain a mixture; homogenize the mixture at 65℃ and 20MPa and then sterilize it at 95℃ for 5 minutes to obtain fermentation raw material; cool the fermentation raw material to 35℃ and inoculate the fermentation agent obtained in step (2) into the fermentation raw material at an inoculation amount of 0.03% (v / v), and ferment it at 35℃ for 16 hours to obtain fermented milk; after coagulating the fermented milk at 42℃, refrigerate it at 4℃ for 24 hours for post-ripening to obtain the finished fermented milk product.

[0182] Example 10: Application of Bifidobacterium longum CCFM5871

[0183] Bifidobacterium longum CCFM5871 can be used to prepare capsule products. The specific preparation process for capsule products is as follows:

[0184] Bifidobacterium longum CCFM5871 was streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. This activation was repeated for two generations to obtain an activated solution. The activated solution was inoculated into MRS liquid medium at a rate of 2% (v / v) and cultured at 37°C for 18 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 8000g for 10 min to obtain a bacterial sludge. The bacterial sludge was washed three times with physiological saline and then resuspended in a cryoprotectant to a concentration of 1×10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was added to a 30 g / L sodium alginate solution to a concentration of 2 × 10⁻⁶. 9 After adding CFU / mL, the mixture was stirred thoroughly to ensure that the Bifidobacterium longum CCFM5871 cells were evenly dispersed in the sodium alginate solution, resulting in a mixture. The mixture was then extruded into a 20 g / L calcium chloride solution to form granules. After the granules were allowed to stand and solidify for 30 minutes, they were filtered and collected. The collected granules were then freeze-dried for 48 hours to obtain a powder. The powder was then filled into pharmaceutical capsules to obtain the capsule product.

[0185] Example 11: Application of Bifidobacterium longum CCFM5871

[0186] Bifidobacterium longum CCFM5871 can be used to prepare tablets. The specific tablet preparation process is as follows:

[0187] Bifidobacterium longum CCFM5871 was streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. This activation was repeated for two generations to obtain an activated solution. The activated solution was inoculated into MRS liquid medium at a rate of 2% (v / v) and cultured at 37°C for 18 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 8000g for 10 min to obtain a bacterial sludge. The bacterial sludge was washed three times with physiological saline and then resuspended in a cryoprotectant to a concentration of 1×10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain Bifidobacterium longum CCFM5871 bacterial powder.

[0188] The protective agent is a skim milk powder solution with a concentration of 130 g / L. 25.7 parts by weight of Bifidobacterium longum CCFM5871 bacterial powder, 55.0 parts by weight of starch, 4.5 parts by weight of cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 parts by weight of talc, 1.0 part by weight of sucrose, and 1.0 part by weight of water were weighed to obtain raw materials. The raw materials were mixed to obtain wet granules. The wet granules were compressed into tablets using a tableting machine from Zhongnan Pharmaceutical Machinery Factory, and then dried using a small pharmaceutical dryer from Qingzhou Yikang Traditional Chinese Medicine Machinery Co., Ltd., to obtain tablets.

[0189] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method of promoting the proliferation of Bacteroides nactimodoriis (B. nactimodoriis) or increasing the abundance of Rikenella (Rikenella), characterized by, Bacteroides nordii Rikenella Bifidobacterium longum ( Bifidobacterium longum CCFM5871 or its microbial preparations were added to an environment containing Bacteroides davidii or Riken bacteria for co-culture. ​​ The Bifidobacterium longum CCFM5871 is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No:63537, and the preservation date is June 7, 2023.

2. The method of claim 1, wherein, The viable cell number of the Bifidobacterium longum CCFM5871 in the microbial preparation is not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

3. The use of Bifidobacterium longum CCFM5871 or microbial preparation thereof in the preparation of health products for helping to regulate intestinal flora.

4. The use according to claim 3, wherein the compound is ###00002### The regulation of intestinal flora is to promote the growth of Bacteroides northernis (… Bacteroides nordii ) proliferation and / or enhancement of Riken bacteria ( Rikenella Abundance.

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