Longissimus bifidus for promoting proliferation of prasrptococcus and application thereof

Abstract

This invention discloses a *Bifidobacterium longum* strain that promotes the proliferation of *Clostridium pluvialis* and its applications, belonging to the fields of microbial technology and pharmaceutical technology. This invention provides a *Bifidobacterium longum* subspecies *CCFM1306*, which can promote the proliferation of *Clostridium pluvialis*, improve *Clostridium pluvialis*'s ability to utilize inulin, and promote the proliferation of *Clostridium pluvialis* CCFM1203, *Clostridium pluvialis* FWXPL145, and *Clostridium pluvialis* A2-165 on inulin culture medium. The *Bifidobacterium longum* subspecies *CCFM1306* of this invention can increase the colony count and butyrate yield of *Clostridium pluvialis* in inulin, thereby improving the utilization rate of inulin and possessing significant application and commercial value.

Description

Technical Field

[0001] This invention relates to a long Bifidobacterium that promotes the proliferation of Clostridium plasmidoides and its applications, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] *Clostridium plasminogen lysate* is an abundant gut microbiota in healthy individuals, playing a crucial role in maintaining gut health, inhibiting the proliferation of harmful microorganisms, and enhancing immunity. *Clostridium plasminogen lysate* primarily exerts its effects by producing short-chain fatty acids and is an important butyrate-producing bacterium in the gut. Butyrate is a major energy source for colonic cells and plays a vital role in regulating gene expression, host cell differentiation, and apoptosis. Studies have found a close relationship between the abundance of *Clostridium plasminogen lysate* in the human gut and the occurrence and development of many diseases. It was initially discovered because its abundance was significantly lower in the gut of patients with IBD than in healthy individuals. Subsequent studies have shown that the abundance of this bacterium is significantly reduced in the guts of patients with obesity, type 2 diabetes, and atopic dermatitis. *Clostridium plasminogen lysate* has important anti-inflammatory effects, promoting the secretion of anti-inflammatory factors and inhibiting the secretion of pro-inflammatory factors. This species is also gradually becoming a biomarker for distinguishing between ulcerative colitis (UC) and Crohn's disease (CD), possessing potential probiotic effects and being hailed as a next-generation probiotic.

[0003] In addition to maintaining gut health, a growing body of research indicates that *Clostridium pluvialis* has alleviating effects on diseases such as Alzheimer's and autism. For example, one study found that a single strain of *Clostridium pluvialis* could alleviate cognitive impairment in Alzheimer's mice.

[0004] Despite increasing evidence of the beneficial effects of Clostridium pluvialis, its application remains a hot topic and challenge due to its extreme sensitivity to oxygen (it dies after just 2 minutes of exposure to air), poor colonization in the gut, and low proliferation rate.

[0005] Since Clostridium pluvialis cannot be consumed directly, dietary intervention and the interaction between gut microbiota have become important methods for proliferating Clostridium pluvialis. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a process method for increasing the number of Clostridium perfringens colonies that is short in cycle and simple in process, and at the same time provide a strain of Bifidobacterium longum subsp. infantis and its application.

[0007] To address the above problems, this invention provides a strain of Bifidobacterium longum infantis (… Bifidobacterium longum subsp. infantisCCFM1306, the Bifidobacterium longum subspecies CCFM1306, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:63216 and deposit date of March 29, 2023.

[0008] The Bifidobacterium longum infant subspecies ( Bifidobacterium longum subsp. infantis CCFM1306 was isolated from a fecal sample derived from an infant. Sequencing analysis of this strain and comparison of its 16S rRNA sequence with GenBank revealed that it belongs to the *Bifidobacterium longum* subsp. *infantii*, and it was named *Bifidobacterium longum* subsp. *infantii*. Bifidobacterium longum subsp. infantis (CCFM1306)

[0009] The Bifidobacterium longum infant subspecies in ( Bifidobacterium longum subsp. infantis The colonies of CCFM1306 on modified MRS solid medium are milky white, semi-circular, raised, with a smooth, moist surface and neat edges.

[0010] The present invention also provides a method containing the above-mentioned Bifidobacterium longum infant subspecies ( Bifidobacterium longum subsp. infantis CCFM1306 microbial preparation.

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

[0012] This invention also provides a method to promote the growth of Clostridium perfringens (Clostridium perfringens) Faecalibacterium prausnitzii The method of proliferation is to co-culture the above-mentioned Bifidobacterium longum subsp. infantis or the above-mentioned microbial preparation with Clostridium plasminoides in a system containing inulin.

[0013] In one embodiment of the present invention, the inulin-containing system is an inulin-containing culture medium: glucose in the modified MRS medium is replaced with inulin.

[0014] In one embodiment of the present invention, the inulin was purchased from Shanghai Chuangsai Scientific Instruments Co., Ltd.

[0015] In one embodiment of the present invention, the *Clostridium pluvialis* includes, but is not limited to, *Clostridium pluvialis* (… Faecalibacterium prausnitzii A2-165, Clostridium plasminoides ( Faecalibacterium prausnitzii CCFM1204 and Clostridium praosporum ( Faecalibacterium prausnitzii (CCFM1203)

[0016] In one embodiment of the present invention, the *Bifidobacterium longum* subsp. *infantitidis* and *Clostridium plasminogen lysate* are added at a live bacteria ratio of 1:1, wherein the concentration of live bacteria is at least 1 × 10⁻⁶. 6 CFU / mL.

[0017] In one embodiment of the present invention, the inulin-containing culture medium comprises: 10.0 g casein peptone, 2.5 g yeast extract, 4.0 g sodium bicarbonate, 1.0 g cysteine ​​hydrochloride, 0.45 g dipotassium hydrogen phosphate, 0.45 g potassium dihydrogen phosphate, 0.9 g sodium chloride, 0.9 g ammonium sulfate, 0.09 g magnesium sulfate heptahydrate, 0.09 g anhydrous calcium chloride, 0.01 g heme, 1 μg biotin, 1 μg cobalamin, 3 μg para-aminobenzoic acid, 5 μg folic acid, and 15 μg pyridoxine, with distilled water added to 1000 mL, pH adjusted to 6.4-6.6, and sterilized at 115°C for 20 min.

[0018] To avoid nutrient loss, the carbon source in the culture medium is 5.0 g inulin. First, dissolve 5.0 g inulin in 10 mL of deionized water and filter it through a 0.22 μm microporous membrane for sterilization. Before use, take 1 mL of inulin solution and add it to the above 9 mL liquid culture medium.

[0019] In one embodiment of the present invention, the culture method is to culture at a constant temperature of 37°C for 48 hours in an anaerobic chamber (80% N2, 10% CO2 and 10% H2).

[0020] In one embodiment of the present invention, the effect of *Bifidobacterium longum* subsp. infantis promoting *Clostridium plasmidonum* proliferation is evaluated by quantitative real-time qPCR, GC-MS determination of short-chain fatty acid content, and determination of the degree of polymerization of inulin molecules in fermentation supernatant.

[0021] The present invention also provides a drug for promoting the proliferation of Clostridium perfringens, the drug containing the above-mentioned Bifidobacterium longum subsp. infantis (… Bifidobacterium longum subsp. infantis CCFM1306, or the above-mentioned microbial preparations.

[0022] In one embodiment of the present invention, the pharmaceutical product contains the above-mentioned Bifidobacterium longum subsp. infantis (… Bifidobacterium longum subsp. infantis The viable count of CCFM1306 in the drug is not less than 1×10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

[0023] This invention also provides the above-mentioned Bifidobacterium longum infant subspecies ( Bifidobacterium longum subsp. child CCFM1306, or the above-mentioned microbial preparations, in the use of medicines to improve gut health.

[0024] The present invention also provides a medicine containing the above-mentioned Bifidobacterium longum subsp. infantis (… Bifidobacterium longum subsp. infantis (CCFM1306 and inulin)

[0025] Beneficial effects

[0026] This invention provides a strain of Bifidobacterium longum infantis subspecies ( Bifidobacterium longum subsp. child CCFM1306 states that this *Bifidobacterium longum* subsp. *infantii* can promote the proliferation of *Clostridium plasmidonum*, specifically in the following ways:

[0027] (1) Bifidobacterium longum infantis subspecies CCFM1306 increases Clostridium perfringens ( Faecalibacterium of Prausnitz The ability of *Clostridium pluvialis* to utilize inulin promotes its activity. Faecalibacterium prausnitzii The proliferation of CCFM1203, FWXPL145, and A2-165 on inulin medium led to the proliferation of Clostridium pratensis (Clostridium perfringens). Faecalibacterium of Prausnitz CCFM1203, FWXPL145, and A2-165 had a viable bacterial count greater than 1×10⁻⁶ in vitro. 7 CFU / mL.

[0028] (2) Bifidobacterium longum infantis subspecies CCFM1306 increases the levels of Clostridium perfringens ( ) Faecalibacterium of Prausnitz The butyrate-producing ability of CCFM1203, FWXPL145, and A2-165 makes Clostridium pluvialis (CCFM1203, FWXPL145, and A2-165) a factor. Faecalibacterium of Prausnitz The production of CCFM1203 butyrate increased tenfold.

[0029] (3) Bifidobacterium longum infantis subspecies ( Bifidobacterium longum subsp. infantis CCFM1306 can promote the proliferation of Clostridium praosporum in inulin, increase the degradation degree of inulin in the fermentation supernatant, and promote the more thorough decomposition and utilization of macromolecular inulin.

[0030] The active ingredient of this invention, Bifidobacterium longum subsp. infantis, can be used to prepare pharmaceuticals and has a very broad application prospect.

[0031] Preservation of biological materials

[0032] A strain of Bifidobacterium longum infantis ( Bifidobacterium longum subsp. child CCFM1306, taxonomically named: Bifidobacterium longum subsp. childIt was deposited on March 29, 2023 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:63216. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0033] Figure 1 Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis CCFM1306 and Clostridium praosporum ( Faecalibacterium prausnitzii Growth curves of CCFM1203 cultured alone and in combination on 0.5% inulin YCFA liquid medium.

[0034] Figure 2 This invention relates to Bifidobacterium longum infantis subsp. ( Bifidobacterium longum subsp. infantis CCFM1306 and Clostridium perfringens (Clostridium perfringens) Faecalibacterium prausnitzii Colony counts of CCFM1203, FWXPL145, and A2-165 cultured individually and in combination on 0.5% inulin YCFA liquid medium.

[0035] Figure 3 This invention relates to Bifidobacterium longum infantis subsp. ( Bifidobacterium longum subsp. infantis CCFM1306 and Clostridium perfringens (Clostridium perfringens) Faecalibacterium prausnitzii The yield of acetic acid by culturing CCFM1203, FWXPL145 and A2-165 individually and in combination on 0.5% inulin YCFA liquid medium.

[0036] Figure 4 This invention relates to Bifidobacterium longum infantis subsp. ( Bifidobacterium longum subsp. infantis CCFM1306 and Clostridium perfringens (Clostridium perfringens) Faecalibacterium prausnitzii The yield of butyric acid by culturing CCFM1203, FWXPL145 and A2-165 individually and in combination on 0.5% inulin YCFA liquid medium.

[0037] Figure 5 The present invention is based on Clostridium praosporum (…). Faecalibacterium prausnitzii The degree of inulin polymerization in the fermentation supernatant of CCFM1203 cultured alone in 0.5% inulin YCFA liquid medium for 48 h was determined.

[0038] Figure 6 This invention relates to Bifidobacterium longum infantis subsp. ( Bifidobacterium longum subsp. infantis The degree of inulin polymerization in the fermentation supernatant of CCFM1306 cultured alone in 0.5% inulin YCFA liquid medium for 48 h was determined.

[0039] Figure 7 This invention relates to Bifidobacterium longum infantis subsp. ( Bifidobacterium longum subsp. infantisCCFM1306 and Clostridium praosporum ( Faecalibacterium prausnitzii The degree of polymerization of inulin in the fermentation supernatant after CCFM1203 was cultured in 0.5% inulin YCFA liquid medium for 48 h was determined. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments.

[0041] The strain involved in the following examples is Clostridium praosporum ( Faecalibacterium prausnitzii CCFM1203 is described in the text of Chinese patent application with publication number CN115093999A.

[0042] The strain involved in the following examples is Clostridium praosporum ( Faecalibacterium prausnitzii ) FWXPL145 was published in the paper "Biodiversity and Physiological Characteristics of NovelFaecalibacterium prausnitzii Strains Isolated from Human Feces".

[0043] The strain involved in the following examples is Clostridium praosporum ( Faecalibacterium prausnitzii Purchased from DSMZ German Collection of Microorganisms and Cell Cultures (A2-165).

[0044] The following examples involve Bifidobacterium longum infantis subsp. ( Bifidobacterium longum subsp. child FGZ23I1M2, Bifidobacterium breve ( Bifidobacterium breve CJ653, Bifidobacterium bifidum ( Bifidobacterium bifidum FXJCJ9M10 is published in the dissertation "Study on the carbon source utilization characteristics and interspecific mutualistic mechanism of Bifidobacteria".

[0045] The animal bifidobacteria involved in the following examples ( Bifidobacterium animalum )JSZJ4M1 is published in the dissertation "An investigation into the interspecific synergistic proliferation patterns of different Bifidobacteria using inulin as a carbon source".

[0046] Information about the materials involved in the following embodiments is as follows:

[0047] The inulin, heme, and microporous membranes used in the following examples were purchased from Shanghai Chuangsai Scientific Instruments Co., Ltd.; the casein peptone, yeast extract, sodium bicarbonate, glucose, cysteine ​​hydrochloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, ammonium sulfate, magnesium sulfate heptahydrate, anhydrous calcium chloride, beef extract, diammonium citrate, sodium acetate, Tween, and manganese sulfate monohydrate used in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd.; the biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine used in the following examples were purchased from Maclean Chemical Reagent Co., Ltd.; the bacterial DNA extraction kit used in the following examples was purchased from Tiangen Biotech Co., Ltd.; and the Clostridium perfringens-specific primers used in the following examples were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0048] The culture media involved in the following examples are as follows:

[0049] YCFA liquid culture medium: 10.0 g casein peptone, 2.5 g yeast extract, 4.0 g sodium bicarbonate, 5.0 g glucose, 1.0 g cysteine ​​hydrochloride, 0.45 g dipotassium hydrogen phosphate, 0.45 g potassium dihydrogen phosphate, 0.9 g sodium chloride, 0.9 g ammonium sulfate, 0.09 g magnesium sulfate heptahydrate, 0.09 g anhydrous calcium chloride, 0.01 g heme, 1 μg biotin, 1 μg cobalamin, 3 μg para-aminobenzoic acid, 5 μg folic acid, 15 μg pyridoxine, add distilled water to 1000 ml, adjust pH to 6.4-6.6, sterilize at 115°C for 20 min.

[0050] 0.5% Inulin YCFA liquid medium: 10.0 g casein peptone, 2.5 g yeast extract, 4.0 g sodium bicarbonate, 1.0 g cysteine ​​hydrochloride, 0.45 g dipotassium hydrogen phosphate, 0.45 g potassium dihydrogen phosphate, 0.9 g sodium chloride, 0.9 g ammonium sulfate, 0.09 g magnesium sulfate heptahydrate, 0.09 g anhydrous calcium chloride, 0.01 g heme, 1 μg biotin, 1 μg cobalamin, 3 μg para-aminobenzoic acid, 5 μg folic acid, 15 μg pyridoxine, add distilled water to 1000 ml, adjust pH to 6.4-6.6, sterilize at 115°C for 20 min. To avoid nutrient loss, the carbon source in the culture medium is 5.0 g inulin. First, dissolve 5.0 g inulin in 10 mL of deionized water and filter it through a 0.22 μm microporous membrane for sterilization. Before use, take 1 mL of inulin solution and add it to the above 9 mL liquid culture medium.

[0051] Modified MRS liquid culture medium formula: 10.0 g peptone, 10.0 g beef extract, 5.0 g yeast powder, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium citrate, 2.0 g sodium acetate, 1.0 mL Tween 80, 0.5 g cysteine, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate monohydrate, add distilled water to 1000 mL, adjust pH to 6.4-6.6, and sterilize at 115°C for 20 min.

[0052] Improved MRS solid culture medium formula: Add 15.0 g of agar powder to the liquid culture medium.

[0053] 0.5% Inulin MRS Liquid Medium: 10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium citrate, 2.0 g sodium acetate, 1.0 mL Tween 80, 0.5 g cysteine, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate tetrahydrate, add distilled water to 1000 mL, adjust pH to 6.4-6.6, and sterilize at 115°C for 20 min. To avoid nutrient loss, the carbon source in the medium is 5.0 g inulin. First, dissolve 5.0 g inulin in 10 mL of deionized water, filter sterilize using a 0.22 μm microporous membrane, and add 1 mL of the inulin solution to 9 mL of the above liquid medium before use.

[0054] The method for preparing the bacterial seed solution involved in the following examples is as follows:

[0055] Preparation of Bifidobacterium seed culture:

[0056] Take 2 mL of bacterial culture that has grown to the logarithmic phase into a preservation tube, centrifuge at 8000 rpm for 2 min, discard the supernatant, add 1 mL of 30% glycerol, mix well, and store at -80°C as bacterial seed culture.

[0057] Preparation of Clostridium praosporum seed culture:

[0058] Take 50 mL of bacterial culture that has grown to the logarithmic phase and filter it in a sterile filter cup to remove the fermentation broth. Dissolve the bacterial sludge adsorbed on the filter membrane with 30% glycerol. The entire operation is carried out in a sterile anaerobic chamber. Store the glycerol tube at -80°C as the inoculum.

[0059] The detection methods involved in the following embodiments are as follows:

[0060] Detection method for Clostridium perfringens proliferation (real-time quantitative qPCR detection method):

[0061] (1) The qPCR primers are Clostridium praosporum-specific primers:

[0062] F: 5'-GGAGGAAGAAGGTCTTCGG-3';

[0063] R: 5'-AATTCCGCCTACCTCTGCACT-3'.

[0064] (2) qPCR reaction system (10 μL): DNA template 1 μL, 10 mM upstream and downstream primers 0.5 μL each, ddH2O 3 μL, SYBR Green Premix Ex TaqII 5 μL.

[0065] (3) The qPCR reaction procedure is shown in Table 1 below:

[0066] Table 1: qPCR reaction procedure

[0067]

[0068] (4) Constructing a standard curve:

[0069] Clostridium plasminoides ( Faecalibacterium prausnitzii CCFM1203 was inoculated onto YCFA agar plates and incubated at 37°C for 48 h. Single colonies were then picked and inoculated into liquid culture medium, and the culture was subcultured three times. One mL of the bacterial suspension was added to 9 mL of sterile physiological saline and vortexed to prepare a 1:100 sample homogenate. Serial dilutions of 10-fold were prepared. One mL of each dilution was pipetted into sterile Petri dishes for decanting and counting.

[0070] The serially diluted bacterial solutions were centrifuged at 12000 r / min for 2 min, washed three times with PBS, and bacterial DNA was extracted for quantitative real-time PCR.

[0071] Clostridium plasminoides ( Faecalibacterium prausnitzii The standard sample and the test sample were simultaneously subjected to real-time quantitative PCR (qPCR) to create a standard curve. The colony count of different concentrations of positive templates was plotted on the ordinate, and the initial cycle number (Ct) of the fluorescence signal during the qPCR reaction was plotted on the abscissa. The number of Clostridium plasmids was obtained by comparing the test sample with the standard curve.

[0072] Methods for detecting short-chain fatty acids:

[0073] (1) The content of short-chain fatty acids in the culture supernatant was determined by gas chromatography-mass spectrometry (GC-MS).

[0074] (2) Chromatographic detection conditions: An Rtx-Wax column (30 m*0.25 μm*0.25 μm) and a mass spectrometer detector (GC-MS-QP2010Vitta, system) were used to separate the short-chain fatty acids in the sample. N2 was used as the carrier gas, the flow rate was 1 mL / min, the injection volume was 1 μL, the split ratio was 10:1, the initial column temperature was 100°C, the temperature was increased to 140°C at a rate of 7.5°C / min, and then increased to 200°C at a rate of 60°C / min. The temperature was held at 200°C for 3 min, the injection temperature was 240°C, and the ionization temperature was 220°C.

[0075] (3) Plotting the standard curve: Take 10 μL each of acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and isobutyric acid into a 2 mL Eppendorf tube, then add diethyl ether to bring the volume to 1 mL and mix well; take 100 μL, dilute it 10 times with diethyl ether, mix well, and then take 200 μL, 100 μL, 50 μL, 25 μL, 15 μL, and 10 μL of the mixture respectively, and bring the volume to 1 mL with diethyl ether. Pass the mixtures of different concentration gradients through a gas chromatography-mass spectrometer into a gas chromatograph vial, and analyze the short-chain fatty acid content using GC-MS. Plot the standard curve with peak area as the abscissa and concentration as the ordinate. Calculate the concentration by measuring the peak area of ​​the corresponding short-chain fatty acid in the sample.

[0076] Method for determining the molecular weight of inulin in fermentation supernatant:

[0077] (1) Chromatographic detection conditions: The molecular weight of inulin in the fermentation supernatant was determined using an ion chromatograph (ICS-5000), high-performance anion exchange chromatography (HPAEC), and a pulsed amperometric detector (PAD).

[0078] The following three eluent gradients are mobile phases:

[0079] Eluent A: Ultrapure water; Eluent B: 1 M NaAc; Eluent C: 250 mM NaOH; Flow rate: 0.5 mL / min.

[0080] Elution gradient: 0 min, 4% B and 38% C; 40 min, 40% B and 38% C; 41 min, 4% B and 30% C; 50 min, 4% A and 38% B.

[0081] (2) Preparation of standard solution: Accurately weigh 1 mg of inulin into ultrapure water, dissolve it in 10 mL volumetric flask, and prepare a standard solution of 1 mg / mL.

[0082] Example 1: Bifidobacterium longum infantis subspecies ( Bifidobacterium longum subsp. infantis Isolation, screening and identification of CCFM1306

[0083] The specific steps are as follows:

[0084] 1. Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis Separation and screening of CCFM1306

[0085] (1) Take 1 mL of feces from a newborn, dilute it in a gradient, spread the feces on a modified MRS solid medium, and incubate it at a constant temperature of 37°C for 48 h in an anaerobic workstation.

[0086] (2) Select strains with different colony morphologies and isolate them by streaking on modified solid medium.

[0087] (3) Pick the single colony isolated from the culture medium in step (2), transfer it to the modified MRS liquid culture medium, and culture it at a constant temperature of 37°C for 48 h in an anaerobic workstation. Gram stain the obtained strain and record the colony morphology.

[0088] (4) Discard the Gram-negative strains and Gram-positive cocci in the colonies from step (3) and select Gram-positive bacilli.

[0089] (5) After catalase analysis, discard catalase-positive strains and retain catalase-negative strains.

[0090] 2. Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis Molecular identification of CCFM1306

[0091] (1) Genome extraction:

[0092] The obtained *Bifidobacterium longum* subsp. infantis was cultured in modified MRS liquid medium for 20 h to prepare a bacterial suspension. 1 mL of the bacterial suspension was transferred to a 2 mL EP tube and centrifuged at 1000 rpm for 3 min. The supernatant was discarded to obtain the bacterial cells.

[0093] Add 1 mL of sterile water to the obtained bacterial cells, wash the cells, and centrifuge at 1000 rpm for 3 min to obtain bacterial cells; add 200 μL of SDS lysis buffer to the bacterial cells, incubate at 80°C for 30 min to obtain bacterial cell lysis buffer; then add phenol-chloroform mixture to the bacterial cell lysis buffer, with a composition and volume ratio of Tris saturated phenol:chloroform:isoamyl alcohol = 25:24:1, then invert and mix well, centrifuge at 12000 rpm for 5 min, and collect 200 μL of supernatant; then add 40 μL of ice-cold ethanol or ice-cold isopropanol to the 200 μL supernatant, let stand at -20°C for 1 h, then centrifuge at 12000 rpm for 3 min, and discard the supernatant; then place in a 60°C oven to dry or air dry naturally; then redissolve the precipitate with 50 μL of ddH2O to obtain template DNA for PCR reaction.

[0094] (2) 16S rDNA PCR

[0095] PCR reaction system 25 μL: Taq enzyme 12.5 μL; 27F 1.0 μL; 1492R 1.0 μL; ddH2O 10 μL; template 0.5 μL.

[0096] PCR reaction program settings: 95°C, 5 min; 95°C, 30 s; 55°C, 30 s; 72°C, 1 min; 34 cycles; 72°C, 10 min; 12°C, 10 min.

[0097] Electrophoresis: Prepare a 1% agarose gel, mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging analysis.

[0098] After completion, the PCR products were sent to a sequencing company for analysis. The obtained sequencing sequences were compared with the NCBI database, and the strain was preliminarily identified as Bifidobacterium longum subsp. infantis.

[0099] (3) Whole genome sequencing

[0100] The extracted whole genome was sent to a professional sequencing company, where a next-generation sequencer was used to sequence the bacterial genome. The obtained sequence results were searched and compared for similarity in GenBank using BLAST. The results showed that the nucleic acid sequence similarity with *Bifidobacterium longum* subsp. *infantii* was 100%, indicating that the strain belongs to *Bifidobacterium longum* subsp. *infantii* of the genus *Bifidobacterium*, and it was named *Bifidobacterium longum* subsp. *infantii*. Bifidobacterium longum subsp. infantis CCFM1306; Store at -80°C for later use.

[0101] Example 2: Determination of *Clostridium plasminogen activator* colony counts in monoculture and co-culture with *Bifidobacterium plasminogen activator* and *Bifidobacterium* in 0.5% inulin YCFA liquid medium.

[0102] Bifidobacterium ( Bifidobacterium ) and Clostridium plasmidoides ( Faecalibacterium prausnitzii Using inulin as the sole carbon source, different Bifidobacteria were cultured individually and in co-cultured in carbon-limited media to evaluate their ability to stimulate the proliferation of Clostridium praosporum.

[0103] (1) Preparation of culture medium:

[0104] YCFA liquid medium: used to activate Clostridium plasminogen activator CCFM1203;

[0105] Modified MRS liquid culture medium: used for activating Bifidobacteria;

[0106] 0.5% inulin YCFA liquid medium: used for single culture and co-culture of Bifidobacterium and Clostridium perfringens, respectively.

[0107] (2) Activation of the strain:

[0108] Bifidobacterium longum infantis subsp. (Bifidobacterium infantis) preserved at -80°C were respectively Bifidobacterium longum subsp. infantis FGZ23I1M2, Bifidobacterium breve ( Bifidobacterium breve CJ653, Bifidobacterium animalis ( Bifidobacterium animalum JSZJ4M1, Bifidobacterium bifidum ( Bifidobacterium bifidum FXJCJ9M10 was inoculated into modified MRS liquid medium and cultured in an anaerobic chamber (80% N2, 10% CO2, 10% H2) at 37°C for 24 h. Then, it was passaged to the 3rd generation at an inoculum rate of 2% (v / v).

[0109] Clostridium plasminoides ( Faecalibacterium prausnitzii CCFM1203 was inoculated into YCFA liquid medium and cultured in an anaerobic chamber (80% N2, 10% CO2, 10% H2) at 37°C for 24 h. Then, it was passaged to the 3rd generation at an inoculum rate of 2% (v / v).

[0110] (3) Single-strain culture (preparation of culture medium):

[0111] The activated Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. child FGZ23I1M2, Bifidobacterium breve ( Bifidobacterium breve CJ653, Bifidobacterium animalis ( Bifidobacterium animalum JSZJ4M1, Bifidobacterium bifidum ( Bifidobacterium bifidumFXJCJ9M10, Clostridium praosporum ( Faecalibacterium prausnitzii CCFM1203 was inoculated at a rate of 2% (v / v) into 0.5% inulin YCFA liquid medium, and three replicates were performed. The cultures were incubated anaerobicly at 37°C for 48 h. Culture media were prepared separately, and the *Clostridium perfringens* strain was tested in single cultures. Faecalibacterium prausnitzii The colony count of CCFM1203 (Table 2).

[0112] (4) Co-culture of strains:

[0113] Bifidobacterium longum infantis subspecies ( Bifidobacterium longum subsp. infantis FGZ23I1M2 culture medium and Clostridium praosporum ( Faecalibacterium prausnitzii CCFM1203 culture medium, containing Bifidobacterium breve ( Bifidobacterium breve CJ653 culture medium and Clostridium plasmid ( Faecalibacterium of Prausnitz CCFM1203 culture medium, containing Bifidobacterium animalis ( Bifidobacterium animalum JSZJ4M1 culture medium and Clostridium plasmid ( Faecalibacterium prausnitzii CCFM1203 culture medium, containing Bifidobacterium bifidum ( Bifidobacterium bifidum FXJCJ9M10 culture medium and Clostridium plasmid ( Faecalibacterium of Prausnitz CCFM1203 culture medium was prepared at a viable cell ratio of 1:1 (total viable cell count: 2 × 10⁻⁶). 6 CFU was inoculated into 0.5% inulin YCFA liquid medium. The inoculation amount of both strains was 1% (v / v). Three replicates were performed. The cultures were mixed and cultured at 37°C for 48 h under anaerobic conditions to prepare bacterial suspensions.

[0114] After culturing, 1 mL of bacterial suspension was taken and DNA was extracted according to the instructions of the Tiangen Bacterial Genomic DNA Extraction Kit. The colony count of *Clostridium praecoxibaris* under different culture conditions was then detected (Table 2). The co-culture colony count / single-culture colony count refers to the number of *Clostridium praecoxibaris* CCFM1203 colonies in the co-culture system divided by the number of *Clostridium praecoxibaris* CCFM1203 colonies in the single-culture system.

[0115] Table 2: Colony counts of Clostridium praosporum under different culture methods

[0116]

[0117] The results showed that co-culturing *Clostridium pluvialis* with different species of Bifidobacterium increased the number of colonies to varying degrees compared with monoculture. Among them, *Bifidobacterium longum* subsp. infantis had the strongest ability to stimulate the proliferation of *Clostridium pluvialis*, with the number of colonies in co-culture being 16.74 times that in monoculture. This was followed by *Bifidobacterium animalis*, with the number of colonies in co-culture of *Clostridium pluvialis* being 15.07 times that in monoculture. *Bifidobacterium bifidum* and *Bifidobacterium breve* had a weaker ability to stimulate the proliferation of *Clostridium pluvialis*, with the number of *Clostridium pluvialis* colonies in co-culture being 11.63 and 6.98 times that in monoculture, respectively.

[0118] Example 3: Growth of Bifidobacterium CCFM1306 and Clostridium plasminoides CCFM1204 in 0.5% inulin YCFA liquid medium

[0119] Bifidobacterium (Bifidobacterium) and Clostridium plasmidon ( Faecalibacterium prausnitzii Using inulin as the research subject and inulin as the sole carbon source, the cells were cultured in micropores in a carbon-defined culture medium to evaluate the utilization of inulin. Figure 1~Figure 2 ).

[0120] The specific steps are as follows:

[0121] (1) Preparation of culture medium:

[0122] YCFA liquid culture medium: used for activating Clostridium plasminogen activator ( Faecalibacterium prausnitzii CCTV-1203;

[0123] 0.5% inulin YCFA liquid medium: used for the determination of Clostridium pratensis (… Faecalibacterium of Prausnitz CCFM1203's ability to utilize inulin;

[0124] Modified MRS liquid culture medium: used for activating Bifidobacteria;

[0125] 0.5% inulin MRS liquid medium: used for the determination of Bifidobacterium longum subsp. infantis (… Bifidobacterium longum subsp. infantis CCFM1306's ability to utilize inulin.

[0126] (2) Activation of the strain:

[0127] Bifidobacterium longum infantis subsp. (preserved at -80°C) Bifidobacterium longum subsp. child CCFM1306 was inoculated into modified MRS liquid medium and cultured for 24 h. Then, it was passaged to the 3rd generation at an inoculation rate of 2% (v / v).

[0128] Clostridium plasminoides ( Faecalibacterium prausnitziiCCFM1203 was inoculated into YCFA liquid medium and cultured for 24 hours. Then, it was passaged to the 3rd generation at an inoculation rate of 2% (v / v).

[0129] (3) Detection of strain growth:

[0130] The activated strain of Bifidobacterium longum subsp. infantis (Bifidobacterium longum) was used to reach the 3rd generation. Bifidobacterium longum subsp. child CCFM1306, Clostridium plasminoides ( Faecalibacterium prausnitzii CCFM1203 cells were inoculated into 0.5% inulin-modified MRS liquid medium and 0.5% inulin-YCFA liquid medium, respectively. Under aseptic conditions, the inoculated culture was transferred into 96-well cell culture plates, 200 μL per well, with three replicates. The cells were incubated at 37°C for 48 h under anaerobic conditions. OD was measured using a multi-functional microplate reader. 600 The system automatically detected the data every 30 minutes and continuously measured it for 36 hours. The results are shown in Table 3.

[0131] CCFM1306+CCFM1203 co-culture: After activating Bifidobacterium infantis subsp. infantis CCFM1306 and Clostridium plasmidolium CCFM1203 to the third generation, Bifidobacterium and Clostridium plasmidolium were simultaneously inoculated at a 1:1 ratio into 0.5% inulin YCFA liquid medium for co-culture, with a total inoculum of 2%. The co-culture solution was transferred to 96-well cell culture plates, 200 μL per well, with 3 replicates. The plates were incubated at 37°C for 48 h under anaerobic conditions. The OD600 value was measured using a Tecan Infinite F50 microplate reader, automatically detected every 30 min for 48 h, to evaluate the growth curve of the co-cultured strains of Bifidobacterium and Clostridium plasmidolium in inulin-based medium.

[0132] Table 3: OD values ​​of different strains cultured in 0.5% inulin liquid medium for 0 h, 12 h, 18 h, 24 h, and 36 h. 600 value

[0133]

[0134] The results showed that Clostridium plasminogen toxicum (C. plasminogen toxicum) Faecalibacterium prausnitzii The growth rate and amount of CCFM1203 in inulin were lower than those of Bifidobacterium longum subsp. infantis CCFM1306. After 36 h of culture, the OD of Clostridium plasmidon CCFM1203 was significantly lower. 600 It can reach 0.3, while the OD of Bifidobacterium longum subsp. infantis CCFM1306 is... 600 It can reach 0.5.

[0135] However, after co-culturing the two strains, the growth rate of the strains was faster, and the OD of the mixed bacterial solution increased after 36 hours of incubation. 600 It can reach 1.10, OD 600 The growth rate was much higher than that of single-strain culture, indicating that the growth effect of mixed culture was far superior to that of single-strain culture.

[0136] Example 4: The effect of Bifidobacterium longum infant subspecies CCFM1306 on the proliferation of Clostridium praosporum in vitro using inulin as a carbon source.

[0137] Bifidobacterium longum infantis subspecies ( Bifidobacterium longum subsp. infantis CCFM1306 and Clostridium plasmidoides (CCFM1306 and Clostridium plasmidoides) Faecalibacterium prausnitzii CCFM1203, FWXPL145, and A2-165 were used as research subjects. Inulin was used as the sole carbon source. The effects of Bifidobacterium longum subsp. infantis on promoting Clostridium praosporum proliferation in vitro were evaluated by quantitative real-time qPCR experiments using single bacterial culture and mixed culture of the two bacteria at a 1:1 ratio.

[0138] The specific steps are as follows:

[0139] (1) Seed liquid preparation:

[0140] Take 2 mL of Bifidobacterium longum subsp. infantis grown to the logarithmic phase (Bifidobacterium longum subsp. infantis). Bifidobacterium longum subsp. child The CCFM1306 bacterial culture was placed in a preservation tube, centrifuged at 8000 rpm for 2 min, the supernatant was discarded, 1 mL of 30% glycerol was added, mixed well, and stored at -80°C as a bacterial seed culture.

[0141] Take 50 mL of Clostridium pluvialis grown to the logarithmic phase ( Faecalibacterium prausnitzii CCFM1203, Clostridium praosporum ( Faecalibacterium prausnitzii FWXPL145 and Clostridium praosporum ( Faecalibacterium of Prausnitz The A2-165 bacterial solution was filtered in a sterile filter cup to remove the fermentation broth. The bacterial sludge adsorbed on the filter membrane was dissolved with 30% glycerol. The entire operation was carried out in an anaerobic chamber. The glycerol tube was stored at -80°C as the inoculum solution.

[0142] Preparation of culture medium for single culture:

[0143] The above seed culture was inoculated into 0.5% inulin YCFA liquid medium at an inoculation rate of 2% (v / v), and three replicates were performed. The culture was then cultured at 37°C for 48 h under anaerobic conditions to prepare the culture solutions.

[0144] (2) Culture of mixed bacteria:

[0145] Bifidobacterium longum infantile subspecies ( Bifidobacterium longum subsp. infantisCCFM1306 culture medium and Clostridium plasmid ( Faecalibacterium prausnitzii The following cultures were prepared: CCFM1203 culture medium, CCFM1306 culture medium of Bifidobacterium longum subsp. infantis, and FWXPL145 culture medium of Clostridium plasmidonii; and CCFM1306 culture medium of Bifidobacterium longum subsp. infantis and A2-165 culture medium of Clostridium plasmidonii, respectively, at a viable ratio of 1:1 (total viable count: 2 × 10⁻⁶). 6 CFU was inoculated into 0.5% inulin YCFA liquid medium, with the inoculation amount of both strains being 1%. Three replicates were performed, and the cultures were mixed and cultured at 37°C for 48 h under anaerobic conditions to prepare bacterial suspensions.

[0146] (3) After the culture was completed, 1 mL of bacterial suspension was taken and DNA was extracted according to the instructions of the Tiangen Bacterial Genomic DNA Extraction Kit to detect the proliferation of Clostridium plasmid under different culture conditions. The results are shown in Table 4.

[0147] Table 4: Colony Count of Clostridium praosporum under Different Culture Methods

[0148]

[0149] The results showed that Clostridium plasminogen toxicum (C. plasminogen toxicum) Faecalibacterium prausnitzii The colony count of a single culture of CCFM1203 was 10. 6.6 CFU / mL, but with Clostridium plasminoides ( Faecalibacterium prausnitzii After co-culturing CCFM1203 and Bifidobacterium longum subsp. infantis CCFM1306, the colony count of Clostridium praosporum can reach 10. 8 The CFU / mL co-culture colony count was 25.12 times that of the monoculture, an increase of 1.38 orders of magnitude, indicating that Bifidobacterium longum subsp. infantis CCFM1306 can significantly promote the proliferation of Clostridium plasmidonum.

[0150] Example 5: Production of SCFAs of Bifidobacterium longum subsp. infantis and Clostridium praosporum in vitro under different culture methods with inulin as carbon source.

[0151] *Clostridium plasminogen lysate* is an important butyrate-producing bacterium in the gut, while *Bifidobacterium* is an acetate-producing bacterium. Furthermore, the acetic acid produced by *Bifidobacterium* can be converted into butyrate as a substrate. *Bifidobacterium longum* subsp. *infantile* (… Bifidobacterium longum subsp. infantis CCFM1306 and Clostridium praosporum ( Faecalibacterium prausnitzii CCFM1203, FWXPL145, and A2-165 were used as research subjects, with inulin as the sole carbon source. Single-cell cultures and 1:1 mixed cultures of the two strains were conducted. The SCFA content in the culture supernatant was determined by GC-MS. Figure 3~Figure 4 ).

[0152] The specific steps are as follows:

[0153] (1) Preparation of seed liquid:

[0154] Following the method in Example 4, *Bifidobacterium longum* infant subsp. *infantii* was prepared respectively. Bifidobacterium longum subsp. infantis CCFM1306 seed culture, Clostridium plasmidoides ( Faecalibacterium prausnitzii Seed liquid.

[0155] Preparation of culture medium for isolated culture: Bifidobacterium longum subsp. infantis (… Bifidobacterium longum subsp. infantis CCFM1306 seed culture, Clostridium plasmidoides ( Faecalibacterium prausnitzii Seed culture was inoculated into 0.5% inulin YCFA liquid medium at an inoculation rate of 2% (v / v), and three replicates were performed. The culture was then cultured at 37°C for 48 h under anaerobic conditions to prepare the culture solutions.

[0156] Preparation of the culture medium for co-culture: Bifidobacterium longum subsp. infantis (… Bifidobacterium longum subsp. infantis CCFM1306 culture medium and Clostridium plasmid ( Faecalibacterium prausnitzii The culture medium was prepared at a viable cell ratio of 1:1 (total viable cell count: 2 × 10⁻⁶). 6 CFU was inoculated into 0.5% inulin YCFA liquid medium, with the inoculation amount of both strains being 1%. Three replicates were performed, and the cultures were mixed and cultured at 37°C for 48 h under anaerobic conditions to prepare bacterial suspensions (CCFM1306+CCFM1203 co-culture bacterial suspension, CCFM1306+FWXPL145 co-culture bacterial suspension, and CCFM1306+A2-165 co-culture bacterial suspension were prepared respectively).

[0157] Take 1 mL of bacterial suspensions of Bifidobacterium longum subsp. infantis CCFM1306, Clostridium plasmidonii CCFM1203, and a mixed bacterial suspension of Bifidobacterium longum subsp. infantis CCFM1306 and Clostridium plasmidonii, respectively, and centrifuge at 4°C and 12000 rpm for 3 min, and collect the supernatant.

[0158] (2) Extraction of SCFA from culture supernatant: Take 500 μL of culture supernatant, add 40 μL of 10% concentrated sulfuric acid for acidification, and shake to mix; add 1 mL of diethyl ether in a fume hood, vortex for 30 s to mix, and centrifuge at 4°C and 12000 rpm for 15 min; take the supernatant, add 0.25 g of anhydrous sodium sulfate to remove water for 15 min, and centrifuge at 4°C and 12000 rpm for 15 min. Transfer the supernatant to a gas chromatography vial, analyze the short-chain fatty acid content by GC-MS, and calculate the short-chain fatty acid content using the external standard method. The results are shown in Table 5.

[0159] Table 5: Short-chain fatty acid content under different culture methods

[0160]

[0161] The results showed that Clostridium plasminogen toxicum (C. plasminogen toxicum) Faecalibacterium prausnitzii The butyrate concentrations produced by CCFM1203, Clostridium plasminogen lysate FWXPL145, and Clostridium plasminogen lysate A2-165 in monoculture were 0.63 mmol / L, 0.34 mmol / L, and 2.59 mmol / L, respectively. However, after co-culturing with Bifidobacterium longum subsp. infantis CCFM1306, the butyrate concentrations produced were approximately 9 times, 6.89 times, and 2.37 times that of monoculture, respectively, significantly increasing the butyrate content.

[0162] Meanwhile, because *Clostridium plasminogen lysate* converts acetic acid produced by *Bifidobacterium* into butyric acid, the acetate content is significantly reduced after co-culturing. The acetic acid produced by *Bifidobacterium longum* subsp. *infantica* CCFM1306 is converted by *Clostridium plasminogen lysate* (…). Faecalibacterium prausnitzii The consumption produces butyric acid.

[0163] Example 6: Determination of the molecular weight of inulin in the fermentation supernatant of Bifidobacterium longum subsp. infantis and Clostridium plasmidonum under different culture methods with inulin as carbon source in vitro.

[0164] Bifidobacterium longum subsp. infantis has a stronger ability to degrade inulin than Clostridium plasminogen lysate. Therefore, in a co-culture system, Bifidobacterium longum subsp. infantis can preferentially degrade long-chain inulin into smaller molecular fragments, facilitating utilization by Clostridium plasminogen lysate and greatly promoting inulin utilization. In this embodiment, the inventors used the Bifidobacterium longum subsp. infantis of the present invention (… Bifidobacterium longum subsp. infantis CCFM1306 and Clostridium praosporum ( Faecalibacterium of Prausnitz Using CCFM1203 as the research object and inulin as the sole carbon source, the molecular weight of inulin in the fermentation supernatant was determined by single-strain culture and mixed culture of the two strains in a 1:1 ratio, using an ion chromatograph (ICS-5000), high-performance anion exchange chromatography (HPAEC), and a pulsed amperometric detector (PAD).

[0165] The specific steps are as follows:

[0166] (1) Preparation of seed liquid:

[0167] Following the method in Example 4, *Bifidobacterium longum* infant subsp. *infantii* was prepared respectively. Bifidobacterium longum subsp. infantis CCFM1306 seed culture, Clostridium plasmidoides ( Faecalibacterium prausnitzii CCFM1203 seed solution.

[0168] Preparation of culture medium for isolated culture: Bifidobacterium longum subsp. infantis (… Bifidobacterium longum subsp. infantis CCFM1306 seed culture, Clostridium plasmidoides ( Faecalibacterium prausnitzii CCFM1203 seed culture was inoculated into 0.5% inulin YCFA liquid medium at an inoculation rate of 2% (v / v), and three replicates were performed. The culture was then cultured at 37°C for 48 h under anaerobic conditions to prepare the culture solutions.

[0169] Preparation of the culture medium for co-culture: Bifidobacterium longum subsp. infantis (… Bifidobacterium longum subsp. infantis CCFM1306 culture medium and Clostridium plasmid ( Faecalibacterium prausnitzii CCFM1203 culture medium was prepared at a viable cell ratio of 1:1 (total viable cell count: 2 × 10⁻⁶). 6 CFU was inoculated into 0.5% inulin YCFA liquid medium, with the inoculation amount of both strains being 1%. Three replicates were performed, and the cultures were mixed and cultured at 37°C for 48 h under anaerobic conditions to prepare bacterial suspensions.

[0170] Take 1 mL of bacterial suspensions of Bifidobacterium longum subsp. infantis CCFM1306, Clostridium plasmidonii CCFM1203, and a mixed bacterial suspension of Bifidobacterium longum subsp. infantis CCFM1306 and Clostridium plasmidonii CCFM1203, respectively, and centrifuge at 4°C and 12000 rpm for 3 min, and collect the supernatant.

[0171] (2) Sample pretreatment: Take 1 mL of culture supernatant after 48 h of fermentation, add an equal volume of acetonitrile, place on ice for 30 min to precipitate proteins, centrifuge at 21036 g / min for 15 min at 4°C, take the supernatant, place it in a vacuum concentrator for 3 h, redissolve with 500 μL of ultrapure water, filter through a 0.22 μm microporous membrane, and put it into a sample vial for testing. The results are as follows: Figure 5~Figure 7 As shown.

[0172] Chromatographic results showed that Clostridium plasmidoides ( Faecalibacterium prausnitziiAfter CCFM1203 was cultured in pure inulin, almost no inulin was degraded and utilized in the fermentation supernatant.

[0173] After pure culture of Bifidobacterium longum subsp. infantis CCFM1306 in inulin, the peak areas of inulin fragments with different degrees of polymerization in the fermentation supernatant were all reduced.

[0174] And when Clostridium plasminoides ( Faecalibacterium prausnitzii When CCFM1203 and Bifidobacterium longum infantis CCFM1306 were co-cultured in inulin, the peak area of ​​the highly polymerized inulin fragments was almost zero, indicating that they were basically completely degraded, thus decomposing to produce more small molecular fragments.

[0175] 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 strain of Bifidobacterium longum subsp. infantis (B. infantis) CCFM1306, characterized in that, Bifidobacterium longum subsp. infantis The *Bifidobacterium longum* subspecies *CCFM1306* is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:63216, and the deposit date is March 29, 2023. ​ 2. A microbial preparation containing the Bifidobacterium longum subsp. infantis CCFM1306 as described in claim 1.

3. The microbial preparation as claimed in claim 2, characterized in that The viable cell count of the B. longum subsp. infantis CCFM1306 in the microbial preparation is not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

4. A method of promoting the proliferation of Faecalibacterium prausnitzii (F. prausnitzii) in a system containing inulin, characterized in that, Faecalibacterium prausnitzii The *Bifidobacterium longum* subsp. *infantitidis* as described in claim 1, or the microbial preparation as described in claim 2 or 3, is co-cultured with *Clostridium plasminoides* in a system containing inulin. ​ 5. A substance that can promote the growth of Clostridium plasmidoides (Clostridium plasmidoides) Faecalibacterium prausnitzii The drug for proliferation, characterized in that, The drug contains Bifidobacterium longum infantis subsp. CCFM1306 as described in claim 1, or the microbial preparation as described in claim 2 or 3.

6. The pharmaceutical product as described in claim 5, characterized in that, The viable cell count of the B. longum subsp. infantis CCFM1306 in the drug product is not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

7. A medicine, characterized in that, The medicine contains Bifidobacterium longum infantis subsp. CCFM1306 as described in claim 1 and inulin.

8. The use of the Bifidobacterium longum subsp. infantis CCFM1306 as described in claim 1, or the microbial preparation as described in claim 2 or 3, in the preparation of a medicine for improving intestinal health.

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