Bifidobacterium longum subsp.infantis FMBL B250020 GXY capable of metabolizing breast milk oligosaccharide and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to bifidobacterium longum subsp.infantis FMBL B250020GXY for metabolizing breast milk oligosaccharide and application of the bifidobacterium longum subsp.infantis FMBL B250020GXY, the bifidobacterium longum subsp.infantis FMBL B250020GXY is preserved in China Center for Type Culture Collection on September 1, 2025, and the preservation number is CCTCC M 20251931; three kinds of breast milk oligosaccharides, namely 2 '-fucosyllactose (2'-FL), lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT), can be metabolized; plant source carbohydrates such as fructo-oligosaccharide and synanthrin can be metabolized; the compound has a relatively strong capability of removing DPPH and ABTS free radicals; a good inhibition effect is achieved on common pathogenic bacteria; good tolerance to acid and cholate is achieved; the strain is sensitive to gentamicin, streptomycin, tetracycline, chloramphenicol and vancomycin; the compound can be used for preparing antioxidant products, medicines for inhibiting pathogenic bacteria, infant foods and fermented foods, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a metabolizing Bifidobacterium longum subsp. infantis FMBL B250020 GXY and its applications. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third most abundant solid component in breast milk, after lactose and fat. They play a crucial role in maintaining gut microbiota balance, regulating immune responses, and promoting infant brain development. Currently, over 200 types of HMOs have been identified in breast milk. However, according to announcements from the National Health Commission, only two HMOs (2'-fucosylated lactose and lactose-N-neotetrasaccharide) are permitted for use in formulated milk powder (limited to infant formula), infant formula, follow-up formula, and formula for special medical purposes. The composition of these two types differs significantly. With societal development, infant formula has become essential for raising infants and young children, and developing formula that closely approximates the functions of breast milk is a pressing technical challenge.

[0003] The most current formula milk solutions that closely resemble the function of breast milk primarily rely on a combination of key technologies such as human milk oligosaccharides (HMOs), probiotics, replication of breast milk lipid structure, and optimization of milk-derived proteins. Adding certain HMOs to infant formula along with probiotics capable of utilizing these HMOs can produce a synergistic effect, more closely mimicking the biological function of breast milk. The probiotics involved include *Bifidobacterium infantis* and *Bifidobacterium breve*, among which *Bifidobacterium longum* subsp. *infantis* colonizes the human infant gut and plays a crucial role in improving infant health. Numerous studies have reported the functional properties of *Bifidobacterium longum* subsp. *infantis* in improving infant disease states, such as inflammatory bowel disease, neonatal diarrhea, and systemic inflammatory responses, thus attracting widespread attention.

[0004] Currently, several strains of *Bifidobacterium longum* subsp. infantis have been reported or applied for in China, and preliminary applications have been explored in the fields of human milk oligosaccharide utilization and infant health. However, due to limitations in strain resources such as geographical location, sample source, and isolation conditions, existing strains still exhibit differences in genetic diversity, HMO utilization profile, and metabolic efficiency, making it difficult to fully meet the needs of different populations and product applications. Therefore, it is still necessary to continue screening and functional evaluation of *Bifidobacterium longum* subsp. infantis strains from different sources to obtain more high-performance, adaptable strains with high application potential. Summary of the Invention

[0005] The primary objective of this invention is to provide a *Bifidobacterium longum* subsp. *infantis* FMBL B250020 GXY that metabolizes human milk oligosaccharides. The invention is characterized in that the *Bifidobacterium longum* subsp. *infantis* FMBL B250020 GXY was deposited at the China Center for Type Culture Collection on September 1, 2025, with accession number CCTCC M20251931.

[0006] A second objective of this invention is to provide a bacterial agent containing the *Bifidobacterium longum* subsp. *infantitidis* FMBL B250020 GXY as described in claim 1.

[0007] Preferably, it can utilize human milk oligosaccharides.

[0008] Preferably, the human milk oligosaccharide is 2'-fucosylated lactose, lactose-N-neotetrasaccharide, or lactose-N-tetrasaccharide.

[0009] Preferably, different carbon sources can be used, wherein the carbon source is one or more of fructose, xylose, lactose, maltose, fructooligosaccharides, galactooligosaccharides and inulin.

[0010] The third objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the anti-oxidation process or in the preparation of anti-oxidation products, wherein the products are food, pharmaceuticals, or health products.

[0011] The fourth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of antibacterial products, wherein the products are food, pharmaceuticals, or health products.

[0012] The fifth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of products that regulate intestinal function, wherein the products are food, pharmaceuticals, or health products.

[0013] The sixth objective of this invention is to provide an infant formula food containing the aforementioned Bifidobacterium longum subsp. infantis FMBL B250020 GXY or its strain fermentation broth or its sterile fermentation supernatant or the aforementioned bacterial agent, as well as prebiotics.

[0014] Preferably, the food includes one or more of the following: dairy products, soy products, probiotic powder, probiotic oil droplets, dietary fiber supplements, nutrition bars, rice cereal, fruit puree, fruit and vegetable juice, solid food beverages, fruit juice, ice cream, candy, and biscuits.

[0015] The beneficial effects of this invention are as follows: This invention provides a *Bifidobacterium longum* subspecies *FMBL* B250020GXY, which was deposited at the China Center for Type Culture Collection on September 1, 2025, with the accession number CCTCC M. 20251931, capable of metabolizing three human milk oligosaccharides: 2'-fucosylated lactose (2'-FL), lactose-N-neotetrasaccharide (LNnT), and lactose-N-tetrasaccharide (LNT); capable of metabolizing plant-derived carbohydrates such as fructooligosaccharides and inulin; its fermentation broth and cell extracts exhibit strong scavenging abilities against DPPH and ABTS free radicals, with DPPH scavenging rates of 65.60% and 22.76%, and ABTS scavenging rates of 85.67% and 6.85%, respectively; it shows good tolerance to acids and bile salts; sensitive to gentamicin, streptomycin, tetracycline, chloramphenicol, and vancomycin; and resistant to diarrhea-causing Escherichia coli CICC-10411 and hemorrhagic Escherichia coli CICC. 21530, enterotoxigenic Escherichia coli CICC-10421, Salmonella enterica subsp. enterica CICC-10420, and Salmonella enteritidis CGMCC1.10754-SM1 exhibit good inhibitory effects; they can be used to prepare antioxidant products and drugs that inhibit pathogenic bacteria, as well as infant foods and fermented foods, and have broad application prospects. Attached Figure Description

[0016] Figure 1 Colony diagram of Bifidobacterium longum subsp. infantis FMBL B250020 GXY

[0017] Note: Colony morphology (a, b), microscopic morphology (c).

[0018] Figure 2 Phylogenetic tree of Bifidobacterium longum subsp. infantis FMBL B250020 GXY Detailed Implementation

[0019] The following embodiments are provided to facilitate a better understanding of the present invention, but are not limited to it. Unless otherwise specified, the experimental methods in the following embodiments are conventional laboratory methods. Unless otherwise specified, the experimental materials used in the following embodiments are conventional biochemical reagents. Quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.

[0020] The culture medium formulations used in the following examples are as follows:

[0021] MRS liquid culture medium (1L): peptone 10g; beef extract 10g; yeast extract 5g; glucose 20g; Tween 80 1mL; K2HPO4 2g; anhydrous sodium acetate 5g; diammonium citrate 2g; MgSO4·7H2O 0.58g; MnSO4·4H2O 0.25g; L-cysteine ​​hydrochloride 0.5g; mupirocin 0.5mg; deionized water 1000mL;

[0022] MRS solid medium (1L): peptone 10g; beef extract 8g; yeast extract 4g; glucose 20g; Tween 80 1mL; K2HPO4 2g; anhydrous sodium acetate 5g; diammonium hydrogen citrate 2g; MgSO4·7H2O 0.29g; MnSO4·4H2O 0.25g; agar 20g; deionized water 1000mL; L-cysteine ​​hydrochloride 0.5g; mupirocin 0.5mg; sterilize at 115℃ for 20min.

[0023] PYG solid medium (1L): 10g peptone; 5g yeast extract; 1g glucose; 15g agar; 1000mL deionized water; adjust pH to 6.8, sterilize at 115℃ for 20min;

[0024] Nutrient gravy agar medium (1L): 5g peptone; 3g beef extract; 5g NaCl; 20g agar; 1000mL deionized water; adjust pH to 7.0, sterilize at 115℃ for 20min;

[0025] TSA solid medium (1L): 15g tryptone; 5g soybean peptone; 5g NaCl; 15g agar; 1000mL deionized water; adjust pH to 7.2, sterilize at 115℃ for 20min;

[0026] IST medium formula (1L): Hydrolyzed casein 11g; Peptone 3g; Glucose 2g; Sodium chloride 3g; Soluble starch 1g; Disodium hydrogen phosphate 2g; Sodium acetate 1g; Magnesium glycerophosphate 0.2g; Calcium gluconate 0.1g; Cobalt sulfate 0.001g; Copper sulfate 0.001g; Zinc sulfate 0.001g; Ferric sulfate 0.001g; Magnesium chloride 0.002g; Vitamin K 0.001g; Vitamin B12 0.001g; L-cysteine ​​hydrochloride 0.02g; L-tryptophan 0.02g; Vitamin B6 0.003g; Pantothenic acid 0.003g; Nicotine 0.003g; Vitamin H 0.000 3g; Vitamin B1 0.000 0.04g; adenine 0.01g; guanine 0.01g; xanthine 0.01g; uracil 0.01g; deionized water 1000mL; sterilize at 115℃ for 20min.

[0027] Unless otherwise specified, the reagents and consumables used in the following examples can be purchased from the market.

[0028] Unless otherwise specified, the methods used in the following embodiments are conventional methods and can be obtained by referring to the corresponding literature.

[0029] It should be noted that the "antibacterial product" mentioned in this invention refers to a product with antibacterial function, which can be in any form to achieve the antibacterial function, such as drugs (all dosage forms that achieve the antibacterial function are acceptable), food, health products, additives, etc. Example 1: Isolation, purification, and identification method of *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY 1. Strain isolation and purification

[0030] Fresh fecal samples were collected from healthy Uyghur infants in Shule County, Kashgar Prefecture. The samples were cultured under anaerobic conditions (80% nitrogen, 10% hydrogen, 10% carbon dioxide) using the gradient dilution plating method and bacterial strains were isolated.

[0031] Fecal samples were diluted to 10 μL with liquid Man-Rogosa-Sharpe (MRS) medium containing 0.5% L-cysteine ​​hydrochloride at room temperature. -2 10 -3 10 -4 Three dilution gradients were used. 100 μl of each dilution of fecal sample was evenly spread onto modified Man-Rogosa-Sharpe (MRS) agar medium (50 mg mupirocin per liter) and incubated at 37°C for 48 h (each sample was repeated three times) in an anaerobic incubator until numerous colonies appeared on the surface of the medium. The target strain was selected based on colony morphology and purified three times using the streak plate method to obtain a single strain, designated FMBL B250020 GXY. Strain FMBL B250020 GXY was stored in MRS liquid medium supplemented with 25% glycerol at -20°C for future use.

[0032] 2. Strain identification

[0033] Identification was performed using GroEL gene sequencing and specific primer PCR.

[0034] DNA was extracted from the strain using a kit, and the GroEL gene was amplified by PCR using primers Bif-GroEL-F (5′-TCCGATTACGAYCGYGAGAAGCT-3′) and Bif-GroEL-R (5′-CSGCYTCG GTSGTCAGGAACAG-3′). The GroEL gene PCR amplification system is shown in Table 1, and the PCR reaction conditions are shown in Table 2. The PCR amplification products were sent to the company for sequencing. The returned sequencing results were uploaded to the NCBI database for BLAST alignment. After alignment, *Bifidobacterium longum* subsp. *infantitidis* was further identified as *Bifidobacterium longum* subsp. *infantitidis* using primers Sia-266F (5′-GACGAGGAGGAATACAGCAG-3′) and Sia-676R (5′-CACGAACAGCGAATCATGGATT-3′). The PCR amplification system is shown in Table 1, and the reaction conditions are shown in Table 3. The corresponding species sequence was obtained from the database, and a phylogenetic tree was constructed using MEGA11.0.

[0035] Table 1. Premixed solution system for PCR amplification (25 μL)

[0036]

[0037] Table 2. PCR reaction conditions for GroEL gene

[0038]

[0039]

[0040] Table 3 PCR reaction conditions for specific primers

[0041]

[0042] like Figure 1 As shown, strain FMBL B250020 GXY has a smooth surface, milky white colonies, and appears rod-shaped under a microscope. It is non-spore-forming and Gram-positive. Its morphological and physiological / biochemical characteristics are similar to those of Bifidobacterium. The phylogenetic tree based on the Gro EL gene is as follows: Figure 2As shown, its groEL gene sequence has 99.99% homology with the groEL sequence of *Bifidobacterium longum*. Specific primer amplification results indicate that strain FMBL B250020 GXY is *Bifidobacterium longum* subsp. *infantis*. According to the classification of the genus *Bifidobacterium* in Bergey's Manual of Systematic Bacteriology, *Bifidobacterium longum* subsp. *infantis* belongs to the phylum *Actinobacteria*, class *Actinobacteria*, subclass *Actinobacteridae*, order *Bifidobacteriates*, family *Bifidobacteriaceae*, genus *Bifidobacterium*, species *Bifidobacterium longum*, and subsp. *infantis*. subsp.infantis).

[0043] The aforementioned *Bifidobacterium longum* subsp. *infantis* FMBLB250020GXY was deposited on September 1, 2025, at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M20251931, located at Wuhan University, Wuhan, China, telephone: 027-68754052, email: cctcc@whu.edu.cn.

[0044] In the following examples, Bifidobacterium longum subsp. infantis FMBL B250020 GXY is abbreviated as Bifidobacterium longum subsp. infantis FMBL B250020 GXY.

[0045] Example 2: Carbohydrate metabolism capacity of Bifidobacterium longum subsp. infantis FMBL B250020 GXY

[0046] 1. Experiment on the metabolism of human milk oligosaccharides

[0047] Activated *Bifidobacterium longum* subsp. *infantii* FMBL B250020 GXY was inoculated at 2% (v / v) into modified MRS liquid medium containing 0.5% L-cysteine ​​hydrochloride and anaerobically cultured at 37°C for 36 h. The bacterial cells were separated by centrifugation (8000 rpm, 5 min, 4°C), the supernatant was discarded, and the cells were washed twice with sterile pH 7.0 phosphate-buffered saline, then resuspended in 1 mL PBS. Next, the bacterial suspension was inoculated at 2% into modified MRS liquid medium containing 2'-fucosylated lactose (2'-FL), lactose-N-neotetrasaccharide (LNnT), and lactose-N-tetrasaccharide (LNT), respectively. Glucose was used as a positive control, and a medium without any carbon source was used as a negative control. The OD600 absorbance (OD1) of each culture was measured before culturing (0 h). After 48 hours of anaerobic culture, the OD600 absorbance of each culture was measured again (denoted as OD2). The final OD600 value is the difference between OD2 and OD1.

[0048] The results are shown in Table 4. The *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY can utilize 2'-fucosylated lactose, lactose-N-neotetrasaccharide, and lactose-N-tetrasaccharide, and the utilization efficiency of 2'-fucosylated lactose and lactose-N-tetrasaccharide is better than that of glucose.

[0049] Table 4. Ability of Bifidobacterium longum subsp. infantis FMBL B250020 GXY to utilize human milk oligosaccharides.

[0050]

[0051] Note: Different letters represent significant differences.

[0052] 2. Experiments on the metabolism of plant-derived compounds

[0053] Activated *Bifidobacterium longum* subsp. *infantii* FMBL B250020 GXY was inoculated at 2% (v / v) into modified MRS liquid medium containing 0.5% L-cysteine ​​hydrochloride and anaerobically cultured at 37°C for 36 h. The bacterial cells were separated by centrifugation (8000 rpm, 5 min, 4°C), the supernatant was discarded, and the cells were washed twice with sterile pH 7.0 phosphate-buffered saline, then resuspended in 1 mL PBS. Next, the bacterial suspension was inoculated at 2% into modified MRS liquid medium containing different carbon sources (xylose, fructose, arabinose, lactose, maltose, fructooligosaccharides, xylooligosaccharides, galactooligosaccharides, xylan, and inulin). Glucose was used as a positive control, and medium without any carbon source was used as a negative control. The OD of each culture was measured before culturing (0 h). 600Absorbance value (denoted as OD1). After 48 hours of anaerobic culture, the OD1 of each culture was measured again. 600 Absorbance value (denoted as OD2). Final OD 600 The value is the difference between OD2 and OD1.

[0054] The results are shown in Table 5. The carbohydrate metabolism experiment results show that the *Bifidobacterium longum* subsp. infantis FMBLB250020GXY strain can utilize fructose, xylose, lactose, maltose, fructooligosaccharides, galactooligosaccharides, and inulin; but it can hardly utilize arabinose, xylooligosaccharides, and xylan.

[0055] Table 5. Ability of Bifidobacterium longum subsp. infantis FMBL B250020 GXY to utilize carbohydrates

[0056]

[0057]

[0058] Example 3: Antioxidant capacity of Bifidobacterium longum subsp. infantis FMBL B250020 GXY

[0059] Activated Bifidobacterium longum subsp. infantis FMBL B250020 GXY at 2% (v / v) (OD 600 An inoculum of 1.0 ± 0.05 g (mg / L) was added to MRS liquid medium. After anaerobic culture at 37°C for 36 h, the culture was centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 0.22 μm aqueous microfiltration membrane to collect the cell-free supernatant. The cells were washed three times with phosphate-buffered saline (PBS) solution at pH 7.4. Subsequently, the cells were resuspended in PBS, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 9 Cells were broken up using a cell disruptor at CFU / mL, centrifuged at 8000×g for about 15 min at 4°C, and the supernatant was filtered through a 0.22 μm aqueous microfiltration membrane to obtain cell extracts.

[0060] (1) Determination of ABTS free radical scavenging ability

[0061] Mix 7.4 mmol / L ABTS working solution and 2.6 mmol / L potassium persulfate aqueous solution at a 1:1 (v / v) ratio and store in a dark environment (room temperature) for 12-16 h. Adjust the concentration by adding anhydrous ethanol solution to ensure that the absorbance of the ABTS working solution at 734 nm is approximately 0.70 ± 0.005. Mix 0.2 mL of the test sample with 0.8 mL of the adjusted ABTS working solution, incubate at room temperature for 6 min, and measure the absorbance. Use an equal volume of blank culture medium as a control.

[0062] ABTS clearance rate (%) = (1 - (Ac - A) / Ac) × 100%

[0063] In the formula, Ac is the absorbance of the blank control, and A is the absorbance of the sample solution.

[0064] (2) DPPH free radical scavenging capacity determination experiment

[0065] Take 0.6 mL of the sample to be tested and mix it with 0.15 mL of the adjusted DPPH working solution. After mixing, react in the dark at room temperature for 30 min and measure its absorbance at a wavelength of 517 nm. Use an equal volume of sterile liquid culture medium as a blank control.

[0066] DPPH clearance rate (%) = (1 - (D1 - D2) / D0) × 100%

[0067] Where: D0: absorbance of PBS buffer; D1: absorbance of sample solution; D2: absorbance of blank control.

[0068] Table 6 Results of Antioxidant Capacity Measurement

[0069]

[0070] Table 6 shows that the scavenging rates of ABTS by the fermentation supernatant of *Bifidobacterium longum* subsp. *infantii* FMBL B250020 GXY and the cell extract were 85.67% and 6.85%, respectively, and the scavenging rates of DPPH were 65.60% and 22.76%, respectively. The results indicate that *Bifidobacterium longum* subsp. *infantii* FMBL B250020 GXY has a strong scavenging ability against DPPH and ABTS free radicals, and its fermentation supernatant showed better antioxidant performance compared with the cell extract.

[0071] Example 4: Probiotic Properties of Bifidobacterium longum Infant Subsp. FMBL B250020 GXY

[0072] 1. Antibiotic resistance test

[0073] The resistance of *Bifidobacterium longum* subsp. *infantitidis* FMBL B250020 GXY to gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin was determined using the microbroth dilution method. Single colonies were picked and placed on LSM medium, and the OD600 was adjusted to 0.16–0.20 before being diluted 500-fold. Microdilution plates were prepared according to the national standard GB31615.2—2025. The bacterial suspension was added to the prepared antimicrobial microdilution plates and anaerobically incubated for 48 h. The minimum inhibitory concentration (MIC) was compared with the breakpoint values ​​listed in GB31615.2—2025. A MIC lower than or equal to the breakpoint value was considered sensitive (S), and a MIC higher than or equal to the breakpoint value was considered resistant (R).

[0074] The resistance of *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY to eight antibiotics is shown in Table 7. The strain was sensitive to gentamicin, streptomycin, tetracycline, chloramphenicol, and vancomycin; but resistant to erythromycin, ampicillin, and clindamycin.

[0075] Table 7. Resistance of Bifidobacterium longum subsp. infantis FMBL B250020 GXY

[0076] Note: R: drug resistance; S: drug sensitivity.

[0077] 2. Determination of performance in inhibiting conditionally pathogenic bacteria

[0078] Escherichia coli CICC-10411 (diarrhea-inducing), Escherichia coli CICC 21530 (hemorrhagic), Escherichia coli CICC-10421 (enterotoxigenic), Salmonella enterica serotype CICC-10420 (typhoid serotype), Listeria monocytogenes CGMCC1.9136-LS1 (monocytozoonosis), and Salmonella enteritidis serotype CGMCC1.10754-SM1 (serotype) were used as indicator bacteria (Table 8). The antibacterial activity of the metabolites of Bifidobacterium longum subsp. infantis FMBL B250020 GXY was determined using the Oxford cup method. Bifidobacterium longum subsp. infantis FMBL B250020 GXY was added to MRS liquid medium at an inoculum of 2% (v / v) (OD600: 1.0±0.05), anaerobically cultured at 37℃ for 36 h, and the bacterial suspension was centrifuged at 8000 rpm for 5 min to obtain the supernatant, which was then used to prepare a cell-free supernatant. After the indicator bacteria are activated, follow the 10 6 ~10 7 CFU / mL concentrations were spread onto the corresponding solid culture medium. Sterile Oxford cups were placed vertically in the culture dishes containing the pathogenic bacteria, and 0.2 mL of cell-free supernatant was added to the Oxford cups. The culture dishes were placed at 4°C for 4 hours for diffusion, and then incubated in a 37°C anaerobic incubator for 24 hours. The diameter of the inhibition zone was then measured.

[0079] Table 8. Sources and culture media of indicator bacteria

[0080]

[0081] Table 9. Determination of the antibacterial activity of Bifidobacterium longum subsp. infantis FMBL B250020 GXY.

[0082]

[0083] Note: The diameter of the inhibition zone includes the outer diameter of the Oxford cup; no inhibition zone: -.

[0084] As shown in Table 9, *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY exhibits good inhibitory activity against *Escherichia coli* CICC-10411 (causing diarrhea), *Escherichia coli* CICC 21530 (causing hemorrhagic diarrhea), *Escherichia coli* CICC-10421 (causing enterotoxigenic diarrhea), *Salmonella enterica* serotype CICC-10420 (causing typhoid fever), and *Salmonella enteritidis* serotype CGMCC1.10754-SM1 (causing enteritis). This indicates that *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY has significant application potential in the development of drugs related to inhibiting opportunistic pathogens, preventing or alleviating diarrhea, and improving intestinal health.

[0085] 3. Acid and bile salt tolerance test

[0086] Two generations of activated *Bifidobacterium longum* subsp. *infantii* FMBL B250020 GXY were inoculated at a 2% inoculum in MRS liquid medium supplemented with 0.5% L-cysteine ​​hydrochloride and anaerobically cultured at 37°C for 48 h. After centrifugation (8000 rpm, 3 min, 4°C), the supernatant was discarded, and the cells were washed twice with PBS at pH 7.0. The cells were then resuspended in PBS at pH 3.0, 3.5, 4.0, and 7.0, respectively, and anaerobically incubated at 37°C for 3 h. Plate counting was then performed, with the pH 7.0 suspension serving as a blank control. The viable cell count was recorded as N (CFU / mL), and the viable cell count of the suspensions treated with different pH PBS was recorded as N1 (CFU / mL). Three independent replicate experiments were performed, and the survival rate was calculated.

[0087]

[0088] The bacterial cells were collected and washed as described above, and resuspended in PBS with no bile salts and bile salt concentrations (w / v) of 0.03% and 0.05%, respectively. After anaerobic incubation at 37°C for 3 hours, samples were taken for plate counting. The bacterial suspension without bile salts served as a blank control, and the viable cell count was recorded as N0 (CFU / mL). The viable cell counts of the bacterial suspensions treated with different concentrations of bile salts were recorded as N2 (CFU / mL). Three independent replicate experiments were performed, and the survival rate was calculated.

[0089]

[0090] Table 10. Tolerance of Bifidobacterium longum subsp. infantis FMBL B250020 GXY to acids and bile salts.

[0091]

[0092] As shown in Table 10, Bifidobacterium longum infant subspecies FMBL B250020 GXY has good tolerance to the infant gastrointestinal environment, which is conducive to its survival in the infant gastrointestinal tract and exerting its probiotic function.

[0093] Application Example 1: Preparation of Bifidobacterium longum subsp. infantis FMBL B250020 GXY bacterial agent

[0094] Preparation of basal culture medium: A culture medium containing 20 g / L glucose, 10 g / L peptone, 8 g / L beef extract, 10 g / L yeast extract, 1 mL / L Tween 80, 2 g / L K₂HPO₄, 5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 0.58 g / L MgSO₄·7H₂O, and 0.25 g / L MnSO₄·4H₂O was prepared using water and culture medium raw materials. The pH was adjusted to 6.8 to obtain the culture medium. High-density culture medium: Malt extract to water ratio 1:4, 50 g / L tryptone, 10 g / L anhydrous sodium acetate, 25 g / L fructooligosaccharides, 10 g / L trehalose, and 1 mL / L Tween 80.

[0095] Preparation of the preservative: A preservative containing 5% skim milk powder, 20 mL / L glycerol, 8% maltodextrin, 2% β-glucan, and 10% lactose was prepared using water and preservative raw materials.

[0096] Bifidobacterium longum subsp. infantis FMBL B250020 GXY was inoculated at a 2% inoculum into basal medium sterilized at 115℃ for 20 min, and activated twice. Then, it was inoculated at a 5% inoculum into high-density medium and cultured at 37℃ for 36 h. The resulting bacterial cells were centrifuged, washed twice with pH 7.2 PBS buffer, and resuspended in a cryoprotectant to achieve a concentration of 10. 11 CFU / mL. The protective agent contains 5% skim milk powder, 20 mL / L glycerol, 8% maltodextrin, 2% β-glucan, and 10% lactose. The suspension is then pre-cultured at 37°C for 60 min, followed by freeze-drying to obtain the *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY bacterial agent.

[0097] Application Example 2: Preparation of fermented milk using Bifidobacterium longum subsp. infantis FMBL B250020 GXY of the present invention.

[0098] Fresh milk is dissolved with sugar and homogenized at 60℃ and 20MPa. Then, it is sterilized at 90-95℃ for 5-8 minutes. When the temperature drops to 35℃, a mixed bacteria consisting of *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY or its inoculum, commercial dry powder starter *Lactobacillus bulgaricus*, and *Streptococcus thermophilus* is added in a mass ratio of 1:1:1. The inoculation amount of the mixed bacteria is 0.03-2.0% of the weight of the fresh milk. The mixture is mixed well and fermented at 37℃ for 4-6 hours. After curdling, it is refrigerated at 4℃ for 16 hours to obtain the fermented milk.

[0099] Application Example 3: Preparation of microcapsules and capsule products containing Bifidobacterium longum subsp. infantis FMBL B250020 GXY

[0100] The *Bifidobacterium longum* subsp. *infantii* FMBL B250020 GXY of this invention was enriched in MRS liquid medium for 24 h, centrifuged at 8000 r / min for 10 min at 4 °C, the supernatant was discarded, and the bacterial cells were collected, washed twice with sterile physiological saline, and resuspended to obtain a concentration of 1×10⁻⁶. 9 -10 10 A bacterial suspension of CFU / mL was added sequentially with an equal volume of sterile core material solution (7.5 g / L fructooligosaccharide solution and 21 g / L inulin solution) and 8 times the volume of wall material solution (a mixed solution of pectin and sodium alginate at a mass ratio of 1.0% and 1.25%, respectively), and the mixture was stirred and mixed thoroughly. The mixture was then extruded dropwise into a 2.0% calcium chloride curing solution to form gel particles. After curing for 30 min, the particles were filtered and washed with sterile water to collect them. The particles were then freeze-dried using vacuum freeze-drying technology to obtain microcapsules of *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY. The microcapsules had a particle size of 2.0-2.5 mm, an encapsulation efficiency ≥80.0%, and exhibited resistance to simulated gastrointestinal fluid and heat stress. These microcapsules were then filled into commercially available pharmaceutical capsules to obtain the described capsule product.

[0101] In summary, this invention provides a *Bifidobacterium longum* subsp. infantis FMBL B250020 GXY, which can metabolize three human milk oligosaccharides: 2'-fucosylated lactose (2'-FL), lactose-N-neotetrasaccharide (LNnT), and lactose-N-tetrasaccharide (LNT); it can also metabolize plant-derived carbohydrates such as fructooligosaccharides and inulin; its fermentation broth and cell extracts exhibit strong scavenging abilities against DPPH and ABTS free radicals, with DPPH scavenging rates of 65.60% and 22.76%, and ABTS scavenging rates of 85.67% and 6.85%, respectively; it shows good tolerance to acids and bile salts; it is sensitive to gentamicin, streptomycin, tetracycline chloramphenicol, and vancomycin; and it is resistant to diarrheal *Escherichia coli* CICC-10411 and hemorrhagic *Escherichia coli* CICC. 21530, enterotoxigenic Escherichia coli CICC-10421, Salmonella enterica subsp. enterica CICC-10420, and Salmonella enteritidis CGMCC1.10754-SM1 exhibit good inhibitory effects; they can be used to prepare antioxidant products and drugs that inhibit pathogenic bacteria, as well as infant foods and fermented foods, and have broad application prospects.

Claims

1. A subsp. *Bifidobacterium longum* FMBL B250020GXY that metabolizes human milk oligosaccharides, characterized in that, The aforementioned *Bifidobacterium longum* subspecies *FMBLB250020GXY* was deposited at the China Center for Type Culture Collection on September 1, 2025, with accession number CCTCC M20251931.

2. A microbial agent, characterized in that, The bacterial agent contains *Bifidobacterium longum* subsp. *infantitidis* FMBLB250020GXY as described in claim 1.

3. The *Bifidobacterium longum* subsp. infantis FMBL B250020GXY or its strain fermentation broth or sterile fermentation supernatant as described in claim 1, characterized in that, It can utilize human milk oligosaccharides.

4. The *Bifidobacterium longum* subsp. *infantitidis* FMBL B250020GXY or its strain fermentation broth or sterile fermentation supernatant as described in claim 3, or the bacterial agent as described in claim 2, characterized in that, The aforementioned human milk oligosaccharides are 2'-fucosylated lactose, lactose-N-neotetrasaccharide, and lactose-N-tetrasaccharide.

5. The *Bifidobacterium longum* subsp. *infantitidis* FMBL B250020GXY or its strain fermentation broth or sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, characterized in that... It can utilize plant-derived carbon sources, wherein the carbon source is one or more of fructose, xylose, lactose, maltose, fructooligosaccharides, galactooligosaccharides, and inulin.

6. The application of *Bifidobacterium longum* subsp. infantis FMBL B250020GXY or its strain fermentation broth or sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in antioxidant processes or the preparation of antioxidant products, characterized in that... The products mentioned are food, medicine, or health products.

7. The application of *Bifidobacterium longum* subsp. infantis FMBL B250020GXY or its strain fermentation broth or sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in the preparation of antibacterial products, characterized in that... The products mentioned are food, medicine, or health products.

8. The application of *Bifidobacterium longum* subsp. infantis FMBL B250020GXY or its strain fermentation broth or sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in the preparation of products for regulating intestinal function, characterized in that... The products mentioned are food, medicine, or health products.

9. An infant formula food, characterized in that, The food contains the *Bifidobacterium longum* subsp. infantis FMBL B250020GXY or its strain fermentation broth or its sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, as well as prebiotics.

10. The infant food as described in claim 9, characterized in that, The food products mentioned include one or more of the following: dairy products, soy products, probiotic powder, probiotic oil droplets, dietary fiber supplements, nutrition bars, rice cereal, fruit puree, fruit and vegetable juice, solid food beverages, fruit juice, ice cream, candy, and biscuits.

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