Oxygen-resistant animal bifidobacterium subsp. Lactis FMBL B23403, microbial inoculum and application of microbial inoculum

Abstract

The invention belongs to the technical field of biology, and particularly relates to a bifidobacterium animalis subsp. Lactis FMBL B23403 which is preserved in the China Center for Type Culture Collection on August 26, 2024, and the preservation number is CCTCC NO: M 20241845. The compound has good tolerance to oxygen; the fermentation liquor of the bacillus subtilis has an inhibition effect on the activity of alpha-glucosidase, alpha-amylase and dipeptidyl peptidase; the compound has a relatively strong capability of removing DPPH and ABTS free radicals; good oxidation resistance is realized; the acid-resistant and cholate-resistant capability, the self-agglutination property and the surface hydrophobicity are good; the compound can be used for preparing antioxidant products, hypoglycemic drugs, fermented foods, health-care products, food additives and feeds, and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an aerobic animal Bifidobacterium lactis subspecies FMBL B23403, its bacterial agent, and its application. Background Technology

[0002] Probiotics are live microorganisms that are safe to consume, colonize the human body, and exert beneficial effects. They are widely used in fermented foods, health products, food additives, and dietary supplements. Probiotics can directly or indirectly affect the gastrointestinal environment of mammals by regulating the internal ecosystem. The most common probiotic strains include Bifidobacteria, Lactobacillus, Streptococcus, Bacillus, and Enterococcus. Among them, Bifidobacteria (Bifidobacterium) are obligate symbiotic bacteria that inhabit the animal gut and belong to the category of intestinal probiotics. They play a key role in the host's metabolism and health, and have functions such as reducing allergy symptoms, improving immunity, inhibiting opportunistic pathogens, alleviating inflammation, and improving gut health. They are widely used in the food and pharmaceutical fields and have become a research hotspot in recent years.

[0003] Bifidobacterium animalis comprises two subspecies: *Bifidobacterium animalis* and *Bifidobacterium animalis* lactis. *Bifidobacterium animalis* lactis is a typical nomadic species, widely distributed in nature, adaptable to various ecological niches, and closely related to human life. It is an important species affecting the digestive system of humans and mammals, playing a crucial role in maintaining intestinal microecological balance, enhancing the body's immune system, alleviating lactose intolerance, and preventing diarrheal infections. It is currently the most widely commercially used Bifidobacterium species. Researchers have also screened out many subspecies of Bifidobacterium lactis every year and studied their functions in detail. For example, the Bifidobacterium lactis subspecies BX-245 disclosed in invention patent CN116747245B has broad-spectrum antibacterial activity, which can effectively inhibit pathogenic bacteria and foodborne spoilage bacteria. It can also synthesize functional active substances such as short-chain fatty acids, organic acids and their derivatives, amino acids and their derivatives, dole-indole metabolites, biogenic amines, and brain-gut axis regulatory products. The Bifidobacterium lactis subspecies BN12 disclosed in invention patent CN118207124A can promote food digestion in the intestine, promote nutrient absorption, help improve appetite, enhance intestinal motility, and protect intestinal health.

[0004] The genetic polymorphism of microorganisms results in extremely complex and diverse physiological, biochemical, and metabolic characteristics among strains of different species and strains of the same species from different sources. *Bifidobacterium animalis* subsp. *lactamase* has poor oxygen tolerance; its activity is significantly affected when exposed to various aerobic environments. Therefore, most existing aerobic-tolerant *Bifidobacterium animalis* subsp. *lactamase* are domesticated, such as the aerobic-tolerant domesticated *Bifidobacterium animalis* subsp. *lactamase* BZ11 disclosed in invention patent CN106167775B; original aerobic-tolerant *Bifidobacterium animalis* subsp. *lactamase* are relatively rare.

[0005] During their research, the inventors isolated a strain of *Bifidobacterium animalis* subsp. *Lactis* FMBL B23403 from fecal samples of infants aged 0-1 years. This strain exhibits good oxygen tolerance, with an oxygen tolerance coefficient approaching 1.0, demonstrating strong survival ability in aerobic environments. Furthermore, its fermentation broth showed inhibition rates of 62.39%, 80.53%, and 48.74% on α-glucosidase, α-amylase, and dipeptidyl peptidase activities, respectively, exhibiting excellent antioxidant and inhibitory effects. It also demonstrates good acid and bile salt resistance, self-polymerization, and surface hydrophobicity. This strain has broad application prospects and can be used to prepare antioxidant products, hypoglycemic and pathogenic bacteria-inhibiting drugs, fermented foods, health products, food additives, and feed. Summary of the Invention

[0006] The primary objective of this invention is to provide an aerobic subsp. *Bifidobacterium animalis* FMBL B23403, which was deposited at the China Center for Type Culture Collection on August 26, 2024, with accession number CCTCC NO: M 20241845.

[0007] A second objective of this invention is to provide a microbial agent containing the aforementioned Bifidobacterium animalis subsp. lactis FMBL B23403.

[0008] A third objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of pharmaceuticals, food, and health products.

[0009] The fourth objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in inhibiting dipeptidyl peptidase activity or preparing dipeptidyl peptidase inhibitors.

[0010] The fifth objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in inhibiting α-glucosidase activity or preparing α-glucosidase inhibitors.

[0011] The sixth objective of this invention is to provide the application of the aforementioned Bifidobacterium animalis subsp. lactis FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in inhibiting α-amylase activity or preparing α-amylase inhibitors.

[0012] The seventh objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of hypoglycemic drugs and health products.

[0013] The eighth objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in antioxidant or preparation of antioxidant products.

[0014] Preferably, the products include pharmaceuticals, food, health products, cosmetics, etc.

[0015] The ninth objective of this invention is to provide the application of the aforementioned Bifidobacterium animalis subsp. lactis FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of yogurt or yogurt starter.

[0016] The tenth objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of feed / feed additives.

[0017] The beneficial effects of this invention are as follows: This invention provides a *Bifidobacterium lactis* subsp. *animal* FMBL B23403, which exhibits good oxygen tolerance and strong survival ability in aerobic environments. Its fermentation broth shows inhibition rates of 62.39%, 90.53%, and 48.74% for α-glucosidase, α-amylase, and dipeptidyl peptidase activities, respectively. Its fermentation broth and cell extracts demonstrate strong scavenging abilities against DPPH and ABTS free radicals, with ABTS scavenging rates of 71.60% and 30.26%, respectively, and DPPH scavenging rates exceeding 50%, reaching 63.45% and 74.34%, respectively. It possesses excellent antioxidant capacity, good acid and bile salt resistance, self-polymerization properties, and surface hydrophobicity. It can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives, and feeds, and has broad application prospects. Attached Figure Description

[0018] Figure 1 Strain growth trend

[0019] Note: a. Growth curves for 10 bacterial strains are anaerobic; b. Growth curves for 10 bacterial strains are aerobic.

[0020] Figure 2 Colony diagram of Bifidobacterium animalis subsp. lactis FMBL B23403

[0021] Note: Colony morphology (a, b), microscopic morphology (c).

[0022] Figure 3 Phylogenetic tree of Bifidobacterium animalis subsp. lactis FMBL B23403 Detailed Implementation

[0023] 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.

[0024] The culture medium formula used is as follows:

[0025] 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;

[0026] 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; L-cysteine ​​hydrochloride 0.5g; mupirocin 0.5mg. Sterilize at 115℃ for 20min.

[0027] Wilkins-Chalgren (MAN) medium (1L): 48g modified Wilkins-Chalgren agar; 5g soybean peptone; 1mL Tween 80; 1mL glacial acetic acid; 6g anhydrous sodium acetate; 20g agar; 0.5g L-cysteine ​​hydrochloride; 0.5mg mupirocin; sterilize at 115℃ for 20min.

[0028] 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;

[0029] 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;

[0030] TSA solid medium (1L): 15g tryptone; 5g / L soybean peptone; 5g NaCl; 15g agar; 1000mL deionized water; adjust pH to 7.2, sterilize at 115℃ for 20min.

[0031] Unless otherwise specified, the reagents and consumables used in the following examples can be purchased from the market.

[0032] Unless otherwise specified, the methods used in the following embodiments are conventional methods and can be obtained by referring to the corresponding literature.

[0033] Example 1: Screening of Bifidobacterium lactis subsp. FMBL B23403 (aerobic animal bifidobacterium)

[0034] 1. Strain isolation and purification

[0035] Fresh fecal samples were collected from Kazakh infants in Ili Prefecture, Xinjiang. The samples were cultured under anaerobic conditions (80% nitrogen, 10% hydrogen, 10% carbon dioxide) using the gradient dilution plating method to isolate bacterial strains.

[0036] 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 different dilutions of fecal samples were evenly spread on modified Man-Rogosa-Sharpe (MRS) agar medium (with 50 mg mupirocin added per liter) and incubated at 37 °C for 48 h in an anaerobic incubator (each sample was repeated 3 times) until a large number of colonies appeared on the surface of the medium. Colonies suspected to be Bifidobacterium were picked and purified 3 times. The isolates were then stored in MRS liquid medium supplemented with 25% glycerol and stored at -20 °C for later use.

[0037] Ten strains were selected based on colony characteristics and cell morphology and named accordingly. Specific information is shown in Table 1.

[0038] Table 1. Information on the 10 selected bacterial strains and their hosts.

[0039]

[0040] Ten bacterial strains were spread on MRS solid medium containing 0.5% L-cysteine ​​hydrochloride and incubated in an anaerobic incubator at 37°C for 48 hours. Then, a single colony was picked and inoculated into MRS liquid medium containing 0.5% L-cysteine ​​hydrochloride and incubated in an anaerobic incubator at 37°C for 24 hours to activate the bacteria.

[0041] 2. Evaluation of oxygen tolerance coefficient

[0042] Adjust the activated bacterial culture medium to OD 600 The inoculum size was 1.0 ± 0.05 μL. The inoculum was 2% (v / v) in MRS liquid medium, and the cultures were incubated at 37°C for 48 h under both anaerobic and aerobic conditions. The OD values ​​of 10 strains were measured at 0 h, 4 h, 16 h, 24 h, and 48 h under both anaerobic and aerobic conditions. 600 Values ​​were calculated, growth curves were plotted, the optimal time for aerobic culture of 10 bacterial strains was determined, and OD values ​​were recorded. 600 value.

[0043] Utilizing OD under aerobic and anaerobic culture conditions at the end of the culture 600 The oxygen tolerance coefficient (specific growth rate, i.e., OD measured under aerobic conditions) of the strain was calculated. 600 Values ​​and OD measured under strictly anaerobic conditions 600 The ratio of the oxygen tolerance coefficient to the aerobic value indicates that the strain has higher growth activity and stronger oxygen tolerance under aerobic conditions. After the culture is completed, the bacterial solution is spread on MRS solid medium and anaerobically cultured at 37℃ for 48 hours. The growth of the strains in aerobic culture is observed. Based on the growth on the plate and the oxygen tolerance coefficient, the strains that are tolerant to oxygen and have the highest growth activity in aerobic environment are screened.

[0044] The growth curves of 10 bacterial strains under aerobic and anaerobic conditions are shown below. Figure 1 a, Figure 1 As shown in b. During anaerobic culture, 10 strains grew well and entered the stationary phase within 16-24 hours. Under aerobic conditions, most strains grew slowly or gradually lost activity, with only strain FMBL B23403 showing good growth and continuous proliferation under aerobic conditions. After the aerobic culture was completed, the bacterial suspension was plated on MRS solid medium and anaerobically cultured at 37°C for 48 hours. The results showed that only strain FMBL B23403 could grow normally on the plate, while strains YLa20x7, FMBL B23452, and FMBL B23416 produced a small number of colonies on the plate. The remaining strains had lost activity and could not grow on the plate.

[0045] The oxygen tolerance coefficients of each strain were calculated at the end of fermentation, and the results are shown in Table 2. The oxygen tolerance coefficient of strain FMBL B23403 was as high as 0.9668, indicating that strain FMBL B23403 has good oxygen tolerance, and its growth under aerobic and anaerobic conditions is similar; while other strains could not grow normally under aerobic conditions, showing poor oxygen tolerance. Furthermore, the results showed that strain FMBL B23403 entered a stationary phase within 16-24 hours during fermentation; therefore, in subsequent experiments, strain FMBL B23403 was cultured for 24 hours.

[0046] Table 210 shows the oxygen tolerance coefficients of 10 bacterial strains under aerobic and anaerobic conditions.

[0047]

[0048] 3. Strain identification

[0049] The strain with the highest oxygen tolerance coefficient, FMBL B23403, was identified.

[0050] DNA was extracted from the strain using a kit, and the GroEL gene was amplified by PCR using the primers Bif-GroEL-F (5′-TCCGATTACGAYCGYGAGAAGCT-3′) and Bif-GroEL-R (5′-CSGCYTCG GTSGTCAGG AACAG-3′). The GroEL gene PCR amplification system is shown in Table 3, and the PCR reaction conditions are shown in Table 4. 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, the strain was further identified as *Bifidobacterium animalis* subsp. *lactamella* using primer recA. The subsp. *lactamella*-specific primers are shown in Table 5, and the PCR reaction conditions are shown in Table 6. The corresponding species sequence was obtained from the database, and a phylogenetic tree was constructed using ME GA11.0.

[0051] Table 3. Premixed solution system for GroEL gene PCR amplification (25 μL)

[0052]

[0053] Table 4 PCR reaction conditions

[0054]

[0055] Table 5. Primers for PCR amplification of subspecies-specific primer recA

[0056]

[0057] Table 6. PCR reaction conditions for primer recA

[0058]

[0059] The colony characteristics and microscopic features of the strain FMBL B23403 are as follows: Figure 2 As shown, the phylogenetic tree is as follows: Figure 3As shown. The morphological and physiological-biochemical characteristics of the above-mentioned strain FMBL B23403 are similar to those of the genus Bifidobacterium. Its groEL gene sequence has 99.99% homology with the groEL sequence of Bifidobacterium animalis subsp. Lactis. According to the classification of the genus Bifidobacterium in Bergey's Manual of Systematic Bacteriology, Bifidobacterium animalis subsp. Lactis FMBL B23403 belongs to the phylum Actinobacteria, class Actinobacteria, subclass Actinobacteriae, order Bifidobacteriales, family Bifidobacteriaceae, genus Bifidobacterium, species Bifidobacterium animalis, and subsp. Lactis of Bifidobacterium animalis.

[0060] After identification, the strain was determined to be *Bifdobacterium animalis* subsp. *Lactis*, and named *Bifdobacterium animalis* subsp. *Lactis* F MBL B23403. It was deposited at the China Center for Type Culture Collection on August 26, 2024, with accession number CCTCC NO: M 20241845. The deposit address is Wuhan University, Wuhan, China, and the contact number is (027)-68752319.

[0061] In the following examples, Bifidobacterium animalis subsp. Lactis FMBL B23403 is abbreviated as Bifidobacterium animalis subsp. Lactis FMBL B23403.

[0062] Example 2: Antioxidant capacity of Bifidobacterium animalis subsp. lactis FMBL B23403

[0063] Activated Bifidobacterium animalis subsp. lactis FMBL B23403 at 2% (v / v) (OD 600An inoculum of 1.0 ± 0.05 g (mg / L) was added to MRS liquid medium. After aerobic incubation at 37°C for 24 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.

[0064] (1) Determination of ABTS free radical scavenging ability

[0065] 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 hours. 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.

[0066] ABTS clearance rate (%) = (1 - (Ac - A) / Ac) × 100%

[0067] In the formula, Ac is the absorbance of the blank control, and A is the absorbance of the sample solution.

[0068] (2) DPPH free radical scavenging capacity determination experiment

[0069] 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.

[0070] DPPH clearance rate (%) = (1 - (D1 - D2) / D0) × 100%

[0071] Where: D0: absorbance of PBS buffer; D1: absorbance of sample solution; D2: absorbance of blank control.

[0072] Table 7 Results of Antioxidant Capacity Measurement

[0073]

[0074] As shown in Table 7, the scavenging rates of ABTS by the fermentation supernatant and cell extract of Bifidobacterium animalis subsp. lactis FMBL B23403 were 71.60% and 30.26%, respectively, and the scavenging rates of DPPH by both exceeded 50%, reaching 63.45% and 74.34%, respectively. The results indicate that Bifidobacterium animalis subsp. lactis FMBL B23403 has a strong scavenging ability against DPPH and ABTS free radicals, and its fermentation supernatant showed better antioxidant performance compared with the cell extract.

[0075] Example 3: Inhibitory effect of Bifidobacterium animalis subsp. lactis FMBL B23403 on α-amylase, α-glucosidase, and dipeptidyl peptidase.

[0076] Activated Bifidobacterium animalis subsp. lactis FMBL B23403 at 2% (v / v) (OD 600 An inoculum of 1.0 ± 0.05 g (mg / L) was added to MRS liquid medium. After aerobic incubation at 37°C for 24 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, and then resuspended in sterile water to adjust the bacterial concentration to 1 × 10⁻⁶. 9 CFU / mL available for use.

[0077] (1) Determination of α-glucosidase activity inhibition capacity

[0078] 50 μL of sample and 100 μL of 0.2 U / mL α-glucosidase solution were mixed in a test tube and reacted at 37 °C for 10 min. Then, 50 μL of 5 mmol / L p-nitrophenol-α-D-glucopyranoside solution was added, mixed, and reacted in a constant temperature water bath at 37 °C for 20 min. Finally, 50 μL of 0.2 mol / L Na₂CO₃ solution was added to terminate the reaction. The absorbance of the reaction solution was measured at 400 nm.

[0079]

[0080] In the formula: A is the sample group, containing the sample solution and α-glucosidase solution; B is the blank sample group, containing the sample solution but not the α-glucosidase solution; C is the control group, containing no sample solution but containing the α-glucosidase solution; D is the blank group, containing neither the sample solution nor the α-glucosidase solution.

[0081] (2) Determination of α-amylase activity inhibition capacity

[0082] Mix 125 μL of sample solution with an equal volume of 1 mg / mL α-amylase solution and incubate at 37 °C for 15 min. Then, add the reaction solution to 250 μL of 1% soluble starch solution and incubate at 37 °C for 15 min. Next, add 500 μL of DNS solution, boil in a water bath for 5 min, and then rapidly cool to room temperature. Dilute 20 times and allow to stand at room temperature. Measure the absorbance at 540 nm. Use PBS solution (0.1 mol / L, pH 6.8) as a blank control for both the α-amylase solution and the test sample.

[0083]

[0084] In the formula: A is the sample group, containing the sample solution and α-amylase solution; B is the blank sample group, containing the sample solution but not the α-amylase solution; C is the control group, not containing the sample solution but containing the α-amylase solution; D is the blank group, not containing the sample solution or the α-amylase solution.

[0085] (3) Determination of dipeptidyl peptidase (DPP-IV) activity inhibition capacity

[0086] The inhibitory activity of DPP-IV was determined using a DPP-IV inhibitor screening kit, and the fluorescence value (FLU) of the samples was measured using a multi-functional microplate reader (output wavelength λex = 360 nm / input λem = 460 nm). DPP-IV and substrate were added to the samples; the control group had no sample but added sample solvent; the DPP-IV in the blank group was replaced with buffer solution.

[0087] The formula for calculating the DPP-IV inhibition rate of the sample is as follows:

[0088]

[0089] In the formula: F 对 The control group contains the sample solvent and DPP-IV enzyme solution; F 样 This is the sample group, containing sample solution and DPP-IV enzyme solution; F 空 This is the blank control group, containing sample solvent but without DPP-IV enzyme solution.

[0090] Alpha-glucosidase and alpha-amylase catalyze the hydrolysis of complex carbohydrates into monosaccharides, which are then absorbed by the intestines. Inhibiting the activity of alpha-glucosidase and alpha-amylase in the intestines can effectively reduce the hydrolysis of polysaccharides and disaccharides, thereby reducing the release and absorption of glucose and thus lowering postprandial blood glucose. DPP-IV is a very stable serine protease on the cell surface, highly expressed in the intestines, and also expressed in the liver, pancreas, placenta, thymus, etc. Some of it exists in soluble form in the circulating blood, thus acting on tissues and organs. DPP-IV can act on physiological hormones, such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). Inhibiting the activity of DPP-IV can increase the half-life of GLP-1, thereby promoting insulin secretion and achieving the effect of lowering blood glucose. The results are shown in Table 8. The fermentation broth of Bifidobacterium animalis subsp. lactis FMBL B23403 showed strong inhibitory effects on α-glucosidase, α-amylase, and dipeptidyl peptidase activities, reaching 62.39%, 80.53%, and 48.74%, respectively. This indicates that Bifidobacterium animalis subsp. lactis FMBL B23403 has great application potential in lowering blood sugar and improving diabetes.

[0091] Table 8 Enzyme activity inhibition ability

[0092]

[0093] Example 4: Probiotic Properties of Bifidobacterium animalis subsp. lactis FMBL B23403

[0094] 1. Determination of acid and bile salt resistance

[0095] Bifidobacterium animalis subsp. lactis FMBL B23403 was added to MRS liquid medium at an inoculum of 2% (v / v) (OD600: 1.0±0.05) and cultured aerobically at 37°C for 24 h. After centrifuging the bacterial suspension at 8000 rpm for 5 min, the supernatant was discarded and the bacterial pellet was collected.

[0096] (1) After washing the bacterial cells three times with PBS buffer (pH=7.4), the bacterial cells were resuspended in PBS solutions at pH 3.5 and pH 4.5 and cultured anaerobically at 37°C for 4 h. Samples at 0 h and 4 h were spread on plates and cultured for 24 h before viable cell counts were performed. Each experiment was performed in triplicate.

[0097] (2) The collected bacterial cells were resuspended in MRS liquid medium containing different concentrations (0.04%, 0.06%) of bile salts and cultured at 37°C for 2.5 h. Samples from 0 h and 2.5 h were spread on plates, and viable cell counts were performed after 24 h. Each sample was replicated in triplicate.

[0098] Table 9. Acid and salt tolerance properties of Bifidobacterium lactis subsp. FMBL B23403

[0099]

[0100] As shown in Table 9, *Bifidobacterium animalis* subsp. *lactamella* FMBL B23403 can survive at pH 3.5, pH 4, and bile salt concentrations of 0.04% and 0.06%. The survival rate is 30.5% at pH 3.5 and 70.28% at pH 4; the survival rate at 0.04% and 0.06% bile salt concentrations ranges from 14.04% to 17.25%. This indicates that *Bifidobacterium animalis* subsp. *lactamella* FMBL B23403 possesses certain acid and bile salt tolerance characteristics. These good acid and bile salt tolerance characteristics enable the strain to survive in the gastrointestinal tract, reach its colonization site, grow, and exert its probiotic effects.

[0101] 2. Determination of self-agglomeration and hydrophobicity

[0102] (1) Determination of self-aggregation ability

[0103] Bifidobacterium animalis subsp. lactis FMBL B23403 was added to MRS liquid medium at an inoculum size of 2% (v / v) (OD600: 1.0 ± 0.05) and cultured aerobically at 37°C for 24 h. After centrifugation at 8000 rpm for 5 min, the supernatant was discarded, and the bacterial pellet was collected. The bacterial cells were washed twice with PBS solution at pH 7.2, and then resuspended in PBS and diluted to a concentration of OD600. 600 =0.6±0.05 (A0). After vortexing for 20 seconds and allowing to stand at room temperature for 2 hours, its OD was measured. 600 (A2) value.

[0104] Self-agglomeration (%) = (1 - A2 / A0) × 100

[0105] (2) Surface hydrophobicity determination

[0106] Bifidobacterium animalis subsp. lactis FMBL B23403 was added to MRS liquid medium at an inoculum size of 2% (v / v) (OD600: 1.0 ± 0.05) and cultured aerobically at 37°C for 24 h. After centrifugation at 8000 rpm for 5 min, the supernatant was discarded, and the bacterial pellet was collected. The bacterial cells were washed twice with PBS solution at pH 7.2, and then resuspended in PBS and diluted to a concentration of OD600. 600 =0.6±0.05(A0). Take 3 mL of cell suspension and mix thoroughly with 1 mL of xylene, vortex for 20 s, let stand at room temperature for 2 h, and then measure the OD of the xylene phase. 600 Value (A1).

[0107] Surface hydrophobicity (%) = (1-A) 1 / A0)×100

[0108] Table 10. Self-aggregation and hydrophobicity of Bifidobacterium animalis subsp. lactis FMBL B23403

[0109]

[0110] Surface characteristics of bacterial strains are key factors affecting their survival and adhesion. The hydrophobicity of the outermost surface of microorganisms is an important factor in bacterial attachment to host tissues, and this characteristic can provide an advantage for bacterial survival in the human gastrointestinal tract. Table 10 shows that the self-aggregation of *Bifidobacterium animalis* subsp. *lactamella* FMBL B23403 is 10.08%, and its surface hydrophobicity is 18.13%, indicating that *Bifidobacterium animalis* subsp. *lactamella* FMBL B23403 possesses certain self-aggregation and hydrophobic properties.

[0111] Application Example 1: Preparation of Bifidobacterium animalis subsp. lactis FMBL B23403 bacterial agent

[0112] Culture medium preparation: 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 diammonium hydrogen citrate, 0.58 g MgSO₄·7H₂O, and 0.25 g MnSO₄·4H₂O was prepared using water and culture medium raw materials. The pH was adjusted to 6.8 to obtain the culture medium.

[0113] Preparation of the preservative: A preservative containing 120 g / L skim milk powder, 20 mL / L glycerol, 22 g / L maltodextrin, 60 g / L trehalose, and 22 g / L galactooligosaccharides was prepared using water and preservative raw materials.

[0114] Bifidobacterium animalis subsp. lactis FMBL B23403 was inoculated at a 2% inoculum into the above-mentioned culture medium sterilized at 115℃ for 20 min. The culture medium contained 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 diammonium citrate, 0.58 g MgSO₄·7H₂O, and 0.25 g MnSO₄·4H₂O, and the pH was adjusted to 6.8. After incubation at 37℃ for 24-48 h, the bacterial cells were centrifuged, washed twice with pH 7.2 PBS buffer, and then resuspended in a protective agent to achieve a concentration of 10. 11CFU / mL. The protective agent contains 120 g / L skim milk powder, 20 mL / L glycerol, 22 g / L maltodextrin, 60 g / L trehalose, and 22 g / L galacto-oligosaccharides. The suspension is then pre-cultured at 37°C for 60 min, followed by freeze-drying to obtain the *Bifidobacterium animalis* subsp. *lactamase* FMBL B23403 bacterial agent.

[0115] Application Example 2: Preparation of Fermented Milk using Bifidobacterium lactis subsp. FMBL B23403 of the present invention

[0116] Fresh milk is dissolved with sugar and homogenized at 60°C and 20 MPa. Then, it is sterilized at 90-95°C for 5-8 minutes. When the temperature drops to 35°C, a mixed culture consisting of Bifidobacterium animalis subsp. lactis FMBL B23403 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 culture is 0.03-2.0% of the weight of the fresh milk. The mixture is mixed well and fermented at 37°C for 4-6 hours. After curdling, it is refrigerated at 4°C for 16 hours to obtain the fermented milk.

[0117] Application Example 3: Preparation of microcapsules and capsule products containing Bifidobacterium animalis subsp. lactis FMBL B23403

[0118] The *Bifidobacterium lactis* subsp. FMBL B23403 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 animalis* subsp. *lactamase* FMBL B23403. The microcapsules had a particle size of 2.0-2.5 mm, an encapsulation efficiency of ≥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.

[0119] In summary, this invention provides a *Bifidobacterium animalis* subsp. *milk* FMBL B23403, which exhibits good oxygen tolerance and strong survival ability in aerobic environments. Its fermentation broth shows inhibition rates of 62.39%, 80.53%, and 48.74% for α-glucosidase, α-amylase, and dipeptidyl peptidase activities, respectively. Its fermentation broth and cell extracts demonstrate strong scavenging abilities against DPPH and ABTS free radicals, with ABTS scavenging rates of 71.60% and 30.26%, respectively, and DPPH scavenging rates exceeding 50%, reaching 63.45% and 74.34%, respectively. It possesses good antioxidant capacity, acid and bile salt resistance, self-polymerization properties, and surface hydrophobicity. It can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives, and feeds, showing broad application prospects.

Claims

1. A subsp. *Bifidobacterium animalis*, FMBLB23403, characterized in that... The aforementioned Bifidobacterium lactis subspecies FMBL B23403 was deposited at the China Center for Type Culture Collection on August 26, 2024, with accession number CCTCC NO: M 20241845.

2. A microbial agent, characterized in that, The bacterial agent contains Bifidobacterium animalis subsp. lactis 23403 as described in claim 1.

3. The application of Bifidobacterium animalis subsp. lactis FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in the preparation of pharmaceuticals, food, and health products.

4. The use of Bifidobacterium animalis subsp. lactis FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in inhibiting dipeptidyl peptidase activity or preparing dipeptidyl peptidase inhibitors.

5. The use of Bifidobacterium animalis subsp. lactis FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in inhibiting α-glucosidase activity or preparing α-glucosidase inhibitors.

6. The use of Bifidobacterium animalis subsp. lactis FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in inhibiting α-amylase activity or preparing α-amylase inhibitors.

7. The application of Bifidobacterium animalis subsp. lactis FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in the preparation of hypoglycemic drugs and health products.

8. The use of Bifidobacterium animalis subsp. lactis FMBL B23403 or its strain fermentation broth or its sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in antioxidation or the preparation of antioxidation products.

9. The use of Bifidobacterium animalis subsp. lactis FMBLB23403 or its strain fermentation broth or its sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in the preparation of yogurt or yogurt starter.

10. The application of Bifidobacterium lactis subsp. FMBL B23403 of claim 1 or its strain fermentation broth or its sterile fermentation supernatant, or the bacterial agent of claim 2, in the preparation of feed / feed additives.

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