Bifidobacterium animalis subsp. Lactis FMBL B23439 AHDM capable of reducing blood sugar, 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 B23439 AHDM capable of reducing blood sugar, which is preserved in the China Center for Type Culture Collection (CCTCC) on November 11, 2024, the preservation number is CCTCC NO: M 20242506, the preservation number is CCTCC NO: M 20242506, the preservation number is CCTCC NO: M 20242506, the preservation number is CCTCC NO: M 20242506, and the preservation number is CCTCC NO: M 20242506. The fermentation liquor of the bacillus subtilis has inhibitory activity on the activity of alpha-glucosidase and dipeptidyl peptidase; the compound has relatively high scavenging capacity on DPPH and ABTS free radicals, and is relatively good in oxidation resistance; good acid-resistant and cholate-resistant capability and antibacterial capability are realized; the strain can be used for preparing hypoglycemic drugs, fermented food and health care products, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a blood sugar-lowering animal Bifidobacterium lactis subspecies FMBLB23439 AHDM, its bacterial agent, and its application. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease affecting the quality of life of hundreds of millions of people worldwide. It can lead to a variety of serious complications and significantly increase the risk of death. Therefore, drug intervention is crucial for the prevention and control of type 2 diabetes and its related hyperglycemic complications. Currently, commonly used antidiabetic drugs, such as thiazolidinediones and biguanides, mainly exert their therapeutic effects by improving insulin sensitivity. However, long-term use of these drugs may lead to adverse reactions such as diarrhea and bloating, and even induce drug resistance. These limitations make the search for safer treatment strategies a hot research topic.

[0003] In recent years, enzyme inhibition therapy has demonstrated significant value in glycemic regulation. Alpha-glucosidase, a key enzyme catalyzing the digestion and breakdown of carbohydrates, can delay intestinal glucose absorption by its inhibitors, effectively alleviating postprandial hyperglycemia. Furthermore, dipeptidyl peptidase-4 (DPP-4) inhibitors can promote insulin secretion and inhibit glucagon release by increasing endogenous glucagon-like peptide-1 (GLP-1) levels, thus achieving stable glycemic control. Given the risks of side effects associated with chemically synthesized inhibitors, natural enzyme inhibitors derived from plants, vegetables, and probiotics have attracted considerable attention due to their safety profile.

[0004] Probiotics, as live microorganisms, can promote nutrient absorption and inhibit pathogenic bacteria in the gut when consumed in appropriate amounts, and have shown potential in the prevention of cardiovascular diseases and the management of chronic diseases such as blood sugar regulation. Among them, *Bifidobacterium animalis* subsp. *lactobacter* (… Bifidobacterium animalis subsp Bifidobacterium lactis (Bifidobacterium lactis) is one of the most commonly used probiotic strains in functional foods and dietary supplements due to its excellent processing properties and health benefits. This strain naturally colonizes the intestines of healthy adults and infants and has been included in the EU's list of safe fermentation bacteria since 1980, boasting a long history of safe use. Studies have found that Bifidobacterium animalis subsp. lactis has a hypoglycemic function. For example, invention patent CN120137852A discloses a hypoglycemic Bifidobacterium animalis subsp. lactis YYSJ001, which can effectively reduce fasting blood glucose levels and insulin resistance index in diabetic mice, improve glucose absorption and metabolism, slow down the damage to pancreatic β-cell function, and improve blood lipid levels and gut microbiota balance. Invention patent CN119372116A discloses a hypoglycemic Bifidobacterium animalis subsp. lactis strain B21247, which exhibits in vitro inhibition of α-glucosidase activity and in vivo hypoglycemic activity, and can improve glucose and lipid metabolism function caused by diabetes.

[0005] Meanwhile, the inventor's research group also applied for invention patent CN120738040A, "A Subspecies of Bifidobacterium animalis FMBL B24304 AHDM and its Applications," based on previous research results. This patent describes how fermentation broth inhibits α-glucosidase, α-amylase, and dipeptidyl peptidase IV, regulating gut microbiota, improving blood glucose regulation and insulin sensitivity in T2DM mice, and preparing hypoglycemic and antioxidant products. Building on previous research, the inventor's research group continued their work, isolating a strain of Bifidobacterium animalis subspecies of lactic acid bacteria (FMBL B24304 AHDM) from the feces of infants aged 0-1 years. Bifidobacterium animalis subsp *Lactis* FMBL B23439 AHDM. Experiments showed that the fermentation broth of this strain exhibited inhibition rates of 80.76% and 19.25% against α-glucosidase and DPP-4, respectively, demonstrating both good antioxidant capacity and acid and bile salt tolerance. This strain can be applied to the development of products related to antioxidation, hypoglycemia, and inhibition of pathogenic bacteria, including fermented foods, health products, food additives, and feed, showing promising application prospects. Summary of the Invention

[0006] The primary objective of this invention is to provide a subspecies of Bifidobacterium lactis that can lower blood sugar. Bifidobacterium animalis subsp Bifidobacterium animalis FMBL B23439 AHDM was deposited at the China Center for Type Culture Collection on November 11, 2024, with accession number CCTCC NO: M 20242506.

[0007] A second objective of this invention is to provide a microbial agent containing the aforementioned Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM.

[0008] Preferably, the dosage form of the microbial agent includes solution, powder, tablet or capsule.

[0009] A third objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23439 AHDM 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.

[0010] The fourth objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23439 AHDM 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.

[0011] The fifth objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23439 AHDM or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of hypoglycemic drugs and health products.

[0012] The sixth objective of this invention is to provide the application of the aforementioned Bifidobacterium lactis subsp. FMBL B23439 AHDM or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in antioxidant processes or in the preparation of antioxidant products.

[0013] The seventh objective of this invention is to provide the application of the aforementioned Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of yogurt or yogurt starter.

[0014] The beneficial effects of this invention are: This invention provides a subspecies of Bifidobacterium lactis that can lower blood sugar ( Bifidobacterium animalis subsp Bifidobacterium animalis FMBL B23439 AHDM fermentation broth exhibits inhibition rates of 80.76% and 19.25% against α-glucosidase 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 84.89% and 30.44%, respectively, and DPPH scavenging rates exceeding 50%, reaching 72.65% and 70.17%, respectively. It possesses excellent antioxidant capacity, good acid and bile salt resistance, and antibacterial properties. It can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives, and feed, showing broad application prospects. Attached Figure Description

[0015] Figure 1 Colony morphology and microscopic features

[0016] Figure 2 Phylogenetic tree of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM Detailed Implementation

[0017] 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, which can be purchased commercially. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0018] The culture medium formula used in this invention is as follows:

[0019] MRS liquid culture medium (1L): peptone 10 g; beef extract 10 g; yeast extract 5 g; glucose 20 g; Tween 80 1 mL; K2HPO4 2 g; anhydrous sodium acetate 5 g; diammonium citrate 2 g; MgSO4·7H2O 0.58 g; MnSO4·4H2O 0.25 g; L-cysteine ​​hydrochloride 0.5 g; mupirocin 0.5 mg; deionized water 1000 mL;

[0020] MRS solid medium (1L): peptone 10 g; beef extract 8 g; yeast extract 4 g; glucose 20 g; Tween 80 1 mL; K2HPO4 2 g; anhydrous sodium acetate 5 g; diammonium hydrogen citrate 2 g; MgSO4·7H2O 0.29 g; MnSO4·4H2O 0.25 g; agar 20 g; L-cysteine ​​hydrochloride 0.5 g; mupirocin 0.5 mg, sterilized at 115℃ for 20 min.

[0021] Wilkins-Chalgren (MAN) medium (1L): Modified Wilkins-Chalgren agar 48 g; soybean peptone 5 g; Tween 80 1 mL; glacial acetic acid 1 mL; anhydrous sodium acetate 6 g; agar 20 g; L-cysteine ​​hydrochloride 0.5 g; mupirocin 0.5 mg; sterilize at 115℃ for 20 min.

[0022] PYG solid medium (1L): 10 g peptone; 5 g yeast extract; 1 g glucose; 15 g agar; 1000 mL deionized water; adjust pH to 6.8, sterilize at 115℃ for 20 min;

[0023] Nutrient gravy agar medium (1L): 5 g peptone; 3 g beef extract; 5 g NaCl; 20 g agar; 1000 mL deionized water; adjust pH to 7.0, sterilize at 115℃ for 20 min;

[0024] TSA solid medium (1L): 15 g tryptone; 5 g / L soybean peptone; 5 g NaCl; 15 g agar; 1000 mL deionized water; adjust pH to 7.2, sterilize at 115℃ for 20 min.

[0025] Example 1: Screening of strains that inhibit α-glucosidase

[0026] 1. Strain isolation and purification

[0027] Fresh fecal samples from infants were collected in Hainan, Wuwei in Gansu, and Kashgar and Nileke in Xinjiang Uygur Autonomous Region. The samples were cultured under anaerobic conditions (80% nitrogen, 10% hydrogen, 10% carbon dioxide) using the gradient dilution plating method and strains were isolated.

[0028] Fecal samples were diluted to 10 μL in liquid Man-Rogosa-Shape (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.

[0029] Six strains were selected based on colony characteristics and cell morphology and named accordingly. Specific information is shown in Table 1.

[0030] Table 1. Information on the 6 selected strains and their hosts.

[0031] Six bacterial strains were spread on MRS solid medium containing 0.5% L-cysteine ​​hydrochloride and cultured 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 cultured in an anaerobic incubator at 37°C for 24 hours to activate the bacteria.

[0032] 2. Determination of α-glucosidase inhibitory activity

[0033] 60 μL of sample and 120 μL of 0.2 U / mL α-glucosidase solution were mixed in a test tube and reacted at 37℃ for 10 min. Then, 60 μL of 5 mmol / L p-nitrophenol-α-D-glucopyranoside solution was added, mixed, and reacted in a constant temperature water bath at 37℃ for 20 min. Finally, 60 μL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction. The absorbance of the reaction solution was measured at 405 nm.

[0034] α-glucosidase inhibition rate (%) =

[0035] 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.

[0036] Alpha-glucosidase is located at the brush border of the small intestine and is responsible for hydrolyzing complex carbohydrates into monosaccharides for absorption by the intestine. Inhibiting the activity of alpha-glucosidase in the intestine can effectively reduce the hydrolysis of polysaccharides and disaccharides, reduce the release and absorption of glucose, thereby lowering postprandial blood glucose.

[0037] Table 2 α-glucosidase inhibitory activity

[0038] The results are shown in Table 2. The fermentation broth of strain FMBL B23439 AHDM showed the strongest inhibitory effect on α-glucosidase, reaching 80.76%, which was significantly higher than that of other strains. This also indicates that strain FMBL B23439 has great application potential in lowering blood sugar and improving diabetes.

[0039] 3. Strain identification

[0040] The strain FMBL B23439 AHDM, which exhibited the highest α-glucosidase inhibitory activity, was identified.

[0041] 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 GTSGTCAGGAACAG-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 MEGA 11.0 as shown below. Figure 2 As shown.

[0042] Table 3. Premixed solution system for GroEL gene PCR amplification (25 µL)

[0043] Table 4 PCR Reaction Conditions

[0044] Table 5. PCR amplification primers for subspecies-specific primer recA

[0045] Table 6. PCR reaction conditions for primer recA

[0046] The colony characteristics and microscopic features of strain FMBL B23439 AHDM are as follows: Figure 1 As shown, the phylogenetic tree is as follows: Figure 2 As shown. The morphological and physiological-biochemical characteristics of the above-mentioned strain FMBL B23439 AHDM 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 B23439 AHDM belongs to the phylum Actinobacteria, class Actinobacteria, subclass Actinobacteridae, order Bifidobacteriales, family Bifidobacteriaceae, genus Bifidobacterium, species Bifidobacterium animalis, and subsp. Lactis of Bifidobacterium animalis.

[0047] After identification, the strain was determined to be *Bifdobacterium animalis* subsp. *Lactis*, and named *Bifdobacterium animalis* subsp. *Lactis* FMBL B23439 AHDM. It was deposited at the China Center for Type Culture Collection (CCTCC) on November 11, 2024, with accession number CCTCC NO: M 20242506. The deposit address is Wuhan University, Wuhan, China, and the contact number is (027)-68754052.

[0048] In the following embodiments, the Bifdobacterium animalis subsp. Lactis FMBL B23439 AHDM is abbreviated as Bifdobacterium animalis subsp. Lactis FMBL B23439 AHDM.

[0049] Example 2: Inhibitory activity of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM against dipeptidyl peptidase IV

[0050] Activated Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM at 2% (v / v) (OD 600 An inoculum of 1.0 ± 0.05 g was added to MRS liquid medium and cultured aerobicly at 37°C for 48 h. The mixture was then centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 0.22 μm aqueous microfiltration membrane to obtain cell-free supernatant.

[0051] 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 wavelength λem = 460 nm). DPP-IV and substrate were added to the samples; the control group had no sample but added sample solvent; the blank group used buffer instead of DPP-IV. The reaction systems for each group are shown in Table 7.

[0052] Table 7 DPP-IV Inhibitory Activity Assay System

[0053] The formula for calculating the DPP-IV inhibition rate of the sample is as follows:

[0054] Dipeptidyl peptidase IV inhibition rate (%) =

[0055] 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.

[0056] Table 8 DPP-IV inhibitory activity

[0057] Example 3: Antioxidant properties of Bifidobacterium animalis subsp. lactis FMBL B23429 AHDM

[0058] (1) Determination of ABTS free radical scavenging ability

[0059] 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.

[0060]

[0061] In the formula, Ac is the absorbance of the blank control, and A is the absorbance of the sample solution.

[0062] (2) DPPH free radical scavenging capacity determination experiment

[0063] 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.

[0064]

[0065] Where: D0: absorbance of PBS buffer; D1: absorbance of sample solution; D2: absorbance of blank control.

[0066] Table 9 Results of Antioxidant Capacity Measurement

[0067] As shown in Table 9, the scavenging rates of ABTS by the fermentation supernatant of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM and the cell extract were 84.89% and 30.44%, respectively, and the scavenging rates of DPPH both exceeded 50%, reaching 72.65% and 70.17%, respectively. The results indicate that Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM has a strong scavenging ability against DPPH and ABTS free radicals, and its fermentation supernatant showed better antioxidant performance compared with the cell extract.

[0068] Example 4: Probiotic Properties of Bifidobacterium animalis subsp. lactis FMBL B23429 AHDM

[0069] 1. Determination of acid and bile salt resistance

[0070] Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM was added to MRS liquid medium at an inoculum of 2% (v / v) (OD600: 1.0±0.05) and cultured at 37℃ for 24 h. After centrifuging the bacterial suspension at 8000 rpm for 5 min, the supernatant was discarded and the bacterial pellet was collected.

[0071] (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.

[0072] (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 at 0 h and 2.5 h were spread on plates and viable counts were performed after 24 h. Each sample was performed in triplicate.

[0073] Table 10. Acid and salt tolerance properties of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM

[0074] As shown in Table 10, *Bifidobacterium animalis* subsp. *lactamella* FMBL B23439 AHDM survived at pH 3.5, pH 4, and bile salt concentrations of 0.04% and 0.06%. The survival rate was 22.26% at pH 3.5 and 31.69% at pH 4; the survival rate ranged from 6.34% to 29.08% at 0.04% and 0.06% bile salt concentrations. This indicates that *Bifidobacterium animalis* subsp. *lactamella* FMBL B23439 AHDM 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.

[0075] 2. Determination of performance in inhibiting conditionally pathogenic bacteria

[0076] Escherichia coli (10411), toxin-producing Escherichia coli (10421), Salmonella typhimurium (10420), Listeria monocytogenes (LS1), and serotype Salmonella enteritidis (SM1) were used as indicator bacteria (Table 11). The Oxford cup method was used to determine the antibacterial activity of metabolites of Bifidobacterium animalis subsp. lactis FMBL B23439. Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM was added to MRS liquid medium at an inoculum of 2% (v / v) (OD600: 1.0±0.05), and anaerobically cultured at 37℃ for 36 h. The bacterial suspension was centrifuged at 8000 rpm for 5 min, and the supernatant was collected to prepare a cell-free supernatant. After activation of the indicator bacteria, the supernatant was prepared according to a 10... 6 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 diffusion for 4 h, and then incubated in a 37°C anaerobic incubator for 24 h. The diameter of the inhibition zone was then measured.

[0077] Table 11 Sources of indicator bacteria

[0078] Table 12. Determination of the antibacterial activity of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM.

[0079] Note: The diameter of the inhibition zone includes the outer diameter of the Oxford cup. No inhibition zone: -; 8-15 mm: +; 15-20 mm: ++; 20-25 mm: +++.

[0080] As shown in Table 12, Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM has good inhibitory ability against diarrhea-causing Escherichia coli (10411), enterotoxigenic Escherichia coli (10421), Listeria monocytogenes (LS1), and serotype Salmonella enteritidis (SM1), indicating that Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM has great potential for the development of drugs related to inhibiting opportunistic pathogens, preventing or alleviating diarrhea, and improving intestinal health.

[0081] 3. Antibiotic resistance test

[0082] The disk diffusion method (KB method) was used, and the information on the drug sensitivity test tablets is shown in Table 13. Bifidobacterium animalis subsp. lactis FMBLB23439 AHDM was added to MRS liquid medium at an inoculum of 2% (v / v) (OD600: 1.0±0.05). After anaerobic culture at 37℃ for 36 h, the culture was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were reconstituted with sterile water to a concentration of 1×10⁻⁶. 7-10 8 A bacterial suspension of CFU / mL was prepared. 100 μL of the suspension was evenly spread onto an MRS-agar plate. The tablets were then evenly placed on the medium and anaerobically incubated at 37°C for 48 h. The diameter of the inhibition zone was then precisely measured using calipers. The experiment was repeated three times, and the average value was taken. The sensitivity of the strain to the susceptibility testing tablets was determined based on the diameter of the inhibition zone. The experimental results were determined according to the CISI data and susceptibility classification standards established by the Clinical Laboratory Standards Institute (CLS).

[0083] Table 13. Antimicrobial susceptibility testing of Bifidobacterium animalis subsp. Lactobacillus FMBL B23439 AHDM.

[0084] Note: R: drug resistance; I: moderate sensitivity; S: sensitive

[0085] To ensure the safety and reliability of probiotics for human consumption, antibiotic resistance has become an important indicator for assessing the in vitro safety of probiotic strains. The results are shown in Table 13. *Bifidobacterium animalis* subsp. *lactamase* FMBL B23439 AHDM was sensitive to minocycline, cefotaxime, rifampin, and ampicillin, but resistant to levofloxacin, gentamicin, streptomycin, kanamycin, and norfloxacin.

[0086] 4. Carbohydrate metabolism experiment

[0087] Activated *Bifidobacterium animalis* subsp. *lactamase* FMBL B23439 AHDM was inoculated at 2% (v / v) into modified MRS liquid medium containing 0.5% L-cysteine ​​hydrochloride and anaerobically cultured at 37 °C for 48 h. The bacterial cells were isolated by centrifugation (5000 rpm, 10 min, 4 °C), the supernatant was discarded, and the cells were washed three times 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 (lactose, galactose, fructose, sucrose, fructooligosaccharides, galactooligosaccharides, D-(+)-trehalose, inulin, maltose, mannose, cellobiose, mannitol, arabinose, L-sorbitol, and resistant starch). Glucose was used as a positive control, and medium without any carbon source was used as a negative control. OD of each culture was measured before incubation (0 h). 600 Absorbance values ​​(denoted as OD1). After 48 h 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. Based on OD... 600 The range of values ​​determines the growth status: OD 600A value less than 0.15 indicates no growth, 0.15 to 0.35 indicates limited growth, and a value greater than 0.35 indicates good growth. Each experimental condition was repeated three times, and the average value was taken as the final result.

[0088] The results are shown in Table 14. The carbohydrate metabolism experiment results show that the animal Bifidobacterium lactis subsp. FMBLB23439 AHDM strain can effectively utilize 13 carbon sources, including resistant starch, pectin, chitosan oligosaccharide, fructooligosaccharide, galactooligosaccharide, mannose, trehalose, mannitol, and arabinose; but it cannot effectively utilize xylooligosaccharide and resistant starch.

[0089] Table 14. Carbohydrate Utilization Capacity of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM

[0090] Application Example 1: Preparation of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM bacterial agent

[0091] Culture medium preparation: Mix malt powder and water at a ratio of 1:4 (m / v), add 0.1-0.2% cellulase, stir at 60-70°C for 1-2 hours, and sonicate for 20-30 minutes until saccharification is complete. After saccharification, centrifuge (8000 rpm / 10 min) or filter through 4-6 layers of gauze to collect the supernatant. The obtained saccharified liquid is the high-density culture medium substrate, malt wort. Add 5% tryptone, 1% anhydrous sodium acetate, and 2.5% fructooligosaccharides to adjust the pH to 6.8 to obtain the culture medium.

[0092] 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.

[0093] Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM was inoculated at a 2% inoculum into the above-mentioned culture medium sterilized at 115℃ for 20 min. The culture medium consisted of malt powder and water at a ratio of 1:4 (m / v), with 0.1-0.2% cellulase added. The mixture was stirred at 60-70℃ for 1-2 h for saccharification, followed by sonication for 20-30 min until complete saccharification. After saccharification, the supernatant was collected by centrifugation (8000 rpm / 10 min) or by filtration through 4-6 layers of gauze. The resulting saccharified liquid was the high-density nutrient medium substrate, malt extract. 5% tryptone, 1% anhydrous sodium acetate, and 2.5% fructooligosaccharides were added to adjust the pH to 6.8. After incubation at 37℃ for 48 h, the resulting bacterial cells were washed three times with pH 7.2 PBS buffer and then resuspended in a protective agent to achieve a concentration of 10. 12CFU / 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* FMBLB23439 AHDM bacterial agent.

[0094] Application Example 2: Preparation of fermented milk using Bifidobacterium lactis subsp. FMBL B23439 AHDM of the present invention.

[0095] 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 bacterial culture consisting of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM or its inoculum, commercial dry powder starter culture 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 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.

[0096] Application Example 3: Preparation of microcapsules and capsule products containing Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM

[0097] The *Bifidobacterium lactis* subsp. FMBL B23439 AHDM of this invention was enriched in MRS liquid medium for 48 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 to 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 B23439 AHDM. 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.

[0098] In summary, this invention provides a blood sugar-lowering animal Bifidobacterium lactis subsp. FMBL B23439AHDM. The fermentation broth of this Bifidobacterium longum subsp. FMBL B23439AHDM exhibits high inhibitory activity against α-glucosidase, with an inhibition rate as high as 80.76%; its fermentation broth also shows an inhibition rate of 19.25% against dipeptidyl peptidase IV; it possesses the ability to utilize various carbohydrates; and it has good inhibitory effects on diarrheal Escherichia coli, enterotoxigenic Escherichia coli, Listeria monocytogenes, and serotype Salmonella enteritidis. It can be used to prepare drugs for lowering blood sugar and inhibiting pathogenic bacteria, fermented foods, health products, food additives, and feed, and has broad application prospects.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A subspecies of Bifidobacterium lactis that can lower blood sugar ( Bifdobacterium animalis subsp . Lactis FMBL B23439 AHDM, characterized in that, The aforementioned Bifidobacterium animalis subspecies Lactobacillus FMBL B23439 AHDM was deposited at the China Center for Type Culture Collection on November 11, 2024, with accession number CCTCC NO: M 20242506.

2. A microbial agent, characterized in that, The bacterial agent contains the animal Bifidobacterium lactis subsp. FMBLB23439 AHDM as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The dosage forms of the bacterial agent include solutions, powders, tablets, or capsules.

4. The use of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM 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.

5. The use of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM 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.

6. The application of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM 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.

7. The use of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM 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.

8. The use of Bifidobacterium animalis subsp. lactis FMBL B23439 AHDM 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.

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