Bifidobacterium longum subsp. Longum FMBL B241768 LS, microbial inoculum and application of microbial inoculum in hypoglycemic products

Abstract

The invention belongs to the technical field of biology, and particularly relates to a bifidobacterium longum subsp. Longum FMBL B241768LS, a microbial inoculum and application of the microbial inoculum in hypoglycemic products, the bifidobacterium longum subsp. Longum FMBL B241768LS is preserved in China Center for Type Culture Collection on October 14, 2024, and the preservation number is CCTCC NO: M 20242173; the inhibitory activity of the compound on dipeptidyl peptidase IV is as high as 79.17%; the compound has a relatively good inhibition effect on the activity of alpha-glucosidase and alpha-amylase; the compound shows drug resistance to ciprofloxacin, ampicillin, penicillin G, clindamycin, kanamycin and polymyxin B; a good inhibition effect is achieved on pathogenic bacteria; the strain can be used for preparing medicines, fermented foods, health-care products and food additives for reducing blood sugar and inhibiting pathogenic bacteria, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a long subspecies of Bifidobacterium longum FMBL B241768 LS, its bacterial agent, and its application in hypoglycemic products. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a common chronic metabolic disease. Its pathogenesis involves decreased sensitivity of the body's cells to insulin, preventing insulin from effectively lowering blood sugar. Alternatively, as the disease progresses, pancreatic β-cell function gradually declines, leading to reduced insulin secretion and an inability to meet the body's needs. Current treatment options for T2DM include dietary control, increased exercise, oral hypoglycemic agents, or direct insulin supplementation. Among the currently used hypoglycemic agents, in addition to stimulating insulin secretion and improving peripheral tissue sensitivity to insulin, some inhibit DPP-4 enzyme activity, increase endogenous glucagon-like peptide-1 (GLP-1) levels, promote insulin secretion, and inhibit glucagon secretion, thus achieving stable blood sugar control. Therefore, dipeptidyl peptidase IV inhibitors can be used to treat type 2 diabetes.

[0003] The human gut contains trillions of microorganisms that play an indispensable role in promoting metabolic function, the development of the nervous system, and the maturation of the immune system, thus influencing human health. Among them, obligate symbiotic bacteria, represented by Bifidobacteria, have formed a close symbiotic relationship with the human body, co-evolving and adapting to each other. This promotes the digestion and absorption of nutrients, inhibits pathogenic bacteria in the gut, and even plays a significant role in preventing cardiovascular diseases and controlling blood sugar levels, among other chronic human diseases. For example, the invention patent CN114350547A discloses Bifidobacterium lactis B-622, which achieves therapeutic effects on diabetic model mice by promoting glycemic peptide-1 secretion, repairing insulin secretion function, regulating lipid metabolism, and reducing chronic low-grade inflammation. Invention patent CN110604749A discloses an animal Bifidobacterium A12 that benefits glucose and lipid metabolism and can be used to develop probiotic products related to lowering blood sugar.

[0004] Bifidobacterium longum subsp. longum is the most abundant Bifidobacterium in the human gut, possessing extremely high genetic diversity and flexible glycan metabolism. It can improve oxidative stress, strengthen the intestinal barrier, regulate the composition of gut microbiota and short-chain fatty acids (SCFAs), and modulate the body's immune system, thereby exerting anti-obesity, anti-diabetic, and alleviating various metabolic diseases effects. It can be widely used as a probiotic supplement in the food and pharmaceutical fields. However, strains from different populations exhibit significantly different probiotic effects and are difficult to culture; therefore, the application of Bifidobacterium longum subsp. longum in the pharmaceutical field is limited.

[0005] During their research, the inventors isolated a strain of *Bifidobacterium longum* subsp. *longum* FMBL B241768 LS from fecal samples of healthy Tajik children. The fermentation broth exhibited inhibition rates of 79.17%, 53.32%, and 96.65% against dipeptidyl peptidase IV, α-glucosidase, and α-amylase activities, respectively. It also demonstrated good inhibitory effects against diarrheal *Escherichia coli*, hemorrhagic *Escherichia coli*, enterotoxigenic *Escherichia coli*, and *Salmonella typhimurium*. Furthermore, it showed resistance to ciprofloxacin, ampicillin, penicillin G, clindamycin, kanamycin, and polymyxin B. This strain has broad application prospects and can be used to prepare hypoglycemic and pathogenic bacteria-inhibiting drugs, fermented foods, health products, and food additives. Summary of the Invention

[0006] The primary objective of this invention is to provide a Bifidobacterium longum subsp. longum FMBL B241768 LS that inhibits dipeptidyl peptidase IV. The Bifidobacterium longum subsp. longum FMBL B241768 LS was deposited at the China Center for Type Culture Collection on October 14, 2024, with accession number CCTCCNO: M 20242173.

[0007] A second objective of this invention is to provide a microbial agent containing the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241768 LS.

[0008] A third objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241768 LS or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of dipeptidyl peptidase IV inhibitors.

[0009] The fourth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241768 LS or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in inhibiting α-glucosidase or preparing α-glucosidase inhibitors.

[0010] The fifth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241768 LS 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.

[0011] The sixth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241768 LS 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 seventh objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241768 LS or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of antibacterial products.

[0013] The eighth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241768 LS or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of fermented foods, pharmaceuticals, health products, or dietary supplements.

[0014] The ninth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241768 LS or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of drugs, foods, and health products that regulate intestinal function.

[0015] The beneficial effects of this invention are as follows: This invention provides a *Bifidobacterium longum* subsp. *longum* FMBL B241768 LS, the fermentation broth of which exhibits high inhibitory activity against dipeptidyl peptidase IV, with an inhibition rate as high as 79.17%; its fermentation broth also shows inhibition rates of 53.32% and 96.65% against α-glucosidase and α-amylase activities, respectively; it has good inhibitory effects against diarrheal *Escherichia coli*, hemorrhagic *Escherichia coli*, enterotoxigenic *Escherichia coli*, and *Salmonella typhimurium*; it exhibits resistance to ciprofloxacin, ampicillin, penicillin G, clindamycin, kanamycin, and polymyxin B; it can be used to prepare hypoglycemic and pathogenic bacteria-inhibiting drugs, fermented foods, health products, and food additives, and has broad application prospects. Attached Figure Description

[0016] Figure 1Phylogenetic tree of Bifidobacterium longum subsp. FMBL B241768 LS 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. Quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.

[0018] The culture medium formulations used in the following examples are as follows:

[0019] 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;

[0020] 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.

[0021] 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;

[0022] 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;

[0023] 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.

[0024] Unless otherwise specified, the reagents and consumables used in the following examples can be purchased from the market.

[0025] Unless otherwise specified, the methods used in the following embodiments are conventional methods and can be obtained by referring to the corresponding literature.

[0026] Dipeptidyl peptidase IV (DPP-IV) is a highly stable serine protease on the cell surface, highly expressed in the intestine, and also expressed in the liver, pancreas, placenta, thymus, etc. Some DPP-IV exists in a 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 DPP-IV activity increases the half-life of GLP-1, thereby promoting insulin secretion.

[0027] It should be noted that the "antibacterial product" mentioned in this invention refers to a product with antibacterial function, which can be any form that achieves the antibacterial function, such as drugs (all dosage forms that achieve the antibacterial function can be used), food, health products, additives, etc.

[0028] Example 1: Screening for Bifidobacterium longum subsp. FMBL B241768 LS with inhibition of dipeptidyl peptidase IV

[0029] 1. Strain isolation and purification

[0030] Fresh fecal samples were collected from healthy Tajik children in Kashgar, Xinjiang. 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 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.

[0032] Four strains were selected based on colony characteristics and cell morphology and named accordingly. Specific information is shown in Table 1.

[0033] Table 1. Information on the four selected bacterial strains and their hosts.

[0034]

[0035] Four 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 h. 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 h to activate the bacteria.

[0036] 2. Determination of dipeptidyl peptidase IV inhibitory activity

[0037] Experimental strains: strains FMBL B241768 LS, FMBL B241769 LS, FMBL B241771 LS, and FMBL B241772 LS were all deposited at the Food Microbiology and Biotechnology Research Center of the College of Food Science and Technology, Shihezi University; Lacticaseibacillus rhamnosus LGG was purchased from the China Industrial Microbial Culture Collection Center.

[0038] After activation, the strain 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 (18 h for Lactobacillus rhamnosus LGG), the culture was centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 0.22 μm aqueous microfiltration membrane to harvest cell-free supernatant.

[0039] 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 blank group used buffer instead of DPP-IV. The reaction systems for each group are shown in Table 2.

[0040] Table 2. DPP-IV Inhibitory Activity Assay System

[0041]

[0042]

[0043] The formula for calculating the DPP-IV inhibition rate of the sample is as follows:

[0044]

[0045] 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.

[0046] Table 3 DPP-IV inhibitory activity

[0047]

[0048] The results are shown in Table 3. The fermentation broth of strain FMBL B241768 LS showed the strongest inhibitory effect on dipeptidyl peptidase IV, reaching 79.17%, which was significantly higher than that of the reference strain Lactobacillus rhamnosus LGG and other strains. This also indicates that strain FMBLB241768 LS has great application potential in lowering blood sugar and improving diabetes.

[0049] 3. Strain identification

[0050] The strain FMBL B241768 LS, which exhibited the highest dipeptidyl peptidase IV inhibitory activity, was identified.

[0051] 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 4, and the PCR reaction conditions are shown in Table 5. 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 longum* subsp. *longum* using BiLON primers. The subsp. *longum*-specific primers are shown in Table 6, and the PCR reaction conditions are shown in Table 7. The corresponding species sequence was obtained from the database, and a phylogenetic tree was constructed using MEGA 11.0.

[0052] Table 4. Premixed solution system for GroEL gene PCR amplification (25 μL)

[0053]

[0054]

[0055] Table 5 PCR reaction conditions

[0056]

[0057] Table 6. Primers for PCR amplification of BiLON using subspecies-specific primers.

[0058]

[0059] Table 7 Primer BiLONPCR Reaction Conditions

[0060]

[0061] The phylogenetic tree of strain FMBL B241768 LS is as follows: Figure 1 As shown, its groEL gene sequence has 99.99% homology with the groEL sequence of *Bifidobacterium longum* subsp. *longum*. According to the classification of the genus *Bifidobacterium* in Bergey's Manual of Systematic Bacteriology, *Bifidobacterium longum* subsp. *longum* belongs to the phylum *Actinobacteria*, class *Actinobacteria*, subclass *Actinobacteridae*, order *Bifidoacteriales*, family *Bifidobacteriaceae*, genus *Bifidobacterium*, species *Bifidobacterium longum*, and subsp. *longum*.

[0062] In summary, the strain FMBL B241768 LS is Bifidobacterium longum subsp. longum, and is named Bifidobacterium longum subsp. longum FMBL B241768 LS. It was deposited at the China Center for Type Culture Collection on October 14, 2024, with accession number CCTCC NO: M 20242173. The deposit address is Wuhan University, Wuhan, China, and the contact number is (027)-68752319.

[0063] In the following examples, Bifidobacterium longum subsp. longum FMBL B241768 LS is abbreviated as Bifidobacterium longum subsp. longum FMBL B241768 LS.

[0064] Example 2: Inhibitory effect of Bifidobacterium longum subsp. FMBL B241768 LS on α-glucosidase and α-amylase.

[0065] Activated Bifidobacterium longum subsp. FMBL B241768 LS at 2% (v / v) (OD 600An 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, and then resuspended in sterile water to adjust the bacterial concentration 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.

[0066] (1) Determination of α-glucosidase inhibitory activity

[0067] 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 405 nm.

[0068]

[0069] 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.

[0070] (2) Determination of α-amylase inhibitory activity

[0071] Mix 125 μL of sample solution with an equal volume of 10 mg / mL α-amylase solution and react at 37 °C for 15 min. Then, add the reaction solution to 250 μL of 1.5% soluble starch solution and react 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.

[0072]

[0073] 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.

[0074] Human blood glucose primarily originates from diet, with the main carbohydrates being starch and sugar molecules, which have relatively large chains. These cannot be directly absorbed into the bloodstream and require hydrolysis by key digestive enzymes in the intestine (α-amylase and α-glucosidase) into glucose monomers before they can be absorbed into the bloodstream and raise blood glucose levels. Therefore, inhibiting the activity of α-glucosidase and α-amylase can reduce glucose production and thus lower blood glucose. The results, as shown in Table 8, indicate that the fermentation broth of *Bifidobacterium longum* subsp. *longum* FMBL B241768 LS exhibits strong inhibitory activity against α-glucosidase and α-amylase, reaching 53.32% and 96.65%, respectively. The cell extract showed inhibition rates of 5.53% and 62.06% against α-glucosidase and α-amylase, respectively. This further demonstrates the significant application potential of *Bifidobacterium longum* subsp. *longum* FMBL B241768 LS in blood glucose regulation.

[0075] Table 8 Enzyme activity inhibition ability

[0076]

[0077] Example 4: Probiotic properties of Bifidobacterium longum subsp. FMBL B241768 LS

[0078] 1. Carbohydrate metabolism experiment

[0079] Activated *Bifidobacterium longum* subsp. *longum* FMBL B241768 LS 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 (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 values ​​of each culture were measured before incubation (0h). 600 Absorbance 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. Based on OD... 600 The range of values ​​determines the growth status: OD 600 A 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.

[0080] The results are shown in Table 9. The carbohydrate metabolism experiment results show that the Bifidobacterium longum subsp. longum FMBL B241768LS strain can effectively utilize 13 carbon sources, including lactose, galactose, fructose, sucrose, fructooligosaccharides, galactooligosaccharides, inulin, maltose, mannose, D-(+)-trehalose, mannitol, L-sorbitol, and arabinose; but it cannot effectively utilize cellobiose and resistant starch.

[0081] Table 9. Ability of Bifidobacterium longum subsp. FMBL B241768 LS to utilize carbohydrates

[0082]

[0083] 2. Antibiotic resistance test

[0084] The disk diffusion method (KB method) was used, and the information on the drug sensitivity test discs is shown in Table 11. *Bifidobacterium longum* subsp. *longum* FMBLB241768 LS was added to MRS liquid medium at an inoculum size 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 hours. 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).

[0085] Table 10. Antimicrobial susceptibility testing of Bifidobacterium longum subsp. FMBL B241768 LS

[0086]

[0087]

[0088] Note: R: drug resistance; I: moderate sensitivity; S: sensitive

[0089] To ensure the safety and reliability of probiotics for human consumption, antibiotic resistance has become an important indicator for the in vitro safety assessment of probiotic strains. The results are shown in Table 10. *Bifidobacterium longum* subsp. *longum* FMBL B241768 LS was sensitive to minocycline and vancomycin; moderately sensitive to erythromycin; and resistant to ciprofloxacin, ampicillin, penicillin G, clindamycin, kanamycin, and polymyxin B.

[0090] 3. Determination of performance in inhibiting conditionally pathogenic bacteria

[0091] Escherichia coli (10411), Escherichia coli hemorrhagicum (21530), Escherichia coli toxin-producing bacteria (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 longum* subsp. *longum* FMBL B241768 LS. *Bifidobacterium longum* subsp. *longum* FMBL B241768 LS was added to MRS liquid medium at an inoculum size of 2% (v / v) (OD600: 1.0±0.05), and cultured anaerobicly at 37°C 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 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.

[0092] Table 11 Sources of indicator bacteria

[0093]

[0094] Table 12 Determination of the antibacterial activity of Bifidobacterium longum subsp. FMBL B241768 LS

[0095]

[0096]

[0097] Note: The diameter of the inhibition zone includes the outer diameter of the Oxford cup. No inhibition zone: -; 8-15mm: +; 15-20mm: ++; 20-25mm: +++; >25mm: ++++.

[0098] As shown in Table 12, *Bifidobacterium longum* subsp. *FMBL* B241768 LS exhibits good inhibitory activity against diarrhea-causing *Escherichia coli* (10411), enterotoxigenic *Escherichia coli* (10421), hemorrhagic *Escherichia coli* (21530), and *Salmonella typhimurium* (10420), indicating that *Bifidobacterium longum* subsp. *FMBL* B241768 LS has great potential for application in the development of drugs related to inhibiting opportunistic pathogens, preventing or alleviating diarrhea, and improving intestinal health.

[0099] Application Example 1: Preparation of Bifidobacterium longum subsp. longum FMBL B241768 LS bacterial agent

[0100] 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.

[0101] 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.

[0102] Bifidobacterium longum subsp. FMBL B241768 LS was inoculated at a 2% inoculum into the above-mentioned culture medium, which had been sterilized at 115°C 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°C for 24-48 h, the bacterial cells were centrifuged, washed twice with pH 7.2 PBS buffer, and then resuspended in a cryoprotectant to achieve a concentration of 10. 11 CFU / 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. Next, the suspension is pre-cultured at 37°C for 60 min, followed by freeze-drying to obtain the *Bifidobacterium longum* subsp. *longum* FMBL B241768LS bacterial agent.

[0103] Application Example 2: Preparation of fermented milk using *Bifidobacterium longum* subsp. *FMBL* B241768 LS of the present invention.

[0104] 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. *longum* FMBL B241768 LS 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.

[0105] Application Example 3: Preparation of microcapsules and capsule products containing Bifidobacterium longum subsp. FMBL B241768 LS

[0106] The *Bifidobacterium longum* subsp. *FMBL* B241768 LS 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. *longum* FMBL B241768 LS. 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.

[0107] In summary, this invention provides a *Bifidobacterium longum* subsp. *longum* FMBL B241768 LS, whose fermentation broth exhibits high inhibitory activity against dipeptidyl peptidase IV, with an inhibition rate as high as 79.17%; its fermentation broth also shows inhibition rates of 53.32% and 96.65% against α-glucosidase and α-amylase activities, respectively; it possesses the ability to utilize various carbohydrates; it exhibits good inhibitory effects against diarrheal *Escherichia coli*, hemorrhagic *Escherichia coli*, enterotoxigenic *Escherichia coli*, and *Salmonella typhimurium*; and it demonstrates resistance to ciprofloxacin, ampicillin, penicillin G, clindamycin, kanamycin, and polymyxin B. It can be used to prepare hypoglycemic and pathogenic bacteria-inhibiting drugs, fermented foods, health products, and food additives, and has broad application prospects.

Claims

1. A Bifidobacterium longum subsp. longum FMBL B241768LS inhibitor of dipeptidyl peptidase IV, characterized in that, The aforementioned *Bifidobacterium longum* subspecies FMBLB241768LS was deposited at the China Center for Type Culture Collection on October 14, 2024, with accession number CCTCC NO: M 20242173.

2. A microbial agent, characterized in that, The bacterial agent contains the long subspecies of Bifidobacterium longum as described in claim 1, FMBLB241768LS.

3. The use of the *Bifidobacterium longum* subsp. *FMBL* B241768LS 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 dipeptidyl peptidase IV inhibitors.

4. The use of the *Bifidobacterium longum* subsp. *FMBL* B241768LS or its strain fermentation broth or sterile fermentation supernatant as described in claim 1, or the bacterial agent as described in claim 2, in inhibiting α-glucosidase or preparing α-glucosidase inhibitors.

5. The use of the *Bifidobacterium longum* subsp. *FMBL* B241768LS or its strain fermentation broth or 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.

6. The application of the *Bifidobacterium longum* subsp. *FMBL* B241768LS 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 hypoglycemic drugs and health products.

7. The application of the *Bifidobacterium longum* subsp. *FMBL* B241768LS 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 antibacterial products.

8. The use of the *Bifidobacterium longum* subsp. *FMBL* B241768LS 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 fermented foods, pharmaceuticals, health products, or dietary supplements.

9. The application of the *Bifidobacterium longum* subsp. *FMBL* B241768LS 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 drugs, foods, and health products for regulating intestinal function.

Images