Bifidobacterium longum subsp. Longum FMBL B241770 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 B241770LS, a microbial inoculum and application of the microbial inoculum in hypoglycemic products, the bifidobacterium longum subsp. Longum FMBL B241770LS is preserved in China Center for Type Culture Collection on September 14, 2024, and the preservation number is CCTCC NO: M 20241993; the inhibitory activity of the compound on dipeptidyl peptidase IV is up to 84.99%; the compound has a relatively good inhibition effect on the activity of alpha-glucosidase and alpha-amylase; the compound has a relatively strong capability of removing DPPH and ABTS free radicals; the lactobacillus plantarum has good carbohydrate utilization capacity, can be used for preparing antioxidant products, hypoglycemic drugs, fermented food, 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 a long subspecies of Bifidobacterium longum FMBL B241770 LS, its bacterial agent, and its application in hypoglycemic agents. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by hyperglycemia, hyperlipidemia, and insulin resistance. With 537 million adults worldwide affected, it has become one of the most serious threats to human health. In T2DM patients, carbohydrates from food are broken down into glucose and fructose by α-amylase and α-glucosidase after meals, leading to elevated blood glucose levels. Therefore, the main mechanism of existing drugs for treating T2DM is to inhibit the activity of α-amylase and α-glucosidase, including acarbose, voglibose, and miglitol. However, these drugs have numerous side effects, which vary from person to person, affecting treatment efficacy. Furthermore, with the increasing number of diabetes patients and their trend towards younger ages, the development of drugs with fewer side effects that can also treat complications has become a key research focus for those in the field.

[0003] Dipeptidylpeptidase 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. It exists in soluble form in the circulating blood, thus acting on tissues and organs. In humans, the only substrates of DPP-IV are glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1). Inhibiting DPP-IV activity can reduce the degradation rate of GIP and GLP-1, increase glucose-dependent insulin secretion, reduce glucagon release, slow gastric emptying, promote pancreatic β-cell proliferation and differentiation, and enhance satiety, thereby achieving a hypoglycemic effect. Therefore, dipeptidylpeptidase IV inhibitors can be used to treat type 2 diabetes.

[0004] 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 with the host. This promotes nutrient digestion and absorption, 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 *Lactobacillus casei* LCZ disclosed in invention patent CN111575202A can intervene and delay weight gain in mice on a high-fat diet; *Bifidobacterium lactis* B-622 disclosed in invention patent CN114350547A achieves therapeutic effects on diabetic model mice by promoting glucagon-1 secretion, repairing insulin secretion function, regulating lipid metabolism, and reducing chronic low-grade inflammation. Invention patent CN110604749A discloses an animal *Bifidobacterium* A12 with beneficial glucose and lipid metabolism functions, which can be used to develop probiotic products related to lowering blood sugar. 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. Due to the difficulty in culturing it, the development of Bifidobacterium is far from sufficient.

[0005] During their research, the inventors isolated a strain of *Bifidobacterium longum* subsp. *longum* FMBL B241770LS from fecal samples of healthy Tajik children. The fermentation broth exhibited inhibition rates of 84.99%, 68.11%, and 97.80% on dipeptidyl peptidase IV, α-glucosidase, and α-amylase activities, respectively. It also demonstrated good antioxidant capacity and the ability to utilize various carbohydrates; good tolerance to simulated gastrointestinal fluids; self-polymerization; and excellent surface hydrophobic properties. This strain has broad application prospects and can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives, and feed. Summary of the Invention

[0006] The primary objective of this invention is to provide a Bifidobacterium longum subsp. longum FMBL B241770LS that inhibits dipeptidyl peptidase IV. The Bifidobacterium longum subsp. longum FMBL B241770LS was deposited at the China Center for Type Culture Collection on September 14, 2024, with accession number CCTCC NO: M 20241993.

[0007] A second objective of this invention is to provide a bacterial agent containing the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241770LS.

[0008] A third objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241770LS 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* B241770LS 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.

[0010] Preferably, the products include pharmaceuticals, food, health products, cosmetics, etc.

[0011] The fifth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241770LS or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in inhibiting α-glucosidase or preparing α-glucosidase inhibitors.

[0012] The sixth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241770LS 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.

[0013] The seventh objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241770LS or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of hypoglycemic drugs and health products.

[0014] The eighth objective of this invention is to provide the application of the aforementioned *Bifidobacterium longum* subsp. *FMBL* B241770LS or its strain fermentation broth or its sterile fermentation supernatant, or the aforementioned bacterial agent, in the preparation of fermented foods.

[0015] The beneficial effects of this invention are as follows: This invention provides a *Bifidobacterium longum* subsp. *longum* FMBL B241770LS, whose fermentation broth exhibits high inhibitory activity against dipeptidyl peptidase IV, with an inhibition rate as high as 84.99%; its fermentation broth shows inhibition rates of 68.11% and 97.80% against α-glucosidase and α-amylase activities, respectively; its fermentation broth and cell extracts have strong scavenging abilities against DPPH and ABTS free radicals, with ABTS scavenging rates of 82.07% and 27.43%, respectively, and DPPH scavenging rates of 68.11% and 29.73%, respectively; it possesses the ability to utilize various carbohydrates and exhibits good antioxidant capacity; it demonstrates good tolerance to simulated gastrointestinal fluids, self-polymerization, and surface hydrophobic properties; it can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives, and feed, and has broad application prospects. Attached Figure Description

[0016] Figure 1 Colony diagram of Bifidobacterium longum subsp. FMBLB241770LS

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

[0018] Figure 2 Phylogenetic tree of Bifidobacterium longum subspecies FMBLB241770LS Detailed Implementation

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

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

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

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

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

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

[0025] 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.

[0026] Unless otherwise specified, the reagents and consumables used in the following examples can be purchased from the market.

[0027] Unless otherwise specified, the methods used in the following embodiments are conventional methods and can be obtained by referring to the corresponding literature.

[0028] Dipeptidylpeptidase 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. It 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.

[0029] Example 1: Screening for Bifidobacterium longum subsp. FMBLB241770LS with inhibition of dipeptidyl peptidase IV

[0030] 1. Strain isolation and purification

[0031] Fresh fecal samples were collected from healthy Tajik children in Kashgar, 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 bacterial strains were isolated.

[0032] 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.

[0033] Four strains were selected based on colony characteristics and cell morphology and named accordingly. Specific information is shown in Table 1.

[0034] Table 1. Information on the four selected bacterial strains and their hosts.

[0035]

[0036] 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.

[0037] 2. Determination of dipeptidyl peptidase IV inhibitory activity

[0038] Experimental strains: strains FMBL B241769LS, FMBL B241770LS, FMBL B241771LS, and FMBL B241772LS 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.

[0039] 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.

[0040] 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.

[0041] Table 2. DPP-IV Inhibitory Activity Assay System

[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 B241770LS showed the strongest inhibitory effect on dipeptidyl peptidase IV, reaching 84.99%, which was significantly higher than that of the reference strain Lactobacillus rhamnosus LGG and other strains. This also indicates that strain FMBLB241770LS has great application potential in lowering blood sugar and improving diabetes.

[0049] 3. Strain identification

[0050] The strain FMBL B241770LS, 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] Table 5 PCR reaction conditions

[0055]

[0056] Table 6. Primers for PCR amplification of BiLON using subspecies-specific primers.

[0057]

[0058] Table 7 Primer BiLONPCR Reaction Conditions

[0059]

[0060]

[0061] The colony characteristics and microscopic features of the strain FMBL B241770LS are as follows: Figure 1 As shown, the morphological and physiological-biochemical characteristics of the above-mentioned strain FMBLB241770LS are similar to those of the genus Bifidobacterium; the phylogenetic tree is as follows. Figure 2As 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 B241770LS is Bifidobacterium longum subsp. longum, and is named Bifidobacterium longum subsp. longum FMBL B241770LS. It was deposited at the China Center for Type Culture Collection on September 14, 2024, with accession number CCTCC NO: M 20241993. 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 B241770LS is abbreviated as Bifidobacterium longum subsp. longum FMBLB241770LS.

[0064] Example 2: Inhibitory ability of Bifidobacterium longum subsp. 241770LS against α-glucosidase and α-amylase.

[0065] Activated Bifidobacterium longum subsp. FMBL B241770LS at 2% (v / v) (OD 600 An inoculum of 1.0 ± 0.05 g (mg / L) was added to MRS liquid medium. After anaerobic culture at 37°C for 36 h, the culture was centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 0.22 μm aqueous microfiltration membrane to collect the cell-free supernatant. The cells were washed three times with phosphate-buffered saline (PBS) solution at pH 7.4, 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. *L.* FMBL B241770LS exhibits strong inhibitory activity against α-glucosidase and α-amylase, reaching 68.11% and 97.80%, respectively. The cell extract showed an inhibition rate of 63.57% against α-amylase, further demonstrating the significant application potential of *Bifidobacterium longum* subsp. *L.* FMBL B241770LS in blood glucose regulation.

[0075] Table 8 Enzyme activity inhibition ability

[0076]

[0077] Note: "ND" indicates no suppression.

[0078] Example 3: Antioxidant capacity of Bifidobacterium longum subsp. FMBLB241770LS

[0079] Activated Bifidobacterium longum subsp. FMBL B241770LS at 2% (v / v) (OD 600 An inoculum of 1.0 ± 0.05 g (mg / L) was added to MRS liquid medium. After anaerobic culture at 37°C for 36 h, the culture was centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 0.22 μm aqueous microfiltration membrane to collect the cell-free supernatant. The cells were washed three times with phosphate-buffered saline (PBS) solution at pH 7.4. Subsequently, the cells were resuspended in PBS, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 9 Cells were broken up using a cell disruptor at CFU / mL, centrifuged at 8000×g for about 15 min at 4°C, and the supernatant was filtered through a 0.22 μm aqueous microfiltration membrane to obtain cell extracts.

[0080] (1) Determination of ABTS free radical scavenging ability

[0081] 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. ABTS scavenging rate (%) = (1 - (Ac - A) / Ac) × 100%

[0082] In the formula, Ac is the absorbance of the blank control, and A is the absorbance of the sample solution.

[0083] (2) DPPH free radical scavenging capacity determination experiment

[0084] 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.

[0085] DPPH clearance rate (%) = (1 - (D1 - D2) / DO) × 100%

[0086] Where: D0: absorbance of PBS buffer; D1: absorbance of sample solution; D2: absorbance of blank control.

[0087] Table 9 Results of Antioxidant Capacity Measurement

[0088]

[0089] As shown in Table 9, the scavenging rates of ABTS by the fermentation supernatant of *Bifidobacterium longum* FMBL B241770LS and the cell extract were 82.07% and 27.43%, respectively, and the scavenging rates of DPPH were 68.11% and 29.73%, respectively. The results indicate that *Bifidobacterium longum* FMBL B241770LS has a strong scavenging ability against DPPH and ABTS free radicals, and its fermentation supernatant showed better antioxidant performance compared with the cell extract.

[0090] Example 4: Probiotic Characteristics of Bifidobacterium longum subsp. FMBLB241770LS

[0091] 1. Carbohydrate metabolism experiment

[0092] Activated *Bifidobacterium longum* subsp. *longum* FMBL B241770LS 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. The OD of each culture was measured before culturing (0 h). 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.

[0093] The results are shown in Table 10. The carbohydrate metabolism experiment results show that the *Bifidobacterium longum* subsp. *longum* strain FMBLB241770LS can effectively utilize 10 carbon sources, including lactose, galactose, fructose, sucrose, fructooligosaccharides, galactooligosaccharides, inulin, maltose, mannose, and arabinose; but it cannot effectively utilize D-(+)-trehalose, cellobiose, mannitol, L-sorbitol, and resistant starch.

[0094] Table 10. Ability of Bifidobacterium longum subsp. FMBL B241770LS to utilize carbohydrates

[0095]

[0096] 2. Simulated gastrointestinal fluid tolerance test

[0097] Prepare simulated gastric juice (pH 3) with 3 g / L pepsin and simulated intestinal juice (pH 8) with 1 g / L ox bile salts and 1 g / L trypsin. Activate *Bifidobacterium longum* subsp. *longum* FMBL B241770LS strain twice. The OD of the activated bacterial solution... 600Adjust to 1.2. Take 1 mL of the adjusted bacterial culture, centrifuge at 6000 r / min at 4℃ for 10 min, discard the supernatant, resuspend the bacteria in 1 mL of artificial gastric fluid, and incubate in an anaerobic environment at 37℃ for 3 h. Perform plate colony counting on the sample at the beginning (0 h) and the end (3 h) of the incubation. Next, centrifuge the bacterial culture from the gastric fluid for 3 h again for 10 min (under the same conditions), discard the supernatant, and resuspend the bacteria in an equal volume of artificial intestinal fluid. Continue incubation at 37℃ for 2 h, followed by plate colony counting. The survival rate is calculated using the following formula:

[0098]

[0099] In the formula: N1 represents the number of surviving colonies of the strain after treatment with artificial gastrointestinal fluid, and N0 represents the number of surviving bacteria of the original strain before treatment.

[0100] Table 11. Simulated gastrointestinal fluid tolerance of *Bifidobacterium longum* subsp. *L.* FMBL B241770LS

[0101]

[0102] As shown in Table 11, the survival rate of *Bifidobacterium longum* subsp. *FMBLB241770LS* was over 70% after treatment with simulated gastric juice for 3 hours and simulated intestinal juice for 2 hours. This indicates that *Bifidobacterium longum* subsp. *FMBLB241770LS* has good tolerance to simulated gastric and intestinal juices, which helps it to successfully colonize and adhere in the intestine, thereby exerting its probiotic effect in the intestinal mucosa.

[0103] 3. Determination of self-agglomeration and hydrophobicity

[0104] (1) Determination of self-aggregation ability

[0105] Bifidobacterium longum subsp. FMBLB241770LS was added to MRS liquid medium at an inoculum size of 2% (v / v) (OD600: 1.0 ± 0.05) and anaerobically cultured at 37°C for 36 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.

[0106] Self-agglomeration (%) = (1 - A2 / A0) × 100

[0107] (2) Surface hydrophobicity determination

[0108] Bifidobacterium longum subsp. FMBLB241770LS was added to MRS liquid medium at an inoculum size of 2% (v / v) (OD600: 1.0 ± 0.05) and anaerobically cultured at 37°C for 36 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 2 mL of cell suspension and mix thoroughly with an equal volume 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).

[0109] Surface hydrophobicity (%) = (1-A) 1 / A0)×100

[0110] Table 12 Self-aggregation and hydrophobicity of Bifidobacterium longum subsp. FMBL B241770LS

[0111]

[0112] 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 12 shows that the self-aggregation of *Bifidobacterium longum* subsp. *fMBLB241770LS* is 49.15%, and its surface hydrophobicity is 78.46%, indicating that *Bifidobacterium longum* subsp. *fMBLB241770LS* has good self-aggregation and hydrophobicity.

[0113] Application Example 1: Preparation of *Bifidobacterium longum* subsp. *longum* FMBLB241770LS bacterial agent

[0114] 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.

[0115] 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.

[0116] Bifidobacterium longum subsp. FMBL B241770LS 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. 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. The suspension is then pre-cultured at 37°C for 60 min, followed by freeze-drying to obtain the *Bifidobacterium longum* subsp. *longum* bacterial agent FMBLB241770LS.

[0117] Application Example 2: Preparation of Fermented Milk Using Bifidobacterium longum subsp. 241770LS of the Present Invention

[0118] 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 longum* subsp. *longum* FMBLB241770LS 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.

[0119] Application Example 3: Preparation of microcapsules and capsule products containing Bifidobacterium longum subsp. longum FMBLB241770LS

[0120] The *Bifidobacterium longum* subsp. *FMBLB241770LS* 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 10A 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 B241770LS. 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.

[0121] In summary, this invention provides a *Bifidobacterium longum* subsp. *longum* FMBL B241770LS, whose fermentation broth exhibits high inhibitory activity against dipeptidyl peptidase IV, with an inhibition rate as high as 84.99%; its fermentation broth also shows inhibition rates of 68.11% and 97.80% against α-glucosidase and α-amylase activities, respectively; its fermentation broth and cell extracts demonstrate strong scavenging abilities against DPPH and ABTS free radicals, with ABTS scavenging rates of 82.07% and 27.43%, respectively, and DPPH scavenging rates of 68.11% and 29.73%, respectively; it possesses the ability to utilize various carbohydrates, exhibits good tolerance to gastrointestinal fluids, self-polymerization, and surface hydrophobic properties; and can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives, and feed, showing broad application prospects.

Claims

1. A Bifidobacterium longum subsp. longum FMBL B241770LS inhibitor of dipeptidyl peptidase IV, characterized in that, The aforementioned *Bifidobacterium longum* subspecies FMBL B241770LS was deposited at the China Center for Type Culture Collection on September 14, 2024, with accession number CCTCC NO: M 20241993.

2. A microbial agent, characterized in that, The bacterial agent contains the long subspecies of Bifidobacterium longum as described in claim 1, FMBLB241770LS.

3. The application of the *Bifidobacterium longum* subsp. *FMBL* B241770LS 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 application of the *Bifidobacterium longum* subsp. *FMBL* B241770LS 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 antioxidative products.

5. The use of the *Bifidobacterium longum* subsp. *FMBL* B241770LS 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.

6. The use of the *Bifidobacterium longum* subsp. *FMBL* B241770LS 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.

7. The application of the *Bifidobacterium longum* subsp. *FMBL* B241770LS 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 application of the *Bifidobacterium longum* subsp. *FMBL* B241770LS 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.

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