A multifunctional lactobacillus plantarum and application thereof

Abstract

This invention discloses a multifunctional strain of *Lactobacillus plantarum* and its applications, belonging to the field of microbial and fermentation engineering technology. This invention screens and obtains a multifunctional strain of *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum DFR-1 has the activity of producing bile salt hydrolase, polyphenol oxidase and other enzymes; it has inhibitory activity on α-glucosidase, xanthine oxidase and adenosine deaminase and other enzymes; it can lower blood sugar, lipids and uric acid, and can be used for the prevention and treatment of nutritional metabolic diseases, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the fields of microbial and fermentation engineering technology, specifically to a multifunctional plant lactobacillus strain and its applications. Background Technology

[0002] In recent years, with the rapid development of the global economy and the continuous updating of people's consumption concepts, improved living standards, overnutrition, and reduced physical activity have led to an increasing number of nutritional and metabolic diseases, which are particularly serious among the middle-aged and elderly population.

[0003] Nutritional metabolic diseases include diabetes, hyperlipidemia, and hyperuricemia. Diabetes is a metabolic disease characterized by high blood sugar, leading to glycosuria and disturbances in the metabolism of proteins, fats, electrolytes, and water. Hyperlipidemia refers to excessively high blood lipid levels, which can directly cause serious health problems. Uric acid is a trioxypurine; abnormally high blood uric acid levels can cause hyperuricemia, leading to joint swelling and pain, frequent urination, hematuria, hydronephrosis, and even kidney failure, endangering life. In recent years, the incidence of diabetes, hyperlipidemia, and hyperuricemia has been increasing annually. Furthermore, hyperuricemia is closely linked to blood sugar and lipid metabolism, and is a risk factor for atherosclerosis, coronary heart disease, and hypertension. Hyperuricemia, hyperlipidemia, and hyperglycemia are mutually reinforcing, creating a vicious cycle. Therefore, nutritional metabolic diseases in middle-aged and elderly people are receiving increasing attention, making the search for appropriate treatment strategies crucial.

[0004] Lactobacillus plantarum ( Lactiplantibacillus plantarum Lactobacillus is a Gram-positive bacterium belonging to the genus Lactobacillus. This bacterium is widely distributed and used in fruit and vegetable production, feed processing, dairy and meat processing, playing a role in inhibiting the growth of putrefactive bacteria, improving food flavor, alleviating chronic metabolic diseases, and enhancing immunity. For example, patent CN 116478864A discloses an acid- and bile-resistant strain of *Lactobacillus plantarum* ASOSIN, which has excellent hypoglycemic, hypotensive, and lipid-lowering effects. Patent CN 115786187A discloses a strain of *Lactobacillus plantarum* YC that can efficiently degrade uric acid. However, reports of *Lactobacillus plantarum* with therapeutic effects on hyperlipidemia, hyperglycemia, and hyperuricemia are still rare. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a multifunctional plant lactobacillus and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a strain of *Lactobacillus plantarum* (… Lactiplantibacillus plantarumDFR-1, this strain was deposited on June 23, 2025 at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number: CCTCC NO: M 20251450.

[0008] The present invention contains *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The main characteristics of DFR-1 compared to existing reports of Lactobacillus plantarum are:

[0009] (1) It has the ability to withstand acid, bile salts and high uric acid, and can survive normally under complex and harsh conditions.

[0010] (2) It has bile salt hydrolase activity, which can degrade cholesterol; it has α-glucosidase inhibitory activity, which can effectively control the body's blood glucose concentration, and can be used to prepare hypoglycemic and lipid-lowering products.

[0011] (3) It has xanthine oxidase inhibitory activity and can be used to prepare products that lower uric acid.

[0012] (4) It can produce polyphenol oxidase and has in vitro antioxidant activity.

[0013] The present invention contains *Lactobacillus plantarum* ( Lactiplantibacillus plantarum DFR-1 integrates multiple functions and has excellent preventive and therapeutic effects on high uric acid, high blood lipids and high blood sugar.

[0014] A second aspect of the present invention provides a microbial agent containing the aforementioned *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum DFR-1.

[0015] Preferably, the bacterial agent contains *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum DFR-1 exists in one or more of the following forms: cultured live bacteria, bacterial suspension, fermentation broth, fermentation supernatant, and cell lysate.

[0016] Furthermore, the fermentation broth refers to the liquid produced after inoculating the microbial strain into a culture medium and culturing it for a period of time.

[0017] The fermentation supernatant refers to the clear liquid at the top after centrifugation of the fermentation broth, which contains abundant metabolic products from the growth and reproduction of the bacterial strain.

[0018] The bacterial suspension refers to the bacterial precipitate obtained by resuspending the precipitate after centrifugation of the fermentation broth.

[0019] The cell lysis buffer refers to the supernatant obtained by ultrasonically breaking up the bacterial suspension and then centrifuging it.

[0020] In a third aspect, the present invention provides the above-mentioned *Lactobacillus plantarum* (… Lactiplantibacillus plantarumThe application of DFR-1 or the microbial agent in at least one of the following (1)-(4):

[0021] (1) Production of bile salt hydrolase;

[0022] (2) Prepare cholesterol-degrading products;

[0023] (3) Preparation of α-glucosidase inhibitors;

[0024] (4) Prepare drugs for lowering blood sugar and / or lowering blood lipids.

[0025] In a fourth aspect, the present invention provides the above-mentioned *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The application of DFR-1 or microbial agents in at least one of the following (A)-(C):

[0026] (A) Preparation of xanthine oxidase inhibitors;

[0027] (B) Preparation of adenosine deaminase inhibitors;

[0028] (C) Prepare products that degrade uric acid.

[0029] In a fifth aspect, the present invention provides the above-mentioned *Lactobacillus plantarum* (… Lactiplantibacillus plantarum Application of DFR-1 or bacterial agents in the production of polyphenol oxidase.

[0030] In a sixth aspect, the present invention provides a medicament for preventing and treating nutritional metabolic diseases, said medicament comprising the above-mentioned *Lactobacillus plantarum* (… Lactiplantibacillus plantarum DFR-1 or bacterial agent is the active ingredient.

[0031] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0032] Preferably, the pharmaceutically acceptable excipient is selected from one or more of wetting agents, emulsifiers, excipients, suspending agents, thickeners, stabilizers, and sweeteners.

[0033] In some preferred embodiments, the pharmaceutically acceptable excipient is selected from one or more of lactose, mannose, starch, cellulose, magnesium stearate, polyvinylpyrrolidone, and mineral oil.

[0034] More preferably, the dosage form of the drug is a solution, suspension, emulsion, pill, tablet or capsule.

[0035] The beneficial effects of this invention are:

[0036] This invention screened and obtained a multifunctional plant lactobacillus strain ( Lactiplantibacillus plantarumDFR-1 has the activity of producing bile salt hydrolase, polyphenol oxidase and other enzymes; it has inhibitory activity on α-glucosidase, xanthine oxidase and adenosine deaminase and other enzymes; it can lower blood sugar, lipids and uric acid, and can be used for the prevention and treatment of nutritional metabolic diseases, and has broad application prospects. Attached Figure Description

[0037] Figure 1 Results of qualitative tests on bile salt hydrolysates of some strains.

[0038] Figure 2 Blood glucose levels measured in each treatment group during the experiment.

[0039] Figure 3 : Oral glucose tolerance test results for each treatment group.

[0040] Figure 4 Results of TC, TG, HDL, and LDL content determination in each treatment group.

[0041] Figure 5 Liver and kidney indices of mice in each treatment group. Detailed Implementation

[0042] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0043] As mentioned earlier, the incidence of diabetes, hyperlipidemia, and hyperuricemia has been increasing year by year in recent years. Moreover, hyperuricemia is closely linked to blood glucose and lipid metabolism and is a risk factor for atherosclerosis, coronary heart disease, and hypertension. Hyperuricemia, hyperlipidemia, and hyperglycemia are mutually causal, creating a vicious cycle. The occurrence of hyperglycemia, hyperlipidemia, and hyperuricemia as a combination is gradually increasing, thereby increasing the difficulty of treatment.

[0044] Most of the lactic acid bacteria reported so far have only one activity that lowers uric acid or only one activity that lowers blood sugar. Lactic acid bacteria that can have all three activities at the same time are rarely reported.

[0045] In view of this, the present invention obtained a multifunctional *Lactobacillus plantarum* DFR-1 strain from lactic acid bacteria preserved in the company's bacterial strain resource bank through screening for hypoglycemic, lipid-lowering, and uric acid-lowering effects. The *Lactobacillus plantarum* DFR-1 of the present invention can produce multiple active enzymes, combining bile salt hydrolase activity, α-glucosidase inhibitory activity, and xanthine oxidase inhibitory activity, thus exhibiting excellent hypoglycemic, lipid-lowering, and uric acid-lowering activities simultaneously. Furthermore, the *Lactobacillus plantarum* DFR-1 of the present invention also has high polyphenol oxidase activity, which may further enhance its uric acid-lowering effect through the following pathways:

[0046] ① By regulating polyphenol activity, the inhibitory effect on xanthine oxidase is indirectly enhanced;

[0047] ② By improving the gut microbiota, the intestine's ability to degrade and excrete uric acid is enhanced;

[0048] ③ It reduces inflammation and oxidative stress, indirectly improving the uric acid metabolic environment.

[0049] Therefore, compared with existing reports of *Lactobacillus plantarum*, the *DFR-1* strain of this invention integrates multiple functions, simultaneously lowering blood sugar, lipids, and uric acid, and also producing high levels of polyphenol oxidase, resulting in unexpected technical effects. The *DFR-1* strain of this invention can be used to develop drugs for treating complications of hyperglycemia, hyperlipidemia, and hyperuricemia, and has broad application prospects.

[0050] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein:

[0051] The 107 strains of lactic acid bacteria used in the experiment were selected from the gut microbiota of healthy local people in Southwest China and were provided by the strain resource bank of the Science and Technology Innovation Center of Shandong Phoenix Biotechnology Co., Ltd.

[0052] MRS liquid culture medium: 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween-80, 0.5g MgSO4▪7H2O, 0.2g MnSO4▪4H2O, bring to a final volume of 1000mL with distilled water, adjust pH to 6.2±0.2, and sterilize at 121℃ for 15min.

[0053] MRS solid medium: Add 1.5% agar to MRS liquid medium and sterilize at 121℃ for 15 min.

[0054] BSH qualitative MRS medium: Add 0.3% (m / v, i.e., 3g / 1000mL) sodium deoxytaurocholate, 0.2% (m / v) sodium thioacetate, and 0.37g / L CaCl2 to freshly prepared MRS solid medium and dissolve them completely. Sterilize at 121℃ for 15min.

[0055] 20mg / mL uric acid stock solution: Dissolve 0.2g uric acid in 10mL 1M NaOH, and use a 45℃ water bath to aid dissolution.

[0056] MRS-UA medium: Take 250µL of 20mg / mL uric acid stock solution and add it to MRS liquid medium (pH=5) to make the final uric acid concentration 1000µg / mL.

[0057] Standard nutrient agar medium: 10g peptone, 3g beef extract, 5g sodium chloride, 15g agar, 1000mL distilled water, final pH 7.2-7.4.

[0058] Preparation method of citric acid-sodium citrate buffer (pH=4.2-4.5): First, prepare a stock solution, and prepare 0.1 mol / L citric acid solution (solution A) and 0.1 mol / L sodium citrate solution (solution B) separately. Then adjust the pH by mixing solutions A and B in proportion (e.g., 16.5 mL of solution A + 13.5 mL of solution B, which can be finely adjusted according to the pH meter) to stabilize the pH of the final mixture at 4.2-4.5.

[0059] STZ solution: Weigh 90mg of STZ powder (weighing should be done in the dark) and quickly add it to 15mL of citrate-sodium citrate buffer solution. Mix gently until completely dissolved. After preparation, immediately wrap the solution in aluminum foil to protect it from light and place it in an ice bath. Use within 30 minutes.

[0060] Sodium deoxytaurocholate, sodium thioglycolate, and cholesterol were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; citric acid and sodium citrate were purchased from Sinopharm Chemical Reagent Co., Ltd. (China); PNPG, α-glucosidase, and acarbose were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and basic feed and high-fat feed were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0061] Example 1: Screening of lactic acid bacteria with hypoglycemic, lipid-lowering and uric acid-lowering functions

[0062] 1. Experimental Methods

[0063] 1.1 Preparation of bacterial strain test samples

[0064] 1.1.1 Preparation of lactic acid bacteria fermentation broth:

[0065] Activated bacterial slants of 107 lactic acid bacteria strains from the bacterial strain resource bank were prepared. Under aseptic conditions, bacterial cells were picked up with an inoculation loop and inoculated into MRS liquid medium. The culture was incubated at 37°C for 18 hours to obtain primary seed culture. The prepared primary seed culture was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v) under aseptic conditions. The culture was incubated at 37°C for 48 hours to obtain lactic acid bacteria fermentation broth.

[0066] 1.1.2 Preparation of lactic acid bacteria fermentation supernatant and bacterial suspension:

[0067] (1) Preparation of fermentation supernatant: Centrifuge the lactic acid bacteria fermentation broth prepared in 1.1.1 at 4℃ and 8000rpm / min for 10min, collect the supernatant, and obtain the lactic acid bacteria fermentation supernatant.

[0068] (2) Preparation of bacterial suspension: The lactic acid bacteria fermentation broth prepared in 1.1.1 was centrifuged at 4℃ and 8000 rpm / min for 10 min, the bacterial precipitate was collected, resuspended in sterile physiological saline, and the viable count was adjusted to 1.0 × 10⁻⁶. 9 CFU / mL available for use.

[0069] 1.2 Screening of lipid-lowering strains

[0070] 1.2.1 Qualitative test of bile salt hydrolysates

[0071] Pour freshly prepared BSH qualitative MRS medium into a sterile agar plate. After solidification, evenly place sterile filter paper discs into the plate. Add 5 μL of the bacterial suspension prepared in 1.1.2 above (viable count 1.0 × 10⁻⁶) to each filter paper disc. 9 The concentration of CFU / mL was measured, and each strain was repeated three times. For the blank control, 5 μL of sterile phosphate buffer (0.2 M, pH 7.0) was added, and the result was repeated three times. The plates were incubated at 37°C for 72 h. The presence of a white precipitate around the filter paper indicates that the strain produces bile salt hydrolase.

[0072] 1.2.2 Cholesterol Degradation Test

[0073] 107 strains of lactic acid bacteria from the strain resource bank were inoculated into MRS liquid medium and cultured at 37℃ for 18 h to obtain seed culture. Freshly prepared MRS liquid medium was mixed with 0.1% (m / v, i.e., 1 g / 1000 mL) cholesterol, 0.2% (m / v) taurine bile salts, 1 mL Tween-80, and 5 mL anhydrous ethanol, dissolved completely, and brought to a final volume of 100 mL. The medium was then sterilized at 121℃ for 15 min. The experimental groups were inoculated with 2% seed culture, while the control group was inoculated with an equal volume of sterile MRS liquid medium. Each group was repeated three times, and the mixture was cultured at 37℃ for 48 h to obtain the fermentation broth. The obtained fermentation broth was centrifuged at 4℃ and 8000 rpm for 10 min to obtain the fermentation supernatant. According to the instructions for the cholesterol test kit (catalog number: A111-2-1, purchased from Nanjing Jiancheng Biotechnology Institute), 1 mL of working solution was taken and 10 μL of fermentation supernatant (sample) of the above-mentioned strains were added to each sample. The mixture was mixed and incubated at 37°C for 10 minutes. The absorbance of each group was measured at a wavelength of 510 nm, a light path of 0.5 cm, and zeroed with distilled water. The calibrator concentration was 5.17 mmol / L.

[0074] Total cholesterol content = (sample OD value - blank OD value) / (calibration OD value - blank OD value) × calibrator concentration

[0075] In the formula, the sample OD value is the OD value measured by the fermentation supernatant of the above 107 lactic acid bacteria strains; the blank OD value is the OD value measured by the supernatant of sterile MRS liquid culture medium; and the calibration OD value is the OD value measured by the calibrator.

[0076] 1.3 Screening of hypoglycemic strains

[0077] 1.3.1 Determination of the half-maximal inhibitory concentration (WHM) of acarbose in the reaction system

[0078] Using 4-nitrophenol-α-D-glucopyranoside (PNPG) as a substrate, different concentrations of acarbose were selected as inhibitors (samples) to determine the inhibitory activity of different concentrations of acarbose (1 mM, 0.1 mM, 0.01 mM, 0.001 mM, 0.0001 mM, 0.00001 mM) on α-glucosidase in the reaction system.

[0079] The reaction was performed using a 96-well plate. The α-glucosidase activity assay consisted of a 100 μL reaction mixture. First, 25 μL of 20 mmol / L substrate 4-nitrophenol-α-D-glucopyranoside (PNPG) and 25 μL of sample were mixed and incubated at 37°C for 10 min. Then, 50 μL of 0.2 U / mL α-glucosidase was added, mixed, and incubated at 37°C for 30 min. The reaction was then terminated by rapidly adding 100 μL of 0.2 mol / L Na₂CO₃ solution. The absorbance at 405 nm was measured using a microplate reader. Each experiment was performed in triplicate, and the experiment was divided into four groups (sample group, sample blank control group, negative control group, and negative blank control group). The order of sample addition for each group was as shown in Table 1.

[0080] Table 1. Dosage and order of reactant addition (unit: μL)

[0081]

[0082] The formula for calculating the inhibition rate is as follows:

[0083] α-glucosidase inhibition rate = [1 - (AB) / (CD)] × 100%

[0084] In the formula: A is the absorbance of the sample at 405 nm; B is the absorbance of the blank control at 405 nm; C is the absorbance of the negative control at 405 nm; D is the absorbance of the negative blank control at 405 nm.

[0085] 1.3.2 Determination of α-glucosidase inhibition rate

[0086] Following the method described in Example 1.3.1, the inhibition rate of α-glucosidase by the fermentation supernatant of the above 107 lactic acid bacteria strains was determined, and the inhibition rate of α-glucosidase by 0.0001 mM acarbose (positive control of the sample) was also determined. The order of sample addition for each group was as shown in Table 1, with three replicates for each group. The formula for calculating the α-glucosidase inhibition rate was the same as in Example 1.3.1.

[0087] 1.4 Screening of lactic acid bacteria with uric acid-lowering ability

[0088] 1.4.1 Screening of strains tolerant to high uric acid

[0089] The seed cultures of the 107 strains to be screened were inoculated into MRS-UA medium at a 2% inoculum and incubated at 37°C for 18 h. MRS liquid medium without added uric acid served as a control. OD values ​​were measured using a UV spectrophotometer. 600nm .

[0090] 1.4.2 Determination of the inhibition rate of xanthine oxidase

[0091] Prepare centrifuge tubes and add 0.05M Tris-HCl buffer, 10mM xanthine solution, inhibitor (fermentation supernatant of the strain), and 0.125U / mL xanthine oxidase solution in sequence. Before use, incubate the xanthine oxidase solution at 25℃ for 30min. The specific amounts of each substance added are shown in Table 2. Mix well immediately after adding the samples in sequence and react in a 25℃ water bath for 30min. Add 100µL HCl (1M) to terminate the reaction and measure the absorbance at 295nm.

[0092] Table 2. Sample addition details for xanthine oxidase inhibition.

[0093]

[0094] Xanthine oxidase inhibition rate (%) = [(AB)−(CD)] / (AB)×100%.

[0095] In the formula, A is the absorbance value of the positive control group; B is the absorbance value of the negative control group; C is the absorbance value of the inhibitor group; and D is the absorbance value of the inhibitor control group.

[0096] 2. Experimental Results

[0097] 2.1 Screening results of lipid-lowering strains

[0098] 2.1.1 Results of qualitative test for bile salt hydrolytic enzymes

[0099] Strains with bile salt hydrolase activity break down conjugated bile acids, which exist in the body mostly in the form of bound bile acids (glycine or taurine). Bile salt hydrolase (BSH) can break them down into free bile acids. On the other hand, it promotes cholesterol conversion: free bile acids are more easily excreted in feces. To maintain the balance of bile acids in the body, the liver accelerates the conversion of cholesterol into new bile acids, thereby lowering cholesterol levels in the body.

[0100] If there is a distinct and clearly defined milky white precipitate around the strain, it indicates that the strain has good bile salt hydrolase activity. The more white precipitate there is, the stronger the enzyme activity. Figure 1 The results of qualitative tests on bile salt hydrolases from some strains are presented.

[0101] Fourteen strains of fungi were selected, namely QTRY-5, DFR-1, R3-3, R1-1, R1-2, R6-1, R6-2, R8-1, R9-1, R9-2, R9-3, R10-1, R14, and R20-2, which had a lot of white precipitate around the paper disc.

[0102] 2.1.2 Results of cholesterol degradation test

[0103] Table 3 Results of cholesterol degradation rate determination of strains

[0104]

[0105] Table 3 shows that 23 lactic acid bacteria strains with a cholesterol degradation rate of over 50% were screened out of 107 strains. Combined with the results of the bile salt hydrolase qualitative test in section 2.1.1, 5 strains with significant lipid-lowering effects were selected: R3-3, DFR-1, R6-2, R9-3, and R10-1.

[0106] 2.2 Screening results of glucose-lowering strains

[0107] 2.2.1 Half-maximal inhibitory concentration of acarbose against α-glucosidase

[0108] The results are shown in Table 4.

[0109] Table 4. Results of the inhibition rate of α-glucosidase by different concentrations of acarbose.

[0110]

[0111] The results are shown in Table 4. Acarbose was used as a positive control and PBS as a negative control in the α-glucosidase inhibition rate assay. In the selected system, the inhibition rate of α-glucosidase was approximately 50% when the acarbose concentration was 0.0001 mM. The half-maximal inhibitory concentration (WHM) of acarbose against α-glucosidase was determined to be 0.0001 mM, indicating a good model reaction system.

[0112] 2.2.2 Results of the test for determination of α-glucosidase inhibition rate

[0113] Table 5 Results of α-glucosidase inhibition rate assay

[0114]

[0115] As shown in Table 5, 25 lactic acid bacteria strains showed an inhibition rate of over 60% against α-glucosidase. Among them, R8-3, DFR-1, and R2-4 showed better hypoglycemic effects, with an inhibition rate of over 78% against α-glucosidase.

[0116] 2.3 Screening results of uric acid-lowering strains

[0117] 2.3.1 Screening results of strains tolerant to high uric acid

[0118] The OD of the bacterial culture was measured using an ultraviolet spectrophotometer. 600nm The results of the five strains that grew best under high uric acid conditions are shown in Table 6.

[0119] Table 6. Results of the test on the strain's tolerance to uric acid.

[0120]

[0121] 2.3.2 Results of the assay of the strain's inhibitory ability on xanthine oxidase

[0122] The results of the assay for the inhibitory ability of the strain against xanthine oxidase are shown in Table 7.

[0123] Table 7. Results of the assay of the inhibitory ability of the strains against xanthine oxidase.

[0124]

[0125] Based on the screening results for lipid-lowering, blood sugar-lowering, and uric acid-lowering effects, strain DFR-1, which showed the best overall performance, was selected for further research.

[0126] Example 2: Study on the relevant characteristics of strain DFR-1

[0127] 1. Experimental Materials and Methods

[0128] 1.1 Polyphenol oxidase activity assay

[0129] A single colony of strain DFR-1 was picked using an inoculation loop and streaked onto a fresh MRS solid slant medium to activate the strain. The activated strain was then inoculated into MRS liquid medium and incubated at 37°C for 48 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was collected for later use. The supernatant was filtered through a 0.22 µm filter membrane to obtain the fermentation supernatant for the experimental strain.

[0130] The polyphenol oxidase activity of the fermentation supernatant of the test strain was determined according to the instructions of the commercial test kit (item number: A136-1-1, Nanjing Jiancheng Biotechnology Research Institute).

[0131] 1.2 Determination of the in vitro antioxidant capacity of the strain

[0132] Commercially available reagent kits (catalog number: A015-1-2, Nanjing Jiancheng Biotechnology Institute) were used to detect the total antioxidant capacity, superoxide anion resistance, total superoxide dismutase activity (T-SOD), DPPH free radical scavenging capacity, and hydroxyl free radical inhibition capacity in the fermentation supernatant of the strain.

[0133] 1.3 Determination of acid resistance of strains

[0134] Activated DFR-1 was inoculated at a 5% inoculum into MRS liquid medium at pH 2.4 and incubated at 37°C for 0.5 h, 1 h, and 2 h. Samples were taken for plate colony counting, and the survival rate was calculated. Survival rate = (number of viable bacteria in the culture at different times at pH 2.4 / initial number of viable bacteria) × 100%. Its tolerance to different incubation times at pH 2.4 was investigated.

[0135] 1.4 Bile salt tolerance test of the strain

[0136] A 10% bile salt stock solution was prepared, sterilized, and then added to sterilized MRS medium at the specified ratio before inoculation to achieve a final concentration of 0.3%. Strain DFR-1 was inoculated at a 5% inoculum into MRS liquid medium containing 0.3% bile salt and incubated at 37°C for 0.5 h, 1 h, and 2 h. Samples were taken for plate colony counting, and the survival rate was calculated. Survival rate = (number of viable bacteria in the culture at different times with 0.3% bile salt / initial number of viable bacteria) × 100%. The tolerance of DFR-1 to different incubation times at a 0.3% bile salt concentration was investigated.

[0137] 1.5 Determination of the antibacterial activity of the strain

[0138] The antibacterial activity of *Lactobacillus plantarum* DFR-1 against indicator bacteria was determined using the perforation method. Cultures of *Escherichia coli*, *Staphylococcus aureus*, *Salmonella*, and *Candida albicans* were diluted to 10⁻⁶. 6CFU / mL. First, add 10 mL of ordinary nutrient agar medium (containing 1.5% agar) to the petri dish. After it solidifies, place the Oxford cup evenly on the surface of the medium. Take 3 mL of the above indicator bacterial suspension and add it to 100 mL of semi-solid ordinary nutrient agar medium (containing 0.75% agar), mix thoroughly, and pour 15 mL into the petri dish containing the Oxford cup. After solidification, carefully remove the Oxford cup. Then, add 150 µL of LDFR-1 fermentation supernatant to each well and incubate overnight at 37°C. Finally, observe and measure the diameter of the inhibition zone.

[0139] 2. Experimental Results

[0140] 2.1 Results of Polyphenol Oxidase Activity Assay

[0141] The polyphenol oxidase activity of the supernatant from the fermentation broth of *Lactobacillus plantarum* DFR-1 was determined to be 23.52 ± 2.18 U / mL.

[0142] 2.2 Results of in vitro antioxidant capacity assay of the strain

[0143] The results of the in vitro antioxidant capacity determination of the strain are shown in Table 8.

[0144] Table 8 Results of in vitro antioxidant capacity determination of the strain

[0145]

[0146] 2.3 Results of acid resistance test of the strain

[0147] The results of the acid resistance test of the strain are shown in Table 9.

[0148] Table 9 Results of acid resistance test for strains

[0149]

[0150] 2.4 Results of the determination of the strain's tolerance to bile salts

[0151] The results of the bile salt tolerance test of the strain are shown in Table 10.

[0152] Table 10 Results of bile salt tolerance test for strains

[0153]

[0154] 2.5 Results of antibacterial activity assay of strain 2.5

[0155] The results of the antibacterial activity study of the strain are shown in Table 11.

[0156] Table 11 Antibacterial activity against indicator bacteria (inhibition zone diameter in mm)

[0157]

[0158] The above results indicate that strain DFR-1 not only has the functions of lowering blood sugar, lowering blood lipids and lowering uric acid, but also produces polyphenol oxidase. It also has acid resistance, bile salt resistance, in vitro antioxidant capacity and antibacterial properties, making it a lactic acid bacterium with multiple functions.

[0159] Example 3: Identification and Preservation of Strains DFR-1

[0160] Strains of strain DFR-1 were picked and inoculated into MRS liquid medium and cultured for 12 h. The cultured bacterial solution was then inoculated into MRS liquid medium at a rate of 2% and cultured at 37°C for 18 h. The culture was collected by centrifugation at 8000 rpm for 5 min at 4°C. Genomic DNA of the target strain was extracted using a bacterial genomic DNA kit. This extracted genomic DNA was used as a template for PCR amplification, employing universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') for 16S rDNA PCR. After PCR amplification, the PCR products were analyzed by agarose gel electrophoresis and photographed. The PCR products were sent to PlatinumBio Biotechnology (Shanghai) Co., Ltd. for sequencing, and the sequencing results were compared with BLAST sequences on the NCBI website.

[0161] Electrophoresis of the 16S rDNA PCR product of strain DFR-1 showed a highly specific band at approximately 1500 bp, consistent with expectations. Sequencing was performed, and the sequence is shown in SEQ ID NO.1. The sequence was compared with the 16S rDNA gene sequences of some strains already registered on the website http: / / www.ncbi.nlm.nih.gov, confirming that strain DFR-1 belongs to *Lactobacillus plantarum* (Lactobacillus). Lactiplantibacillus plantarum ) .

[0162] In summary, strain DFR-1 is deposited at the China Center for Type Culture Collection, and the deposit information is as follows:

[0163] Reference biological material (strain): strain DFR-1;

[0164] Suggested classification and nomenclature: Lactobacillus plantarum ( Lactiplantibacillus plantarum );

[0165] Accession number: CCTCC NO: M 20251450;

[0166] Preservation period: June 23, 2025.

[0167] Example 4: The ameliorative effect of Lactobacillus plantarum DFR-1 on a mouse model of hyperglycemia and hyperlipidemia.

[0168] 1. Experimental Methods

[0169] One hundred 4-5 week old SPF-grade Kunming mice (19±2g) were selected and housed under specific pathogen-free conditions. After 7 days of acclimatization feeding, they were randomly divided into 5 groups of 20 mice each: control group (CK group), high-fat model group (HFD group), high-fat and high-glucose model group (M group), acarbose group (positive drug group), and DFR-1 group. The control group was fed a basal diet, while the other groups were fed a high-fat diet. The experimental groupings and treatments are shown in Table 12. After grouping, the DFR-1 group mice were orally administered a suspension of *Lactobacillus plantarum* DFR-1 via gavage at a volume of 1×10⁻⁶. 9 CFU / 0.2mL / mouse / day; Acarbose group (positive control group) was administered 50 mg / kg·BW acarbose solution by gavage; The model group was established by high-fat diet combined with low-dose streptozotocin STZ (streptozotocin, purchased from Shanghai Yuanye Biotechnology Co., Ltd.). Mice were fed a high-fat diet for 3 weeks. In the 4th week, after fasting for 18 hours, the modeling mice were injected with STZ (40 mg / kg·BW) once daily for 5 consecutive days via intraperitoneal injection. STZ was prepared fresh each time. The control group received intraperitoneal injections of citrate-sodium citrate buffer for 5 consecutive days. Specific grouping and treatment methods are shown in Table 12. All mice had free access to water and food during the experiment.

[0170] Table 12 Experimental Groups and Treatments

[0171]

[0172] 2. Indicator Measurement

[0173] Blood glucose levels in each group of mice were tested weekly. The blood glucose meters and test strips used for the tests were purchased from Sinocare Biosensor Co., Ltd.

[0174] One day before the end of the experiment, an oral glucose tolerance test (OGTT) was performed on mice. Mice were given 2 g / kg body weight of glucose orally after fasting for 8 hours. Blood glucose levels were measured at 0 h, 0.5 h, 1 h, and 2 h to observe changes in blood glucose and assess the mice's ability to adapt to glucose. In normal mice, glucose is rapidly absorbed into the bloodstream from the gastrointestinal tract after oral administration, reaching a peak blood glucose level within 30-60 minutes, but generally not exceeding 10 mmol / L. The area under the curve (AUC) was calculated using GraphPad Prism software. A statistically significant decrease in the AUC indicated improved glucose tolerance.

[0175] After the experimental period, blood was collected from the orbital cavity of anesthetized mice. The blood was placed at 37°C for half an hour, then transferred to a 4°C refrigerator for 1-2 hours, centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected and frozen at -20°C for later use to measure blood lipid levels. Triglyceride, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol assay kits (purchased from Nanjing Jiancheng Biotechnology Institute) were used to measure blood lipid levels (TG, TC, HDL, and LDL).

[0176] 3. Experimental Results

[0177] The changes in blood glucose levels in each group of mice during the experiment are as follows: Figure 2 As shown, during weeks 5 to 11 of the experiment, the fasting blood glucose level in group M was significantly higher than that in other groups. P <0.01). At the end of week 11 of the experiment, compared with group M, both the positive drug group and the DFR-1 group significantly reduced fasting blood glucose levels in mice ( P <0.01), compared with group M, the fasting blood glucose in the DFR-1 group decreased significantly by 28.25%, and there was no significant difference compared with the positive drug group. P >0.05). These results indicate that *Lactobacillus plantarum* DFR-1 can alleviate hyperglycemia in diabetic mice.

[0178] An oral glucose tolerance test was performed at the end of week 11 of the trial, and the results were as follows: Figure 3 As shown, in mice injected with STZ, blood glucose levels rose rapidly within 0.5 hours after oral administration of glucose solution, and then slowly decreased over the next 1.5 hours. At all time points, blood glucose levels in the M group were significantly higher than those in the CK group. P <0.01). Based on the oral glucose tolerance curve, the area under the glucose curve was calculated and a bar chart was plotted. A larger value indicates more severe impairment of glucose tolerance. The results showed that compared to group M, the *Lactobacillus plantarum* DFR-1 group showed a significant decrease of 25.05% (…). P <0.01), indicating no significant difference compared to the positive drug group. P >0.05). These results indicate that DFR-1 can effectively alleviate impaired glucose tolerance symptoms in type 2 diabetic mice.

[0179] The results of TC, TG, HDL, and LDL levels in each group of mice are as follows: Figure 4 As shown, the results indicated that a high-fat diet led to higher levels of TC, TG, HDL, and LDL in mice in the HFD group, M group, DFR-1 group, and positive drug group compared to the CK group. Compared to the M group, the DFR-1 group showed a significant decrease in TG of 11.21% (…). P<0.05), TC decreased by 6.8%, and HDL decreased significantly by 22.15% ( P <0.05), and LDL decreased by 18.34%. This indicates that prophylactic feeding of Lactobacillus plantarum DFR-1 can improve TC, TG, HDL and LDL levels to some extent.

[0180] In summary, *Lactobacillus plantarum* DFR-1 can reduce fasting blood glucose levels in mice and alleviate symptoms of impaired glucose tolerance; it can also significantly reduce TG and HDL levels, indicating that prophylactic feeding of *Lactobacillus plantarum* DFR-1 can, to some extent, reduce blood glucose and improve TC, TG, HDL and LDL levels.

[0181] Example 5: The ameliorative effect of Lactobacillus plantarum DFR-1 on a mouse model of hyperuricemia

[0182] 1. Experimental Grouping and Design

[0183] Eighty-four 4-5 week old SPF-grade Kunming mice (19±2g) were selected and acclimatized for one week before being randomly divided into four groups: blank group, model group, positive group, and sample group. Each group had three replicates, with seven mice in each replicate. The experiment began on the day of grouping and lasted for 28 days.

[0184] From day 1 to day 14 of the experiment, the modeling phase was conducted. Except for the control group, mice in all other groups were administered 250 mg / kg potassium oxonate by gavage daily, while the control group was administered an equal volume of physiological saline by gavage daily. From day 15 to day 28 of the experiment, the intervention phase was conducted. While maintaining the model, the positive control group and the sample group underwent intervention treatment. Mice in the model group were administered 250 mg / kg potassium oxonate by gavage daily, followed by an equal volume of physiological saline 1 hour later. Mice in the positive control group were administered 250 mg / kg potassium oxonate by gavage daily, followed by febuxostat 1 hour later. Mice in the sample group were administered 250 mg / kg potassium oxonate by gavage daily, followed by an equal volume of DFR-1 bacterial suspension 1 hour later. The control group was administered an equal volume of physiological saline by gavage daily. During the experiment, all mice were fed a standard laboratory diet with free access to food and water. The treatment methods for different groups are shown in Table 13.

[0185] Table 13 Intervention Treatments for Different Groups

[0186]

[0187] 2. Indicator Detection

[0188] After the experiment, mice were fasted overnight and treated the following day. After recording mouse weight, blood was collected from the eyes into 1.5 mL centrifuge tubes, incubated at 37°C for 30 min, and then centrifuged at 3500 rpm for 10 min to prepare serum. Serum uric acid (UA), adenosine deaminase (ADA), xanthine oxidase (XOD), blood urea nitrogen (BUN), and creatinine (Cr) were measured using an enzyme-linked immunosorbent assay (ELISA) reader. After necropsy, the liver and kidneys were harvested and their weights recorded. Liver and kidney indices were calculated. Liver index = liver weight (g) / mouse body weight (g) × 100%. Kidney index = kidney weight (g) / mouse body weight (g) × 100%.

[0189] 3. Experimental Results

[0190] 3.1 Effects on the levels of adenosine deaminase (ADA) and xanthine oxidase (XOD)

[0191] The results of serum adenosine deaminase and xanthine oxidase levels in mice under different treatment groups are shown in Table 14.

[0192] Table 14 Serum adenosine deaminase and xanthine oxidase levels in mice from different groups

[0193]

[0194] Note: Different lowercase letters in the same column's subscript indicate significant differences. P <0.05), different capital letters in the same column's shoulder indices indicate extremely significant differences ( P <0.01).

[0195] As shown in Table 14, compared with the model group, the serum adenosine deaminase level in mice was significantly reduced by 12.47% after intervention with *Lactobacillus plantarum* DFR-1. P <0.05). Adenosine deaminase is involved in purine metabolism and is an enzyme related to the production of uric acid; its level is elevated in hyperuricemia. Therefore, inhibiting its activity and expression can effectively reduce uric acid production.

[0196] Compared with the model group, the level of xanthine oxidase in mouse serum was significantly reduced by 10.19% after intervention with *Lactobacillus plantarum* DFR-1. P The concentration of xanthine oxidase in the sample group was <0.01, indicating that *Lactobacillus plantarum* DFR-1 could inhibit xanthine oxidase activity. Compared with the positive group, the xanthine oxidase activity in the sample group was 1.63% higher, but the difference was not statistically significant. Xanthine oxidase is a key enzyme in purine metabolism to uric acid. Under the action of xanthine oxidase, hypoxanthine is oxidized to xanthine, and xanthine is further oxidized to uric acid. Therefore, the reduction of xanthine oxidase activity can reduce uric acid production, thereby reducing serum uric acid levels.

[0197] 3.2 Effect on uric acid (UA) content

[0198] The results of the determination of uric acid content in the serum of mice in different treatment groups are shown in Table 15.

[0199] Table 15. Serum uric acid (UA) content in mice from different groups

[0200]

[0201] Note: Different capital letters in the same column's top label indicate extremely significant differences. P <0.01).

[0202] As shown in Table 15, compared with the model group, the serum uric acid level of mice in the sample group was significantly reduced by 40.13% after intervention with Lactobacillus plantarum DFR-1 (p<0.01), which was consistent with the uric acid level in the positive group.

[0203] 3.3 Effects on blood urea nitrogen (BUN) content, creatinine (Cr) content, kidney index, and liver index

[0204] The results of serum urea nitrogen and creatinine levels in mice under different treatment groups are shown in Table 16; the results of liver and kidney index measurements are shown in Table 16. Figure 5 As shown.

[0205] Table 16. Blood urea nitrogen and creatinine levels in the serum of mice from different groups.

[0206]

[0207] Note: Different lowercase letters in the same column's subscript indicate significant differences. P <0.05), the same or no letter subscript indicates no significant difference ( P >0.05).

[0208] As shown in Table 16, compared with the model group, the serum urea nitrogen content of mice in the sample group was significantly reduced by 6.78% ( P <0.05), creatinine levels decreased significantly by 14.90%. Therefore, Lactobacillus plantarum DFR-1 helps alleviate kidney damage caused by hyperuricemia.

[0209] Depend on Figure 5 It can be seen that there were no significant differences in liver and kidney indices among the groups. Compared with the model group, both liver and kidney indices were reduced in the sample group, indicating that *Lactobacillus plantarum* DFR-1 helps improve liver and kidney tissue abnormalities induced by hyperuricemia.

[0210] In summary, in the potassium oxonate-induced hyperuricemia mouse model, *Lactobacillus plantarum* DFR-1 can inhibit the activity of adenosine deaminase (ADA) and xanthine oxidase (XOD), reduce uric acid levels, and alleviate kidney damage caused by hyperuricemia to a certain extent.

[0211] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A strain of *Lactobacillus plantarum* ( Lactiplantibacillus plantarum DFR-1, with accession number CCTCCNO: M 20251450.

2. A microbial agent, characterized in that, The microbial agent contains *Lactobacillus plantarum* as described in claim 1. Lactiplantibacillus plantarum DFR-1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent contains *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum DFR-1 exists in the form of cultured live bacteria or bacterial suspensions.

4. The *Lactobacillus plantarum* as described in claim 1 ( Lactiplantibacillus plantarum The use of DFR-1 or the bacterial agent according to claim 2 or 3 in the production of bile salt hydrolase.

5. The *Lactobacillus plantarum* as described in claim 1 ( Lactiplantibacillus plantarum The use of DFR-1 or the bacterial agent according to claim 2 or 3 in the preparation of a medicament for treating hyperglycemia, hyperlipidemia or hyperuricemia.

6. The *Lactobacillus plantarum* as described in claim 1 ( Lactiplantibacillus plantarum The use of DFR-1 or the microbial agent according to claim 2 or 3 in the production of polyphenol oxidase.

7. A drug for preventing and treating nutritional metabolic diseases, characterized in that, The drug is derived from the *Lactobacillus plantarum* as described in claim 1. Lactiplantibacillus plantarum DFR-1 or the bacterial agent according to claim 2 or 3 is the active ingredient; The nutritional metabolic diseases mentioned are hyperglycemia, hyperlipidemia, or hyperuricemia.

8. The medicament according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The medicament according to claim 8, characterized in that, The pharmaceutically acceptable excipients are selected from one or more of the following: wetting agents, emulsifiers, excipients, suspending agents, thickeners, stabilizers, and sweeteners.

10. The medicament according to any one of claims 7-9, characterized in that, The dosage form of the drug is a solution, suspension, emulsion, pill, tablet or capsule.

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