Lactobacillus plantarum WZ-9, and application and product thereof

Abstract

The application discloses a plant lactobacillus WZ-9, application and product thereof, and belongs to the technical field of microorganisms. Lactiplantibacillus plantarum The plant lactobacillus WZ-9 is preserved in China General Microbiological Culture Collection Center on April 23, 2025, and the preservation number is CGMCC NO.34318.

Description

[0001] Priority Claim: This invention claims priority to Chinese Patent Application No. 2025111995878, filed on August 26, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of microbial technology, specifically relating to a plant lactobacillus WZ-9 and its applications and products. Background Technology

[0003] For understanding the technical content of this invention: Lactobacillus plantarum ( Lactiplantibacillus plantarum *Lactobacillus plantarum* is a Gram-positive bacterium with rod-shaped cells (0.9-1.2 μm × 3.0-8.0 μm), occurring singly, in pairs, or in chains. It is a facultative anaerobic bacterium with an optimal growth temperature of 30-37℃ and an optimal pH of around 6.5. It commonly ferments α-methyl-D-glucosinolates and menotriose; some strains ferment α-methyl-D-mannosinolates; others ferment arabinose and xylose. It typically does not reduce nitrates. *Lactobacillus plantarum* is a recognized probiotic, possessing strong acid and bile salt tolerance, as well as beneficial properties such as enhancing immunity, improving intestinal flora structure and function, maintaining intestinal flora balance, and lowering cholesterol. [1] .

[0004] Compared to other lactobacilli, *Lactobacillus plantarum* exhibits excellent resistance to acid, bile salts, and osmotic pressure, ensuring its survival in the intestines. [2] Its excellent cell adhesion and biofilm formation capabilities make it easy to colonize the human gut. After colonizing the intestines, *Lactobacillus plantarum* produces antibacterial substances such as bacteriocins, organic acids, and diacetyl, thereby inhibiting the growth of harmful bacteria, maintaining intestinal flora balance, and improving intestinal diseases such as constipation and diarrhea. *Lactobacillus plantarum* can also effectively regulate blood sugar, blood lipids, and blood pressure balance, and has a role in improving metabolic disorders. [3,4] It can also inhibit the effects of glucose metabolism-related enzymes (α-glucosidase or α-amylase), thereby alleviating pathological indicators associated with diabetes. Studies using a hyperlipidemic rat model have found that *Lactobacillus plantarum* NCU116 can significantly increase serum high-density lipoprotein cholesterol (HDL-C) levels, decrease serum total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels, and improve dyslipidemia. [3] Studies by Huang et al. have found that supplementing with *Lactobacillus plantarum* K68 can significantly reduce the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in rats, helping to alleviate hyperglycemia, hyperinsulinemia, and hyperlipidemia in rats. [5]Studies by Lin et al. have shown that long-term oral administration of fermented milk containing *Lactobacillus plantarum* SR37-3 and SR61-2 can significantly reduce blood pressure in hypertensive model rats and alleviate kidney damage. [6] Chen et al.'s research found that *Lactobacillus plantarum* P-8 can significantly enhance the hypoglycemic effect of metformin and can be used as an adjunct treatment for type 2 diabetes. [7] A series of metabolic diseases characterized by hyperglycemia, especially type 2 diabetes, are known to be characterized by insulin resistance and decreased insulin secretion. Ingested carbohydrates are broken down into monosaccharides such as glucose by various carbohydrate-degrading enzymes, such as α-amylase, glucosidase, sucrase, and α-glucosidase, and these monosaccharides are absorbed into the bloodstream, raising blood glucose levels. The two main digestive enzymes used in carbohydrate digestion are α-amylase and α-glucosidase. Some active components in the intestine can inhibit the activity of α-amylase and α-glucosidase, thus slowing down the breakdown of polysaccharides into monosaccharides in the intestine and helping carbohydrates to be absorbed more slowly into the body, thereby lowering blood glucose levels.

[0005] References: [1] Xiao Zijun, Zhong Ruimin, Chen Huiyin, et al. Physiological functions and applications of Lactobacillus plantarum [J]. China Food Additives, 2005(2):87-89. [2] Haghshenas B, Nami Y, Haghshenas M, et al. Bioactivity characterization of Lactiplantibacillus strains isolated from dairy products[J]. MicrobiologyOpen, 2015, 4(5): 803-813. [3] LI C, NIE SP, ZHU KX, et al. Lactiplantibacillus plantarum NCU116 improves liver function, oxidative stress and lipid metabolism in rats with high fat diet induced non-alcoholic fatty liver disease[J]. Food&Function, 2014, 5(12):3216-3223. [4] LI X, WANG N, YIN B, et al. Effects of Lactiplantibacillus plantarumCCFM0236 on hyperglycaemia and insulin resistance in high-fat and streptozotocin-induced type 2 diabetic mice[J]. Journal of Applied Microbiology. 2016, 121(6):1727-1736. [5] HUANG H Y, KORIVI M, TSAI C H, et al. Supplementation of Lactiplantibacillus plantarum K68 and fruit-vegetable ferment along with high fat-fructose diet attenuates metabolic syndrome in rats with insulin resistance[J]. Evidence-Based Complementary and Alternative Medicine, 2013(1):943020. [6] Yuan L, Li Y, Chen M, et al. Antihypertensive activity of milk fermented by Lactiplantibacillus plantarum SR37-3 and SR61-2 in L-NAME-induced hypertensive rats[J]. Foods, 2022, 11(15): 2332. [7] Chen Y, et al. Effect of combination treatment with metformin and probiotics Probio-Fit® on clinical symptoms and gut microbiota structure in type 2 diabetes[J]. Journal of Chinese Institute of Food Science and Technology, 2019, 19(04):16-26. Related patent documents retrieved: The document, published in China (CN116478888A) on July 25, 2023, discloses that the fermentation supernatant of *Lactobacillus plantarum* LY4 exhibits a 96.54% inhibition rate against α-glucosidase. The amylase inhibition rate was 100%.

[0006] Relevant non-patent literature retrieved: The journal title is "Food Industry Technology", the article title is "Screening and Probiotic Characteristics Study of Lactic Acid Bacteria that Inhibit α-Amylase and α-Glucosidase Activity in Camel Milk Products", Volume 43 No. 19, Publication Date: October 2022. This article discloses four strains of *Lactobacillus plantarum*, with the highest α-amylase inhibition rate of 83%, the highest α-glucosidase inhibition rate of 15%, the hydroxyl radical scavenging rate of all strains exceeding 90%, and the superoxide anion scavenging rate of all strains exceeding 93%.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: There is a lack of probiotics with strong free radical scavenging ability and high inhibitory activity against α-amylase and α-glucosidase. Evidence for this includes: The study, "Screening and Probiotic Characteristics of Lactic Acid Bacteria that Inhibit α-Amylase and α-Glucosidase Activity in Camel Milk Products," disclosed four strains of *Lactobacillus plantarum*. The highest α-amylase inhibition rate was 83%, and the highest α-glucosidase inhibition rate was 15%. All strains exhibited hydroxyl radical scavenging rates exceeding 90% and superoxide anion scavenging rates exceeding 93%. Although the free radical scavenging ability was strong, the α-glucosidase inhibition rate was particularly low.

[0008] The fermentation supernatant of *Lactobacillus plantarum* LY4 (GDMCC NO:63373) showed a 96.54% inhibition rate against α-glucosidase. The amylase inhibition rate was 100%. Although it showed strong inhibitory activity against α-amylase and α-glucosidase, its free radical scavenging ability was not disclosed. Summary of the Invention

[0009] The purpose of this invention is to provide: A plant lactobacillus ( Lactiplantibacillus plantarum WZ-9, which was deposited at the China Microbial Culture Collection Center on April 23, 2025, with accession number CGMCC NO.34318, and related technologies, are intended to solve the technical problem of lacking a probiotic with strong free radical scavenging ability and high inhibitory activity against both α-amylase and α-glucosidase, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms, the definition provided in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definition of standard chemical terms can be found in the reference "Microbiology Experiments, Higher Education Press, 4th Edition, 2016".

[0013] Unless otherwise specified, conventional methods within the scope of the art, such as plate coating, spectrophotometry (turbidimetry), amylase inhibitory activity assay, glucosidase inhibitory activity assay, etc., shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The term "colony" as used in this article refers to a visible, structurally defined group of daughter cells formed by the growth and reproduction of a single microbial cell on or inside a suitable solid culture medium.

[0016] The term “CFU” used in this article refers to colony-forming unit.

[0017] The term "pure culture" as used in this article refers to a cell population consisting of a single microorganism, typically formed by the growth and reproduction of a single cell.

[0018] The term "culture medium" used in this article refers to the nutrient substrate for the growth and reproduction of microorganisms. Based on the different contents of the solidifying agent, it can be divided into three types: solid culture medium, semi-solid culture medium (semi-liquid culture medium), and liquid culture medium.

[0019] To achieve the objective, the present invention provides the following technical solution: In a first aspect, the present invention provides: *Lactobacillus plantarum* (… Lactiplantibacillus plantarumWZ-9 was deposited at the China Microbial Culture Collection Center on April 23, 2025, with accession number CGMCC NO.34318.

[0020] Secondly, the present invention provides: a bacterial agent or product containing Lactobacillus plantarum WZ-9.

[0021] These include technical features such as: microbial compositions, compound microbial agents, preparations, cultures, post-biotics, secondary metabolites, fermented foods, health products, and pharmaceuticals.

[0022] The technical features are selected from: fermentation broth, fermentation broth precipitate, and fermentation broth supernatant.

[0023] The preferred preparation of the technical feature is: fermentation broth.

[0024] The preferred preparation of the technical feature is: live bacteria fermentation broth.

[0025] Thirdly, the present invention provides a technology using Lactobacillus plantarum WZ-9.

[0026] These include technical features such as: microbial culture methods, post-biotic preparation methods, product preparation methods, secondary metabolite production methods, secondary metabolite purification methods, and methods corresponding to specific applications.

[0027] The cultivation method for technical characteristics is selected from: cultivation using liquid or solid culture media.

[0028] The preferred method for cultivating the technical characteristics is to use bacterial culture media such as MSR medium or LB medium for cultivation.

[0029] The preparation methods for the technically characteristic products are selected from preparation methods for products such as food, health products, pharmaceuticals, and fertilizers.

[0030] The preferred method for preparing the technically characteristic product is a food preparation method.

[0031] The preferred method for preparing the product with technical features is a method for preparing oral liquid or powder.

[0032] Fourthly, this invention provides the application of Lactobacillus plantarum WZ-9.

[0033] This includes technical features: applications in specific directions.

[0034] Among them, the applications of specific technical features are selected from: applications in food preparation, applications in pharmaceutical preparation, and applications in health product preparation.

[0035] The preferred application of the technical features in food preparation is the preparation of food additives.

[0036] The preferred application of the technical features in drug preparation is the preparation of drugs for lowering blood sugar, lowering blood lipids, treating stomach diseases, fighting cancer, and fighting inflammation.

[0037] The preferred application of the technical features in drug preparation is the preparation of hypoglycemic drugs.

[0038] The present invention has at least the following beneficial effects: Compared with the prior art, the *Lactobacillus plantarum* WZ-9 of the present invention has better technical effects in terms of the inhibition rate of α-glucosidase and α-amylase.

[0039] According to experimental tests, the *Lactobacillus plantarum* WZ-9 of this invention exhibits a hydroxyl radical scavenging rate of 85.65%, a DPPH radical scavenging rate of 80.34%, an ABTS radical scavenging rate of 98.21%, and a superoxide anion scavenging rate of 85.47%, demonstrating strong antioxidant activity.

[0040] With similar free radical scavenging capabilities, according to experimental tests, the *Lactobacillus plantarum* WZ-9 of the present invention increases the α-amylase inhibition rate from 83% in the prior art to 99.17%, and from 15% in the prior art to 98.09%.

[0041] Preservation Instructions Preserved strain: Lactobacillus plantarum WZ-9; Classification and nomenclature: Lactobacillus plantarum Lactiplantibacillus plantarum ; Accession number: CGMCC No. 34318; Preservation period: April 23, 2025; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0042] Figure 1 The effect of Lactobacillus plantarum WZ-9 on FBG in T2DM mice was investigated. Among them, NC: normal control group, MC: diabetic model group; MET: metformin hydrochloride group, WZ9: Lactobacillus plantarum WZ-9 group. Figure 2 The effect of *Lactobacillus plantarum* WZ-9 on oral glucose tolerance in type 2 diabetes mellitus (T2DM) mice is shown in the figure. Figure A represents the dynamic curve of blood glucose changes over time; Figure B represents the corresponding area under the curve (AUC). glucose ); ### indicates a significant difference compared to the NC group ( P<0.001), This indicates a significant difference compared to the MD group. P<0.001 ); NC: normal control group, MC: diabetes model group, MET: metformin hydrochloride group, WZ9: Lactobacillus plantarum WZ-9 group; Figure 3 The figure shows the effects of *Lactobacillus plantarum* WZ-9 on insulin and glucagon in T2DM mice. In the figure, A represents fasting insulin (FINS) results; B represents glucagon results; ### indicates a significant difference compared to the NC group. P<0.001 ), This indicates a significant difference compared to the MD group. P<0.001 ); Figure 4 The effect of Lactobacillus plantarum WZ-9 on insulin resistance in T2DM mice; Figure 5 The effect of *Lactobacillus plantarum* WZ-9 on glycated hemoglobin (HbA1c) in T2DM mice; where NC: normal control group, MC: diabetic model group, MET: metformin hydrochloride group, and WZ9 *Lactobacillus plantarum* WZ-9 group; ### indicates a significant difference compared to the NC group ( P<0.001 ), This indicates a significant difference compared to the MD group. P<0.001 ); Figure 6 The effects of *Lactobacillus plantarum* WZ-9 on ALT and AST in T2DM mice; where A in the figure represents ALT results; B in the figure represents AST results; ### indicates a significant difference compared to the NC group ( P<0.001 ), This indicates a significant difference compared to the MD group. P<0.001 ); Figure 7 Effect of Lactobacillus plantarum WZ-9 on GLP-1 in T2DM mice; ### indicates a significant difference compared to the NC group. P<0.001 ), This indicates a significant difference compared to the MD group. P<0.001 ); Figure 8The effects of *Lactobacillus plantarum* WZ-9 on TC, TG, LDL-C, and HDL-C in T2DM mice are shown in the figure. In the figure, A represents the effect on TC in T2DM mice; B represents the effect on TG in T2DM mice; C represents the effect on HDL-C in T2DM mice; and D represents the effect on LDL-C in T2DM mice. ### indicates a significant difference compared to the NC group. P<0.001 ), This indicates a significant difference compared to the MD group. P<0.001 ), This indicates a significant difference compared to the MD group. P<0.01 ); Figure 9 The effects of Lactobacillus plantarum WZ-9 on liver histopathology in T2DM mice; where A in the figure represents the NC group; B represents the MC group; C represents the MET group; and D represents the WZ9 group. Figure 10 The effects of Lactobacillus plantarum WZ-9 on pancreatic histopathology in T2DM mice are shown in the figure. In the figure, A is the NC group; B is the MC group; C is the MET group; and D is the WZ9 group. Detailed Implementation

[0043] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0044] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0045] Example 1: Preparation of bacterial agent containing Lactobacillus plantarum WZ-9 This invention screens *Lactobacillus plantarum* (Lactobacillus plantarum) from kimchi. Lactiplantibacillus plantarum WZ-9, It was deposited at the China Culture Collection Center for Microbial Cultures on April 23, 2025, with accession number CGMCCNO.34318.

[0046] This invention provides a microbial inoculant containing the active ingredient *Lactobacillus plantarum* WZ-9, and its preparation method is as follows: Step 1: Take out the *Lactobacillus plantarum* WZ-9 stored at -80℃, use an inoculation loop to take a small amount of bacterial solution and streak it on MRS solid medium, and incubate at 37℃ for 36h; use an inoculation loop to pick a single colony on the solid medium and inoculate it into a 100 mL Erlenmeyer flask of MRS liquid medium, and incubate at 37℃ for 12h to obtain activated bacterial solution.

[0047] Step 2: Inoculate the primary cultured bacteria at a rate of 10% v / v into MRS liquid culture medium for expansion culture, and culture at 37°C for 12 hours to obtain the bacterial agent.

[0048] The composition and concentration per liter of MRS liquid culture medium are as follows: peptone 10 g / L, beef extract 10 g / L, Tween 80 1 mL / L, dipotassium hydrogen phosphate 1.16 g / L, yeast extract 5 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, glucose 20 g / L, manganese sulfate 0.03 g / L, sodium acetate 3.02 g / L; pH 6.2. Sterilization is performed at 121°C for 15 minutes.

[0049] Example 1: Determination of physicochemical properties (1) Acid resistance Reagents and instruments: bacterial suspension, liquid culture medium, sterile equipment, 1 mol / L hydrochloric acid, pH meter, spectrophotometer.

[0050] Specific steps: After adjusting the pH of the MRS medium to 2, 3, 4, or 5 with hydrochloric acid, it was sterilized in an autoclave at 121°C for 15 minutes and then cooled for later use. The activated strains were inoculated into different culture media at 2% v / v and incubated at 37°C for 6 hours. Samples were taken at 0, 2, 4, and 6 hours and counted using viable bacteria plates.

[0051] Calculation formula: , The control group consisted of individuals who had been cultured in MRS medium (without pH adjustment) for 0, 2, 4, and 6 hours, and the viable cell count was determined using the viable cell plate counting method.

[0052] (2) Bile salt tolerance Reagents and instruments: bacterial suspension, liquid culture medium, sterile equipment, ox bile salts, spectrophotometer Specific steps: After adjusting the bile salt concentration in the MRS medium to 0, 0.15%, 0.30%, and 0.60%, the medium was sterilized in an autoclave at 121°C for 15 minutes and then cooled for later use. The activated strains were inoculated into different culture media at 1% v / v and incubated at 37°C for 6 hours. Samples were taken at 0, 2, 4, and 6 hours and counted using viable bacteria plates.

[0053] Calculation formula: , The control group consisted of individuals who had been cultured in MRS medium (without bile salts) for 0, 2, 4, and 6 hours, and the viable bacteria count was determined using the viable bacteria plate count method.

[0054] (3) Tolerance to artificial gastrointestinal fluid Reagents and instruments: 9.5%-10.5% hydrochloric acid, pepsin, trypsin, potassium dihydrogen phosphate, 0.22μm filter membrane, syringe, bacterial solution, sodium hydroxide.

[0055] Specific steps: Preparation of artificial gastric fluid: Dilute 9.5%-10.5% hydrochloric acid with water to pH=3.0, weigh 10g of pepsin and add it to the hydrochloric acid, and finally sterilize it with a 0.22μm filter membrane.

[0056] Preparation of artificial intestinal fluid: Weigh 6.8g of potassium dihydrogen phosphate and dissolve it in 500mL of distilled water. Adjust the pH to 8 with 0.4% sodium hydroxide. Dilute with water and bring the volume to 1000mL. Add 10g of trypsin and sterilize with a 0.22μm filter membrane.

[0057] The activated strain was inoculated into artificial gastric or intestinal fluid at a 10% v / v inoculation rate and incubated at 37°C. Samples were taken at 0, 30, and 180 min and counted using viable cell plates.

[0058] Calculation formula: , The control group consisted of viable bacteria counts obtained by plate counting after culturing in MRS medium for 0, 30, and 180 minutes.

[0059] (4) Determination of hypoglycemic capacity 4.1 Determination of α-amylase inhibition rate Reagents and instruments: α-amylase, bacterial culture, starch solution, PBS buffer (0.1 mol / L, pH=6.8), DNS solution, Lactobacillus rhamnosus.

[0060] Specific steps: Solution preparation: 1 mg / mL α-amylase solution: Place 0.1 g of α-amylase powder into a container and add 100 mL of PBS buffer.

[0061] 1.5% soluble starch solution: Add 1.5g of starch to 80mL of purified water, dissolve, and then add to 100mL.

[0062] DNS solution: Weigh 10g of 3,5-dinitrosalicylic acid and add it to 600mL of water. After dissolving, add 16g of sodium hydroxide, then add 300g of sodium potassium tartrate. After it is completely dissolved, add water to make up to 1L. Store in a brown bottle and let it stand at room temperature for one week before use.

[0063] The activated bacterial culture was inoculated into MRS medium at an inoculation rate of 2% v / v, and activated at 37℃ for 12 hours. After three generations of activation, it was ready for use. Adjust the bacterial concentration to 10 9 After obtaining cfu / mL, centrifuge at 4℃ and 6000rpm for 10min, collect the supernatant, and sterilize it by passing it through a 0.22μm filter membrane to obtain the bacterial supernatant for later use; Add 0.25 mL of α-amylase solution to 0.25 mL of sample (bacterial supernatant), react at 37 °C for 10 min, then add 0.5 mL of 1.5% soluble starch solution at 37 °C, and react for 5 min. Add 1 mL of DNS solution, boil in water for 5 min, then rapidly cool to room temperature, dilute tenfold, let stand for 30 min, and measure the OD value at 540 nm using an ELISA reader. Calculation formula: .

[0064] Among them, sample group (A) had sample and α-amylase; sample blank group (B) had sample and no α-amylase; control group (C) had no sample and α-amylase solution; blank (D) had an equal amount of PBS solution, no sample and no α-amylase solution.

[0065] 4.2 Determination of α-glucosidase inhibition rate Reagents and instruments: α-glucosidase, PBS buffer (0.1 mol / L, pH=6.8), PNPG (p-nitrophenyl-α-D-glucosidase solution), sodium carbonate, 96-well plate, spectrophotometer.

[0066] Specific steps: Solution preparation: The concentration was adjusted to 0.5 U / mL based on the activity of α-glucosidase; 2.5 mol / L PNPG: For example, dissolve 7.53 mg of PNPG in 9 mL of PBS buffer, and then bring the total volume to 10 mL.

[0067] 1 mol / L sodium carbonate solution: For example, dissolve 10.599 g of sodium carbonate in distilled water to make 100 mL of solution.

[0068] Add 50 μL of PBS solution, 50 μL of PNPG solution, and 25 μL of sample (same as the bacterial supernatant in 4.1) to a 96-well plate, and react at 37°C for 10 min. Add 30 μL of α-glucosidase solution and react at 37°C for 30 min. Finally, 50 μL of sodium carbonate solution was added to terminate the reaction, and the OD was measured at 405 nm.

[0069] Calculation formula:

[0070] Among them, sample group (A) had sample and α-glucosidase; sample blank group (B) had sample and no α-glucosidase; control group (C) had no sample and α-glucosidase; blank (D) had an equal amount of PBS solution, with no sample and no α-glucosidase.

[0071] (5) Antioxidant capacity determination 5.1 Determination of ABTS+• Scavenging Rate ① Preparation of the initial stock solution: 1.0 M Sodium Acetate: Dissolve 82 g of accurately weighed sodium acetate in 1 L of distilled water.

[0072] 1.0 M glacial acetic acid: Pour 59 mL of accurately measured glacial acetic acid into a 1 L volumetric flask, dilute to volume with distilled water, and shake well.

[0073] ② Sodium acetate buffer (pH=4.5): First, mix 1.0 M sodium acetate and 1.0 M glacial acetic acid in a ratio of 47:63, and then adjust to the required pH value using a pH meter.

[0074] ③ ABTS solution (7 mM): Weigh 0.38 g of ABTS and dissolve it in 100 mL of sodium acetate buffer (pH=4.5). ④ K2S2O8 solution (2.5 mM): Weigh 0.07 g of K2S2O8 and dissolve it in 100 mL of distilled water.

[0075] Before use, the ABTS solution was placed in the dark at room temperature for 16 hours. Then, 1.0 mL of ABTS solution was taken and diluted with ethanol to an absorbance of 0.7 ± 0.1 at 752 nm. 30 μL of sample (same as the bacterial supernatant in 4.1) was added to 3.0 mL of diluted ABTS+ solution. After reacting for 6 minutes, ethanol was used as a blank control. Finally, the absorbance was measured at 734 nm. Each treatment was repeated in triplicate, and all measurements were the average of the three data. The control group was replaced with distilled water instead of the test sample.

[0076] Calculation formula: Clearance rate (%) = (1-As / Ac) × 100%.

[0077] Where As is the absorbance of the sample, and Ac is the absorbance of the control.

[0078] 5.2 Determination of DPPH• Scavenging Rate ① DPPH solution: Weigh 3.9 mg of DPPH and dissolve it in 100 mL of anhydrous ethanol. After calculating the required amount, prepare the solution according to this ratio.

[0079] Prepare a 0.1 mM ethanol-DPPH solution and measure its initial absorbance at 517 nm. Add 0.1 mL of sample (same as the bacterial supernatant from section 4.1) to 3.0 mL of ethanol-DPPH solution, incubate at 30°C in the dark for 30 min, and then measure the absorbance at 517 nm. The control group is a DPPH solution without sample. The DPPH free radical scavenging rate is calculated using the following formula: Clearance rate (%) = (1-As / Ac) × 100%.

[0080] Where As is the absorbance of the sample, and Ac is the absorbance of the control.

[0081] 5.3 O2−• Scavenging Capacity Determination 50 μL of sample solution (same as the bacterial supernatant in 4.1), 50 μL of NBT solution (78 μM), 50 μL of NADH solution (78 μM), and 50 μL of PMS solution (10 μM) were dissolved in phosphate buffer (pH 7.8) and mixed in a 96-well plate. The mixture was incubated at 25°C for 5 min, and the absorbance was measured at 560 nm. The superoxide anion radical scavenging rate was calculated using the following formula: Superoxide anion radical scavenging rate (%) )×100%.

[0082] Where A0 is the absorbance of the control group (using distilled water instead of the test sample); A1 is the absorbance of the sample group; and A2 is the absorbance of the sample only (using phosphate buffer instead of NBT solution).

[0083] 5.4 Determination of OH−• scavenging ability Add 0.1 mL of the sample to be tested (same as the bacterial supernatant in 4.1), 1 mL of 5 mM ferrous sulfate solution, 1 mL of 5 mM salicylic acid-ethanol solution, and 1 mL of 3 mM H2O2 to the test tube in sequence, make up to 10 mL, react in a water bath at 37℃ for 20 min, and measure the absorbance at a wavelength of 510 nm. Perform the test three times for each sample. Blank group: Distilled water was used instead of the test sample; Control group: Distilled water was used instead of H2O2 solution; Calculation formula: OH- radical scavenging rate (%) )×100% Where A2 is the absorbance of the sample solution; A1 is the absorbance of the control group; and A0 is the absorbance of the blank group.

[0084] The results of the physicochemical property determination are as follows: This strain exhibits remarkable antioxidant capacity, effectively scavenging free radicals and reducing oxidative damage. Experimental data show that its hydroxyl radical scavenging rate is 85.65%, DPPH radical scavenging rate is 80.34%, ABTS radical scavenging rate is 98.21%, and superoxide anion scavenging rate is 85.47%, demonstrating strong antioxidant activity and helping to alleviate diabetes-related oxidative stress. Furthermore, this strain also demonstrates significant hypoglycemic ability, slowing glucose absorption and improving glucose metabolism by inhibiting the activity of α-glucosidase and α-amylase. Experimental results show that its inhibition rates against α-glucosidase and α-amylase are as high as 99.17% and 98.09%, respectively, providing a new approach for the adjunctive treatment of diabetes. Based on this, the strain exhibits good tolerance, surviving in acidic environments at pH 3, 4, and 5, and maintaining significant growth under 0.15% and 0.3% bile salt conditions. Its survival rate exceeds 85% in a simulated gastric juice environment at pH 3, and it maintains high activity even after 4 hours of gastric juice treatment. These characteristics ensure its stability in the digestive tract, making it promising for functional foods and clinical applications. Therefore, *Lactobacillus plantarum* WZ-9 combines strong antioxidant capacity, excellent hypoglycemic effect, and good intestinal tolerance, demonstrating its broad application prospects in diabetes management and intestinal health regulation. *Lactobacillus plantarum* WZ-9 exhibits significant hypoglycemic potential by inhibiting the activity of α-glucosidase and α-amylase.

[0085] The two main digestive enzymes used to break down carbohydrates into monosaccharides are α-amylase and α-glucosidase. Some active substances in the intestine inhibit the activity of α-amylase and α-glucosidase, thus slowing down the breakdown of polysaccharides into monosaccharides and helping carbohydrates to be absorbed more slowly into the body, thereby lowering blood sugar.

[0086] The combination of antioxidant capacity, tolerance characteristics, and hypoglycemic mechanism of *Lactobacillus plantarum* WZ-9 provided by this invention demonstrates significant hypoglycemic potential, showcasing its broad application prospects in diabetes management and gut health regulation. Regarding the efficacy verification protocol, refer to "Zhou Baiyi, Cai Youshan, Lin Shiwei, et al. *Leuconostoc mesenteroides* (…)" Leuconostoc mesenteroides subsp. cremoris The experimental protocol described in "Evaluation of the hypoglycemic activity of GKM5 [J]. Food and Nutrition Science, 2022" is incorporated herein by reference. Those skilled in the art are capable of designing and implementing experiments based on this reference and other existing experimental protocols. Specific efficacy verification protocols include, but are not limited to, the following: Detection example I. Materials and Methods 1. Test strain Lactobacillus plantarum WZ-9, stored at -80 degrees Celsius.

[0087] 2. Animal Management Forty 4-week-old male C57BL / 6J mice (16-20g in weight) were provided by Beijing Weishang Lide Biotechnology Co., Ltd. They were housed in a standardized laboratory at room temperature (22±2)℃, relative humidity (50±5)%, and 12 h light / 12 h dark conditions. The animal experimental protocol was approved by the Experimental Animal Ethics Review Committee of Beijing University of Agriculture.

[0088] 3. Animal grouping and T2DM model establishment After one week of acclimatization, mice were randomly divided into four groups: normal control group (NC), diabetic model group (MC), metformin hydrochloride group (MET), and Lactobacillus plantarum WZ-9 group (WZ9), n=10. For the first four weeks before modeling, NC group mice were fed a standard diet, while MC, MET, and WZ9 group mice were continuously fed a high-fat diet (HFD). All mice had free access to water during this period.

[0089] Four weeks after a high-fat diet intervention, all mice were fasted for 12 hours. Mice in the MC, MET, and WZ9 groups were intraperitoneally injected with streptozotocin (STZ) (dissolved in sodium citrate buffer, freshly prepared) at a dose of 50 mg / kg body weight for one week. Mice in the NC group were injected with an equal volume of sodium citrate buffer as a control. Three days after the end of the continuous injections, all mice were fasted for 12 hours, and fasting blood glucose (FBG) was measured using a blood glucose meter after tail clipping. If the fasting blood glucose level of a mouse was ≥ 11.1 mmol / L, the T2DM mouse model was considered to have been successfully established.

[0090] 4. Gavage intervention and fasting blood glucose (FBG) monitoring After successful modeling, week 0 of drug administration was recorded. Each group received the drug via gavage for 4 consecutive weeks. Group WZ9 received 1×10⁻⁶ doses. 9The bacterial culture concentration was 1 mL / 100 g body weight, administered via gavage. The MET group was administered metformin hydrochloride aqueous solution via gavage at a dose of 200 mg / kg body weight. The NC and MC groups were administered an equal volume of sterile PBS buffer via gavage as controls. FBG was measured weekly in each mouse during the intervention period (week 0, week 1, week 2, week 3, week 4). All mice were strictly fasted for 12 h before measurement, but had free access to water.

[0091] 5. Oral glucose tolerance test (OGTT) After the intervention period, to avoid metabolic stress caused by continuous fasting, mice were fed normally for 3 days before undergoing an oral glucose tolerance test (OGTT). Mice in each group were weighed and fasted for 12 hours, and their basal blood glucose levels were measured (recorded as 0 min). Subsequently, a single administration of 20% glucose solution at a dose of 2 g / kg body weight was given, and blood glucose levels were measured at 30 min, 60 min, 90 min, and 120 min after glucose administration. Blood glucose levels at each time point were recorded, and curves were plotted to calculate the area under the blood glucose curve (AUC).

[0092] 6. Sample Collection and Tissue Processing After the OGTT test, the mice resumed normal diet and water intake for 48 hours. Before the end of the experiment, the mice in each group were fasted again for 12 hours, anesthetized with an appropriate amount of CO2, and their eyeballs were immediately removed to collect blood. After the blood was allowed to stand at room temperature for 2 hours, it was centrifuged at 3500 r / min for 10 min at 4 ℃. After centrifugation, the blood was quickly aliquoted and stored in a -80 ℃ freezer.

[0093] After blood collection, mice were euthanized, and the liver, pancreas, ileum, epididymal fat, spleen, and kidneys were quickly dissected and separated. The liver was weighed. The pancreatic tissue was rinsed with pre-cooled physiological saline. Partial tissue samples from the liver, pancreas, and ileum were fixed in 4% paraformaldehyde solution for histological examination; all remaining tissues were rapidly flash-frozen in liquid nitrogen and then transferred to -80 °C for storage.

[0094] 7. Detection of serum biochemical and lipid metabolism indicators (1) Glucose metabolism and liver function indicators: The levels of insulin, glucagon, glucagon-like peptide-1 (GLP-1) and glycated hemoglobin (HbA1c) in serum were measured using an ELISA kit, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured according to the instructions.

[0095] (2) Blood lipid level measurement: Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were measured using a biochemical kit.

[0096] (3) Insulin resistance assessment: The HOMA-IR index is calculated using the following formula: HOMA-IR = [Fasting blood glucose (mmol / L) × Fasting serum insulin (mIU / L)] / 22.5 8. Histopathological observation Pancreatic and liver tissues fixed in 4% paraformaldehyde solution were removed and rinsed with tap water for 10 min. The tissues were then sequentially dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and cut into sections approximately 4 μm thick. After routine hematoxylin-eosin (H&E) staining, the sections were mounted and placed under an optical microscope for histopathological observation and photographic recording.

[0097] II. Test Results 1. Effects of Lactobacillus plantarum WZ-9 on fasting blood glucose (FBG) in type 2 diabetes mellitus (T2DM) mice Depend on Figure 1 It was found that before the intervention (Week 0), the fasting blood glucose levels of mice in the MC, MET, and WZ9 groups were significantly higher than those in the NC group, indicating that the type 2 diabetes mouse model was successfully established. During the four-week intervention, the fasting blood glucose level in the MC group remained high; however, after WZ9 intervention, the fasting blood glucose level of the mice showed a significant and continuous downward trend. By the end of the fourth week, the blood glucose level of the WZ9 group mice was significantly lower than that of the MC group, and the hypoglycemic effect was similar to that of the positive drug metformin (MET) group.

[0098] 2. Effects of Lactobacillus plantarum WZ-9 on oral glucose tolerance in T2DM mice like Figure 2 The dynamic curves of blood glucose over time (A) show that after glucose administration, blood glucose levels in all groups of mice peaked at 30 minutes. The MC group mice not only exhibited abnormally high initial blood glucose levels, but also experienced a rapid spike in blood glucose after glucose loading, maintaining an extremely high level for 120 minutes, indicating severely impaired blood glucose regulation. In contrast, after WZ9 intervention, the blood glucose peak in mice was significantly suppressed and rapidly decreased within 120 minutes, exhibiting a blood glucose regulation trend highly consistent with the positive drug MET group.

[0099] Depend on Figure 2 As shown in the AUC bar chart for group B, the blood glucose AUC of mice in the MC group was significantly higher than that in the NC group. Compared with the MC group, the AUC of mice in the WZ9 group was significantly reduced, and its improvement effect was comparable to that of the positive drug MET group.

[0100] 3. Effects of *Lactobacillus plantarum* WZ-9 on fasting insulin (FINS) and glucagon (Glucagon) in type 2 diabetes mellitus (T2DM) mice Depend on Figure 3 As shown in section A, compared to the NC group, the fasting insulin level in the MC group mice was significantly higher, exhibiting typical hyperinsulinemia. However, after intervention with the WZ9 strain, the fasting insulin level in the mice was significantly lower than that in the MC group, and its level returned to near the levels of the normal group and the positive drug MET group. As shown in section B, the glucagon level in the MC group mice was also significantly higher than that in the NC group, suggesting a severe hormonal secretion disorder in the model mice. Compared to the MC group, the glucagon level in the WZ9 group mice was significantly lower, effectively inhibiting abnormal glucagon secretion.

[0101] 4. Effects of Lactobacillus plantarum WZ-9 on insulin resistance HOMA-IR in type 2 diabetes mellitus (T2DM) mice Depend on Figure 4 It was found that the HOMA-IR index of the MC group mice was significantly higher than that of the NC group, indicating that there was extremely severe insulin resistance in the type 2 diabetes model mice. After intervention in the WZ9 group, the HOMA-IR index of the mice was significantly lower than that of the MC group, and the index dropped sharply to near normal physiological levels, with the degree of improvement comparable to that of the positive drug MET group.

[0102] 5. Effects of *Lactobacillus plantarum* WZ-9 on glycated hemoglobin (HbA1c) in type 2 diabetes mellitus (T2DM) mice Depend on Figure 5 The results showed that the HbA1c level in the MC group was significantly higher than that in the NC group, indicating that the untreated type 2 diabetes model mice were in a state of severe hyperglycemia. However, after WZ9 intervention, the HbA1c level in the mice was significantly lower than that in the MC group. The improvement effect of the WZ9 group on HbA1c was not only comparable to that of the positive drug MET group, but also showed a better trend in terms of mean reduction.

[0103] 6. Effects of Lactobacillus plantarum WZ-9 on ALT and AST in T2DM mice Depend on Figure 5 A and Figure 5 As shown in B, compared with the NC group, the serum ALT and AST levels in the MC group mice were significantly elevated. ALT and AST are sensitive indicators of hepatocyte damage, and this result indicates severe liver damage or liver dysfunction in the type 2 diabetic model mice. After WZ9 intervention, the serum ALT and AST levels in the mice were significantly reduced compared with the MC group. Moreover, the levels of the two transaminases in the WZ9 group mice not only decreased significantly, but the improvement effect was comparable to that in the MET group, and they basically returned to the healthy levels close to those of normal mice.

[0104] The liver is a key organ for maintaining glucose homeostasis in the body. The above results indicate that WZ9 not only possesses excellent blood sugar-lowering and insulin resistance-alleviating abilities, but also significantly reduces liver damage caused by type 2 diabetes, exerting a superior liver-protective effect.

[0105] 7. Effects of Lactobacillus plantarum WZ-9 on GLP-1 levels in T2DM mice GLP-1 is a core gut-derived hormone that regulates glucose metabolism, promotes insulin secretion, and inhibits glucagon secretion. Figure 7 It was found that, compared with the NC group, the GLP-1 level in the MC group mice was significantly decreased, indicating a severe deficiency in incretin secretion in the type 2 diabetes model mice. After WZ9 intervention, the GLP-1 level in the mice was significantly increased compared with the MC group, and its effect on promoting the increase of endogenous GLP-1 was comparable to that of the MET group.

[0106] 8. Effects of Lactobacillus plantarum WZ-9 on lipid metabolism in type 2 diabetes mellitus (T2DM) mice Lactobacillus plantarum WZ-9 on serum TC, TG, and LDL in type 2 diabetic mice C and HDL The effect of C level is shown in [see] Figure 8 .

[0107] Depend on Figure 8 It was found that, compared with the NC group, the serum levels of TC, TG, and LDLC in the MC group mice were significantly elevated, while the level of HDLC, which has a cardiovascular protective effect, was significantly reduced. These results indicate that type 2 diabetic model mice have extremely severe lipid metabolism disorders and a very high risk of cardiovascular complications. After intervention with WZ9, the serum levels of LDLC, TC, and TG in the mouse group decreased significantly compared with the MC group; at the same time, HDLC significantly increased compared with the MC group. The comprehensive improvement effect of *Lactobacillus plantarum* WZ-9 on the above four lipid indicators was comparable to that of the MET group.

[0108] The above results fully demonstrate that WZ9 not only has excellent blood glucose regulation capabilities, but also has a strong lipid-lowering effect, which can comprehensively improve the glucose and lipid metabolism syndrome caused by type 2 diabetes.

[0109] 9. Effects of Lactobacillus plantarum WZ-9 on liver histopathology in T2DM mice H&E staining morphology of mouse liver is shown in Figure 9In the NC group, the liver tissue of mice was intact and clearly morphological, with hepatocytes arranged radially and neatly without abnormal vacuoles. Compared with the NC group, the hepatocytes of mice in the MC group were extremely disordered, exhibiting severe diffuse macrovesicular steatosis, with numerous large lipid vacuoles compressing the cell nuclei and accompanied by severe damage such as ballooning cell swelling. Both the MET group and the WZ9 group significantly improved the above-mentioned pathological damage. In particular, the large lipid droplet vacuoles in the hepatocytes of the WZ9 group were almost completely eliminated, and the cell morphology was basically restored to normal. The improvement effect was even better than that of the positive MET group, indicating that WZ9 has a very significant repair and protective effect on severe lipid accumulation and pathological damage to the liver caused by T2DM.

[0110] 10. Effects of Lactobacillus plantarum WZ-9 on pancreatic histopathology in T2DM mice H&E staining of pancreatic tissue sections was used to evaluate the effects of *Lactobacillus plantarum* WZ-9 on the pancreas of T2DM mice. Figure 10 As shown, the pancreatic tissue structure of mice in the NC group was intact, with regular arrangement of exocrine acini. The islets of Langerhans were normal in morphology, regularly round or oval in shape, with clear boundaries; the number of endocrine cells in the islets was abundant and tightly arranged, with deeply stained nuclei, and no obvious cell degeneration, necrosis, or inflammatory cell infiltration was observed. Compared with the normal group, the islet structure in the MC group was significantly damaged. The islets were irregular in shape with blurred boundaries; the cells within the islets were loosely and disordered, with a significantly reduced number of cells, and obvious enlarged intercellular spaces and vacuolar degeneration, presenting a typical state of islet damage and atrophy. After metformin treatment, the pathological damage to the pancreas was significantly improved. In the MET group, the islet morphology basically recovered to a regular shape, and the degree of atrophy was reduced; the cell density within the islets was significantly increased compared to the model group, with a relatively regular arrangement and only slight intercellular spaces. The pancreatic tissue morphology of the WZ9 treatment group showed significant recovery. The islets had clear outlines, were full, and their morphology was close to that of the normal control group; the cells within the islets were abundant and tightly arranged, and pathological phenomena such as vacuolar degeneration were significantly reduced.

[0111] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum WZ-9, characterized in that, It was deposited at the China Culture Collection Center for Microbial Cultures on April 23, 2025, with accession number CGMCC NO.34318.

2. A microbial agent, characterized in that, The microbial agent comprises *Lactobacillus plantarum* as described in claim 1 or a preparation of *Lactobacillus plantarum* as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The preparations of *Lactobacillus plantarum* include cultures of *Lactobacillus plantarum*, culture extracts, freeze-dried powders, fermentation broths, fermentation broth precipitates, fermentation broth supernatants, or fermentation broth extracts.

4. The microbial agent according to claim 3, characterized in that, The bacterial agent is the fermentation broth of *Lactobacillus plantarum*, preferably a live bacterial fermentation broth.

5. A product comprising the *Lactobacillus plantarum* as described in claim 1 or the microbial agent as described in any one of claims 2-4.

6. The product according to claim 5, characterized in that, The products include fertilizers, food, pharmaceuticals, and health products.

7. The product according to claim 6, characterized in that, The product is a pharmaceutical product, which also includes pharmaceutically acceptable excipients.

8. The method for culturing *Lactobacillus plantarum* according to claim 1, characterized in that, This includes culturing the plant lactobacillus using a culture medium.

9. The cultivation method according to claim 8, characterized in that, The culture medium includes bacterial culture media, which include MSR medium, LB medium and SS medium.

10. The application of *Lactobacillus plantarum* according to claim 1, the microbial agent according to any one of claims 2-4, or the product according to any one of claims 5-7, characterized in that, The applications include those in the preparation of hypoglycemic products.

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