A rubia cordifolia fermentation liquor, a preparation method and application thereof

Abstract

This invention provides a fermentation broth for *Polygonum viviparum*, its preparation method, and its application, belonging to the field of natural product extraction technology. This invention provides the application of *Lactobacillus plantarum* in the preparation of fermented *Polygonum viviparum* products. This invention uses *Lactobacillus plantarum* to ferment *Polygonum viviparum* fruits. The fermentation product shows a significant decrease in α-glucosidase and acetylcholinesterase activities, a significant increase in total antioxidant capacity, and a significant improvement in the scavenging rate of superoxide anions and hydroxyl radicals. The fermentation process enriches flavor substances such as nitrogen oxides, aromatic components, and organosulfur compounds, forming a unique salty taste that effectively masks the unpleasant bitter and fishy aftertaste of unfermented samples, greatly improving palatability and product acceptability. The fermentation of *Polygonum viviparum* by *Lactobacillus plantarum* promotes the efficient release and structural transformation of active ingredients, achieving a three-dimensional simultaneous improvement in hypoglycemic, antioxidant, and sensory quality, providing key technical support for the high-value application of *Polygonum viviparum* in the functional food field.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to a fermentation broth of Polygonum bulbiferum, its preparation method, and its application. Background Technology

[0002] Polygonum bulbifera ( Bistorta vivipara Polygonum bulbiferum (Polygonum villosa) is a perennial herbaceous plant belonging to the Polygonaceae family and the Polygonum genus. It is widely distributed in the Qinghai-Tibet Plateau and the cold, high-altitude regions of southwest my country and is an important medicinal plant used in traditional medicine. Its bulbils and rhizomes are rich in active ingredients such as flavonoids, polyphenols, anthraquinones, and polysaccharides. Traditional Tibetan and Mongolian medicine records its effects of clearing heat and detoxifying, stopping bleeding and dysentery, and reducing inflammation and diarrhea. Modern pharmacological studies have also preliminarily confirmed its antioxidant, anti-inflammatory, antibacterial, and immunomodulatory biological activities. In recent years, with the increasing demand for the in-depth development of natural plant resources, the application potential of Polygonum bulbiferum in functional foods, dietary supplements, and veterinary medicine has gradually attracted attention.

[0003] However, existing methods of utilizing *Polygonum villosa* still have the following drawbacks: 1. Low bioavailability of active ingredients; traditional processing methods struggle to efficiently release the active ingredients. Currently, the utilization of *Polygonum villosa* mainly relies on physical or simple chemical extraction processes such as direct crushing, water extraction, or alcohol extraction. While these methods are simple to operate, their penetration into the dense cell wall structure of *Polygonum villosa* tissue is limited, resulting in low release rates and slow dissolution rates of active ingredients such as flavonoids and polysaccharides, and unstable content of effective ingredients in the extract. Furthermore, secondary metabolites of alpine plants are often tightly bound to structural polysaccharides such as cellulose and lignin, and traditional extraction methods struggle to break these bonds, leading to resource waste. 2. Unclear active components; lack of in-depth development targeting specific diseases. Existing research mainly focuses on broad-spectrum activity screening of crude extracts of *Polygonum villosa*, while research on the material basis and mechanism of action against modern metabolic diseases such as glucose metabolism disorders and oxidative damage to pancreatic β-cells remains lacking. 3. Limited product forms; poor taste and stability; limited industrial application. Currently, most Polygonum villosa products are in the form of coarse powder, decoction, or simple extracts, which suffer from problems such as bitter taste, poor palatability, easy oxidation and spoilage, and short shelf life. Consumer acceptance is low, making it difficult to meet the modern functional beverage market's requirements for taste, stability, and portability. Furthermore, crude extracts that have not undergone biotransformation may contain anti-nutritional factors such as tannins, affecting the safety and efficacy of the products.

[0004] In summary, existing technologies for utilizing Polygonum viviparum suffer from key problems such as low extraction efficiency, unclear efficacy targets, and low product added value, which severely restrict its application in high-value development. Therefore, developing a novel Polygonum viviparum formulation capable of efficiently releasing and transforming its active ingredients has significant scientific importance and broad market prospects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide *Lactobacillus plantarum* (… Lactiplantibacillus plantarum This invention relates to the application of *Polygonum viviparum* fermentation products. Using *Lactobacillus plantarum* to ferment *Polygonum viviparum* can more effectively promote the release of its active ingredients and improve its efficacy. This invention also provides a *Polygonum viviparum* fermentation broth, its preparation method, and its applications.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides a plant lactobacillus ( Lactiplantibacillus plantarum Application of Polygonum bulbiferum fermentation products.

[0007] Preferably, the *Lactobacillus plantarum* includes *Lactobacillus plantarum* XZFMCC101.23155, with accession number GDMCCNo: 65783.

[0008] This invention provides a method for preparing Polygonum bulbiferum fermentation broth, comprising: After mixing Polygonum bulbil powder with water, Lactobacillus plantarum was added for fermentation to obtain Polygonum bulbil fermentation broth.

[0009] Preferably, the *Lactobacillus plantarum* includes *Lactobacillus plantarum* XZFMCC101.23155, with accession number GDMCCNo: 65783.

[0010] Preferably, the bulbils powder is obtained by crushing bulbils fruit; the ratio of bulbils powder to water is 1g:(10~50)mL.

[0011] Preferably, the fermentation time is 24-120 hours; the fermentation temperature is 20-36°C.

[0012] Preferably, the *Lactobacillus plantarum* is added using a *Lactobacillus plantarum* seed culture method; the viable count of the *Lactobacillus plantarum* seed culture is 1 × 10⁻⁶. 8 CFU / mL; the amount of Lactobacillus plantarum seed solution added is 1.0%~3.0% (V / V) of the volume of the mixture of bulbils and Polygonum hydropiper.

[0013] This invention provides a fermentation broth of Polygonum bulbiferum prepared by the preparation method described in the above technical solution.

[0014] This invention provides a fermented product of Polygonum bulbiferum, including the fermented broth of Polygonum bulbiferum described in the above technical solution.

[0015] This invention provides the application of the above-described Polygonum bulbil fermentation broth or the above-described Polygonum bulbil fermentation product in the preparation of treatments or improvements for glucose metabolism diseases and / or enhancement of oxidative damage repair.

[0016] The beneficial effects of this invention are: This invention provides *Lactobacillus plantarum* ( Lactiplantibacillus plantarum Application of *Polygonum viviparum* fermentation products in this invention. This invention utilizes *Lactobacillus plantarum* for targeted fermentation of *Polygonum viviparum* fruits, achieving a synergistic improvement in efficacy and quality compared to unfermented *Polygonum viviparum* stock solution. The use of *Lactobacillus plantarum* for fermentation of *Polygonum viviparum* fruits in this invention can more effectively promote the release of active ingredients from the fruits, improving their application efficacy. In this invention, *Polygonum viviparum* fruits significantly release and transform bound functional components through specific biotransformation and cell wall degradation by *Lactobacillus plantarum*. The fermentation products obtained using *Lactobacillus plantarum* for *Polygonum viviparum* fruits show significantly reduced α-glucosidase and acetylcholinesterase activities, thereby effectively delaying carbohydrate breakdown, improving insulin resistance, and demonstrating a clear and potent hypoglycemic potential. The fermentation products showed a significant increase in total antioxidant capacity and a marked improvement in the scavenging rate of superoxide anions and hydroxyl radicals. This indicates that the fermentation process not only released antioxidants such as polyphenols and flavonoids but also produced microbial-derived antioxidant components, which in turn helped to synergistically enhance the oxidative damage repair capacity of pancreatic β-cells. Furthermore, fermentation significantly enriched flavor compounds such as nitrogen oxides, aromatic components, and organosulfur compounds, creating a unique salty taste and effectively masking the unpleasant bitter and fishy aftertaste of the unfermented samples, greatly improving palatability and product acceptability. In summary, the fermentation of Polygonum viviparum fruits by Lactobacillus plantarum promotes the efficient release and structural transformation of active ingredients, achieving a three-dimensional simultaneous improvement in blood sugar reduction, antioxidant effects, and sensory quality, providing key technical support for the high-value application of Polygonum viviparum in the functional food field. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The graph shows the effects of different bacterial strains on relevant indicators of Polygonum viviparum fermentation broth. In this graph, A represents the blood sugar lowering indicator; B represents the blood lipid lowering indicator; and C represents the antioxidant indicator. Figure 2 The figure shows the effect of different material-to-liquid ratios on relevant indicators of Polygonum bulbiferum fermentation broth. In the figure, A represents the blood sugar lowering index; B represents the blood lipid lowering index; and C represents the antioxidant index. In the figure, 10 corresponds to the treatment group with a material-to-liquid ratio of 1:10, 20 corresponds to the treatment group with a material-to-liquid ratio of 1:20, 30 corresponds to the treatment group with a material-to-liquid ratio of 1:30, 40 corresponds to the treatment group with a material-to-liquid ratio of 1:40, and 50 corresponds to the treatment group with a material-to-liquid ratio of 1:50. Figure 3 The graph shows the effects of different amounts of bacterial strains on relevant indicators of Polygonum viviparum fermentation broth. In this graph, A represents the blood sugar lowering indicator; B represents the blood lipid lowering indicator; and C represents the antioxidant indicator. Figure 4The graph shows the effects of different fermentation times on relevant indicators of Polygonum viviparum fermentation broth. In this graph, A represents the blood sugar lowering indicator; B represents the blood lipid lowering indicator; and C represents the antioxidant indicator. Figure 5 The graph shows the effect of different fermentation temperatures on relevant indicators of Polygonum viviparum fermentation broth. In this graph, A represents the blood sugar lowering indicator; B represents the blood lipid lowering indicator; and C represents the antioxidant indicator. Figure 6 Figure A shows the orthogonal experimental results of Polygonum bulbiferum fermentation broth; Figure B shows the orthogonal experimental results of blood sugar lowering index; Figure C shows the orthogonal experimental results of blood lipid lowering index; Figure C shows the orthogonal experimental results of antioxidant index. Figure 7 The graph shows the results of the relevant experimental indicators; A represents the blood sugar lowering indicator; B represents the blood lipid lowering indicator; and C represents the antioxidant indicator. Figure 8 The images show the electronic nose detection results for samples 1-3. A is a multi-index radar chart for samples 1-3; B is a box plot of the W1C index for samples 1-3. Different letters in the figures indicate significant differences. Figure 9 Box plots for the sensor responses of samples 1-3 are shown below; C is the box plot for the W5S sensor response; D is the box plot for the W3C sensor response; E is the box plot for the W6S sensor response; F is the box plot for the W5C sensor response; G is the box plot for the W1S sensor response; H is the box plot for the W1W sensor response; I is the box plot for the W2S sensor response; J is the box plot for the W2W sensor response; K is the box plot for the W3S sensor response. Different letters in the figures indicate significant differences. Figure 10 Figure 1 shows the PCA results of different electronic nose samples. Figure 11 Radar graphs showing the electronic tongue detection results for samples 1-3; Figure 12 Box plots showing different taste response values ​​of the electronic tongue; B represents the sour taste response value; C represents the bitter taste response value; D represents the astringent taste response value; E represents the astringent aftertaste response value; F represents the bitter aftertaste response value; G represents the umami taste response value; H represents the richness response value; I represents the salty taste response value; J represents the sweet taste response value. Figure 13 The following is a graph showing the PCA results of samples 1-3 using electronic tongues; Figure 14 The figure shows the effects of Polygonum viviparum fermentation broth on NO and GLUT4 levels in a STZ-induced MIN6 cell hyperglycemia model; A represents NO content; B represents GLUT4 content; compared with the normal control group, the model group showed #p<0.05, ##p<0.01, and ###p<0.001; compared with the model control group, the drug intervention group showed... p<0.05, p<0.01, p<0.001; Figure 15 Figure 1 shows the effects of Polygonum viviparum fermentation broth on the levels of BIS, GSIS, and ISI in a STZ-induced MIN6 cell hyperglycemia model; A represents BIS; B represents GSIS; C represents ISI; Compared with the normal control group, the model group showed p<0.05, p<0.01, and p<0.001; Compared with the model control group, the drug intervention group showed... p<0.05, p<0.01, p<0.001. Detailed Implementation

[0019] This invention provides *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The application of *Polygonum viviparum* in the preparation of fermented products. As an optional embodiment of the present invention, the *Lactobacillus plantarum* includes *Lactobacillus plantarum* XZFMCC101.23155, with accession number GDMCC No: 65783.

[0020] This invention utilizes *Lactobacillus plantarum* to ferment *Polygonum viviparum* fruits, which more effectively promotes the release of active ingredients and improves the efficacy of *Polygonum viviparum* fruits. Through specific biotransformation and cell wall degradation by *Lactobacillus plantarum*, *Polygonum viviparum* fruits significantly release and transform bound functional components. Fermentation of *Polygonum viviparum* fruits with *Lactobacillus plantarum* significantly reduces the activities of α-glucosidase and acetylcholinesterase in the fermentation products, thereby effectively delaying carbohydrate breakdown, improving insulin resistance, and demonstrating a clear and potent hypoglycemic potential. The total antioxidant capacity of the fermentation products is significantly improved, with a significant increase in the scavenging rate of superoxide anions and hydroxyl radicals, indicating that the fermentation process not only releases antioxidants such as polyphenols and flavonoids but also produces microbial-derived antioxidant components, which in turn help to synergistically enhance the oxidative damage repair capacity of pancreatic β-cells. Furthermore, fermentation significantly enriches flavor substances such as nitrogen oxides, aromatic components, and organosulfur compounds, forming a unique salty taste and effectively masking the unpleasant bitter and fishy aftertaste of unfermented samples, greatly improving palatability and product acceptability. This invention uses Lactobacillus plantarum to perform targeted fermentation of Polygonum bulbils, achieving a synergistic improvement in efficacy and quality compared to unfermented Polygonum bulbils extract.

[0021] This invention provides a method for preparing Polygonum bulbiferum fermentation broth, comprising: After mixing Polygonum bulbil powder with water, Lactobacillus plantarum was added for fermentation to obtain Polygonum bulbil fermentation broth.

[0022] In an optional embodiment of the present invention, the *Polygonum bulbiferum* powder is obtained by pulverizing *Polygonum bulbiferum* fruits; the particle size of the *Polygonum bulbiferum* powder is preferably ≤60 mesh. The present invention does not specifically limit the preparation method of the *Polygonum bulbiferum* powder; any conventional preparation method in the art can be used. In an optional embodiment of the present invention, the preparation method of the *Polygonum bulbiferum* powder can be to wash, dry, and then pulverize the *Polygonum bulbiferum* fruits to obtain the powder. In the present invention, the *Polygonum bulbiferum* fruits are preferably dried products. After obtaining the dried *Polygonum bulbiferum* fruits, the present invention preferably washes the dried *Polygonum bulbiferum* fruits. The present invention does not specifically limit the washing method; any conventional washing method in the art can be used. The washing in the present invention mainly removes impurities such as dirt from the surface of the *Polygonum bulbiferum* fruits. The present invention does not specifically limit the drying method; any conventional drying method in the art can be used. In an optional embodiment of the present invention, the drying method can be air-drying and / or sun-drying. The drying in the present invention mainly involves air-drying and / or sun-drying the surface moisture of the *Polygonum bulbiferum* fruits. This invention does not specifically limit the pulverization method; any conventional pulverization method in the art can be used. As an optional embodiment of this invention, the pulverization method can be grinding using a multi-functional pulverizer. After pulverization, this invention obtains pulverized Polygonum bulbiferum. Preferably, this invention sieves the obtained pulverized Polygonum bulbiferum and collects the undersize material to obtain Polygonum bulbiferum powder. In this invention, the sieve aperture can be 60 mesh.

[0023] After obtaining Polygonum bulbiferum powder, the present invention preferably mixes the Polygonum bulbiferum powder with water. In the present invention, the water can be ultrapure water. As an optional embodiment of the present invention, the material-to-liquid ratio of the Polygonum bulbiferum powder to water can be 1g:(10~50)mL, or 1:10, 1:20, 1:30, 1:40 or 1:50g / mL. The present invention obtains a Polygonum bulbiferum-water mixture system by mixing Polygonum bulbiferum powder with water.

[0024] After obtaining the bulbils and Polygonum hydropiper mixed system, this invention adds *Lactobacillus plantarum* to the mixed system for fermentation. As an optional embodiment of this invention, the *Lactobacillus plantarum* includes *Lactobacillus plantarum* XZFMCC101.23155, with accession number GDMCC No: 65783. In this invention, the *Lactobacillus plantarum* is preferably added using a *Lactobacillus plantarum* seed culture method. This invention does not specifically limit the preparation method of the *Lactobacillus plantarum* seed culture; any conventional preparation method in the art can be used. In this invention, the viable count of the *Lactobacillus plantarum* seed culture can be 1 × 10⁻⁶. 8CFU / mL; the amount of *Lactobacillus plantarum* seed culture added can be 1.0%~3.0% (V / V) of the mixed volume of *Polygonum bulbiferum* and water, or it can be 1.0%, 1.5%, 2.0%, 2.5% or 3.0% (V / V). As an optional embodiment of the present invention, the fermentation temperature can be 20~36℃, or it can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36℃; the fermentation time can be 24~120h, or it can be 24, 48, 72, 92 or 120h. As an optional embodiment of the present invention, the fermentation process is preferably accompanied by oscillation, and the oscillation speed can be 150 rpm. After fermentation, the present invention obtains *Polygonum bulbiferum* fermentation broth.

[0025] As an optional embodiment of the present invention, the *Polygonum bulbiferum* fermentation broth can be further centrifuged to collect the supernatant; the centrifugation speed can be 4000 r / min; the centrifugation time can be 10-15 min. The obtained supernatant of the *Polygonum bulbiferum* fermentation broth is further aseptically filled and sterilized in a water bath to obtain the *Polygonum bulbiferum* fermentation broth.

[0026] This invention provides a *Polygonum viviparum* fermentation broth prepared by the method described above. The fermentation broth exhibits significantly reduced α-glucosidase and acetylcholinesterase activities, significantly increased total antioxidant capacity, and significantly enhanced scavenging rates against superoxide anions and hydroxyl radicals.

[0027] This invention provides a fermented product of Polygonum bulbiferum, including the fermented broth of Polygonum bulbiferum described in the above technical solution.

[0028] This invention provides the application of the above-described Polygonum bulbil fermentation broth or product in the preparation of drugs for treating or improving glucose metabolism disorders and / or enhancing oxidative damage repair. As an optional embodiment of this invention, the glucose metabolism disorders include diabetes.

[0029] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0030] 1. Experimental materials involved in the following technical solutions The fruits of Polygonum villosa were harvested from Tibet (dried product); commercially available Lactobacillus plantarum was purchased from DuPont Danisco. Strain 793 specifically refers to *Lactobacillus plantarum* (…). Lactiplantibacillus plantarumXZFMCC101.23155 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65783, and has been disclosed in patent CN 120485044 A; Strain 794 is Pediococcus pentosaceus / Lactococcus lactis: XZFMCC107.2307; Strain 795 is Pediococcus pentosaceus / Lactococcus lactis: XZFMCC107.2308; Strains 793, 794, and 795 were isolated from the Institute of Agricultural Product Development and Food Science, Tibet Academy of Agricultural and Animal Husbandry Sciences; α-Glucosidase (α-GC) Activity Assay Kit (Catalog No.: BC2550), α-Amylase (α-AL) Activity Assay Kit (Catalog No.: BC4570), Acetylcholinesterase (AChE) Activity Assay Kit (Catalog No.: BC2020), Total Antioxidant Capacity (T-AOC) Assay Kit (Catalog No.: BC1315, FRAP Method), ABTS Free Radical Scavenging Capacity Assay Kit (Catalog No.: BC4770), Superoxide Anion Scavenging Capacity Assay Kit (Catalog No.: BC1415), Hydroxyl Free Radical Scavenging Capacity Assay Kit (Catalog No.: BC1325), DPPH Free Radical Scavenging Capacity Assay Kit (Catalog No.: BC4750) (Solebo Biotech Co., Ltd.)

[0031] 2. Main instruments and equipment Multifunctional grinder (2500A, Yongkang Hongtaiyang Electromechanical Co., Ltd.); Analytical balance (BS223S, Beijing Sartorius Instrument Systems Co., Ltd.); Full-wavelength microplate reader (K6600-B, Beijing Kai'ao Technology Development Co., Ltd.); Thermostatic magnetic stirring water bath (HJ-A6, Shandong Oulaibo Instrument Co., Ltd.); Thermostatic shaker (SW23, JULABO); Thermostatic incubator (BPH-9162, Shanghai Yiheng Scientific Instrument Co., Ltd.); Ultraviolet spectrophotometer (UV1700PC, Shanghai Aoxi Scientific Instrument Co., Ltd.); Benchtop low-speed centrifuge (TD-6M, Sichuan Shuke Instrument Co., Ltd.); Rotary water bath thermostatic shaker (SHY-2, Changzhou Jintan Dadi Automation Instrument Factory); Ultrapure water production system (UPH-II-10T, Sichuan Youpu Ultrapure Technology Co., Ltd.); Pipettes of various volumes (Eppendorf).

[0032] 3. Methods for determining relevant indicators of Polygonum bulbiferum fermentation broth involved in the following technical solutions. (1) α-glucosidase (α-GC) activity assay was performed in accordance with the instructions of the α-glucosidase (α-GC) activity assay kit.

[0033] A standard curve was plotted by diluting a 5 µmol / mL p-nitrophenol solution with distilled water to prepare standard solutions of 100, 50, 25, 12.5, 6.25, and 0 nmol / mL. The equation of the standard curve is as follows: y =0.0032 x -0.0123, R 2 ==0.999 9. Dilute the product 5 times, then dilute it proportionally according to the kit instructions and add it to a 96-well plate. Mix thoroughly, incubate at a constant temperature for 0.5 h, let stand at room temperature for 2 min, and then measure the absorbance at 400 nm. ΔA is determined according to the standard curve ( y ΔA determination) is substituted into the formula to calculate the sample mass concentration ( x (nmol / mL). Calculated based on cell number, the formula is shown in (1): α-GC activity (U / 10) 4 cell) = ( x × V (Total) ÷ ​​(1000 ×) V sample ÷ V Total sample size) ÷ T = 0.02 x F (1); V Total reaction volume: 1 mL; V Sample: Add 0.1 mL of the sample volume to the reaction system; V Total sample: 1 mL of extraction solution added; T: reaction time, 0.5 h. F Dilution factor: 5.

[0034] (2) The α-amylase (α-AL) activity was determined according to the instructions of the α-amylase (α-AL) activity test kit (iodine-starch colorimetric method).

[0035] A 1 mg / mL starch standard solution was diluted with distilled water to prepare standard solutions of 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, and 0.0015625 mg / mL. The solutions were measured at 570 nm, and a standard curve was plotted. The equation for the standard curve is: y =1.7492 x +0.0039, R 2 ==0.996 1.

[0036] Dilute the product 5-fold, then dilute proportionally according to the kit instructions and add to a 96-well plate. Mix thoroughly and measure the absorbance at 570 nm. ΔA is determined based on the standard curve. y ΔA measurement) Substitute into the formula to calculate (x (mg / mL). Calculated based on liquid volume, the formula is shown in (2): Unit definition: One unit of enzyme activity is defined as 1 mg of starch consumed per mL of liquid per minute.

[0037] α-Amylase activity (U / mL) = x × V sample ÷ V sample ÷ T F =0.1× x F (2); V Sample volume added to the reaction system: 0.25 mL; V Total sample volume; T: reaction time, 10 min; F Dilution factor: 5.

[0038] (3) Acetylcholinesterase (AChE) activity assay was performed in accordance with the instructions of the Acetylcholinesterase (AChE) activity assay kit.

[0039] For direct product determination, add the sample and reagent II to the 96-well plate in sequence according to the kit instructions, react accurately in a 37°C water bath for 5 min, then add the remaining reagents according to the kit instructions, mix thoroughly, and measure the absorbance at 412 nm after standing for 2 min. Calculate the AchE activity of the sample according to formula (3): Definition of an activity unit: 1 nmol TNB produced per minute per milliliter of serum is defined as 1 enzyme activity unit.

[0040] AchE enzyme activity (U / mL) = [ΔA ÷ (ε × d) × V Color rendering ×10 9 ]÷( V Sample × V Shangqing ÷ V (enzyme-catalyzed) ÷ T = 2255 × ΔA (3); ε: TNB molar extinction coefficient, 13.6 × 10⁻⁶ 3 L / mol / cm; d: optical path length of the cuvette, 1 cm; Vdevelopment: total volume of the colorimetric reaction system, 1 mL = 0.001 L; 10 9 Unit conversion factor, 1 mol = 1 × 10 9 nmoL; V Enzymatic reaction: Total volume of the enzymatic reaction, 0.23 mL; V Supernatant: Take 0.05 mL of the supernatant. VSample: Add sample volume, 0.03 mL; T: Reaction time, 5 min.

[0041] (4) Determination of antioxidant index of Polygonum bulbil fermentation broth Total antioxidant capacity (T-AOC) was determined according to the instructions of the Total Antioxidant Capacity (T-AOC) Detection Kit.

[0042] Prepare FeSO4 standard solution according to the kit instructions and plot the standard curve, based on Fe 2+ Final concentration ( x , μ mol / mL) and absorbance ΔA standard ( y Establish a standard curve. The equation of the standard curve is: y =11.136 x -0.0041, R 2 =0.9979.

[0043] Dilute the test solution 30-fold (D=30) with the kit buffer, mix thoroughly, and label as diluted test solution. Add all reagents to the 96-well plate according to the instructions, mix thoroughly, and incubate at room temperature for 10 min. Measure the absorbance at 593 nm. Determine ΔA based on the standard curve. y Substitute into the formula to calculate the sample concentration. x (μmol / mL). Calculated based on sample mass, the calculation formula is shown in (4): Total antioxidant capacity (μmol / g) = x × V Total ÷ ( V sample ÷ V Sample total × W ) = 34 × X ÷ W (4); In the formula: V Total sample volume: 1 mL of the extraction solution added; V Total reaction volume: 0.204 mL; V Sample volume in the reaction: 0.006 mL; W : Sample quality, g.

[0044] (5) The hydroxyl radical (·OH) scavenging rate was determined according to the instructions of the hydroxyl radical scavenging ability test kit.

[0045] Take samples of Polygonum viviparum fermentation broth and determine them directly or after dilution by 5-fold or 10-fold according to the optimized process. Add each reagent sequentially to a 1.5 mL EP tube according to the kit instructions. Incubate at 37℃ for 60 min. After the reaction, centrifuge at 10000 r / min for 10 min at room temperature. Take 200 μL of the incubator. μ The absorbance of the supernatant at 536 nm was measured in each well of a 96-well plate. ·OH scavenging rate ( D Calculate as shown in equation (5).

[0046] D =( A Measurement - A (Comparison) / ( A blank- A (5) × 100%.

[0047] (6) ABTS + • The scavenging rate was determined according to the instructions of the ABTS Free Radical Scavenging Ability Test Kit.

[0048] The fermentation broth of *Polygonum viviparum* was taken for direct measurement or diluted 5-fold according to the process optimization progress. Reagents were added as per the kit instructions, and the mixture was allowed to stand at room temperature in the dark for 6 minutes. The absorbance at 405 nm was then measured. (ABTS) + • Clearance rate ( D ) Calculate the reference formula (6).

[0049] D=[ A blank-( A Measurement - A (Comparison) A Blank × 100% (6).

[0050] (7) Superoxide anion (O 2- • The free radical scavenging rate was determined according to the instructions of the superoxide anion scavenging ability test kit.

[0051] Take the sample to be tested and measure it directly according to the kit instructions or dilute it 5 times before measurement. Add each reagent to the 96-well plate according to the order and steps in the instructions, gently shake to mix, and place it in the microplate reader. Complete the reaction process according to the instructions. After the reaction, measure the absorbance value of each well at a wavelength of 530 nm. The calculation formula is as follows: Superoxide anion scavenging rate (%) = ( A blank- A (Measurement) ÷ A Blank × 100% (7).

[0052] (8) The DPPH free radical scavenging rate was determined according to the instructions of the DPPH free radical scavenging ability test kit.

[0053] Take the sample to be tested, and take 100 μL of each sample. μ Add 900 ml of each sample solution to L. μ L extract, vortex to mix, centrifuge at 10000 rpm for 10 min at room temperature, collect the supernatant, and set aside for testing. Add reagents according to the kit instructions, and incubate at room temperature in the dark for 30 min. After the reaction, measure the absorbance of each well at 515 nm using a microplate reader. DPPH free radical scavenging rate ( D The calculation formula is as shown in equation (8): D =[[ A blank-( A Measurement - A (Compare) ÷ A [Blank] × 100% (8).

[0054] (9) Electronic nose analysis The electronic nose analysis was performed using the direct headspace sampling method. 10 mL of product was pipetted into a 30 mL screw-cap glass vial and incubated in a 30℃ water bath for 40 min before testing. The sampling interval was 1 s, washing for 60 s, zero-point adjustment for 10 s, pre-sampling for 5 s, and testing for 120 s. The injection flow rate was 300 mL / min. Each treatment group was measured in triplicate, and the average value was used for data analysis. Detailed information on each sensor of the electronic nose is shown in Table 1.

[0055] Table 1. Characteristics of the PEN3 sensor array

[0056] (10) Electronic tongue measurement Electronic tongue analysis was performed using the TS-5000Z taste analysis system. The fermented broth of Polygonum bulbiferum, stored at 4℃, was removed and allowed to stand at room temperature for 12 hours. Approximately 30 mL of sample was then placed in a dedicated sample cup for the electronic tongue and tested. Each treatment group was measured in triplicate. The raw data were converted into taste values ​​using the electronic tongue's built-in data processing software and then exported for analysis. Detailed information on each sensor of the electronic tongue is shown in Table 2.

[0057] Table 2 Characteristics of the Electronic Tongue Sensor Array

[0058] 4. Data Statistical Analysis Methods In GraphPad Prism (version 6.0, GraphPad Software Inc., San Diego, CA), one-way ANOVA was used to analyze the significance of differences in means between groups. Experimental data are expressed as mean ± standard deviation. P(0.01) indicates a highly significant difference, while P(0.05) indicates a significant difference.

[0059] Example 1 Single-factor experiment on the preparation of fermented beverage from Polygonum bulbiferum 1. Process flow for preparing fermented beverages from Polygonum bulbiferum: Dried Polygonum bulbiferum fruit (dried product) → Washing → Drying → Grinding → Sieving → Adding ultrapure water → Mixing → Autoclaving (conditions: 121℃, 15 min) → Cooling → Inoculation → Mixing → Constant temperature fermentation → Centrifugation → Aseptic filling → Sterilization (72℃, 30 min) → Refrigeration → For later use 2. Key Operating Points The Polygonum bulbiferum fruit samples used in the following tests were tested by Shanghai Huace Testing Technology Co., Ltd. The results were as follows: Energy 1526 kJ / 100g; Protein 12.6g / 100g; Fat 0.7g / 100g; Carbohydrates 75.63g / 100g; Calcium 99.3mg / 100g; Ash 2.7g / 100g; Moisture 8.37g / 100g; Crude polysaccharide (calculated as glucose) 4180mg / 100g; Total flavonoids (calculated as rutin) 8.08g / 100g; Total triterpenes 0.328g / 100g; Total saponins (calculated as ginsenoside Re) 2.39%.

[0060] Raw material processing: Clean the dried Polygonum bulbils (dried product) to remove surface dirt and other impurities, dry the surface moisture, grind into powder using a multi-functional pulverizer, pass through a 60-mesh sieve, and collect the sieved material to obtain Polygonum bulbils powder for later use.

[0061] Sample preparation: Accurately weigh 10 g of Polygonum bulbiferum fruit powder into a fermenter, add ultrapure water according to the material-liquid ratio (1 g: 10 mL, 1 g: 20 mL, 1 g: 30 mL, 1 g: 40 mL, or 1 g: 50 mL), mix well to obtain a Polygonum bulbiferum-water mixture system.

[0062] Sterilization: The bulbils-water mixture was sterilized by high-pressure steam at 121°C for 15 minutes and then cooled to room temperature in a clean bench for later use.

[0063] Strain activation: Commercially available Lactobacillus plantarum and laboratory-preserved strains 793, 794 and 795 were pre-cultured in MRS broth under the following conditions: 150 rpm, 36℃, and 48 h, to obtain bacterial suspensions of each strain.

[0064] Add bacterial strains: Measure the OD of each bacterial culture. 600 Adjust the bacterial concentration of each strain to 1×10⁻⁶. 8CFU / mL was used to obtain seed solutions for each strain for inoculation. Depending on the amount of seed solution added (e.g., 1%, 1.5%, 2%, 2.5%, or 3% (V / V), a certain amount of bacterial solution was centrifuged (4000 r / min, 10 min), the supernatant was discarded, and the solution was washed twice with sterile physiological saline. This was then added to the sterile trial product for fermentation. The fermentation time could be 24, 48, 72, 96, or 120 h, and the fermentation temperature could be 20, 24, 28, 32, or 36℃. The fermentation process was accompanied by shaking at a speed of 150 rpm. After fermentation, the fermentation product, or fermentation product, was obtained.

[0065] Centrifugation: Centrifuge the fermentation product at 4000 r / min for 10-15 min to separate the sample supernatant.

[0066] Filling: The sample supernatant is aseptically filled in a clean bench.

[0067] Sterilization: The bottled samples were sterilized in a 72℃ water bath for 30 min and stored at 4℃ for later use to obtain the Polygonum bulbil fermentation broth. The Polygonum bulbil fermentation broth measured or used in subsequent experiments was the sterilized Polygonum bulbil fermentation broth after bottling.

[0068] 3. Single-factor experimental design Using different bacterial strains, material-to-liquid ratios, strain dosage, fermentation time, and fermentation temperature as single factors, and α-glucosidase (α-GC), α-amylase (α-AL), and acetylcholinesterase (AChE) activities as the main indicators, and total antioxidant capacity (T-AOC) and free radical scavenging capacity as auxiliary indicators, the hypoglycemic and hypolipidemic activity of Polygonum viviparum fermentation broth was optimized. The single-factor experimental factors and level design are shown in Table 3.

[0069] Table 3. Factors and Levels in Single-Factor Experiments

[0070] Note: The mixed strains are seed culture of strain 794 (1×10⁻⁶). 8 CFU / mL), 793 strain seed culture (1×10) 8 CFU / mL), 795 strain seed culture (1×10) 8 CFU / mL), commercially available Lactobacillus plantarum strain seed solution (1×10) 8 The mixture (CFU / mL) was prepared by mixing in a volume ratio of 1:1:1:1.

[0071] Experimental Procedure: Accurately weigh 10 g of Polygonum viviparum powder into an Erlenmeyer flask, add pure water at a ratio of 1 g: 10 mL, mix well, and obtain the sample; sterilize the sample by high-pressure steam at 121℃ for 15 min, and cool it to room temperature in a clean bench to obtain a sterile trial product for later use; commercially available Lactobacillus plantarum and laboratory-preserved strains (793, 794, 795) were pre-cultured in MRS broth (150 rpm, 36℃) for 48 h; the OD of the bacterial culture was measured. 600 Adjust the concentration of each bacterial culture to 1×10⁻⁶. 8 CFU / mL was used to obtain seed cultures of each strain, and mixed strain seed cultures were prepared using these seed cultures. A certain amount of bacterial culture was centrifuged (4000 r / min, 10 min) at a seed culture addition rate of 1% (V:V volume ratio), and the supernatant was discarded. After washing twice with sterile physiological saline, different strains were added to the aseptically prepared product for fermentation for 72 h (150 rpm, 36℃). A blank control group was set up without any added strains, a positive control group was inoculated with commercially available *Lactobacillus plantarum*, and a mixed strain group was inoculated. After fermentation, the fermentation product was centrifuged at 4000 r / min for 10-15 min, and the sample supernatant was aseptically packaged in a clean bench. The packaged samples were sterilized in a 72℃ water bath for 30 min and stored at 4℃ for later use.

[0072] The effects of different bacterial strains on α-GC, α-AL, AChE properties and antioxidant-related indicators of Polygonum viviparum fermentation broth were investigated, and the optimal fermentation strain was determined based on the experimental results. Each experiment was conducted in triplicate, and other single-factor experiments were performed using the same method, employing the controlled single-variable method.

[0073] Specifically: (1) Single-factor experiments with different strains. The variable factors were strains (blank group without strains, 794, 793, 795, mixed strains, commercially available Lactobacillus plantarum). The fixed conditions were: material-to-liquid ratio 1g:10mL; strain addition amount 1.0%; fermentation time 72h; fermentation temperature 36℃.

[0074] (2) Single-factor experiments with different material-liquid ratios: the variable factor was the material-liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50 g / mL), and the fixed factors were 793 strains; the strain addition amount was 1.0%; the fermentation time was 72h; and the fermentation temperature was 36℃.

[0075] (3) Single-factor experiments with different strain addition amounts: the variables were strain addition amounts of 1.0%, 1.5%, 2.0%, 2.5%, and 3.0% (V / V), and the fixed factors were strain 793; material-liquid ratio of 1:20 g / mL; fermentation time of 72 h; and fermentation temperature of 36 ℃.

[0076] (4) Single factor for different fermentation times, with the variable being fermentation time (24, 48, 72, 92, 120 h) and the fixed factors being strain 793; material-liquid ratio of 1:20 g / mL; 3.0% strain addition; and fermentation temperature of 36℃.

[0077] (5) Single-factor experiments at different fermentation temperatures: the variable was the fermentation temperature (20, 24, 28, 32, 36℃), and the fixed factors were strain 793; the material-liquid ratio of 1:20 g / mL; the amount of 3.0% strain added; and the fermentation time of 24 h.

[0078] 4. Results of single-factor experiments (1) Results of single-factor experiments on different bacterial species Results of different bacterial strains on the hypoglycemic, hypolipidemic and antioxidant indicators of Polygonum viviparum fermentation broth are as follows: Figure 1 As shown in the figure, compared with no added bacteria, strains 793, 794, and 795 significantly reduced α-GC activity, and there was no significant difference in α-GC (α-glucosidase) activity compared with commercially available *Lactobacillus plantarum*. Figure 1 (A). Compared with the blank control group, there was no significant difference in the activities of α-AL (α-amylase) and AChE (acetylcholinesterase) in the fermentation broth of different Polygonum viviparum strains. Figure 1 (A and B). The antioxidant indices of different *Polygonum viviparum* strains differed significantly from the blank control group. The fermentation broth of strain 793 showed the highest ·OH (hydroxyl radical) scavenging rate, which was not significantly different from that of commercially available *Lactobacillus plantarum* fermentation broth. The DPPH and O2 levels of different strains of *Polygonum viviparum* fermentation broth were also significantly different. 2- • There was no significant difference in free radical scavenging rate. The 794 strain, mixed strain, and commercially available *Lactobacillus plantarum* strain showed strong total antioxidant capacity, with no significant difference compared to strains without addition. The 793, 795, and mixed strains exhibited good ABTS (antioxidant activity levels). + • The clearance rate was significantly higher than that of commercially available Lactobacillus plantarum. Figure 1 (C). Therefore, based on its good hypoglycemic and lipid-lowering effects and certain antioxidant effects, strain 793 was initially selected as the strain for further optimization of the Polygonum viviparum fermentation broth process.

[0079] (2) Results of single-factor experiments with different feed-to-liquid ratios The effects of different feed-to-liquid ratios on the blood sugar, lipid-lowering, and antioxidant indicators of Polygonum bulbiferum fermentation broth are as follows: Figure 2 As shown. By Figure 2 It can be seen that with the increase of the material-to-liquid ratio, the α-GC activity of Polygonum bulbiferum fermentation broth showed a trend of first remaining unchanged, then increasing, then decreasing, and finally remaining unchanged. When the material-to-liquid ratio was 1:10, 1:20, 1:40, and 1:50, the α-GC activity did not change significantly, and the α-GC activity was strongest at a material-to-liquid ratio of 1:30; however, the α-AL activity did not change significantly with the increase of the material-to-liquid ratio. Figure 2(A). The activity of AchE also gradually decreased with the material-liquid ratio. When the material-liquid ratio was 1:10 and 1:20, 1:20 and 1:30, and 1:30 and 1:40, the activity of AchE did not change significantly. When the material-liquid ratio was 1:10 and 1:30, and 1:30 and 1:50, the activity of AchE decreased significantly, and the activity of AchE was lowest at a material-liquid ratio of 1:50. Figure 2 (B). The antioxidant index also showed a certain trend with the change of the feed-to-liquid ratio. ABTS + • The removal rate initially increases with the feed-to-liquid ratio, then decreases significantly, and finally increases again. 2- The free radical scavenging rate initially remained constant, then decreased significantly, and finally remained constant with increasing feed-to-liquid ratio. The DPPH and ·OH scavenging rates initially decreased significantly, then remained constant, then decreased significantly again, and finally remained constant with increasing feed-to-liquid ratio. However, the total antioxidant capacity initially decreased significantly with increasing feed-to-liquid ratio and then remained constant. Figure 2 (C). Overall, the antioxidant capacity of *Polygonum viviparum* fermentation broth gradually decreased with increasing material-to-liquid ratio. This may be because as the material-to-liquid ratio increases, the concentration of *Polygonum viviparum* fermentation broth decreases, resulting in fewer effective antioxidant active ingredients and thus a gradual decrease in antioxidant capacity. Therefore, based on the significant changes in blood sugar and lipid-lowering and antioxidant indicators with increasing material-to-liquid ratio, material-to-liquid ratios of 1:10, 1:30, and 1:50 were determined as three levels for further optimization of the *Polygonum viviparum* fermentation broth process. Considering both blood sugar and lipid-lowering effects and antioxidant effects, the material-to-liquid ratio for the subsequent single-factor experiments was determined to be 1:20 g / mL.

[0080] for Figure 1 and Figure 2 Explanation of the differences in the median values: In addition to controllable factors, environmental factors, bacterial activity and density factors can affect the experimental results in each experiment; the determination of hydroxyl radicals can be referred to the method description. In this experiment, because the sample was directly measured, the hydroxyl radical scavenging rate was as high as 80% or more; when measuring the hydroxyl radical scavenging rate of the material-to-liquid ratio, the same batch of samples should be diluted in the same proportion before measurement, otherwise the data will exceed 100% or be negative.

[0081] (3) Results of single-factor experiments with different amounts of bacterial strains The effects of different strain addition amounts on the blood sugar, lipid-lowering and antioxidant indicators of Polygonum viviparum fermentation broth are as follows: Figure 3 As shown. By Figure 3 It can be seen that as the amount of inoculum added increases, the α-GC activity of the Polygonum villosa fermentation broth initially remains unchanged and then decreases. When the inoculum addition amount is 3.0%, the α-GC activity is the lowest, but the α-AL activity does not change significantly with the increase of the inoculum addition amount. Figure 3(A). AchE activity showed a trend of first decreasing, then remaining constant, then decreasing again, and finally remaining constant with increasing strain concentration. When the strain concentration was 1.0% and 1.5%, 1.5%, 2.0% and 2.5%, and 2.5% and 3.0%, AchE activity showed no significant change. When the strain concentration was 1.0% and 2.0%, and 2.0% and 3.0%, AchE activity decreased significantly. The lowest AchE activity was observed at a strain concentration of 3.0%. Figure 3 (B) The antioxidant index did not change significantly with the amount of bacteria added. Figure 3 From C, we can see that ABTS + ·、O 2- The scavenging rates of ·, DPPH, and ·OH showed no significant change with increasing inoculum dosage, while T-AOC initially remained constant and then decreased with increasing inoculum dosage. Therefore, based on the significant changes in blood sugar, lipid-lowering, and antioxidant indicators with increasing inoculum dosage, inoculum dosages of 1.0%, 2.0%, and 3.0% were determined as the three levels for further optimization of the Polygonum viviparum fermentation broth process. Since the product exhibited the best blood sugar, lipid-lowering, antioxidant, and free radical scavenging effects when the inoculum dosage was 3.0%, the inoculum dosage for the subsequent single-factor experiments was determined to be 3.0%.

[0082] right Figure 2 and Figure 3 Explanation of the differences in median values: In addition to the controllable factors, environmental factors, bacterial viability and density factors can affect the experimental results in each experiment. It cannot be guaranteed that the bacterial viability and density will be equal in each experiment. It is only possible to control the conditions of the same batch of experiments. Moreover, during the optimization process, the content of some indicators will change as the optimization conditions change.

[0083] (4) Results of single-factor experiments at different fermentation times The effects of different fermentation times on the blood sugar, lipid-lowering and antioxidant indicators of Polygonum viviparum fermentation broth are as follows: Figure 4 As shown. By Figure 4 It can be seen that with the increase of fermentation time, the α-GC activity of Polygonum bulbiferum fermentation broth showed a trend of first significantly increasing and then decreasing. The α-GC activity was lowest when the fermentation time was 24 h, and the α-GC activity was strongest when the fermentation time was 120 h. However, the α-AL activity did not change significantly with the extension of fermentation time. Figure 4 (A). AchE activity did not change significantly with increasing fermentation time. Figure 4 (B) DPPH, O 2- • The free radical scavenging rate initially remained constant, then decreased, and finally remained constant with increasing fermentation time (ABTS). + • The free radical scavenging rate initially remained constant and then decreased with increasing fermentation time; T-AOC initially increased significantly, then remained constant, and finally decreased significantly with increasing fermentation time; while the ·OH scavenging rate showed no significant change with increasing fermentation time. Figure 4(C). Therefore, based on the significant changes in blood sugar and lipid-lowering and antioxidant indicators with the amount of strain added, 24, 72, and 120 h were determined as the three levels for optimizing the fermentation time of the Polygonum viviparum fermentation broth. Since the product exhibited the best blood sugar and lipid-lowering, antioxidant, and free radical scavenging abilities at 24 h of fermentation, we determined the fermentation time for the subsequent single-factor experiments to be 24 h.

[0084] right Figure 3 and Figure 4 Explanation of the differences in median values: In addition to the controllable factors, environmental factors, bacterial viability and density factors can affect the experimental results in each experiment. It cannot be guaranteed that the bacterial viability and density will be equal in each experiment. It is only possible to control the conditions of the same batch of experiments. Moreover, during the optimization process, the content of some indicators will change as the optimization conditions change.

[0085] (5) Results of single-factor experiments at different fermentation temperatures The effects of different fermentation temperatures on the glucose-lowering, lipid-lowering, and antioxidant indicators of Polygonum viviparum fermentation broth are as follows: Figure 5 As shown. By Figure 5 It can be seen that with the increase of fermentation temperature, the α-GC activity of Polygonum bulbiferum fermentation broth first decreased significantly and then remained unchanged. The α-GC activity was strongest at a fermentation temperature of 20℃, but the α-AL activity did not change significantly with the increase of fermentation temperature. Figure 5 (A). AchE activity showed a trend of first decreasing, then remaining constant, then decreasing again, and finally remaining constant with increasing fermentation temperature. At fermentation temperatures of 24℃ and 28℃, and 32℃ and 36℃, AchE activity showed no significant change. At fermentation temperatures of 20℃ and 28℃, and 28℃ and 36℃, AchE activity decreased significantly. The lowest AchE activity was observed at fermentation temperature of 36℃. Figure 5 (B) ABTS + • The clearance rate did not change significantly with increasing fermentation temperature. 2- • The scavenging rate initially remained constant and then decreased significantly with increasing fermentation temperature; the DPPH scavenging rate initially decreased significantly and then remained constant with increasing fermentation temperature; the OH scavenging rate initially increased and then remained constant with increasing fermentation temperature; while the T-AOC scavenging rate initially remained constant, then increased, then decreased significantly, and finally remained constant with increasing fermentation temperature. Figure 5 (B). Therefore, based on the significant changes in blood sugar, lipid-lowering and antioxidant indicators with fermentation temperature, 20, 28, and 36℃ were determined as the three levels for optimizing the fermentation temperature of Polygonum bulbiferum fermentation broth.

[0086] right Figure 4 and Figure 5Explanation of the differences in median values: In addition to the controllable factors, environmental factors, bacterial viability and density factors can affect the experimental results in each experiment. It cannot be guaranteed that the bacterial viability and density will be equal in each experiment. It is only possible to control the conditions of the same batch of experiments. Moreover, during the optimization process, the content of some indicators will change as the optimization conditions change.

[0087] Example 2 orthogonal experiment 1. Based on the results of single-factor experiments, fermentation broth of Polygonum viviparum was prepared using strain 793, and the material-to-liquid ratio was selected ( A ), inoculum quantity ( B ), fermentation time ( C ), fermentation temperature ( D ) 4 factors set L9 (3 4 An orthogonal experiment was conducted, with experimental factors and levels designed as shown in Table 4. The experiment was carried out according to the procedure in Example 1. Since the results of the single-factor experiment did not meet the conditions for the response surface methodology and did not exhibit a parabolic trend, the response surface methodology was not chosen to optimize the conditions.

[0088] Table 4. Factors and Levels in Orthogonal Experiments

[0089] 2. Results of the orthogonal experiment Based on the results of the single-factor experiment, the material-liquid ratio was selected ( A ), inoculum quantity ( B ), fermentation time ( C ), fermentation temperature ( D ) 4 factors L 9 (3) 4 Orthogonal experiments were conducted to further optimize the extraction process. Each group of experiments was performed in triplicate, and the average value was used for calculation. Range analysis was performed on the results. Influencing factors and levels are shown in Table 4 of the experimental methods. The results of the orthogonal experiments and analysis of variance are shown in Tables 5-8. Figure 6 .

[0090] Table 5. Results of the orthogonal experiment on Polygonum bulbiferum fermentation broth

[0091] Table 6. Analysis of variance of α-AL content in orthogonal experiments

[0092] Table 7. Analysis of variance of α-GC content in orthogonal experiments

[0093] Table 8. Analysis of variance of AChE content in orthogonal experiments

[0094] Table 5 shows that the order of factors affecting α-AL activity is as follows: A > C > D > B The order of factors affecting α-GC and AchE activity is: substrate-to-liquid ratio > fermentation time > fermentation temperature > inoculum size. A > D > B > C The optimal extraction conditions were: solid-liquid ratio > fermentation temperature > inoculum size > fermentation time. Analysis of variance showed that all four factors had a significant impact on α-AL and AchE activities (Tables 6 and 8), while the solid-liquid ratio, inoculum size, and fermentation temperature had a significant impact on α-GC activity (Table 7). The optimal extraction conditions for α-AL were... A 1 B 2 C 1 D 3(1), i.e., a material-to-liquid ratio of 1:10, an inoculum size of 2.0%, and fermentation at 36℃ for 24 h; the optimal extraction conditions for α-GC are: A 1 B 1 C 3 D 2(2), i.e., a material-to-liquid ratio of 1:10, an inoculum size of 1.0%, and fermentation at 28℃ for 120 h; the optimal extraction conditions for AchE are: A 3 B 3 C 3 D 3(3), i.e., material-liquid ratio 1:50, 3.0% inoculum, fermentation at 36℃ for 120 h.

[0095] 3. Verify the experimental results To further determine the optimal process conditions, verification experiments were conducted on three optimal conditions: (1), (2), and (3). The experimental conditions and results are shown in Table 9. Figure 7 As shown.

[0096] (1) A method for preparing Polygonum bulbil fermentation broth, comprising the following steps: Wash the dried Polygonum bulbils to remove surface dirt and other impurities, dry the surface moisture, grind them into powder using a multi-functional pulverizer, pass them through a 60-mesh sieve, and collect the sieved material to obtain Polygonum bulbils powder.

[0097] Accurately weigh 10g of Polygonum bulbiferum powder into a fermenter, add 100mL of ultrapure water at a material-to-liquid ratio of 1:10 (g / mL), mix well, and obtain a Polygonum bulbiferum-water mixed system.

[0098] The bulbils-water mixture was sterilized by high-pressure steam at 121°C for 15 minutes and then cooled to room temperature in a clean bench for later use.

[0099] The activated culture of strain 793 was pre-expanded in MRS broth under the following conditions: 150 rpm, 36℃, and 48 h, to obtain a bacterial suspension of strain 793. The OD value of the bacterial suspension of strain 793 was then measured. 600 Adjust the bacterial concentration to 1×10⁻⁶. 8 CFU / mL was used to obtain the seed culture of strain 793. At a dosage of 2% (V / V), 2 mL of the 793 strain seed culture was added, centrifuged (4000 r / min, 10 min), and the supernatant was discarded. After washing twice with sterile physiological saline, the supernatant was added to a sterile Polygonum viviparum-water mixture for fermentation. The fermentation time was 24 h, the fermentation temperature was 36℃, and the fermentation was accompanied by shaking at 150 rpm. After fermentation, the fermentation product was obtained. The fermentation product was centrifuged at 4000 r / min for 10-15 min to separate the sample supernatant. Packaging: The sample supernatant was aseptically packaged in a laminar flow hood. Sterilization: The packaged sample was sterilized in a 72℃ water bath for 30 min and stored at 4℃ for later use, yielding the Polygonum viviparum fermentation broth.

[0100] (2) A method for preparing Polygonum bulbil fermentation broth, with the same steps as (1), but with a specific material-to-liquid ratio of 1:10, an inoculum amount of 1%, a fermentation time of 120 h, and a fermentation temperature of 28 °C.

[0101] (3) A method for preparing Polygonum bulbil fermentation broth, with the same steps as (1), but with a specific material-to-liquid ratio of 1:50, an inoculum amount of 3%, a fermentation time of 120 h, and a fermentation temperature of 36 °C.

[0102] Table 9. Verification Experiment Conditions and Results

[0103] The results showed that under the optimal conditions (1), there was no significant difference in the α-AL activity of Polygonum bulbiferum fermentation broth ( Figure 7 Under optimal conditions (2), the α-GC activity of Polygonum bulbiferum fermentation broth was the lowest, and there was no significant difference between it and the α-GC activity under optimal conditions (1). Furthermore, the α-GC activity under conditions (1) and (2) was significantly lower than that under (3). Figure 7 Under optimal conditions (3), the AchE activity of Polygonum bulbiferum fermentation broth was the lowest, and there was no significant difference from the AchE activity under condition (1). The AchE activity under conditions (1) and (3) was lower than that under condition (2). Figure 7 (A). Under optimal conditions (1), the ·OH scavenging rate and T-AOC of Polygonum bulbiferum fermentation broth were significantly higher than those under optimal conditions (2), and (2) was higher than that under optimal conditions (3); DPPH and O 2- • The clearance rate was not significantly different from that under optimal condition (2), but was significantly higher than that under optimal condition (3); ABTS +• The clearance rate was not significantly different from that of the optimal condition (2), but was significantly higher than that of the optimal condition (3), while the clearance rate of the optimal condition (2) was ABTS. + • The clearance rate was not significantly different from that under optimal condition (3). Figure 7 In summary, under the optimal conditions (1), namely a material-to-liquid ratio of 1:10, an inoculum amount of 2.0%, and fermentation at 36℃ for 24 h, the activities of α-AL, α-GC, and AchE in the fermentation broth of Polygonum bulbiferum are the lowest, and at this time, the total antioxidant capacity and free radical scavenging rate of the fermentation broth of Polygonum bulbiferum are the strongest.

[0104] 4. The fermentation broth of Polygonum bulbiferum prepared under the conditions in (1) of section 3 was analyzed by electronic nose.

[0105] Electronic nose analysis was performed on three types of samples.

[0106] Sample 1 is a non-fermented Polygonum bulbil stock solution. The specific preparation method is as follows: Polygonum bulbil powder that has passed through a 60-mesh sieve is prepared (method is the same as 3 above). 10g of Polygonum bulbil powder is weighed into a fermenter, and 100mL of ultrapure water is added at a material-to-liquid ratio of 1:10 (g / mL). The mixture is mixed to obtain a Polygonum bulbil-water mixed system. The mixture is extracted at a constant temperature of 36℃ for 24 h to obtain the Polygonum bulbil stock solution.

[0107] Sample 2 is a Polygonum bulbil fermentation broth prepared using the conditions in (1) of section 3.

[0108] Sample 3 is a cherry and raspberry flavored bulbil fermentation broth, which is a compounded sample of Sample 2. The ratio of raspberry concentrate to Sample 2 to purified water is 1:5:4 (volume ratio). The raspberry concentrate was newly purchased from Kro Foods (China) Co., Ltd.

[0109] Samples 1-3 were subjected to electronic nose flavor compound determination, and the results are as follows: Figures 8-9 As shown in the figure. The PCA results of different electronic nose samples are illustrated in the figure below. Figure 10 As shown in the figure. Among them, Fermented Original Flavor is sample 2; Fermented Raspberry Flavor is sample 3; and Unfermented is sample 1.

[0110] From radar chart ( Figure 8 As shown in section A), sensors W5S, W1S, W1W, W2S, and W2W exhibit significant responses, indicating that the three samples contain relatively high levels of nitrogen oxides, short-chain alkanes, inorganic sulfides, ethanol, aromatic components, and organic sulfides. These six types of compounds are also a significant reason for the different odors among the three samples. A bar chart analysis of each sensor yielded the following results: Figure 8 B and Figure 9The values ​​C~K are shown in the diagram. Sample 3, the raspberry-flavored Polygonum bulbil fermentation broth, was compared with samples W1C, W3C, W6S, W5C, W1S, and W3S. Figure 8 B, and Figure 9 The highest response values ​​were found for D, E, F, G, and K, indicating that sample 3 contained a high amount of aromatic and alkane compounds. Sample 2, the original flavor Polygonum bulbil fermentation broth, showed the highest response values ​​for W5S, W1W, W2S, and W2W (…). Figure 9 The highest response values ​​for C, H, I, and J indicate that sample 2 contains more nitrogen oxides, inorganic sulfides, ethanol, aromatic components, organic sulfides, and alkanes. Sample 1, the non-fermented blank group, only has slightly higher response values ​​for W1C and W6S than sample 2. Figure 8 B, and Figure 9 The response values ​​of the samples in the middle (E) were lower than those of the other sensors in the samples 2 and 3, indicating that the hydrogen content in the sample 1 may be slightly higher than that in the sample 2, but the other flavor substances were lower than those in the samples 2 and 3.

[0111] Principal component analysis (PCA) is a statistical method that transforms a set of potentially correlated observed variables into linearly uncorrelated variables (i.e., principal components) through orthogonal transformation. PCA was performed on the response values ​​of the electronic nose sensor for three samples, and the results are as follows: Figure 10 As shown, the contribution rates of PC1 and PC2 were 82.1% and 17.0%, respectively, with a cumulative contribution rate of 99.1%. All data were within the 95% confidence interval, indicating good data that effectively reflects the sample organization information. Furthermore, the groups were effectively separated, indicating that the three samples could be significantly distinguished and that the flavor substances of the electronic nose were significantly different.

[0112] 5. Electronic tongue flavor analysis was performed on the three types of samples in section 4, and the results are as follows: Figures 11-12 As shown in the figure. The PCA results analysis of the electronic tongue for the three types of samples are shown in the figure below. Figure 13 As shown.

[0113] Simultaneously, electronic tongue flavor analysis was performed on the three samples, and the results are as follows: Figures 11-12 As shown in the radar chart ( Figure 11 It can be seen that the electronic tongues of the three samples are relatively rich in flavor compounds. One-way ANOVA of the electronic tongues of different samples shows that sample 3 has the highest levels of sourness and sweetness. Figure 12 The levels of B and J in the sample were higher than those in samples 1 and 2. Sample 1, the non-fermented blank group, had higher levels of bitterness, astringency, and aftertaste. Figure 12 The levels of C and E in the sample were higher than those in samples 2 and 3. Sample 2, the original fermentation broth of Polygonum bulbiferum, had the highest saltiness. Figure 12 (I). There was no significant difference in astringency and richness among the three samples. Figure 12(D, H). Furthermore, there was no significant difference between Sample 1 and Sample 2 in terms of sourness and sweetness, and both were significantly lower than those of Sample 3. Figure 12 Samples B and J showed no significant differences in bitterness, aftertaste, and umami, with both being significantly better than sample 3 in these aspects. Figure 12 (F, G).

[0114] PCA was performed on the taste values ​​of the three samples, and the results are as follows: Figure 13 As shown, the contribution rates of PC1 and PC2 were 92.1% and 6.8%, respectively, with a cumulative contribution rate of 98.9%. All data points were within the 95% confidence interval, indicating good data quality and effective reflection of sample organization information. Samples 1 and 2 largely overlapped, indicating that the two samples had roughly the same taste. However, Sample 3 was clearly distinguishable from Samples 1 and 2, indicating that Sample 3, the raspberry-flavored Polygonum bulbil fermented broth, had a significantly different taste from the original Polygonum bulbil fermented broth.

[0115] This invention relates to a plant-based beverage product developed using Tibetan Polygonum villosa fruit as raw material and fermented with locally sourced Tibetan strains selected independently. After optimization of the processing technology, the α-glucosidase activity can be significantly reduced from 1.24 U / 10⁻⁶. 4 The cell count decreased to 0.54 U / 104 cells, with enzyme activity decreasing by approximately 62.9%. Acetylcholinesterase activity decreased from 242.2 U / mL to 35.4 U / mL, a decrease of approximately 85.4%. Total antioxidant capacity increased from 66.3 μmol / mL to 88.6 μmol / mL, an increase of approximately 33.6%. Furthermore, the optimized product significantly improved the scavenging rate of free radicals such as superoxide anions and hydroxyl radicals, and the product showed a good trend in lowering blood sugar and lipids.

[0116] Different samples exhibited different flavor compounds. Sample 3, the raspberry-flavored Polygonum bulbil fermented broth, contained more aromatic and alkane compounds. Sample 2, the original Polygonum bulbil fermented broth, contained more nitrogen oxides, inorganic sulfides, ethanol, aromatic components, organic sulfides, and alkane compounds. Sample 1, the unfermented control sample, may have slightly higher hydrogen content than Sample 2, but its other flavor compounds were lower than those of Samples 2 and 3. Furthermore, Sample 3 had more pronounced sour and sweet tastes, Sample 1 had more pronounced bitter and astringent aftertastes, while Sample 2 had the strongest salty taste.

[0117] 6. Sample 2, namely the fermentation broth of Polygonum bulbiferum, was sent to SGS-CSTC Standards Technical Services (Qingdao) Co., Ltd. for testing. The results are shown in Tables 10-14.

[0118] Table 10 Sensory Detection Results Table 11 Microbial Detection Results Table 12 Physicochemical test results (1) Table 13 Physicochemical test results (1) Note: The conversion factor for nitrogen to protein is 6.25.

[0119] Table 14 Calculation Results Note: In the table above, ND indicates not detected; # indicates that the test item is not within the scope of CNAS accreditation of this laboratory; This indicates that the testing was performed by the laboratory of SGS-CSTC Standards Technical Services (Tianjin) Co., Ltd.

[0120] Example 3 Hypoglycemic Agent Fermentation Broth as a Blood Sugar Lowering Experiment for Hypoglycemic Agent Fermented Beverage 1. Test materials Test sample: Polygonum bulbiferum fermentation broth from Sample 2 in Example 2. The solvent was MIN6 cell culture medium, prepared by serial dilution according to the specific experimental concentration requirements.

[0121] 2. Experimental cells MIN6 mouse islet tumor cells. The experimental cells were obtained from Jiangsu Kaiji Biotechnology Co., Ltd., and cultured and passaged by Huante Mammalian Technology Center.

[0122] 3. Major Instruments, Consumables and Reagents Micropipette (Eppendorf, Germany); Biosafety cabinet (HFsafe-1500LC, Likang Biomedical Technology Holding Co., Ltd., China); RNase bag tips (EXTRAGENE, USA); Microplate reader (Multiskan FC, Thermo, USA).

[0123] Sterile KRBB buffer (XYL6560, Shanghai Xinyu Biotechnology Co., Ltd.); MIN6 cell culture medium (CM-0674, Wuhan Pronosei Biotechnology Co., Ltd.); Nitric oxide content assay kit (JL-T1270, Shanghai Future Industry Co., Ltd.); Mouse insulin assay kit (JL11459, Shanghai Future Industry Co., Ltd.); Mouse glucose transporter 4 assay kit (JL20505, Shanghai Future Industry Co., Ltd.); Naringenin (CAS: 480-41-1, purity ≥97%, catalog number: N164488-5g, purchased from Aladdin).

[0124] 4. Experimental Methods (1) Cell resuscitation and passage preparation After thawing, cryopreserved MIN6 cells were seeded into T25 culture flasks. Cells were passaged twice and sufficient cells were collected for subsequent assays.

[0125] (2) Evaluation experiment on the efficacy of improving glucose metabolism and repairing oxidative damage A. The experiment was divided into 6 groups: normal control group, model control group, positive control group, low-dose experimental group, medium-dose experimental group, and high-dose experimental group. Each group was tested in 6 replicates.

[0126] B. Treatment of the normal control group: MIN6 cells grown to the logarithmic growth phase were digested, counted, and prepared into a 1×10⁻⁶ concentration using complete culture medium. 5 Cell suspension of cells / mL, at 1×10 4 MIN6 cells / well were seeded into 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then completely replaced with MIN6 cell-specific medium, and the cells were cultured for another 12 hours. After the second 24 hours, the culture medium was completely replaced again with MIN6 cell-specific medium, and the cells were cultured for another 24 hours. The supernatant of MIN6 cells from the normal control group was collected into 1.5 mL sterile centrifuge tubes, and NO and GLU4 levels were measured according to the kit instructions (three replicates per group for each indicator).

[0127] C. Treatment of the model control group: MIN6 cells grown to the logarithmic growth phase were digested, counted, and prepared into a 1×10⁻⁶ concentration using complete culture medium. 5 Cell suspension of cells / mL, at 1×10 4 MIN6 cells / well were seeded into 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then completely replaced with MIN6 cell-specific medium containing 200 μg / mL STZ, and cultured for another 12 hours. After the second culture, the culture medium was replaced again with MIN6 cell-specific medium containing 200 μg / mL STZ, and cultured for another 24 hours. The supernatant of MIN6 cells from the model control group was collected into 1.5 mL sterile centrifuge tubes, and NO and GLU4 levels were measured according to the kit instructions (three replicates per group for each indicator).

[0128] D. Treatment of the positive control group: MIN6 cells grown to the logarithmic growth phase were digested, counted, and prepared into a 1×10⁻⁶ concentration using complete culture medium. 5 Cell suspension of cells / mL, at 1×10 4MIN6 cells were seeded per well in 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then completely replaced with MIN6 cell-specific medium containing 200 μg / mL STZ, and cultured for another 12 hours. After the second culture, the culture medium was replaced again. For the positive control group, MIN6 cell-specific medium containing 200 μg / mL STZ and 25 μM naringenin was added, and the cells were cultured for another 24 hours. The supernatant of the MIN6 cells in the positive control group was collected into 1.5 mL sterile centrifuge tubes, and NO and GLU4 levels were measured according to the kit instructions (three replicates per group for each indicator).

[0129] E. Treatment of low, medium, and high dose experimental groups: MIN6 cells grown to the logarithmic growth phase were digested, counted, and prepared into a 1×10⁻⁶ concentration using complete culture medium. 5 Cell suspension of cells / mL, at 1×10 4 MIN6 cells / well were seeded into 96-well cell culture plates and incubated at 37°C and 5% CO2 for 24 hours. The culture medium was then completely replaced with MIN6 cell-specific medium containing 200 μg / mL STZ, and cultured for another 12 hours. After the second culture, the culture medium was replaced again. For the low, medium, and high dose experimental groups, MIN6 cell-specific medium containing 200 μg / mL STZ and 2.5%, 5%, and 10% concentrations of plant fruit fermentation broth (Polygonum bulbiferum fermentation broth) was added, and the cells were cultured for another 24 hours. The supernatant of MIN6 cell culture from each experimental group was collected into 1.5 mL sterile centrifuge tubes, and NO and GLU4 levels were detected according to the kit instructions (three replicates per group for each indicator).

[0130] (3) Evaluation experiment on improving insulin function A. The experiment was divided into 6 groups: normal control group, model control group, positive control group, low-dose experimental group, medium-dose experimental group, and high-dose experimental group. Each group was tested in 6 replicates.

[0131] B. Treatment of the normal control group: MIN6 cells grown to the logarithmic growth phase were digested, counted, and prepared into a 1×10⁻⁶ concentration using complete culture medium. 5 Cell suspension of cells / mL, at 1×10 4MIN6 cells / well were seeded into 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then completely replaced with MIN6 cell-specific medium, and cultured for another 12 hours. After this second incubation, the culture medium was completely replaced again with MIN6 cell-specific medium, and cultured for another 24 hours. After 24 hours, the medium was discarded, and sterile KRBB buffer (without glucose) was added, followed by another 30 minutes of incubation. After another 30 minutes, the supernatant was discarded. Three replicates were then added to each well with sterile KRBB buffer containing 3 mM glucose, and another three replicates were added to sterile KRBB buffer containing 30 mM glucose. After 1 hour of incubation, the supernatant was collected, and insulin levels were measured according to the kit's instructions.

[0132] C. Treatment of the model control group: MIN6 cells grown to the logarithmic growth phase were digested, counted, and prepared into a 1×10⁻⁶ concentration using complete culture medium. 5 Cell suspension of cells / mL, at 1×10 4 Cells / well were seeded in 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then completely replaced with MIN6 cell-specific medium containing 200 μg / mL STZ, and cultured for another 12 hours. After this, the medium was replaced again with MIN6 cell-specific medium containing 200 μg / mL STZ, and cultured for another 24 hours. After 24 hours, the medium was discarded, and sterile KRBB buffer (without glucose) was added, followed by another 30 minutes of culture. After another 30 minutes, the supernatant was discarded. Then, sterile KRBB buffer containing 3 mM glucose was added to three replicates, and sterile KRBB buffer containing 30 mM glucose was added to the other three replicates. After 1 hour of culture, the supernatant was collected, and insulin levels were measured according to the kit's instructions.

[0133] D. Treatment of the positive control group: MIN6 cells grown to the logarithmic growth phase were digested, counted, and prepared into a 1×10⁻⁶ concentration using complete culture medium. 5 Cell suspension of cells / mL, at 1×10 4Cells were seeded per well in 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. The medium was then completely replaced with MIN6 cell-specific medium containing 200 μg / mL STZ, and cultured for another 12 hours. After this, the medium was replaced again. The positive control group was incubated with MIN6 cell-specific medium containing 200 μg / mL STZ and 25 μM naringenin, and cultured for another 24 hours. After 24 hours, the medium was discarded, and sterile KRBB buffer (without glucose) was added, followed by another 30 minutes of culture. After another 30 minutes, the supernatant was discarded. Three replicates were then incubated with sterile KRBB buffer containing 3 mM glucose, and another three replicates were incubated with sterile KRBB buffer containing 30 mM glucose. After 1 hour of culture, the supernatant was collected, and insulin levels were measured according to the kit's instructions.

[0134] E. Treatment of low, medium, and high dose experimental groups: MIN6 cells grown to the logarithmic growth phase were digested, counted, and prepared into a 1×10⁻⁶ concentration using complete culture medium. 5 Cell suspension of cells / mL, at 1×10 4 MIN6 cells / well were seeded into 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. The medium was then completely replaced with MIN6 cell-specific medium containing 200 μg / mL STZ, and cultured for another 12 hours. After this, the medium was replaced again. For the low, medium, and high dose experimental groups, MIN6 cell-specific medium containing 200 μg / mL STZ and 2.5%, 5%, and 10% concentrations of plant fruit fermentation broth (Polygonum bulbiferum fermentation broth) was added, and the cells were cultured for another 24 hours. After 24 hours, the medium was discarded, and sterile KRBB buffer (without glucose) was added, followed by another 30 minutes of culture. After another 30 minutes, the supernatant was discarded. Then, sterile KRBB buffer containing 3 mM glucose was added to three replicates of each group, and sterile KRBB buffer containing 30 mM glucose was added to the other three replicates. After 1 hour of culture, the supernatant was collected, and insulin levels were measured according to the kit's instructions.

[0135] Calculate the following three insulin secretion-related indices using the following formulas: Insulin levels in the supernatant after co-culturing with sterile KRBB buffer containing BIS=3mM glucose; Insulin levels in the supernatant after co-culturing with sterile KRBB buffer containing GSIS=30mM glucose; ISI = GSIS / BIS.

[0136] 5. Experimental Results (1) Experimental results on improving glucose metabolism and oxidative damage repair are shown in Table 15 and Figure 14 As shown.

[0137] Table 15 Effects of Polygonum villosa fermentation broth on NO and GLUT4 levels in STZ-induced MIN6 cell hyperglycemia model (n=3)

[0138] Compared with the normal control group, the model group showed p < 0.05, p < 0.01, and p < 0.001; compared with the model control group, the drug intervention group showed... p<0.05, p<0.01, p<0.001.

[0139] From Table 15 and Figure 14 It was found that the fermentation broth of *Polygonum viviparum* improved glucose metabolism and enhanced the ability to repair oxidative damage. Specifically, at a concentration of 2.5%, it significantly improved NO levels in the STZ-induced MIN6 cell hyperglycemia model (p<0.05), while at concentrations of 5% and 10%, it showed highly significant improvement in NO levels (p<0.001). The fermentation broth of *Polygonum viviparum* showed a trend of improvement in GLUT4 levels at concentrations of 2.5%, 5%, and 10%, but the differences were not statistically significant.

[0140] (2) Results of experiments on improving insulin secretion are shown in Table 16 and Figure 15 As shown.

[0141] Table 16 Effects of Polygonum villosa fermentation broth on BIS, GSIS, and ISI levels in STZ-induced MIN6 cell hyperglycemia model (n=3)

[0142] Note: Compared with the normal control group, the model group showed #p<0.05, ##p<0.01, and ###p<0.001; compared with the model control group, the drug intervention group showed... p<0.05, p<0.01, p<0.001 From Table 16 and Figure 15As shown, the fermentation broth of Polygonum bulbiferum has the effect of improving insulin secretion. Specifically, at a concentration of 5%, it significantly improved the GSIS level in the STZ-induced MIN6 cell hyperglycemia model (p<0.05); at a concentration of 10%, it significantly improved the BIS level in the STZ-induced MIN6 cell hyperglycemia model (p<0.05), and had a more significant effect on both GSIS and ISI levels (p<0.01).

[0143] The fermentation broth of *Polygonum villosa* has the effect of improving glucose metabolism and enhancing oxidative damage repair capacity. Specifically, at a concentration of 2.5%, it significantly improved NO levels in a STZ-induced MIN6 cell hyperglycemia model (p<0.05), while at concentrations of 5% and 10%, it showed extremely significant improvement in NO levels (p<0.001). The fermentation broth of *Polygonum villosa* showed a trend of improvement in GLUT4 levels at concentrations of 2.5%, 5%, and 10%, but the differences were not statistically significant. At a concentration of 5%, it significantly improved GSIS levels in the STZ-induced MIN6 cell hyperglycemia model (p<0.05); at a concentration of 10%, it significantly improved BIS levels in the STZ-induced MIN6 cell hyperglycemia model (p<0.05), and showed even more significant improvements in GSIS and ISI levels (p<0.01), indicating that the fermentation broth of *Polygonum villosa* has hypoglycemic effects by improving glucose metabolism, insulin secretion, and oxidative damage repair.

[0144] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of Lactobacillus plantarum ( Lactiplantibacillus plantarum Application of Polygonum bulbiferum fermentation products.

2. The application according to claim 1, characterized in that, The Lactobacillus plantarum mentioned includes Lactobacillus plantarum XZFMCC101.23155, with accession number GDMCC No: 65783.

3. A method for preparing a fermentation broth of Polygonum bulbiferum, characterized in that, include: After mixing Polygonum bulbil powder with water, Lactobacillus plantarum was added for fermentation to obtain Polygonum bulbil fermentation broth.

4. The preparation method according to claim 3, characterized in that, The Lactobacillus plantarum mentioned includes Lactobacillus plantarum XZFMCC101.23155, with accession number GDMCC No: 65783.

5. The preparation method according to claim 3, characterized in that, The bulbils powder is obtained by crushing bulbils fruit; the ratio of bulbils powder to water is 1g:(10~50)mL.

6. The preparation method according to claim 3, characterized in that, The fermentation time is 24~120h; the fermentation temperature is 20~36℃.

7. The preparation method according to claim 3, characterized in that, The *Lactobacillus plantarum* was added using a *Lactobacillus plantarum* seed culture method; the viable count of the *Lactobacillus plantarum* seed culture was 1 × 10⁻⁶. 8 CFU / mL; the amount of Lactobacillus plantarum seed solution added is 1.0%~3.0% (V / V) of the volume of the mixture of bulbils and Polygonum hydropiper.

8. A Polygonum bulbil fermentation broth prepared by the preparation method according to any one of claims 3 to 7.

9. A fermented product of Polygonum bulbiferum, characterized in that, Includes the Polygonum bulbil fermentation broth as described in claim 8.

10. The use of the Polygonum bulbil fermentation broth of claim 8 or the Polygonum bulbil fermentation product of claim 9 in the preparation of treatments or improvements for glucose metabolism diseases and / or enhancement of oxidative damage repair.

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