Enoki mushroom polysaccharide, and preparation method and application thereof

Abstract

The present application belongs to the technical field of natural product extraction and preparation, and particularly relates to a Flammulina velutipes polysaccharide, a preparation method and application thereof. The Flammulina velutipes polysaccharide extracted and prepared from Flammulina velutipes processing by-products, i.e. mushroom roots, can simultaneously achieve significant antioxidant, blood supplementing, blood sugar lowering and blood lipid lowering effects, and some effects are even better than those of polysaccharides prepared from edible parts of Flammulina velutipes. Thus, a series of health problems caused by metabolic syndrome, such as oxidative stress damage, anemia tendency and abnormal sugar and lipid metabolism, are solved, so that the Flammulina velutipes polysaccharide is particularly suitable for being used as an active raw material component in many fields such as health foods, medicines and the like for regulating metabolism syndrome people, and has a good market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and preparation technology, specifically relating to enoki mushroom polysaccharide, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Metabolic syndrome (MetS) is a clinical syndrome characterized by oxidative stress, glucose and lipid metabolism disorders, and hematological abnormalities. It mainly includes insulin resistance, hyperglycemia, dyslipidemia, and central obesity, often accompanied by a tendency towards anemia. This syndrome is more common in people with high-calorie diets and those under chronic stress, and has a higher incidence in middle-aged and elderly individuals and obese individuals. Pathologically, oxidative stress is present throughout MetS, directly damaging pancreatic β-cell function and causing hyperglycemia, while also interfering with lipoprotein lipase activity, leading to dyslipidemia. Anemia or decreased erythrocyte function further reduces tissue oxygen supply, forcing increased anaerobic metabolism and exacerbating the metabolic load, which in turn intensifies oxidative inflammation, creating a vicious cycle. Therefore, intervention for MetS cannot target only a single aspect. If an active substance could simultaneously exert a quadruple synergistic effect—scavenging excess free radicals (antioxidant), inhibiting hyperglycemia (hypoglycemic), regulating lipid accumulation (hyperlipidemia), and promoting hematopoiesis (blood replenishment)—then the progression of MetS could be blocked simultaneously at three levels: oxidative stress, glucose and lipid metabolism, and the hematological system.

[0004] Therefore, exploring naturally sourced polysaccharides to achieve the aforementioned multifunctional integration is of significant practical importance for the comprehensive prevention and control of Metabolic Syndrome (MetS). Enoki mushrooms are a widely cultivated edible fungus in my country, and the large amount of mushroom roots generated during their processing is often disposed of as waste, resulting in resource waste. How to utilize this byproduct in a high-value manner and develop active ingredients targeting the aforementioned metabolic syndrome is an urgent technical problem to be solved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides *Flammulina velutipes* polysaccharides, their preparation methods, and applications. Through experimental research, this invention has discovered that *Flammulina velutipes* root polysaccharides, extracted and prepared from the root of *Flammulina velutipes*, a byproduct of processing, can simultaneously achieve significant antioxidant, anemia-enhancing, blood sugar-lowering, and blood lipid-lowering effects, with some effects even surpassing those of polysaccharides prepared from the edible parts of *Flammulina velutipes*. This specifically addresses a series of health problems caused by metabolic syndrome, such as oxidative stress damage, anemia tendency, and abnormal glucose and lipid metabolism, making it particularly suitable as an active ingredient in health foods, pharmaceuticals, and other fields for the treatment of metabolic syndrome. Based on the above research results, this invention has been completed.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the invention provides the use of enoki mushroom polysaccharide in the preparation of products with antioxidant, blood-tonifying, blood sugar-lowering and / or blood lipid-lowering effects.

[0007] In this invention, the enoki mushroom polysaccharide is particularly the enoki mushroom root polysaccharide. The enoki mushroom root refers to the part of the enoki mushroom fruiting body that is cut off from the base of the stipe during harvesting or processing. It is the main by-product generated during the processing of enoki mushrooms and is usually cut off and discarded as waste.

[0008] A second aspect of the present invention provides a method for preparing polysaccharides from *Flammulina velutipes*, the method comprising: S1. Mix the enoki mushroom sample with water, add pectinase for enzymatic hydrolysis, and then extract with hot water to obtain the supernatant. S2. Add ethanol to the supernatant obtained in step S1 for alcohol precipitation, let stand, centrifuge to collect the precipitate, and dry to obtain crude polysaccharide of Enoki mushroom.

[0009] The present invention has proven through experiments that the enoki mushroom root polysaccharide obtained by the above method has excellent antioxidant, blood-tonifying, blood sugar-lowering and blood lipid-lowering effects.

[0010] The antioxidant effect is manifested at least in having good reducing power, total antioxidant capacity and free radical scavenging capacity, which can effectively combat oxidative stress damage in the body and thus improve abnormal blood glucose and blood lipids caused by oxidative stress.

[0011] The blood-replenishing effect is at least manifested in promoting red blood cell production, significantly increasing the hemoglobin-related optical density value, restoring the red blood cell level in anemic states, thereby effectively improving oxidative inflammation caused by anemia.

[0012] The hypoglycemic effect is at least manifested by inhibiting α-amylase activity.

[0013] The lipid-lowering effect is at least manifested in the inhibition of pancreatic lipase activity and the binding with sodium glycocholate and sodium taurocholate, thereby helping to prevent abnormal glucose and lipid metabolism induced by metabolic syndrome.

[0014] A third aspect of the present invention provides a product having antioxidant, blood-tonifying, blood sugar-lowering and / or blood lipid-lowering effects, wherein the product comprises at least the enoki mushroom polysaccharide obtained by the above preparation method.

[0015] The products mentioned above include, but are not limited to, food, pharmaceuticals, and animal feed. In particular, the products may be health supplements for people with metabolic syndrome, without specific limitations.

[0016] The beneficial technical effects of one or more of the above technical solutions are as follows: The above technical solution utilizes the mushroom roots, a byproduct of enoki mushroom processing, as raw material, achieving high-value utilization of waste. Pectinase-assisted hot water extraction combined with alcohol precipitation was used to obtain enoki mushroom root polysaccharides. Experimental verification showed that these polysaccharides possess multiple biological activities: they can effectively scavenge various free radicals, combat oxidative stress, and have anti-aging potential; they can significantly promote erythrocyte production in a zebrafish anemia model, effectively improving blood deficiency; simultaneously, they can significantly inhibit α-amylase and pancreatic lipase activity, and bind with bile salts to assist in regulating glucose and lipid metabolism.

[0017] The above technical solution closely integrates the various pharmacological activities of *Flammulina velutipes* root polysaccharides with the health management needs of metabolic syndrome, providing a safe, natural, and multifunctional solution. The polysaccharide preparation process of this solution is simple and has high extraction efficiency. The resulting polysaccharides are natural active ingredients with high safety and can be widely used in the preparation of foods (including health foods) with antioxidant, anemia-enhancing, blood sugar-lowering, and blood lipid-lowering effects for people with metabolic syndrome. It can also be applied in pharmaceuticals and animal feed, demonstrating promising market prospects. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 This invention illustrates the effects of material-to-liquid ratio (A), enzyme concentration (B), enzymatic hydrolysis time (C), enzymatic hydrolysis temperature (D), hot water bath time (E), and ethanol concentration (F) on the yield of shiitake mushroom root polysaccharide (FRP). Note: Different letters indicate significant differences. P <0.05).

[0020] Figure 2 These are contour plots and response surface plots showing the effects of various factors on the yield of enoki mushroom root polysaccharides in this invention; where A and D are the contour plots and response surface plots for enzymatic hydrolysis temperature and enzymatic hydrolysis time, respectively; B and E are the contour plots and response surface plots for enzymatic hydrolysis temperature and hot water bath time, respectively; and C and F are the contour plots and response surface plots for enzymatic hydrolysis time and hot water bath time, respectively.

[0021] Figure 3 The images show the FT-IR spectra of the root polysaccharide (FRP) and edible fraction polysaccharide (FEP) of *Flammulina velutipes* in this invention.

[0022] Figure 4 This is the UV-Vis spectrum of the reaction product of enoki mushroom root polysaccharide and edible polysaccharide with I2-KI in this invention.

[0023] Figure 5The experimental spectra of FRP, FEP, and Congo Red in this invention are shown.

[0024] Figure 6 The figures show the HPLC chromatograms of FRP and FEP in this invention; (A) HPLC chromatogram of standard monosaccharides; (B) HPLC chromatogram of FRP; and (C) HPLC chromatogram of FEP. The numbers in the figures represent: 1: Man; 2: Glc-N; 3: Rib; 4: Rha; 5: Glc-UA; 6: Gal-UA; 7: Gal-N; 8: Glc; 9: Gal; 10: Xyl; 11: Ara; and 12: Fuc.

[0025] Figure 7 This is the HPGPC map of FRP (A) and FEP (B) in this invention.

[0026] Figure 8 The reducing power (A), total antioxidant capacity (B), and scavenging rate of ABTS (C), OH (D), and DPPH (E) free radicals of the enoki mushroom root polysaccharide and edible part polysaccharide in this invention are given.

[0027] Figure 9 The inhibition rate of α-amylase by polysaccharides from the roots and edible parts of *Flammulina velutipes* in this invention is shown.

[0028] Figure 10 The inhibition rate of lipase by polysaccharides from the root and edible parts of *Flammulina velutipes* in this invention. Figure 11 The binding rates of the polysaccharides from the roots and edible parts of *Flammulina velutipes* to bile salts; (A) binding rate to sodium glycocholate; (B) binding rate to sodium taurocholate.

[0029] Figure 12 The images show zebrafish erythrocyte staining and data analysis in Example 9 of this invention; (A) Schematic diagram of the intervention effect of FRP and FEP on zebrafish erythrocyte production; (B) Effect of FRP and FEP on the IOD value of zebrafish erythrocytes. The groups were: NC group (no drug added); MC group (0.1 μg / mL phenylhydrazine); Groups I, II, and III (0.1 μg / mL phenylhydrazine + FRP, FRP concentrations of 50, 100, and 200 μg / mL, respectively); Groups IV, V, and VI (0.1 μg / mL phenylhydrazine + FEP, FEP concentrations of 50, 100, and 200 μg / mL, respectively). Compared with NC, ### P <0.0001; compared with MC, P <0.01, P <0.0001; group-to-group comparison,a P <0.05; no difference in ns. Detailed Implementation

[0030] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0032] In a typical embodiment of the present invention, the application of enoki mushroom polysaccharide in the preparation of products with antioxidant, blood-tonifying, blood sugar-lowering and / or blood lipid-lowering effects is provided.

[0033] In this invention, the *Flammulina velutipes* polysaccharide is particularly *Flammulina velutipes* root polysaccharide. The *Flammulina velutipes* root refers to the portion of the fruiting body cut off from the base of the stipe during harvesting or processing. It is a major byproduct of *Flammulina velutipes* processing and is usually removed and discarded as waste. The *Flammulina velutipes* root is defined as a portion cut upwards from the base of the stipe with a length of 1... The stipe base segment is 4cm, more preferably 3cm.

[0034] The products include, but are not limited to, food, pharmaceuticals, and animal feed.

[0035] It should be noted that the term "food" used in this invention should be interpreted broadly, and can be understood as any form that can be eaten. For example, food in this invention includes ordinary food and special food. The special food mentioned in this invention includes health food and food for special medical purposes. Ordinary food, in contrast to special food, is food suitable for everyone.

[0036] The food includes, but is not limited to, solid food and liquid food; the solid food includes, but is not limited to, baked goods, candy, and solid beverages; the liquid food includes, but is not limited to, liquid beverages.

[0037] The drug can be administered in unit doses, and the dosage form can be liquid or solid. Liquid dosage forms can be true solutions, colloids, microparticles, emulsions, or suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, emulsions, granules, suppositories, lyophilized powder for injection, inclusion complexes, implants, patches, and liniments.

[0038] The feed refers to the food for animals raised in agriculture or animal husbandry. The *Flammulina velutipes* polysaccharide of this invention can be added as a feed additive to any type of feed, including but not limited to complete compound feed, concentrated feed, and premixed feed.

[0039] The average molecular weights of the polysaccharides in the root and edible parts of *Flammulina velutipes* are 28,418 Da and 953,670 Da, respectively. Both exhibit typical absorption peaks characteristic of polysaccharides. They are pyranoid polysaccharides with α-glycosidic bonds, and both contain numerous side chains and branched structures. Both possess a triple helix structure and contain nine monosaccharide residues: Man, Glc-N, Rha, Glc-UA, Gal-UA, Glc, Gal, Ara, and Fuc. Glc is the main component of both the root and edible polysaccharides of *Flammulina velutipes*. In another specific embodiment of the present invention, a method for preparing enoki mushroom polysaccharide is provided, the method comprising: S1. Mix the enoki mushroom sample with water, add pectinase for enzymatic hydrolysis, and then extract with hot water to obtain the supernatant. S2. Add ethanol to the supernatant obtained in step S1 for alcohol precipitation, let stand, centrifuge to collect the precipitate, and dry to obtain crude polysaccharide of Enoki mushroom.

[0040] Furthermore, the preparation method also includes a protein removal treatment of the crude polysaccharide from *Flammulina velutipes* obtained in step S2. The protein removal treatment can be performed using Sevag reagent, and no specific limitation is made here.

[0041] In step S1, the enoki mushroom sample can be the edible part of enoki mushroom and the root of enoki mushroom, preferably the root of enoki mushroom.

[0042] The ratio of the enoki mushroom sample to water is 1:20-40, preferably 1:30.

[0043] The amount of pectinase added is 2%-6% (w / w), preferably 4%; the enzymatic hydrolysis temperature is 40-60℃, preferably 50-55℃; and the enzymatic hydrolysis time is 60-120 min, preferably 90-100 min.

[0044] The hot water extraction temperature is 80-100℃, preferably 85-90℃; the hot water extraction time is 90-150 min, preferably 120-125 min. The hot water extraction can be performed 1-3 times. When multiple extractions are performed, the supernatants obtained are combined for subsequent steps.

[0045] In step S2, the ethanol is high-concentration ethanol, which can be ethanol with a concentration of 80% or higher, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ethanol. In one specific embodiment of the present invention, the ethanol is 95% ethanol. In one specific embodiment of the present invention, the final concentration of ethanol precipitation is controlled at 80%. The standing time is 6-24 hours (preferably 12 hours).

[0046] The present invention has proven through experiments that the enoki mushroom root polysaccharide obtained by the above method has excellent antioxidant, blood-tonifying, blood sugar-lowering and blood lipid-lowering effects.

[0047] The antioxidant effect is manifested at least in having good reducing power, total antioxidant capacity and free radical scavenging capacity, which can effectively combat oxidative stress damage in the body and thus improve abnormal blood glucose and blood lipids caused by oxidative stress.

[0048] The blood-replenishing effect is at least manifested in promoting red blood cell production, significantly increasing the hemoglobin-related optical density value, restoring the red blood cell level in anemic states, thereby effectively improving oxidative inflammation caused by anemia.

[0049] The hypoglycemic effect is at least manifested by inhibiting α-amylase activity.

[0050] The lipid-lowering effect is at least manifested in the inhibition of pancreatic lipase activity and the binding with sodium glycocholate and sodium taurocholate, thereby helping to prevent abnormal glucose and lipid metabolism induced by metabolic syndrome.

[0051] Therefore, in another specific embodiment of the present invention, a product with antioxidant, blood-tonifying, blood sugar-lowering and / or blood lipid-lowering effects is provided, wherein the product contains at least the enoki mushroom polysaccharide obtained by the above preparation method.

[0052] The products mentioned above include, but are not limited to, food, pharmaceuticals, and animal feed. In particular, the products may be health foods designed to treat metabolic syndrome, without specific limitations.

[0053] The following embodiments further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these embodiments are for illustrative purposes only and not for limiting the scope of the invention. In each embodiment of the present invention, the enoki mushroom root is a 3 cm long segment of the stipe base cut upwards; the remaining portion is the edible part of the enoki mushroom.

[0054] Example 1: Determination of components in the root and edible parts of enoki mushroom. Experimental methods: The moisture content of the edible parts and roots of enoki mushrooms was determined according to GB 5009.3-2016, "National Food Safety Standard - Determination of Moisture in Food". The samples were chopped and a suitable amount was placed in a pre-weighed weighing dish. The dish was heated in a forced-air drying oven at 101.3 kPa and 105 ℃ ± 2 ℃ for 4 h. After cooling, the sample was weighed, and drying continued for another h until the difference between two weighings did not exceed 2 mg, at which point the weight was considered constant. The moisture content (%) was calculated based on the mass loss.

[0055] The total sugar, protein, crude fiber, reducing sugar, glucose, fat, iron, and zinc content of the edible parts and roots of enoki mushrooms were determined according to the following standards. The following standards are applicable to food safety standards: * **Determination of Total Sugar Content in Edible Fungi** (GB / T 15672—2009); * **Determination of Protein in Food** (GB 5009.5—2025); * **Determination of Crude Fiber in Plant-Based Foods** (GB / T 5009.10—2003); * **Direct Titration Method in Determination of Reducing Sugars in Food** (GB 5009.7—2016); * **Enzyme-Electrode Method in Determination of Glucose in Food** (GB / T 16285—2008); * **Determination of Fat in Food** (GB 5009.6—2016); * **Determination of Iron in Food** (GB 5009.90—2016); * **Determination of Zinc in Food** (GB 5009.14—2017).

[0056] Flavonoid content determination: The edible parts and roots of *Flammulina velutipes* were dried to constant weight in an oven at 55 ℃, pulverized, and passed through an 80-mesh sieve to obtain the test samples. 2 g of each powder was weighed, added to 20 mL of 70% ethanol, sonicated at 500 W for 60 min, centrifuged at 8000 r / min for 20 min, and the supernatant was collected and diluted to 25 mL to obtain the test solution. Using a pipette, 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of rutin standard solution (0.3 mg / mL) were pipetted into test tubes, and 70% ethanol was added to make a total volume of 1.0 mL. 0.6 mL of 50.0 g / L sodium nitrite solution was added, and the mixture was allowed to stand for 6 min. Then, 0.4 mL of 100.0 g / L aluminum chloride solution was added, and the mixture was allowed to stand for 6 min. Finally, 2 mL of 40.0 g / L sodium hydroxide solution was added, and the mixture was allowed to stand for 12 min. The absorbance was measured at a wavelength of 510 nm. Plot a standard curve with rutin concentration on the x-axis and absorbance on the y-axis. Take 1 mL of the sample to be tested in a test tube, add 70% ethanol, and follow the same procedure as for plotting the standard curve. Calculate the flavonoid content of the mushroom root and edible parts based on the standard curve.

[0057] Determination of total phenolic content: The edible parts and roots of *Flammulina velutipes* were ground into powder. 2 g of each powder was weighed out and added to 20 mL of 70% ethanol. The mixture was sonicated at 500 W for 60 min, centrifuged at 8000 r / min for 20 min, and the supernatant was collected and diluted to volume to obtain the extract. 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of gallic acid standard solution (0.1 mg / mL) were transferred to test tubes. Deionized water was added to bring the total volume to 1 mL. 0.5 mL of Folin-Ciocalteu was added, and the mixture was thoroughly mixed and allowed to stand for 5 min. Then, 1.5 mL of 20.0 g / L sodium carbonate solution was added, and the volume was diluted to 10 mL with deionized water. The mixture was shaken well, stored in the dark for 2 h, and the absorbance was measured at 760 nm. A standard curve was plotted with gallic acid concentration on the x-axis and absorbance on the y-axis. 1 mL of the sample to be tested was placed in a test tube, and 0.5 mL of Folin-Ciocalteu was added. The subsequent procedures were the same as for plotting the standard curve. The total phenol content of mushroom roots and edible parts was calculated based on the standard curve.

[0058] Experimental results: The moisture content of the enoki mushroom root and edible portion was 77.52% and 86.74%, respectively. The composition of the dry matter in the enoki mushroom root and edible portion is shown in Table 1.

[0059] Table 1. Results of analysis of the root and edible components of enoki mushrooms.

[0060] Note: Data with the same letter markings in the same row do not show significant differences. P>0.05, data with different letter labels in the same row showed significant differences ( P <0.05).

[0061] As shown in Table 1, there were no significant differences in the total sugar, protein, fat, and total phenol content between the root and edible dry matter of enoki mushrooms. P >0.05), while crude fiber, reducing sugar, glucose, iron, zinc, and flavonoids showed significant differences ( P <0.05). The roots of enoki mushrooms are higher in crude fiber, reducing sugar, glucose, and flavonoids, exceeding the edible portions by 40.63%, 181.46%, 305.59%, and 37.96%, respectively. The edible portions of enoki mushrooms are also higher in zinc and iron, exceeding the roots by 23.73% and 24.61%, respectively. Therefore, the roots of enoki mushrooms are also rich in nutrients, although their composition differs from that of the edible portions.

[0062] Example 2: Optimization of Extraction Process of Polysaccharides from the Roots of Enoki Mushrooms Experimental methods: (1) Pectinase activity assay: Galacturonic acid standard solution and pectinase solution were prepared using citrate-phosphate buffer (pH 5.0). 1 mL of different concentrations of galacturonic acid standard solution (0, 0.2, 0.4, 0.6, 0.8, 1 mg / mL) were placed in test tubes, and 1 mL of DNS reagent was added (18.2 g of potassium sodium tartrate was dissolved in 50 mL of distilled water, heated, and 0.63 g of 3,5-dinitrosalicylic acid and 2.4 g of sodium hydroxide were added sequentially to the hot solution; after cooling, the solution was diluted to 100 mL with distilled water to prepare DNS reagent). The solution was boiled in a water bath for 5 min, cooled, and then diluted to 10 mL. A standard curve was plotted with the galacturonic acid standard solution as the x-axis and the absorbance measured at 540 nm as the y-axis. Take 1 mL of pectin solution (10.0 g / L) preheated to 50 ℃ in a test tube, add 0.5 mL of pectinase solution, and react in a 50 ℃ water bath for 10 min. Immediately after the reaction, add 1.5 mL of DNS reagent, boil in a water bath for 5 min, cool, and bring the volume to 10 mL. Centrifuge at 4000 r / min for 5 min, and measure the absorbance of the supernatant at 540 nm. Calculate the galacturonic acid content in the solution according to the standard curve. Enzyme activity definition: 1 unit of enzyme activity (U): the amount of enzyme required to catalyze the hydrolysis of pectin to produce 1 μmol of galacturonic acid per minute at 50 ℃ and pH 5.0.

[0063] (2) Single-factor experimental design ① Basic process flow for polysaccharide extraction: Mix enoki mushroom root powder with deionized water at a material-to-liquid ratio of 1:30, add pectinase (4%), and perform enzymatic hydrolysis in a water bath (enzymatic hydrolysis temperature 50℃, enzymatic hydrolysis time 90 min), followed by hot water bath extraction (time 120 min, hot water bath temperature 90℃). Repeat the extraction twice and combine the supernatants. Add 95% ethanol to the supernatant to make the final ethanol concentration 80%. Let stand for 12 h, centrifuge (10000 r / min, 10 min), and dry the precipitate in a 55 ℃ oven to obtain crude polysaccharide from enoki mushroom roots.

[0064] ② Single-factor experimental design for polysaccharide extraction: Based on the basic process of polysaccharide extraction, the effects of each single factor on the yield of polysaccharides from the roots of *Flammulina velutipes* were analyzed by changing various variables. ① Material-to-liquid ratio: 1:10, 1:20, 1:30, 1:40, 1:50; ② Pectinase addition: 0, 20, 30, 40, 50 mg / mL; ③ Enzymatic hydrolysis time: 30, 60, 90, 120, 150 min; ④ Enzymatic hydrolysis temperature: 40, 45, 50, 55, 60 ℃; ⑤ Hot water bath extraction time: 60, 90, 120, 150, 180 min; ⑥ Final ethanol precipitation concentration: 50%, 60%, 70%, 80%, 90%.

[0065] (3) Determination of polysaccharide yield: The polysaccharide of *Flammulina velutipes* root was dissolved in deionized water, and the polysaccharide content of FRP was determined by the phenol-sulfuric acid method. Standard curve plotting: Accurately pipette 0, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mL of glucose standard solution (0.1 mg / mL) into 10 mL volumetric flasks, add deionized water to dilute to volume, and shake well to obtain standard solutions with concentrations of 0, 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL, respectively. Take 2 mL of each solution into a test tube, add 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid, shake to mix, incubate in a 90℃ water bath for 20 min, and measure the absorbance at 490 nm. Plot a standard curve with glucose concentration (x) as the abscissa and absorbance (y) as the ordinate, and calculate the regression equation. Take 2 mL of the sample to be tested into a test tube. The following operations are the same as for plotting the standard curve. Measure the absorbance at a wavelength of 490 nm and calculate the polysaccharide concentration (c) in the solution. Polysaccharide yield (%) = (c × n × V) / m. Where c is the crude polysaccharide concentration in the solution (mg / mL), n is the dilution factor of the solution, V is the volume of the solution (mL), and m is the mass of the enoki mushroom root powder (mg).

[0066] (4) Response surface methodology: Based on the single-factor experiments, and according to the Box-Behnken central composite design principle, the main factors affecting the extraction rate of polysaccharides from the roots of *Flammulina velutipes* were selected as experimental factors: enzymatic hydrolysis temperature, enzymatic hydrolysis time, and hot water extraction time. The polysaccharide extraction rate was used as the response value to conduct a three-factor, three-level response surface methodology experiment. Other extraction conditions were: a material-to-liquid ratio of 1:30, a pectinase addition of 4%, a hot water extraction temperature of 90℃, and a final alcohol precipitation concentration of 80%. The experimental factors and levels are shown in Table 2.

[0067] Table 2 Factors and levels affecting the extraction of polysaccharides from the roots of *Flammulina velutipes*.

[0068] Experimental results: (1) Effects of single factors on the extraction rate of polysaccharides from the roots of *Flammulina velutipes*: The pectinase activity used for extracting polysaccharides from the roots of *Flammulina velutipes* was 0.722 U / mL, and the specific activity was 0.903 U / mg. The effects of material-to-liquid ratio, enzyme concentration, hydrolysis time, hydrolysis temperature, hot water bath time, and alcohol precipitation concentration on the yield of polysaccharides from the roots of *Flammulina velutipes* are shown in [the table below]. Figure 1 .

[0069] Depend on Figure 1 It was observed that under five material-to-liquid ratio gradients from 1:10 to 1:50, the polysaccharide yield initially increased and then stabilized, with the optimal material-to-liquid ratio being 1:30. At this ratio, the polysaccharide yield was the highest, and further increases in solvent yield did not significantly improve the extraction rate. This phenomenon may be due to the polysaccharide saturation at a material-to-liquid ratio of 1:30. The polysaccharide extraction rate increased with increasing enzyme dosage, reaching its maximum at 4%, possibly due to substrate saturation caused by excessive enzyme. Therefore, 4% enzyme concentration is considered the optimal enzyme concentration. With increasing enzymatic hydrolysis time, the polysaccharide yield initially increased and then decreased, with 90 min being the optimal hydrolysis time. Excessive hydrolysis time may lead to polysaccharide degradation and a decrease in yield. With increasing enzymatic hydrolysis temperature, the polysaccharide yield initially increased and then decreased. 50℃ was the optimal hydrolysis temperature. When the temperature exceeded 50℃, enzyme protein denaturation occurred, catalytic activity decreased, and the polysaccharide yield decreased. The optimal hot water bath time is 120 min. Shorter bath times may result in incomplete polysaccharide dissolution, while longer bath times may lead to polysaccharide degradation or reduced dissolution efficiency. The polysaccharide yield gradually increases with increasing ethanol concentration. Once the ethanol concentration reaches 80%, the polysaccharide yield plateaus, indicating that complete precipitation of polysaccharides is possible at an ethanol concentration of 80%.

[0070] (2) Response surface methodology Table 3 Response Surface Experimental Design and Results

[0071] Regression fitting was performed on the data in Table 3, and the quadratic polynomial regression equation for the independent variable and the extraction rate (Y) of polysaccharides from the roots of *Flammulina velutipes* was obtained as follows: Y = 6.12 + 0.3838X1 + 0.1738X2 + 0.2025X3 + 0.1025X1X2 - 0.0850X1X3 + 0.0150X2X3 - 0.8253X1 2 -0.3602X2 2 -0.6477X3 2 In the formula, Y is the polysaccharide extraction rate (%); X1 is the enzymatic hydrolysis temperature (°C); X2 is the enzymatic hydrolysis time (min); and X3 is the hot water time (min).

[0072] The data in Table 3 were fitted using Design Expert 13 software, and the significance was tested. The results are shown in Table 4.

[0073] Table 4. Analysis of variance of the regression model for response surface methodology.

[0074] Note: Significant difference ( P <0.05); The difference is extremely significant ( P <0.01) For the polysaccharide yield model, the significance test P <0.01, indicating highly significant; while the lack of fit term P =0.849>0.05 indicates no statistical significance, meaning the model is statistically significant and the polysaccharide yield model has good stability. Coefficient of determination R0.849 2 The coefficient of determination (R²) was 0.9723, and the corrected coefficient of determination (R²) Adj was 0.9366, indicating that the established model has a good fit and high reliability. Therefore, it is appropriate to use this model equation to predict the yield of crude polysaccharide from enoki mushroom roots.

[0075] As shown in Table 4, among the process parameters for the enzymatic extraction of polysaccharides from the roots of *Flammulina velutipes*, the factors affecting the polysaccharide extraction rate, in order of importance, are: enzymatic hydrolysis temperature (X1) > hot water bath time (X3) > enzymatic hydrolysis time (X2).

[0076] Analysis using Design Expert 13 revealed the optimal extraction conditions for enoki mushroom root polysaccharides as follows: enzymatic hydrolysis temperature 51.212 ℃, enzymatic hydrolysis time 98.372 min, and hot water extraction time 124.327 min, yielding a enoki mushroom root polysaccharide yield of 6.203%. For ease of operation, the extraction conditions were modified to: enzymatic hydrolysis temperature 51 ℃, enzymatic hydrolysis time 98 min, and hot water extraction time 124 min. Substituting the modified optimized conditions into the regression equation yielded a theoretical polysaccharide extraction rate of 6.20%. Experimental verification showed a root polysaccharide yield of 6.15%, demonstrating the significant optimization effect of this process.

[0077] Example 3: Preparation and determination of polysaccharide content of mushroom root polysaccharides and edible part polysaccharides. Experimental methods: Based on the results of the extraction process optimization experiment, the edible parts and crude polysaccharides of Enoki mushrooms were extracted under the following conditions: a material-to-liquid mass-to-volume ratio of 1:30 (g / mL), a pectinase mass fraction of 4%, an enzymatic hydrolysis temperature of 51℃, an enzymatic hydrolysis time of 98 min, a hot water bath extraction time of 124 min, and a hot water bath temperature of 90℃. The extraction was repeated twice, and the supernatants were combined. 95% ethanol was added to the supernatant to make the final ethanol concentration 80%, and the mixture was allowed to stand for 12 h. Then, it was centrifuged (10000 r / min, 10 min), and the precipitate was dried in a 55℃ oven to obtain the edible parts and crude polysaccharides of Enoki mushrooms. Both were dissolved separately in deionized water and centrifuged (10000 r / min, 10 min). The supernatant was mixed with Sevag reagent (chloroform:n-butanol = 5:1, v / v) at a volume ratio of 4:1, shaken for 15 min, and centrifuged (8000 r / min, 10 min) to remove the organic phase and the white emulsion layer at the interface between the organic and aqueous phases. The operation was repeated until the emulsion layer disappeared. After freeze-drying the aqueous phase, phytorexiferol polysaccharide (FRP) and edible part polysaccharide (FEP) of enoki mushrooms were obtained, respectively.

[0078] The polysaccharide content of the root polysaccharide and edible part polysaccharide of *Flammulina velutipes* was determined by the phenol-sulfuric acid method (the method is the same as 1.2 in Example 2). Polysaccharide content (mg / g) = (c × n × V) / m, where c is the polysaccharide concentration in the solution (mg / mL), n is the dilution factor of the solution, V is the volume of the solution (mL), and m is the mass of the polysaccharide (g).

[0079] Experimental results: The polysaccharide content of the root polysaccharide of *Flammulina velutipes* was determined by the phenol-sulfuric acid method to be 453.7 mg / g, and the polysaccharide content of the edible part polysaccharide was 508.9 mg / g.

[0080] Example 4 Structural characterization of polysaccharides from the root of *Flammulina velutipes* and its edible fraction. Experimental methods: (1) FT-IR analysis of polysaccharides from the roots and edible parts of *Flammulina velutipes*: The sample was mixed with potassium bromide and ground until homogeneous, then pressed into transparent thin films. Fourier transform infrared spectroscopy was used to scan the 4000-500 cm⁻¹ area. -1 Range, to obtain characteristic absorption spectra.

[0081] (2) Determination of branch structure of polysaccharides in the root and edible parts of enoki mushroom: 2 mL of sample solution (0.5 mg / mL) and 1.2 mL of iodine-potassium iodide reagent (containing 0.2 g / L I2 and 2 g / L KI) were mixed evenly in a test tube and scanned at 230~600 nm using an enzyme-linked immunosorbent assay reader.

[0082] (3) Determination of the triple helix structure of polysaccharides in the root and edible parts of *Flammulina velutipes*: 1 mL each of the sample solution (0.5 mg / mL) and Congo red solution (80 mmol / L) were mixed thoroughly. 1 mL of sodium hydroxide solution of different concentrations was added to make the final sodium hydroxide concentrations 0, 0.5, 1.5, 2.0, 2.5, and 3.0 mol / L. The mixture was reacted at room temperature for 10 min. The maximum absorbance was measured at 485–520 nm using a microplate reader, and the results were plotted.

[0083] Experimental results: (1) FT-IR analysis of polysaccharides in the root and edible parts of Enoki mushroom. like Figure 3 As shown, the FT-IR spectra of FRP and FEP indicate that both polysaccharides exhibit typical polysaccharide characteristic peaks in the 3200–3600 cm⁻¹ range. -1 The strong and broad absorption peaks between these peaks are due to the stretching vibrations of the O-H atoms in the polysaccharide molecules; 2900 cm⁻¹ -1 The nearby absorption peaks are due to the stretching vibrations of C-H; 1601.670 and 1629.495 cm⁻¹ -1 The absorption peak at 1400 cm⁻¹ is related to the stretching vibration of C=O; -1 The nearby absorption peak corresponds to the bending vibration of the C-H bond; 1200 cm⁻¹ -1 ~1000cm -1 The nearby absorption peak is caused by the superposition of C-O-C glycosidic bond vibration, sugar ring vibration, and stretching vibration of C-OH side groups; 840 cm⁻¹ -1 The nearby absorption peaks are caused by the C-H bonds in α-pyran. FRP and FEP exhibit typical absorption peaks of polysaccharides; both are pyranoid polysaccharides with α-glycosidic bonds.

[0084] (2) Determination of the branching structure of polysaccharides in the root and edible parts of enoki mushrooms The iodine-potassium iodide test is used to identify the presence of long side chains and numerous branches in polysaccharide molecules. When a polysaccharide reacts with iodine-potassium iodide reagent, if a significant absorption peak is observed in the wavelength range of 240–400 nm, while no maximum absorption peak is detected at 565 nm, this indicates that the polysaccharide molecule contains numerous side chains and branched structures. Figure 4 It can be seen that the maximum absorption peaks of the polysaccharides in the root and edible parts of *Flammulina velutipes* reacting with I2-KI are concentrated in the range of 240-400 nm, while there is no maximum absorption peak at 565 nm. This indicates that the polysaccharides in the root and edible parts of *Flammulina velutipes* contain a large number of side chains and branched structures.

[0085] (3) Determination of the triple helix structure of polysaccharides in the root and edible parts of enoki mushrooms Congo red, a typical acid dye, possesses a sulfonic acid group in its molecular structure that specifically recognizes and binds to polysaccharide molecules with a triple-helix conformation, forming a stable Congo red-polysaccharide complex through hydrophobic interactions and hydrogen bonding. Under alkaline conditions, the maximum absorption wavelength of this Congo red-polysaccharide complex undergoes a red shift due to complex formation. Figure 5 It can be seen that under weakly alkaline conditions, the maximum absorption wavelength of the mixture of enoki mushroom root polysaccharide and edible part polysaccharide with Congo red increases with the increase of NaOH concentration, indicating that both enoki mushroom root polysaccharide and edible part polysaccharide have a triple helix structure.

[0086] Example 5: Determination of monosaccharide composition of enoki mushroom root polysaccharides and edible fraction polysaccharides. Experimental methods: The monosaccharide composition of the samples was determined by high performance liquid chromatography (HPLC).

[0087] Weigh 0.1 g (accurate to 0.1 mg) of polysaccharide sample into a 20 mL clamp bottle, add 5 mL of 2 mol / L trifluoroacetic acid (TFA), seal the bottle with N2, and hydrolyze in a 120 °C oven for 2 h. After cooling, open the cap, take out 1 mL, add 1 mL of methanol, and dry with N2 in a 70 °C water bath. Repeat this process of adding methanol and drying with N2 twice to remove TFA. Add 1 mL of 0.3 mol / L NaOH solution to fully dissolve the residue to obtain the polysaccharide hydrolysate, and perform derivatization analysis after a certain dilution.

[0088] Take 400 μL of the mixed monosaccharide standard solution or polysaccharide hydrolysate into a 5 mL stoppered test tube, add 400 μL of PMP methanol solution, and vortex to mix. React in a 70 ℃ water bath for 2 h. Remove and allow to cool to room temperature, add 400 μL of 0.3 mol / L HCl to neutralize (pH 6-7), add 1200 μL of water, then add an equal volume of chloroform, vortex to mix, shake, let stand, discard the chloroform phase, and repeat the extraction twice. Filter the aqueous phase through a 0.45 μm microporous membrane (water-based) for HPLC analysis.

[0089] Instrumentation: High-performance liquid chromatography (HPLC); Column: C18 column (50 mm × 4.6 mm, 5 μm); Mobile phase A: PBS (0.1 M, pH 6.4); Mobile phase B: Acetonitrile; Detection wavelength: 245 nm; Column temperature: 30℃; Flow rate: 1 mL / min; Injection volume: 20 μL. Linear gradient elution: 0–30 min, 95%–80% A; 30.1–45 min, 60% A; 45.1–60 min, 95% A. The monosaccharide composition of each sample was calculated by comparing it with monosaccharide standards. The standard monosaccharides are as follows: mannose (Man), glucosamine (Glc-N), ribose (Rib), rhamnose (Rha), glucuronic acid (Glc-UA), galacturonic acid (Gal-UA), galactoseamino (Gal-N), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc).

[0090] Experimental results: Table 5. Molar percentage of monosaccharide residues in the root polysaccharide and edible part polysaccharide of Enoki mushroom.

[0091] like Figure 6Table 5 shows that both the root polysaccharide and the edible polysaccharide of *Flammulina velutipes* contain nine monosaccharide residues: Man, Glc-N, Rha, Glc-UA, Gal-UA, Glc, Gal, Ara, and Fuc. Glc is the main component of both root and edible polysaccharides, with molar percentages of 80.71% and 85.58%, respectively. Compared to the edible polysaccharide, the proportion of Glc residues in the root polysaccharide is relatively low. Furthermore, Man and Gal residues are the most abundant monosaccharide residues in both root and edible polysaccharides. In FRP and FEP, the proportions of other monosaccharide residues besides Glc, Man, and Gal are relatively low. The presence of small amounts of Glc-UA and Gal-UA residues in both root and edible polysaccharides indicates that both contain uronic acid.

[0092] Example 6: Molecular weight determination of polysaccharides from the root and edible parts of *Flammulina velutipes*. Experimental methods 10 mg of enoki mushroom root polysaccharides and edible fraction polysaccharides were dissolved in 1 mL of 0.1 mol / L NaNO3. Molecular weight was determined using High Performance Gel Permeation Chromatography (HPGPC). The determination was performed using an e2695 Waters HPLC system equipped with a 2414 Waters differential refractive index detector and a HPLC column (7.8 × 300 mm, TSKgel GMPW x L). Mobile phase: 0.1 mol / L NaNO3; flow rate: 0.5 mL / min; column temperature: 30 ℃; injection volume: 30 μL; processing software: Empower3. A standard curve was plotted using a series of narrow-distribution pullulan polysaccharides as standards, with retention time on the x-axis and the reciprocal of weight-average molecular weight (Mw) on the y-axis. The Mw of the samples was calculated based on the standard curve.

[0093] Experimental results: Table 6. Weight-average molecular weight of enoki mushroom mycelium polysaccharide (FRP) and edible part polysaccharide (FEP)

[0094] As shown in Table 6, the average weight-average molecular weight (Mw) of FRP is 28,418 Da, while that of FEP is 953,670 Da. Therefore, FRP and FEP have different molecular weights, with FRP having a lower average weight-average molecular weight compared to FEP.

[0095] Example 7: Determination of the in vitro antioxidant capacity of polysaccharides from the root and edible parts of *Flammulina velutipes*. Experimental methods (1) Reducing power experiment of polysaccharides from the root and edible parts of *Flammulina velutipes*: The Prussian blue method was used for determination. 0.2 mL of polysaccharide sample solutions of different concentrations (100–5000 mg / L) were mixed thoroughly with 0.5 mL of phosphate buffer (0.2 mol / L, pH 6.6) and 0.2 mL of potassium ferricyanide solution (10 g / L) in test tubes, and then reacted in a 50℃ water bath for 20 min. 0.4 mL of trichloroacetic acid (100 g / L) and 0.24 mL of ferric chloride solution (1 g / L) were added, with vitamin C (Vc) as a positive control. After mixing, the absorbance of the solution was measured at 700 nm.

[0096] (2) Experiment on the total antioxidant capacity of polysaccharides from the root and edible parts of *Flammulina velutipes*: 0.2 mL of polysaccharide sample solutions of different concentrations (100~5000 mg / L) were placed in test tubes, and 2 mL of P solution (containing 0.6 mol / L sulfuric acid, 28 mmol / L trisodium phosphate, and 4 mmol / L ammonium molybdate) was added. The mixture was placed in a 95 ℃ water bath for 90 min, and after cooling, the absorbance of the solution was measured at 695 nm. Vitamin C was used as a positive control.

[0097] (3) Scavenging experiment of -OH free radicals of polysaccharides from the roots and edible parts of *Flammulina velutipes*: The Fenton method was used for determination. 0.2 mL of ferrous sulfate solution (9 mmol / L), 0.2 mL of salicylic acid ethanol solution (9 mmol / L), 0.2 mL of polysaccharide sample solutions of different concentrations (100~5000 mg / L), and 0.2 mL of hydrogen peroxide solution (8.8 mmol / L) were mixed thoroughly in a test tube. After reacting in a 37℃ water bath for 30 min, the mixture was centrifuged at 8000 r / min for 10 min, and the absorbance of the supernatant was measured at 510 nm. Vitamin C was used as a positive control. -OH free radical scavenging rate (%) = (A0 - A) / A0 × 100. Where: A0 is the absorbance of the blank control (deionized water instead of the sample), and A is the absorbance of the sample solution mixed with the reagent.

[0098] (4) DPPH free radical scavenging experiment of polysaccharides from the roots and edible parts of *Flammulina velutipes*: 0.6 mL of polysaccharide sample solutions of different concentrations (100~5000 mg / L) were placed in test tubes, and 0.6 mL of DPPH ethanol solution (0.2 mmol / L) was added. After mixing thoroughly, the mixture was reacted in the dark for 30 min, and then the absorbance (A) of the solution was measured at 517 nm. Vitamin C was used as a positive control. DPPH free radical scavenging rate (%) = (1 - (A - A) / (A ...对 ) / A 空 ) × 100. Where: A is the absorbance of the sample solution mixed with the DPPH ethanol solution, A 对 A represents the absorbance of the sample solution after mixing with ethanol. 空 The absorbance is the result of mixing deionized water with DPPH ethanol solution.

[0099] (5) ABTS free radical scavenging experiment of polysaccharides from the roots and edible parts of *Flammulina velutipes*: An equal volume of ABTS solution (7 mmol / L) and potassium persulfate solution (4.9 mmol / L) was mixed and allowed to stand in the dark for 20 h to obtain an ABTS stock solution. The ABTS stock solution was diluted with phosphate buffer (0.1 mol / L, pH 7.4) to obtain an absorbance of 0.7 ± 0.02 at 734 nm, which is the ABTS working solution. 0.3 mL of polysaccharide sample solutions of different concentrations (100 ~ 5000 mg / L) were mixed with 0.9 mL of ABTS working solution and reacted at room temperature in the dark for 6 min. The absorbance of the solution was measured at 734 nm. Vitamin C was used as a positive control. ABTS free radical scavenging rate (%) = (1 - (A - A) / (A ... 对 ) / A 空 ) × 100. Where: A is the absorbance of the sample solution mixed with the ABTS working solution, A 对 A represents the absorbance of the sample solution after mixing with phosphate buffer. 空 The absorbance is the result of mixing deionized water with ABTS working solution.

[0100] Experimental results: Table 7. Antioxidant capacity of polysaccharides from the root and edible parts of Enoki mushroom.

[0101] Note: Absorbance at 5000 mg / L is used as the reducing power and total antioxidant capacity; data with different letter markings in the same journal show significant differences. P <0.05).

[0102] The antioxidant capacity of polysaccharides in the root and edible parts of enoki mushrooms, such as... Figure 8 As shown in Table 7, the antioxidant capacity of *Flammulina velutipes* root polysaccharides and edible part polysaccharides gradually increased with increasing polysaccharide concentration, exhibiting a significant dose-dependent effect. At a concentration of 5000 mg / L, the reducing power and total antioxidant capacity of *Flammulina velutipes* root polysaccharides were 227.50% and 99.94% higher than those of the edible part polysaccharides, respectively. (EC) 50The value represents the half-maximal effective concentration (MCC), i.e., the sample concentration at which the in vitro antioxidant activity reaches 50%. A lower value indicates stronger activity. When both the root polysaccharide and edible polysaccharide of *Flammulina velutipes* reached the MCC, the concentrations required for *Flammulina velutipes* root polysaccharide to scavenge ABTS, hydroxyl, and DPPH free radicals were 121.70%, 25.30%, and 271.94% lower than those for the edible polysaccharide, respectively. P <0.05). Therefore, both the root polysaccharide and the edible portion polysaccharide of *Flammulina velutipes* have good antioxidant capacity, and the antioxidant capacity of the root polysaccharide is significantly higher than that of the edible portion polysaccharide. P <0.05). The strong antioxidant capacity of enoki mushroom root polysaccharides may be related to their low molecular weight.

[0103] Example 8: Determination of the hypoglycemic and hypolipidemic effects of polysaccharides from the root and edible parts of *Flammulina velutipes*. Experimental methods (1) Determination of hypoglycemic capacity: Polysaccharide solutions (0.2, 0.6, 1.2, 1.4, 1.8, 2 mg / mL) and soluble starch solutions (10 mg / mL) at different concentrations were prepared using PBS buffer (0.1 mol / L, pH 6.9). α-Amylase solution (1 U / mL) was prepared using 6.7 mmol / L NaCl solution. 50 μL of polysaccharide solution and 50 μL of α-amylase solution were mixed and incubated at 37℃ for 10 min. Then, 50 μL of soluble starch solution was added and incubated for another 10 min. Subsequently, 100 μL of DNS reagent (DNS reagent: 18.2 g of potassium sodium tartrate was dissolved in 50 mL of distilled water, heated, and 0.63 g of 3,5-dinitrosalicylic acid and 2.4 g of sodium hydroxide were added sequentially to the hot solution. After cooling, the solution was diluted to 100 mL with distilled water and boiled in boiling water for 5 min. After cooling to room temperature, dilute with 1 mL of distilled water and measure absorbance at 520 nm. Acarbose was used as a positive control. The inhibition rate of α-amylase (%) = (1 - (A1 - A2) / A0) × 100. Where: A1 is the absorbance of the sample group; A2 is the absorbance of PBS buffer instead of α-glucosidase solution; A0 is the absorbance of the blank control group with PBS buffer instead of the sample.

[0104] (2) Measurement of lipid-lowering ability ① Determination of the inhibition rate of polysaccharides from the roots and edible parts of *Flammulina velutipes* on lipase: Polysaccharide solutions of different concentrations (1, 5, 10, 15, 20 mg / mL) and a 1250 U / mL porcine pancreatic lipase solution were prepared using PBS buffer (0.1 mol / L, pH 6.9). A 0.8 mg / mL laurate-4-nitrobenzene ester substrate solution was prepared using 5 mmol / L sodium acetate solution (containing 1% Trition X-100) as the substrate solution. 50 μL of polysaccharide sample solutions of different concentrations were accurately pipetted into test tubes, and 150 μL of porcine pancreatic lipase solution (1250 U / mL) and 350 μL of PBS buffer (0.1 mol / L, pH 7.4) were added respectively. The mixture was preheated at 37℃ for 10 min, followed by the addition of 450 μL of substrate solution. After thorough mixing, the mixture was incubated in a 37℃ water bath for 2 h. The sample was then centrifuged at 11000 r / min for 10 min, and the absorbance of the supernatant was measured at 405 nm. Pancreatic lipase inhibition rate (%) = ((A1-A2)-(A3-A4)) / (A1-A2)×100. A1 is the absorbance of PBS buffer instead of the polysaccharide solution; A2 is the absorbance of PBS buffer instead of both the polysaccharide and pancreatic lipase solutions; A3 is the absorbance of the sample group; A4 is the absorbance of PBS buffer instead of the pancreatic lipase solution.

[0105] ② Determination of the bile salt binding capacity of polysaccharides from the root and edible parts of *Flammulina velutipes*: Construction of the standard curve: 1 mL of different concentrations of sodium glycocholate (0.04, 0.10, 0.20, 0.30, 0.40 mmol / L) or sodium taurocholate (0.05, 0.10, 0.20, 0.30, 0.40, 0.50 mmol / L) standard solutions were placed in stoppered test tubes. 3 mL of 60% (w / v) concentrated H₂SO₄ solution was added, and the mixture was incubated in a 70 ℃ water bath for 20 min, followed by an ice bath for 5 min. The absorbance was then measured at 387 nm. A standard curve of bile salt content was plotted with bile salt content on the x-axis and absorbance on the y-axis.

[0106] 10 mg / mL pepsin and 10 mg / mL trypsin were prepared using PBS buffer (0.1 mol / L, pH 6.3). 1.5 mL of polysaccharide solutions of different concentrations were placed in centrifuge tubes, and 1.5 mL of pepsin and 0.5 mL of 0.01 mol / L HCl were added. The solutions were digested at 37 °C and 120 r / min for 1 h with shaking to simulate gastric digestion. The pH of the solution was then adjusted to 6.3 with 0.1 mol / L NaOH solution, and 2 mL of trypsin was added. The solutions were then digested at 37 °C with shaking for 1 h to simulate intestinal digestion. After digestion, 2 mL of 0.4 mmol / L sodium glycocholate or 2 mL of 0.5 mmol / L sodium taurocholate were added to the samples, and the solutions were incubated at 37 °C with shaking for 1 h, centrifuged at 4000 r / min for 20 min, and the supernatant was collected. The contents of sodium glycocholate and sodium taurocholate were determined according to the standard curve method. Calculate the binding rates of sodium glycocholate and sodium taurocholate using the formula: Binding rate (%) = (m1 - m2) / m1 × 100. Where: m1 is the mass of bile salt before binding, mg; m2 is the mass of bile salt after binding, mg.

[0107] Experimental results: (1) The hypoglycemic ability of polysaccharides from the roots and edible parts of Enoki mushrooms The mechanism by which polysaccharides inhibit α-amylase activity is that they bind to or competitively occupy the enzyme's binding site, thus hindering the hydrolytic reaction of the substrate by the amylase. Figure 9 It was found that the inhibition rates of α-amylase by both the root polysaccharides and edible polysaccharides of *Flammulina velutipes* increased with increasing sample concentration, exhibiting a clear concentration-dependent relationship. The half-maximal inhibitory concentrations (EC50) of *Flammulina velutipes* root polysaccharides and edible polysaccharides against α-amylase were also determined. 50 The values ​​were 1.42 and 1.46 mg / mL, respectively. At a concentration of 2 mg / mL, the inhibition rates of α-amylase by the root polysaccharide and edible part polysaccharide of *Flammulina velutipes* were 73.90% and 63.44%, respectively. Under this concentration condition, the inhibition rate of α-amylase by the root polysaccharide of *Flammulina velutipes* was significantly higher than that by the edible part polysaccharide. P <0.05).

[0108] Therefore, both the root polysaccharide and the edible polysaccharide of enoki mushrooms have the ability to lower blood sugar, and the root polysaccharide of enoki mushrooms has a stronger ability to lower blood sugar.

[0109] (2) The lipid-lowering ability of polysaccharides from the roots and edible parts of Enoki mushrooms ① Determination of the inhibition rate of lipase by polysaccharides from the root and edible parts of enoki mushrooms. Polysaccharides can inhibit the hydrolysis of lipid substrates by pancreatic lipase by binding to it or its cofactors, thereby altering the enzyme's conformation. Figure 10 It was found that the inhibitory effects of enoki mushroom root polysaccharides and edible portion polysaccharides on lipase exhibited a significant concentration-dependent relationship. The EC50 values ​​of the inhibition rates of enoki mushroom root polysaccharides and edible portion polysaccharides on lipase were [not specified in the original text]. 50 The concentrations were 14.42 mg / mL and 14.04 mg / mL, respectively; at a concentration of 20 mg / mL, the inhibition rates of lipase by the polysaccharides in the rhizome of *Flammulina velutipes* and the polysaccharides in the edible part were 55.97% and 56.65%, respectively. Under this concentration condition, there was no significant difference in the inhibitory ability of the two on lipase. P >0.05). Therefore, both the root polysaccharide and the edible polysaccharide of enoki mushrooms have inhibitory effects on lipase, and their inhibitory effects on lipase are basically the same.

[0110] ② The binding rate of FRP and FEP with sodium glycocholate and sodium taurocholate Polysaccharides specifically bind to bile salts in the intestine, forming insoluble complexes and blocking their reabsorption. This promotes the excretion of bile salts in feces, thereby disrupting the enterohepatic bile acid cycle and reducing bile salt reserves in the body. To maintain homeostasis, the liver upregulates cholesterol 7α-hydroxylase (CYP7A1), accelerating the conversion of serum cholesterol to bile salts, thus promoting a decrease in low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC), achieving a lipid-lowering effect. Figure 11 It was found that the binding rates of both the root polysaccharides and edible portion polysaccharides of *Flammulina velutipes* to sodium glycinecholate and sodium taurocholate increased with increasing polysaccharide concentration, exhibiting a clear dose-dependent effect. At a concentration of 20 mg / mL, the binding rates of root polysaccharides and edible portion polysaccharides to sodium glycinecholate were 33.24% and 30.52%, respectively, while the binding rates to sodium taurocholate were 44.10% and 40.33%, respectively. The binding rates of root polysaccharides to sodium glycinecholate and sodium taurocholate were significantly higher than those of the edible portion polysaccharides. P <0.05). Therefore, both the root polysaccharide and the edible polysaccharide of *Flammulina velutipes* can bind sodium glycinate and sodium taurocholate, and at a concentration of 20 mg / mL, the binding capacity of *Flammulina velutipes* root polysaccharide to sodium glycinate and sodium taurocholate is significantly higher than that of the edible polysaccharide. P <0.05).

[0111] Example 9 Evaluation of the anemia activity of polysaccharides from the roots and edible parts of *Flammulina velutipes*. Experimental methods: Healthy wild-type AB zebrafish that had reached 56 hpf (hour post-fertilization) were placed in 6-well plates and divided into 8 groups of 8 fish per group. The blank control group (NC) was given culture water; the anemia model group (MC) was supplemented with 0.1 μg / mL phenylhydrazine; the low, medium, and high dose *Flammulina velutipes* root polysaccharide intervention groups (I, II, III) were supplemented with 0.1 μg / mL phenylhydrazine and different concentrations of *Flammulina velutipes* root polysaccharide (50, 100, 200 μg / mL); the low, medium, and high dose edible polysaccharide intervention groups (IV, V, VI) were supplemented with 0.1 μg / mL phenylhydrazine and different concentrations of edible polysaccharide (50, 100, 200 μg / mL). The zebrafish in each group were incubated for 3 dpf (day post-fertilization). The samples were treated with an anesthetic (0.16% tricaine) for 1 min, and the embryos were stained with o-anisidine solution in the dark for 20 min. Images were acquired under a microscope (Carl Zeiss, AXIO-V16, Germany). The optical density (IOD) values ​​of the stained areas were quantified using ImageJ (1.0, USA) software.

[0112] Experimental results: Phenylated hydrazine can damage red blood cells, inducing an anemia model in zebrafish. Hemoglobin within mature red blood cells catalyzes the oxidation of o-anisidine by hydrogen peroxide, producing a dark red product. As the number of red blood cells in zebrafish decreases, the average optical density of blood cell staining in the heart region of juvenile fish also decreases. Figure 12 It can be seen that after modeling with 0.1 μg / mL phenylhydrazine, the IOD value at the heart of zebrafish juveniles in the MC group was significantly lower than that in the NC group. P <0.0001), phenylhydrazine effectively inhibited erythropoiesis, indicating the successful establishment of the zebrafish anemia model. Under low, medium, and high doses of *Flammulina velutipes* root polysaccharide and edible part polysaccharide intervention, the IOD value at the heart of juvenile zebrafish gradually increased, showing a clear dose-response relationship. The IOD values ​​of zebrafish in groups I, II, and III were 0.31, 0.48, and 0.70, respectively, representing increases of 47.62%, 128.57%, and 233.33% compared to the MC group. The IOD values ​​of zebrafish in groups IV, V, and VI were 0.25, 0.41, and 0.63, respectively, representing increases of 19.05%, 95.24%, and 200.00% compared to the MC group; the low and medium dose intervention groups of FRP were significantly superior to FEP. P <0.05). Therefore, the polysaccharides from the root of Enoki mushroom and the polysaccharides from its edible portion exhibit a hematopoietic effect in a zebrafish anemia model.

[0113] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of Enoki mushroom polysaccharide in the preparation of products with antioxidant, blood-tonifying, blood sugar-lowering and blood lipid-lowering effects.

2. The application as described in claim 1, characterized in that, The enoki mushroom polysaccharide is a polysaccharide from the root of enoki mushroom. The products include food, pharmaceuticals, and animal feed.

3. A method for preparing polysaccharides from *Flammulina velutipes*, characterized in that, The preparation method includes: S1. Mix the enoki mushroom sample with water, add pectinase for enzymatic hydrolysis, and then extract with hot water to obtain the supernatant. S2. Add ethanol to the supernatant obtained in step S1 for alcohol precipitation, let stand, centrifuge to collect the precipitate, and dry to obtain crude polysaccharide of Enoki mushroom.

4. The preparation method according to claim 3, characterized in that, The preparation method further includes deproteinizing the crude polysaccharide of *Flammulina velutipes* obtained in step S2; further, the deproteinization is performed using Sevag reagent.

5. The preparation method according to claim 3, characterized in that, In step S1, the enoki mushroom sample is the edible part of enoki mushroom or the root of enoki mushroom, preferably the root of enoki mushroom; further, the root of enoki mushroom is a segment of the base of the stipe cut upwards from the base of the stipe, with a length of 1-4 cm, more preferably 3 cm.

6. The preparation method according to claim 3, characterized in that, In step S1, the ratio of the enoki mushroom sample to water is 1:20-40.

7. The preparation method according to claim 3, characterized in that, In step S1, the amount of pectinase added is 2%-6% (w / w), preferably 4%; the enzymatic hydrolysis temperature is 40-60℃, preferably 50-55℃; and the enzymatic hydrolysis time is 60-120 min, preferably 90-100 min.

8. The preparation method according to claim 3, characterized in that, In step S1, the hot water extraction temperature is 80-100℃, preferably 85-90℃; the hot water extraction time is 90-150min, preferably 120-125min; and the hot water extraction is performed 1-3 times.

9. The preparation method according to claim 3, characterized in that, In step S2, the ethanol is ethanol with a concentration of 80% or higher; the final concentration of ethanol precipitation is controlled at 80%; and the standing time is 6-24 h.

10. A product with antioxidant, blood-tonifying, blood sugar-lowering, and blood lipid-lowering effects, characterized in that, The product contains at least one enoki mushroom polysaccharide obtained by the preparation method according to any one of claims 3-9.

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