Green extraction method and application of auricularia auricula polysaccharide

Abstract

The invention provides a green extraction method and application of auricularia auricula polysaccharide, and relates to the technical field of plant active ingredient extraction. The method comprises the following steps: mixing a black fungus raw material with water, carrying out enzymolysis, carrying out extraction in a bionic environment in which the pH value is adjusted to 4.0-7.0, and carrying out solid-liquid separation to obtain a crude extract and dregs; treating the crude extract through a magnetic induction electric field, filtering through an ultrafiltration membrane system, and collecting polysaccharide trapped fluid; and concentrating and drying the polysaccharide trapped fluid to obtain the auricularia auricula polysaccharide. The method comprises the following steps: washing and drying the obtained dregs, extracting by adopting an enzyme-ultrasonic synergistic method, and drying to obtain a dietary fiber byproduct. Through cooperation of enzyme-semi-bionic extraction and magnetoelectric-membrane purification, efficient extraction and green purification of the auricularia auricula polysaccharide are achieved under mild conditions, meanwhile, resource utilization of by-products is completed, and the whole process is green and environmentally friendly. The obtained auricularia auricula polysaccharide is high in purity and good in activity, and has a good application prospect in the aspects of glycolipid metabolism regulation and oxidation resistance.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction technology, specifically a green extraction method for polysaccharides from black fungus and its application. Background Technology

[0002] Black fungus ( Auricularia auricula (L. ex Hook.) Underw. is an important medicinal and edible fungus. Its core active ingredient, black fungus polysaccharide, has been proven by modern research to possess multiple physiological functions, including immunomodulation, antioxidation, antitumor activity, lipid-lowering effects, and regulation of intestinal flora. With the rapid development of the health industry, the market demand for high-purity, high-activity black fungus polysaccharide is increasing. Therefore, developing efficient and green black fungus polysaccharide extraction and purification technologies is of great significance.

[0003] Currently, the main extraction methods for polysaccharides from black fungus include hot water extraction, acid-base extraction, enzymatic hydrolysis, and various physical-assisted extraction methods. Hot water extraction is simple to operate, but suffers from high extraction temperatures, long extraction times, low yields, and easy thermal degradation of active ingredients. While acid-base extraction can improve yields, strong acid or alkali conditions easily damage the glycosidic bonds and spatial conformation of polysaccharides, leading to a significant decrease in their biological activity. Furthermore, subsequent neutralization treatment introduces inorganic salt impurities, which is inconsistent with green production principles. Enzymatic hydrolysis utilizes the specific degradation of cell walls by biological enzymes, providing mild conditions that are conducive to maintaining activity. However, single-enzyme methods often result in incomplete cell wall disruption and limited extraction efficiency; while compound enzyme methods are costly and produce complex hydrolysis products, burdening subsequent purification. Physical-assisted techniques such as ultrasound, microwave, and high-pressure homogenization can effectively disrupt cells and increase extraction rates, but they generally have high equipment requirements, high energy consumption, and the risk of localized overheating. Moreover, the impact on polysaccharide molecular weight is difficult to control precisely, potentially affecting its functional properties. In recent years, some studies have attempted to use chemical cell-wall disruptors to improve efficiency. For example, Chinese patent CN113549161A discloses a method for extracting polysaccharides from black fungus using hydrogen peroxide. This method achieves a high polysaccharide yield, exceeding 65%, by using hydrogen peroxide to oxidize and break down cell walls at high temperatures. While this method improves efficiency, it is essentially a chemical oxidation method with harsh reaction conditions. The high temperature and oxidative environment pose a potential risk of damaging the fine structure and active groups of the polysaccharides. More importantly, the purification process still relies on ethanol precipitation and the Sevag method for protein removal, which is not only cumbersome but also introduces safety and environmental hazards related to residual organic solvents.

[0004] In summary, current black fungus polysaccharide extraction technology faces a core contradiction: the difficulty in simultaneously achieving extraction efficiency, activity retention, and environmental friendliness. Traditional hot water methods result in low yields; acid-base methods destroy activity; and enzymatic or physical-assisted methods each have limitations in efficiency, cost, or activity control. While existing high-efficiency methods improve yields, the harsh conditions threaten the polysaccharide structure, and subsequent purification relies on toxic organic solvents, posing safety and environmental risks. Furthermore, existing processes often neglect the resource utilization of extraction residues. Therefore, there is an urgent need in this field to develop a method for preparing black fungus polysaccharides that can simultaneously achieve high extraction efficiency, high activity retention, a completely green and pollution-free process, and comprehensive utilization of the full value of raw materials. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a green extraction method for black fungus polysaccharides. The black fungus polysaccharides obtained by the extraction method have high purity and good activity, and have good application prospects in regulating glucose and lipid metabolism and anti-oxidation.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a green extraction method for polysaccharides from black fungus, comprising the following steps: (1) Mix the black fungus raw material with water and add enzyme for enzymatic hydrolysis; after the enzymatic hydrolysis is completed, adjust the pH to a biomimetic environment of 4.0-7.0 and extract at 40-60℃. After extraction, separate the solid and liquid to obtain crude extract and residue. (2) The crude extract is subjected to magnetic induction electric field treatment, and then filtered through an ultrafiltration membrane system to collect the polysaccharide retentate; (3) The polysaccharide retentate is concentrated and dried to obtain black fungus polysaccharide.

[0007] Preferably, the enzyme in step (1) includes one or more of cellulase, pectinase and papain.

[0008] Preferably, in step (2), the electric field strength of the magnetic induction electric field treatment is 5-15V / cm, the frequency is 50-200Hz, and the treatment time is 10-30 minutes.

[0009] Preferably, the ultrafiltration membrane used in step (2) has a molecular weight cutoff of 5-50 kDa.

[0010] Preferably, the residue obtained in step (1) is washed with water, dried, and then extracted using an enzyme-ultrasound synergistic method; the extracted liquid is dried to obtain dietary fiber by-products.

[0011] More preferably, the enzyme-ultrasound synergistic method includes the following steps: mixing the dried residue with water, adjusting the pH, adding cellulase for enzymatic hydrolysis, and then performing ultrasound-assisted extraction.

[0012] More preferably, the conditions for ultrasound-assisted extraction are: ultrasound power 200-500W, ultrasound time 20-60 minutes.

[0013] More preferably, the temperature of the ultrasound-assisted extraction is controlled at 50-65℃.

[0014] The present invention also provides a black fungus polysaccharide prepared by the above method.

[0015] The present invention also provides the application of the aforementioned black fungus polysaccharide in the preparation of products with functions of regulating glucose and lipid metabolism and / or antioxidation.

[0016] Compared with the prior art, the present invention has the following advantages: (1) This invention combines enzyme-semi-biomimetic extraction technology with magnetic induction electric field-ultrafiltration membrane coupling purification technology, achieving efficient extraction and highly selective purification of black fungus polysaccharides under mild conditions, avoiding the use of high temperature, strong acids and alkalis and organic solvents, and making the whole process green and environmentally friendly. This invention promotes the controlled aggregation of polysaccharide molecules through magnetic induction electric field treatment, effectively improving the efficiency and selectivity of subsequent ultrafiltration membrane separation. The purification steps do not require traditional cumbersome processes such as alcohol precipitation and deproteinization, and the obtained polysaccharides have high purity, complete structure and better preservation of biological activity.

[0017] (2) This invention utilizes the residue after the main product is extracted to extract water-soluble dietary fiber byproducts through an enzyme-ultrasound synergistic method, realizing the full value and zero-waste comprehensive utilization of black fungus raw materials, and significantly improving the economy and environmental protection of the process.

[0018] (3) The black fungus polysaccharide obtained by the method of the present invention exhibits excellent biological activity in regulating cellular glycolipid metabolism and scavenging free radicals, which is significantly better than the products obtained by traditional hot water extraction and alkaline extraction methods. Compared with the products obtained by existing efficient but harsh methods, it shows better biological activity retention at similar extraction rates and has greater potential for development and application. Detailed Implementation

[0019] This invention provides a green extraction method for polysaccharides from black fungus, wherein the extraction method preferably includes the following steps: (1) Mix the black fungus raw material with water and add enzyme for enzymatic hydrolysis; after the enzymatic hydrolysis is completed, adjust the pH to a biomimetic environment of 4.0-7.0 and extract at 40-60℃. After extraction, separate the solid and liquid to obtain crude extract and residue. (2) The crude extract is subjected to magnetic induction electric field treatment, and then filtered through an ultrafiltration membrane system to collect the polysaccharide retentate; (3) The polysaccharide retentate is concentrated and dried to obtain black fungus polysaccharide.

[0020] In this invention, dried black fungus raw materials are washed, pulverized, and sieved through a 60-200 mesh sieve to obtain dried black fungus powder. The dried black fungus powder is weighed and mixed with water at a material-to-liquid ratio of 1:15 to 1:30 (g / mL), preferably soaked and swollen at room temperature for 0.5-2 hours. Subsequently, an enzyme comprising 0.1%-0.8% of the dry powder mass is added to the system; the enzyme includes one or more of cellulase, pectinase, and papain. Enzymatic hydrolysis is carried out for 1-3 hours at pH 4.5-6.0 and a temperature of 45-55℃. After enzymatic hydrolysis, the pH of the system is adjusted to the range of 4.0-7.0 using dilute hydrochloric acid or dilute sodium hydroxide solution. If simulating a gastric environment, the pH is adjusted to 4.0-5.0; if simulating an intestinal environment, the pH is adjusted to 6.0-7.0. Then, extraction is continued at 50-60℃ with stirring for 1-2 hours. After extraction, solid-liquid separation is performed by centrifugation or filtration to obtain a crude extract containing soluble polysaccharides and a residue mainly composed of cell wall debris.

[0021] In this invention, the obtained crude extract is transferred to a reaction cell equipped with a magnetic induction electric field treatment device with parallel plate electrodes. The device is turned on, and the electric field strength is set to 5-15 V / cm, preferably 8-10 V / cm, and the frequency to 50-200 Hz, preferably 80-100 Hz. Treatment is carried out at room temperature for 10-30 minutes, preferably 15-20 minutes. During this process, the magnetic induction electric field directs the polysaccharide molecules in the solution, promoting their controlled aggregation, which facilitates the initial separation from small molecule impurities. After treatment, the liquid is introduced into an ultrafiltration membrane system. This system can use an ultrafiltration membrane module made of polyethersulfone or ceramic material with a molecular weight cutoff of 5-50 kDa, preferably 10-20 kDa. Cross-flow filtration or dead-end filtration is performed at an operating pressure of 0.1-0.5 MPa. The permeate passing through the membrane mainly contains water, inorganic salts, and small molecule impurities, while the concentrate retained by the membrane is the target polysaccharide retained solution, which is collected and used in subsequent steps. The purification process of this invention requires no addition of any organic solvents.

[0022] In this invention, the collected polysaccharide retentate is placed in a rotary evaporator and concentrated under reduced pressure at a water bath temperature of 50-65℃ and a vacuum of 0.06-0.09 MPa until the liquid volume is reduced to 1 / 5 to 1 / 10 of its original volume, resulting in a viscous concentrate. The concentrate is then transferred to a freeze-drying tray and pre-frozen at -40℃ to -55℃ for 3-6 hours to ensure complete solidification. Finally, the freeze-drying tray is placed in a freeze dryer and dried for 18-36 hours at a cold trap temperature below -50℃ and a vacuum degree below 15 Pa until a loose, porous, and brittle black fungus polysaccharide solid is obtained. This invention maximizes the preservation of the polysaccharide's biological activity.

[0023] In this invention, the enzyme in step (1) includes one or more of cellulase, pectinase, and papain. The enzyme can be a commercially available food-grade or industrial-grade enzyme. Cellulase is mainly used to hydrolyze the β-1,4-glucan backbone in the cell wall of black fungus; pectinase targets pectin-like substances in the intercellular matrix; and papain is a broad-spectrum protease that helps hydrolyze structural proteins in the cell wall. These three can be combined according to their functions to form a synergistic cell wall-breaking system. As a preferred embodiment, cellulase, pectinase, and papain are combined at a total addition amount of 0.1%-0.5% of the dry powder mass, and the preferred mass ratio of cellulase, pectinase, and papain is 2:1:1. For example, when processing 100 grams of dried black fungus powder, 0.3 grams of a compound enzyme can be added, containing 0.15 grams of cellulase, 0.075 grams of pectinase, and 0.075 grams of papain. As an optional embodiment, a combination of cellulase and pectinase is used, with a preferred mass ratio of 3:1. As an alternative implementation method, cellulase can be used alone. Compared with the control experiment using only a single cellulase, the above-mentioned preferred complex enzyme scheme can increase the extraction rate of subsequent polysaccharides by 15%-25% under the same conditions, and the molecular weight distribution of the obtained polysaccharides is more concentrated, indicating that its cell wall disruption is more gentle, efficient and selective.

[0024] In this invention, the residue obtained after solid-liquid separation in step (1) is washed 2-3 times with 3-5 times its weight of warm water at 50-60℃, stirring and separating after each wash until the supernatant is nearly colorless, in order to fully remove residual polysaccharides, pigments and soluble impurities. The washed residue is spread evenly on a tray and placed in a forced-air drying oven at 60-70℃ for 6-10 hours until the moisture content is less than 8%, and then pulverized through an 80-mesh sieve to obtain dried residue powder. A certain amount of the above dried residue powder is weighed and added to an acetate-sodium acetate buffer solution or deionized water with a pH of 4.5-5.5 at a material-to-liquid ratio of 1:10 to 1:20 (g / mL). Cellulase is added at 0.5%-2.0% of the dry residue powder mass, and enzymatic hydrolysis is performed at 50-60℃ with low-speed stirring for 1.5-3 hours. After enzymatic hydrolysis, the system temperature is maintained and ultrasonic-assisted extraction is performed immediately. The reaction vessel is placed in an ultrasonic processor, and the ultrasonic power is set to 200-500W. Treatment is carried out at 50-65℃ for 20-60 minutes. After treatment, the mixture is centrifuged at 8000-10000 rpm for 10-15 minutes, and the supernatant is collected; this is the dietary fiber extract. Finally, the extract is spray-dried to obtain a powdered product. The preferred spray-drying conditions are: inlet air temperature 160-180℃, outlet air temperature 80-90℃, and the feed pump speed is adjusted according to the material viscosity. A light-colored, loose, water-soluble dietary fiber powder byproduct is obtained. This process achieves a dietary fiber recovery rate of 40%-60% from the residue, realizing the full value utilization of the raw materials.

[0025] The present invention also provides a black fungus polysaccharide prepared by the above method.

[0026] The present invention also provides the application of the aforementioned black fungus polysaccharide in the preparation of products with functions of regulating glucose and lipid metabolism and / or antioxidation.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0029] Example 1 A green extraction method for polysaccharides from black fungus, comprising the following steps: (1) Raw material pretreatment: Take 100g of dried black fungus, wash and remove impurities, dry in an oven at 50℃ until constant weight, pulverize and pass through a 180-mesh sieve to obtain black fungus powder (moisture content 8.5%).

[0030] (2) Enzyme-semi-bionic extraction: Black fungus powder was placed in an extraction tank, and deionized water was added at a material-to-liquid ratio of 1:20 (g / mL). The mixture was soaked and swollen at room temperature for 2 hours. 0.3% (w / w, based on dry powder) of a compound enzyme (cellulase:pectinase:papain = 2:1:1) was added, and the mixture was enzymatically hydrolyzed at 50℃ and pH 5.5 for 2 hours. After hydrolysis, the pH of the system was adjusted to 6.0 with 1M HCl solution, and the extraction was continued at 55℃ with stirring for 1.5 hours. After extraction, the mixture was centrifuged at 8000 rpm for 15 minutes to separate the supernatant (crude extract) and precipitate (residue I). The volume of the crude extract (V1) was accurately measured.

[0031] (3) Magnetic induction electric field-ultrafiltration membrane coupling purification: The crude extract was transferred to a magnetic induction electric field treatment device and treated for 20 minutes under an electric field strength of 10 V / cm and a frequency of 100 Hz. Subsequently, the treated solution was passed through a polyether sulfone (PES) ultrafiltration membrane system with a molecular weight cutoff of 10 kDa at an operating pressure of 0.3 MPa, and the retentate was collected.

[0032] (4) Product drying: The retentate was concentrated to 1 / 10 of its original volume by vacuum rotary evaporation at 60℃, and then freeze-dried (-50℃, 10Pa) to obtain a light brown, flocculent black fungus polysaccharide product (denoted as APP-1). The mass (m1) was weighed and recorded.

[0033] (5) Waste Resource Utilization (By-product Preparation): Wash the above-mentioned Waste I with deionized water until the filtrate is colorless, and dry it at 60℃. Take 50g of the dried waste, add deionized water at a material-to-liquid ratio of 1:15, and adjust the pH to 5.0 with NaOH solution. Add 1.0% (w / w, based on dry waste) of cellulase and enzymatically hydrolyze at 55℃ for 1.5 hours. Subsequently, perform ultrasonic-assisted extraction (power 350W, pulse mode: 3s working, 2s intermittent) at the same temperature for 40 minutes. Centrifuge (8000rpm, 10min) to collect the supernatant, and spray dry (inlet air temperature 180℃, outlet air temperature 85℃) to obtain light yellow dietary fiber powder (denoted as APP-F1).

[0034] Example 2 A green extraction method for polysaccharides from black fungus, comprising the following steps: (1) Raw material pretreatment: Same as in Example 1, take 100g of dried black fungus and prepare black fungus powder with a water content of 8.5%.

[0035] (2) Enzyme-semi-bionic extraction: Black fungus powder was placed in an extraction tank, and deionized water was added at a material-to-liquid ratio of 1:20 (g / mL). The mixture was soaked and swollen at room temperature for 2 hours. 0.5% (w / w, based on dry powder) of a compound enzyme (cellulase:pectinase = 1:1) was added, and enzymatic hydrolysis was performed at 50℃ and pH 5.5 for 2 hours. After enzymatic hydrolysis, the pH of the system was adjusted to 5.0 with 1M HCl solution, and the extraction was continued at 55℃ with stirring for 2 hours. After extraction, the mixture was centrifuged at 8000 rpm for 15 minutes to separate the supernatant (crude extract) and precipitate (residue II). The volume of the crude extract (V2) was accurately measured.

[0036] (3) Magnetic induction electric field-ultrafiltration membrane coupling purification: The crude extract was transferred to a magnetic induction electric field treatment device and treated for 15 minutes under an electric field strength of 8V / cm and a frequency of 100Hz. Subsequently, the treated liquid was passed through a polyethersulfone (PES) ultrafiltration membrane system with a molecular weight cutoff of 30kDa at an operating pressure of 0.25MPa, and the retentate was collected. Product drying: The retentate was concentrated to 1 / 10 of its original volume by vacuum rotary evaporation at 60℃, and then freeze-dried (-50℃, 10Pa) to obtain a light brown, flocculent black fungus polysaccharide product (denoted as APP-2). The mass (m2) was weighed and recorded.

[0037] (4) Resource utilization of waste (byproduct preparation): Wash the above-mentioned waste II with deionized water until the filtrate is colorless, and dry it at 60°C. Take 50g of the dried waste, add deionized water at a material-to-liquid ratio of 1:15, and adjust the pH to 5.0 with NaOH solution. Add 1.0% (w / w, based on dry waste) of cellulase and enzymatically hydrolyze at 55°C for 1.5 hours. Subsequently, perform ultrasonic-assisted extraction (power 350W, pulse mode: 3s working, 2s intermittent) at the same temperature for 40 minutes. Centrifuge (8000rpm, 10min) to collect the supernatant, and spray dry (inlet air temperature 180°C, outlet air temperature 85°C) to obtain light yellow dietary fiber powder (denoted as APP-F2).

[0038] Example 3 A green extraction method for polysaccharides from black fungus, comprising the following steps: (1) Raw material pretreatment: Same as in Example 1, take 100g of dried black fungus and prepare black fungus powder with a water content of 8.5%.

[0039] (2) Enzyme-semi-bionic extraction: Black fungus powder was placed in an extraction tank, and deionized water was added at a material-to-liquid ratio of 1:15 (g / mL). The mixture was soaked and swollen at room temperature for 2 hours. 0.4% (w / w, based on dry powder) of cellulase was added, and enzymatic hydrolysis was carried out at 50℃ and pH 5.5 for 2 hours. After enzymatic hydrolysis, the pH of the system was adjusted to 7.0 with 1M NaOH solution, and the extraction was continued at 60℃ with stirring for 1 hour. After extraction, the mixture was centrifuged at 8000 rpm for 15 minutes to separate the supernatant (crude extract) and precipitate (residue III). The volume of the crude extract (V3) was accurately measured.

[0040] (3) Magnetic induction electric field-ultrafiltration membrane coupling purification: The crude extract was transferred to a magnetic induction electric field treatment device and treated for 20 minutes under an electric field strength of 12V / cm and a frequency of 150Hz. Subsequently, the treated solution was passed through a polyethersulfone (PES) ultrafiltration membrane system with a molecular weight cutoff of 10kDa at an operating pressure of 0.3MPa, and the retentate was collected.

[0041] (4) Product drying: The retentate was concentrated to 1 / 10 of its original volume by vacuum rotary evaporation at 60℃, and then freeze-dried (-50℃, 10Pa) to obtain a light brown, flocculent black fungus polysaccharide product (denoted as APP-3). The mass (m3) was weighed and recorded.

[0042] (5) Resource utilization of waste (byproduct preparation): Wash the above-mentioned waste III with deionized water until the filtrate is colorless, and dry it at 60°C. Take 50g of the dried waste, add deionized water at a material-to-liquid ratio of 1:15, and adjust the pH to 5.0 with NaOH solution. Add 1.0% (w / w, based on dry waste) of cellulase and enzymatically hydrolyze at 55°C for 1.5 hours. Subsequently, perform ultrasonic-assisted extraction (power 250W, pulse mode: 2s working, 1s intermittent) at the same temperature for 30 minutes. Centrifuge (8000rpm, 10min) to collect the supernatant, and spray dry (inlet air temperature 180°C, outlet air temperature 85°C) to obtain light yellow dietary fiber powder (denoted as APP-F3).

[0043] Comparative Example 1: Traditional Hot Water Extraction Method Take 100g of black fungus powder (same as in Example 1), add deionized water at a material-to-liquid ratio of 1:20, and reflux extract in a 90℃ water bath for 3 hours. Centrifuge and collect the supernatant, add 4 times the volume of 95% ethanol, and precipitate overnight at 4℃. Centrifuge to collect the precipitate, redissolve it in water, and repeatedly deproteinize using the Sevag method (chloroform:n-butanol = 4:1) until no absorption peaks are observed at 260nm and 280nm under UV scanning. Dialyze, concentrate, and freeze-dry to obtain the polysaccharide product (denoted as C-1).

[0044] Comparative Example 2: Alkali Extraction Method Take 100g of black fungus powder and add 0.1 M NaOH solution at a material-to-liquid ratio of 1:20. Extract in a 60℃ water bath for 2 hours. Neutralize the extract with 1 M HCl to pH 7.0. Subsequent alcohol precipitation, deproteinization, and drying steps are the same as in Comparative Example 1 to obtain the polysaccharide product (denoted as C-2).

[0045] Comparative Example 3 Based on the hydrogen peroxide-vitamin C method described in CN113549161A, 100g of black fungus powder was added to deionized water at a material-to-liquid ratio of 1:104 and swollen for 12 hours. Hydrogen peroxide solution was added to achieve a final concentration of 1.41%, and vitamin C (at a mass ratio of 1:3 to H2O2) was added to adjust the pH to 7.0. Extraction was carried out in a 90℃ water bath with shaking for 2 hours. Subsequent alcohol precipitation, Sevag deproteinization, and drying steps were the same as in Comparative Example 1, yielding the polysaccharide product (denoted as C-3).

[0046] Comparative Example 4 Biomimetic-enzymatic raw material pretreatment based on CN106188331A: (1) Raw material pretreatment: Take 100g of black fungus powder, the same as in Example 1.

[0047] (2) Enzymatic hydrolysis: Black fungus powder was added to deionized water at a material-to-liquid ratio of 1:50 (g / mL) and soaked for 4 hours. Then, 15% (w / w, based on dry powder) of compound cellulase (Novozymes) was added, and enzymatic hydrolysis was carried out at 45℃ and natural pH for 120 minutes. After enzymatic hydrolysis, the enzyme was inactivated in a 100℃ water bath for 10 minutes to obtain the enzymatic hydrolysate.

[0048] (3) Bionic extraction: The pH of the enzymatic hydrolysate was adjusted to 2.0 with 1M HCl solution, and extracted with stirring at 45℃ for 120 minutes. Subsequently, the pH was adjusted to 8.0 with 1M NaOH solution, and extraction was continued with stirring at 45℃ for another 120 minutes. After extraction, the solution was filtered through gauze and the filtrate was collected. The filtrate was concentrated to about 1 / 4 of its original volume by rotary evaporation at 50℃.

[0049] (4) Alcohol precipitation and drying: Add 4 times the volume of 95% (v / v) ethanol to the concentrate and let it stand overnight at 4℃ for alcohol precipitation. Centrifuge (10000 rpm, 15 min) to collect the precipitate. Freeze-dry the precipitate (-50℃, 10 Pa) to obtain the black fungus polysaccharide product, denoted as C-4.

[0050] Example 4: Evaluation of Polysaccharide Extraction Rate and Purity This embodiment aims to evaluate the polysaccharide extraction rate and polysaccharide purity of the preparation processes in Examples 1-3 and Comparative Examples 1-4.

[0051] 1. Extraction rate calculation: ① Determination of total sugar content using the phenol-sulfuric acid method: Prepare a glucose standard curve. Accurately weigh 10 mg of the polysaccharide sample obtained in each example and comparative example, dissolve it in a 100 mL volumetric flask, and dilute to volume. Take an appropriate amount of dilution, and measure the absorbance at 490 nm using the phenol-sulfuric acid method. Calculate the total sugar concentration (C, mg / mL) in the sample solution based on the standard curve.

[0052] ② Extraction rate formula: Extraction rate (%) = [(C×Vd×n) / (M×(1-W%))]×100%. In the formula, C represents the total sugar concentration in the sample solution (mg / mL); Vd represents the sample volume (mL); n represents the dilution factor; M represents the mass of dried black fungus powder added (mg); W% represents the moisture content of the black fungus powder (%).

[0053] 2. Calculation of polysaccharide purity: ① Determination of uronic acid content using the sulfuric acid-carbazole method: A standard curve was established using galacturonic acid as the standard. The sample solution was taken, and the absorbance was measured at 530 nm using the sulfuric acid-carbazole method. The uronic acid content was then calculated.

[0054] ②Purity evaluation formula: Polysaccharide purity (%) is expressed as total sugar content, and uronic acid content is used as a reference indicator for acidic polysaccharide components.

[0055] Table 1. Polysaccharide extraction rate and polysaccharide purity for each group.

[0056] As shown in Table 1, the extraction rates of black fungus polysaccharides (APP-1, APP-2, APP-3) obtained by the method of this invention (19.1%-22.5%) are significantly higher than those obtained by the traditional hot water method. Although lower than those obtained by the high-intensity hydrogen peroxide-vitamin C method (68.5%), the total sugar content (88.5%-91.3%) is higher, indicating superior purity. This demonstrates that the present invention, through a mild enzyme-semi-biomimetic extraction combined with a green purification process, better preserves the integrity and high purity of the polysaccharides without excessively sacrificing the extraction rate, achieving an optimized balance between extraction efficiency and product quality.

[0057] Example 5 Assessment of Glucose and Lipid Metabolism Regulation Capacity 1. Model Establishment: An insulin-resistant HepG2 cell model was used. Cells containing 33 mM glucose and 10... -7 HepG2 cells were cultured in M ​​insulin medium for 24 hours to induce insulin resistance.

[0058] 2. Grouping and drug administration: A normal control group, a model control group, a positive control group (Metformin, 2 mM), and various polysaccharide sample groups (APP-1, APP-2, APP-3, and C-3, all at a concentration of 200 μg / mL) were set up. Each group had 6 replicates.

[0059] 3. Detection indicators: 24 hours after drug intervention, the cells were incubated in serum-free medium containing 2-NBDG (a fluorescent glucose analog) for 30 minutes. Flow cytometry was used to detect the intracellular 2-NBDG fluorescence intensity, reflecting the cells' glucose uptake capacity.

[0060] 4. Results Analysis Table 2. Glucose uptake capacity of each treatment group

[0061] Note: Compared to the Normal group. Compared to the Model group, ##p<0.01.

[0062] As shown in Table 2, the black fungus polysaccharides (APP-1, APP-2, APP-3) prepared by the method of this invention can significantly improve the glucose uptake capacity of insulin-resistant cells, with effects close to those of the positive control drug metformin, and all superior to the hydrogen peroxide-vitamin C method product of Comparative Example 3 (C-3). This indicates that the polysaccharides extracted under mild conditions in this invention can better retain their biological activity in regulating glucose and lipid metabolism.

[0063] Example 6: In vitro antioxidant capacity assessment 1. Sample solution: Accurately weigh each polysaccharide sample (APP-1, APP-2, APP-3, C-1, C-3, C-4) and the positive control vitamin C (Vc), and prepare a test solution with a concentration of 1.0 mg / mL using deionized water. (Comparative Example 2, product C-2 obtained by alkaline extraction, was not included in this antioxidant comparison due to its low purity and unsatisfactory expected activity).

[0064] 2. ABTS + • Scavenging Experiment: React an appropriate amount of test solution with ABTS working solution and measure the absorbance at a wavelength of 734 nm. Use an equal volume of deionized water as a blank control instead of the sample. The scavenging rate is calculated using the formula: Scavenging rate (%) = [1 - (As ample / A blank )]×100%. Among them, A sample A represents the absorbance of the sample group. blank The absorbance is for the blank group.

[0065] 3. DPPH Scavenging Assay: React an appropriate amount of test solution with DPPH ethanol solution and measure the absorbance at 517 nm. Use an equal volume of anhydrous ethanol instead of DPPH solution as a background control, and use an equal volume of deionized water instead of the sample as a blank control. The scavenging rate is calculated as follows: Scavenging rate (%) = [1 - (As...] ample -A background ) / A blank ]×100%. Among them, A sample A represents the absorbance of the sample after the reaction with DPPH. background A represents the background absorbance of the sample. blank The absorbance is for the blank control. All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation.

[0066] 4. Results: At a sample concentration of 1.0 mg / mL, each sample showed good response to ABTS. + The clearance rates of · and DPPH· are shown in the table below.

[0067] Table 3. Treatment of each group on ABTS + Scavenging rate of · and DPPH·

[0068] As shown in Table 3, the results indicate that the black fungus polysaccharides (APP-1, APP-2, APP-3) obtained by the method of this invention exhibit strong antioxidant capacity, significantly superior to the traditional hot water method (C-1) and the biomimetic-enzymatic hydrolysis method (C-4). Although the hydrogen peroxide method product of Comparative Example 3 (C-3) also showed high antioxidant data, its extraction conditions were harsh, which may have a potential negative impact on the long-term stability of the polysaccharides.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A green extraction method of Auricularia auricular polysaccharide, characterized in that, The method comprises the following steps: (1) mixing Auricularia auricula raw material with water, and adding enzyme for enzymolysis; after the enzymolysis is completed, the pH is adjusted to 4.0-7.0 to simulate a biomimetic environment, and extraction is carried out at 40-60°C; after the extraction, solid-liquid separation is carried out, and a crude extract and dregs are obtained; (2) the crude extract is subjected to magnetic induction electric field treatment, and then filtered through an ultrafiltration membrane system to collect a polysaccharide interception liquid; (3) the polysaccharide interception liquid is concentrated and dried to obtain Auricularia auricula polysaccharide.

2. The extraction method according to claim 1, characterized in that, The enzyme in step (1) comprises one or more of cellulase, pectinase and papain.

3. The extraction method of claim 1, wherein, In step (2), the electric field strength of the magnetic induction electric field treatment is 5-15 V / cm, the frequency is 50-200 Hz, and the treatment time is 10-30 minutes.

4. The extraction method of claim 1, wherein, The ultrafiltration membrane used in step (2) has a molecular weight cut-off of 5-50 kDa.

5. The extraction method of claim 1, wherein, The dregs obtained in step (1) are washed with water and dried, and then extracted by an enzyme-ultrasound synergistic method; the extract obtained by extraction is dried to obtain a dietary fiber byproduct.

6. The extraction method according to claim 5, characterized in that, The enzyme-ultrasound synergistic method comprises the following steps: mixing the dried dregs with water, adjusting the pH, adding cellulase for enzymolysis, and then performing ultrasound-assisted extraction.

7. The extraction method according to claim 6, characterized in that, The ultrasound-assisted extraction is carried out at an ultrasonic power of 200-500 W and an ultrasonic time of 20-60 minutes.

8. The extraction method of claim 6, wherein, The ultrasound-assisted extraction is carried out at a temperature of 50-65°C.

9. Auricularia auricula polysaccharide prepared by the method of any one of claims 1-8.

10. Use of the Auricularia auricula polysaccharide of claim 9 in the preparation of a product for regulating glycolipid metabolism and / or antioxidant function.

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