Preparation method and application of poria cocos water-soluble polysaccharide based on enzyme-alkali synergy

Abstract

The invention discloses a poria cocos water-soluble polysaccharide preparation method based on enzyme-alkali synergy. The poria cocos water-soluble polysaccharide preparation method comprises the following steps: S1, crushing poria cocos to obtain poria cocos powder; mixing with ethanol, stirring and extracting; mixing with distilled water, stirring and extracting, and finally drying; s2, mixing the obtained system with a sodium hydroxide solution, stirring at room temperature, and centrifuging to obtain supernate; adjusting the pH value of the supernate by using an acetic acid solution, standing, repeatedly washing by using distilled water, and freeze-drying to obtain the alkali-soluble polysaccharide of the poria cocos; and S3, dissolving the obtained system in a sodium hydroxide solution, adding a compound enzyme, carrying out an enzymatic hydrolysis reaction, and carrying out enzyme deactivation, centrifugation, tetraploid volume ethanol precipitation, dialysis and freeze-drying on an enzymatic hydrolysate to obtain the poria cocos water-soluble polysaccharide. The invention provides a poria cocos water-soluble polysaccharide preparation method based on enzyme-alkali synergy, poria cocos alkali-soluble polysaccharide is used as a substrate, the poria cocos water-soluble polysaccharide is prepared by complex enzyme, and the extraction rate and activity of the poria cocos water-soluble polysaccharide are improved. The invention further discloses application of the poria cocos water-soluble polysaccharide.

Description

Technical Field

[0001] This invention relates to the field of plant active polysaccharide extraction technology, and in particular to a method for preparing water-soluble polysaccharides from Poria cocos based on enzyme-base synergy and its application. Background Technology

[0002] Chronic kidney disease (CKD) is a chronic disease characterized by progressive decline in kidney function and renal fibrosis, with a global prevalence of approximately 10%-15%. Existing treatments have limited effectiveness in intervening in renal fibrosis, necessitating the development of novel natural interventions.

[0003] Poria cocos is a traditional medicinal and edible fungus, rich in active ingredients such as polysaccharides, triterpenes, and steroids. Among them, polysaccharides are its main functional components. The polysaccharides in Poria cocos have significant anti-inflammatory and antioxidant effects, which can reduce the production of inflammatory factors and lower the level of oxidative stress, thereby alleviating the inflammatory response and oxidative damage to the kidneys.

[0004] Poria cocos polysaccharides can be divided into two categories: water-soluble and alkali-soluble. Among them, water-soluble polysaccharides in Poria cocos can reduce inflammatory factors and oxidative stress levels by inhibiting the NF-κB-NOX4 signaling pathway, thereby alleviating lipopolysaccharide-induced acute kidney injury in mice. Although the activity is high, the content is low (<3%). The traditional hot water extraction method commonly used to extract water-soluble polysaccharides from Poria cocos has an extraction rate of only 1.3%, while physical enhancement extraction methods are accompanied by high energy consumption and other side effects. The extraction rate of single / compound enzyme methods using Poria cocos as a substrate is limited to 15%, and the substrate utilization rate is low.

[0005] Therefore, the infusion of water-soluble polysaccharides from Poria cocos possesses anti-fibrotic and renal protective functions, but its low extraction rate and poor water solubility limit its application. Thus, it is necessary to improve the extraction method of water-soluble polysaccharides from Poria cocos to enhance its water solubility and bioactivity, thereby expanding its application in the intervention of chronic kidney disease. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing water-soluble polysaccharides from Poria cocos based on enzyme-base synergy and its application. The method uses alkali-soluble polysaccharides from Poria cocos as substrates and combines enzymes to prepare water-soluble polysaccharides from Poria cocos, thereby improving their extraction rate and activity.

[0007] The technical solution disclosed in this invention is as follows: A method for preparing water-soluble polysaccharides from Poria cocos based on enzyme-base synergy and its application. A method for preparing water-soluble polysaccharides from Poria cocos based on enzyme-base synergy includes the following steps: S1, Raw material pretreatment: Pulverize the dried Poria cocos and pass it through a 50-100 mesh sieve to obtain Poria cocos powder; mix it with 50%-70% ethanol at a material-to-liquid ratio of 1:5-1:10 g / mL, and extract it by stirring at room temperature 2-3 times, each time for 1.5-2.5 hours; Then mix with distilled water at a material-to-liquid ratio of 1:6-1:8 g / mL, and extract by stirring at 85-95℃ 2-3 times, each time for 1.5-2.5 hours. Finally, dry at a temperature of 55-65℃. S2, Extraction of alkali-soluble polysaccharides from Poria cocos: The above-obtained system was mixed with sodium hydroxide solution at a material-to-liquid ratio of 1:15-1:17 g / mL, stirred at room temperature for 1-2 hours, and then centrifuged to obtain the supernatant; Adjust the pH of the supernatant to 6.5-7.0 with 36%-40% acetic acid solution, let it stand, wash it repeatedly with distilled water 3-4 times, and freeze-dry it to obtain alkali-soluble polysaccharide from Poria cocos. S3, Enzymatic extraction of water-soluble polysaccharides from Poria cocos: The above-obtained system was dissolved in sodium hydroxide solution at a ratio of 1:1 to 1:3 g / mL, and a compound enzyme was added. Enzymatic hydrolysis was carried out under different compound enzyme ratios, substrate concentrations, hydrolysis pH, hydrolysis temperature, hydrolysis time, and enzyme dosage. The hydrolysate was then subjected to enzyme inactivation, centrifugation, precipitation with four times the volume of ethanol, dialysis, and freeze-drying to obtain water-soluble polysaccharides from Poria cocos.

[0008] As a preferred embodiment, in step S3, the conditions for enzymatic hydrolysis are determined by single-factor and response surface methodology.

[0009] As a preferred embodiment, in step S3, the complex enzyme is cellulase and β-glucanase.

[0010] As a preferred embodiment, the ratio of cellulase to β-glucanase is 1:3-3:1, the substrate concentration is 1-5%, the enzymatic hydrolysis pH is 4-8, the enzymatic hydrolysis temperature is 30-50℃, the enzymatic hydrolysis time is 0.5-2.5h, and the enzyme dosage is 9000-13000U / mL.

[0011] As a preferred option, based on the single-factor experiment, the Box-Benhken experimental design in Design-Expert13 statistical analysis software was used to establish a three-factor, three-level central composite design with the extraction rate of water-soluble polysaccharides from Poria cocos as the response value and the three factors with the greatest influence in the single-factor experiment—enzyme dosage, substrate concentration, and enzymatic hydrolysis time—as independent variables.

[0012] As a preferred embodiment, the extraction rate of the water-soluble polysaccharide from Poria cocos can reach 40%-42%.

[0013] An application of a water-soluble polysaccharide from Poria cocos: The water-soluble polysaccharide from Poria cocos prepared above is used in the intervention of chronic kidney disease to delay the progression of renal fibrosis.

[0014] As a preferred embodiment, the chronic kidney disease includes one or more diseases such as renal function decline, kidney lesions caused by inflammatory response, and kidney damage caused by decreased antioxidant capacity.

[0015] The beneficial effects of the enzyme-base synergistic method for preparing water-soluble polysaccharides from Poria cocos disclosed in this invention are: through a multi-step extraction process, water-soluble polysaccharides can be efficiently extracted from Poria cocos with high extraction efficiency and purity, while retaining the active ingredients of the polysaccharides, providing high-quality raw materials for subsequent pharmacological research and applications.

[0016] The enzyme-alkali synergistic extraction method not only improves the extraction rate and purity of water-soluble polysaccharides from Poria cocos, but also improves the structure and functional properties of polysaccharides through enzymatic hydrolysis, making them more suitable for pharmacological research and development. By combining the specific catalytic effect of enzymes, the method achieves the goal of efficient, green and high-purity extraction, improves the activity of polysaccharides, thereby increasing the extraction rate, and is suitable for large-scale production of functional foods and pharmaceuticals. Attached Figure Description

[0017] Figure 1 The curves showing the changes in polysaccharide extraction rate by the compound enzyme in Example 1 are shown, where Q is cellulase and X is β-glucanase.

[0018] Figure 2 The curve showing the effect of substrate concentration on polysaccharide extraction rate in Example 1 is shown.

[0019] Figure 3 The curve showing the effect of enzymatic hydrolysis pH on polysaccharide extraction rate in Example 1 is shown.

[0020] Figure 4 The curve showing the effect of enzymatic hydrolysis temperature on polysaccharide extraction rate in Example 1 is shown.

[0021] Figure 5 The curve showing the effect of enzymatic hydrolysis time on polysaccharide extraction rate in Example 1 is shown.

[0022] Figure 6 The curve shows the effect of enzyme dosage on polysaccharide extraction rate in Example 1.

[0023] Figure 7 This is a response surface plot of enzymatic hydrolysis time versus enzyme dosage in Example 1.

[0024] Figure 8 This is a response surface plot of substrate concentration versus enzyme dosage in Example 1.

[0025] Figure 9 This is a response surface plot of enzymatic hydrolysis time versus substrate concentration in Example 1.

[0026] Figure 10 The kidney sections stained with HE in Example 2 are shown, where Normal is the normal group, Model is the model group, HKSXC is the positive control group, and EPCP is the Poria cocos water-soluble polysaccharide group.

[0027] Figure 11The effect of Poria cocos water-soluble polysaccharide in Example 2 on blood biochemical indicators of CKD mice, where Normal is the normal group, Model is the model group, HKSXC is the positive drug group, and EPCP is the Poria cocos water-soluble polysaccharide group. This indicates a comparison with the model group. p < 0.01.

[0028] Figure 12 The effect of Poria cocos water-soluble polysaccharide on inflammatory factor indicators in CKD mice in Example 2 is shown in the figure. Normal is the normal group, Model is the model group, HKSXC is the positive drug group, and EPCP is the Poria cocos water-soluble polysaccharide group. This indicates a comparison with the model group. p < 0.01, p < 0.05.

[0029] Figure 13 The effect of Poria cocos water-soluble polysaccharide in Example 2 on the renal antioxidant index of CKD mice is shown in the figure. Normal is the normal group, Model is the model group, HKSXC is the positive drug group, and EPCP is the Poria cocos water-soluble polysaccharide group. This indicates a comparison with the model group. p < 0.01. Detailed Implementation

[0030] The present invention will be further described and illustrated below with reference to specific embodiments and the accompanying drawings: Example 1: Preparation method of Poria cocos enzymatic hydrolysate (1) Raw material pretreatment: The dried Poria cocos was pulverized and passed through a 100-mesh sieve to obtain Poria cocos powder; 200g of Poria cocos powder was weighed and mixed with 60% ethanol at a material-to-liquid ratio of 1:10g / mL, and extracted twice at room temperature for 2 hours each time; then it was mixed with distilled water at a material-to-liquid ratio of 1:8g / mL, and extracted twice at 90℃ for 2 hours each time; and then dried at 60℃. (2) Extraction of alkali-soluble polysaccharides from Poria cocos: The above-obtained system was mixed with sodium hydroxide solution at a material-to-liquid ratio of 1:16 g / mL, stirred at room temperature for 1 h, and then centrifuged to obtain supernatant; the pH of the supernatant was adjusted to 7 with 37% acetic acid solution, allowed to stand, washed repeatedly with distilled water 4 times, and then freeze-dried to obtain alkali-soluble polysaccharides from Poria cocos. (3) Enzymatic extraction of water-soluble polysaccharides from Poria cocos: The alkali-soluble polysaccharides from Poria cocos were dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. A compound enzyme was added, and the enzymatic hydrolysis reaction was carried out under different compound enzyme ratios, substrate concentrations, hydrolysis pH, hydrolysis temperature, hydrolysis time, and enzyme dosage. The hydrolysate was inactivated by enzyme, centrifuged, precipitated with four times the volume of ethanol, dialyzed, and freeze-dried to obtain water-soluble polysaccharides from Poria cocos. The optimal conditions for enzymatic hydrolysis were determined by single-factor and response surface methodology.

[0031] 2. Determination of the extraction rate of water-soluble polysaccharides from Poria cocos Preparation of a standard curve: Accurately weigh 0.1 g of glucose, dissolve and dilute to 100 mL to prepare a 0.1 mg / mL standard solution. Pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL into 10 mL centrifuge tubes, add water to each tube to bring the volume to 1.0 mL, add 1 mL of 3% phenol, mix well, and finally slowly add 4.0 mL of concentrated sulfuric acid while shaking. React at room temperature for 30 min, and then measure the absorbance at 490 nm. Plot the polysaccharide concentration on the x-axis and the absorbance on the y-axis to obtain a standard curve. Calculate the regression equation and correlation coefficient.

[0032] Determination of water-soluble polysaccharide content in enzymatic hydrolysate of Poria cocos: Take a certain amount of Poria cocos enzymatic hydrolysate, dilute it by a certain factor, and measure its absorbance according to the standard curve determination method. Substitute it into the regression equation to obtain the polysaccharide concentration in the enzymatic hydrolysate.

[0033] The polysaccharide concentration (mg / mL) in the enzymatic hydrolysate of Poria cocos = n c and n are the dilution factors of the enzyme hydrolysate; c is the polysaccharide concentration corresponding to the corresponding absorbance in the regression equation.

[0034] Yield of water-soluble polysaccharides from Poria cocos (%) = .

[0035] Extraction rate of water-soluble polysaccharides from Poria cocos (%) = (%).

[0036] 3. Single-factor optimization experiment ① Effect of the ratio of compound enzymes on the yield of water-soluble polysaccharides from Poria cocos The alkali-soluble polysaccharide from Poria cocos was dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. Under conditions of substrate concentration of 4%, hydrolysis pH of 6, hydrolysis temperature of 50℃, hydrolysis time of 2 h, and enzyme dosage of 9000 U / mL, the ratio of the complex enzyme (cellulase:β-glucanase) was varied (3:1, 2:1, 1:1, 1:2, 1:3). The polysaccharide extraction rate was used as the indicator, and the results are as follows: Figure 1 As shown, according to Figure 1 The optimal ratio of the complex enzyme (cellulase: β-glucanase) was determined to be 3:1.

[0037] ② Effect of substrate concentration on the yield of water-soluble polysaccharides from Poria cocos The alkali-soluble polysaccharide from Poria cocos was dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. Under conditions of a compound enzyme ratio of 3:1, a hydrolysis pH of 6, a hydrolysis temperature of 50℃, a hydrolysis time of 2 h, and an enzyme dosage of 9000 U / mL, the substrate concentration was varied (1%, 2%, 3%, 4%, 5%). The polysaccharide extraction rate was used as the indicator, and the results are as follows: Figure 2 As shown, according to Figure 2 The optimal substrate concentration for enzymatic hydrolysis was determined to be 4%.

[0038] ③ Effect of enzymatic hydrolysis pH on the yield of water-soluble polysaccharides from Poria cocos The alkali-soluble polysaccharide from Poria cocos was dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. Under the conditions of a compound enzyme ratio of 3:1, a substrate concentration of 4%, a hydrolysis temperature of 50℃, a hydrolysis time of 2 h, and an enzyme dosage of 9000 U / mL, the pH of the hydrolysis was varied (4, 5, 6, 7, 8). The polysaccharide extraction rate was used as the indicator, and the results are as follows: Figure 3 As shown, according to Figure 3 The optimal pH for enzymatic hydrolysis was determined to be 6.

[0039] ④ Effect of enzymatic hydrolysis temperature on the yield of water-soluble polysaccharides from Poria cocos The alkali-soluble polysaccharide from Poria cocos was dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. Under the conditions of a compound enzyme ratio of 3:1, substrate concentration of 4%, hydrolysis pH of 6, hydrolysis time of 2 h, and enzyme dosage of 9000 U / mL, the hydrolysis temperature was varied (30℃, 35℃, 40℃, 45℃, 50℃). The polysaccharide extraction rate was used as the indicator, and the results are as follows: Figure 4 As shown, according to Figure 4 The optimal enzymatic hydrolysis temperature was determined to be 35℃.

[0040] ⑤ Effect of enzymatic hydrolysis time on the yield of water-soluble polysaccharides from Poria cocos The alkali-soluble polysaccharide from Poria cocos was dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. Under the conditions of a compound enzyme ratio of 3:1, a substrate concentration of 4%, a hydrolysis pH of 6, a hydrolysis temperature of 35℃, and an enzyme dosage of 9000 U / mL, the hydrolysis time was varied (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h). The polysaccharide extraction rate was used as the indicator, and the results are as follows: Figure 5 As shown, according to Figure 5 The optimal enzymatic hydrolysis time was determined to be 1.5 hours.

[0041] ⑥ Effect of enzyme dosage on the yield of water-soluble polysaccharides from Poria cocos The alkali-soluble polysaccharide of Poria cocos was dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. Under the conditions of a compound enzyme ratio of 3:1, a substrate concentration of 4%, a hydrolysis pH of 6, a hydrolysis temperature of 35℃, and a hydrolysis time of 1.5 h, the enzyme dosage was varied (9000 U / mL, 10000 U / mL, 11000 U / mL, 12000 U / mL, and 13000 U / mL). The polysaccharide extraction rate was used as the indicator, and the results are as follows: Figure 6 As shown, according to Figure 6 The optimal enzyme dosage for enzymatic hydrolysis was determined to be 11000 U / mL.

[0042] 4. Response Surface Methodology Optimization Experiment Based on the results of the single-factor experiments above, the response surface methodology was used to further refine the extraction process of water-soluble polysaccharides from Poria cocos. A three-factor, three-level response surface experiment was designed using DesignExpert13 with three factors: enzyme dosage, substrate concentration, and enzymatic hydrolysis time. The design and results of the response surface experiment are shown in Table 1.

[0043] Table 1. Response surface methodology experimental design and results The water-soluble polysaccharide extracted from Poria cocos by enzymatic hydrolysis was named EPCP.

[0044] The operating parameters and experimental results studied in the response surface methodology are shown in Table 2. Table 2 summarizes the effects of independent factors on the extraction rate of water-soluble polysaccharides from Poria cocos. The quadratic regression equation model for the extraction rate of water-soluble polysaccharides from Poria cocos as the response value is as follows: Y=-244.40996+0.031761A+23.17564B+96.43357C+0.000354AB-0.006089AC+3.67656BC+0.000001045A2-5.17268B2-116.24690C2; Where Y is the extraction rate of water-soluble polysaccharides from Poria cocos, A is the amount of enzyme used, B is the substrate concentration, and C is the enzymatic hydrolysis time.

[0045] The quadratic regression model showed good significance, and the lack-of-fit term was not significant, indicating that the model can be used in the experiment.

[0046] Table 2 Analysis of Response Surface Experiment Results Based on the response surface results ( Figure 7-9 The feasibility of the model was confirmed. Therefore, the experimental parameters were further optimized using Design-Expert software. The optimal conditions were found to be: enzyme dosage of 12000 U / mL, substrate concentration of 3.03%, and enzymatic hydrolysis time of 1.06 h. Under these conditions, the extraction rate of water-soluble polysaccharides from Poria cocos could reach 40.99%, which is close to the predicted value of 40.04%.

[0047] Example 2 Grouping and administration of experimental animals Twenty-eight male BALB / c mice were randomly divided into four groups of seven each: a normal control group (0.25% standard diet), a model group (0.25% purified diet), a positive control group (0.25% purified diet + Haikun Shenxi capsules), and a Poria cocos water-soluble polysaccharide group (0.25% purified diet + Poria cocos water-soluble polysaccharide). Mice were fed either adenine-containing purified or standard diets for four consecutive weeks, with the purified diet inducing a chronic kidney injury mouse model. Mice in the 0.25% purified diet + polysaccharide group (100 mg / kg) received polysaccharide intervention via gavage, while mice in the 0.25% purified diet + Haikun Shenxi capsules (100 mg / kg) group received positive control intervention via gavage. The intervention lasted for 28 days. Urine and feces were collected and stored at -80℃. Mice were sacrificed after 8 hours of fasting, and blood, kidney tissue, and colon contents were collected. The colon tissue was soaked in paraformaldehyde for subsequent histopathological analysis.

[0048] Chronic kidney disease related indicator testing ① Kidney section - HE staining Mouse kidneys were removed, immersed in a protective solution, and then processed into sections. The sections were then scanned and analyzed. Results are as follows: Figure 10 As shown, the matrix components in CKD mice were significantly increased, indicating widespread tubular necrosis and dilation. After adding enzymatically hydrolyzed Poria cocos water-soluble polysaccharides, the matrix components decreased, the damage was significantly reduced, and the structure was largely preserved. This suggests that the sample can delay glomerular damage.

[0049] ② Blood biochemical indicators The levels of blood urea nitrogen, ammonia, creatinine, and uric acid in plasma were determined using a biochemical reagent kit. Mice were anesthetized after fasting for 8 hours, and blood was collected via orbital sampling. The collected blood samples were allowed to stand at room temperature for 2 hours, then centrifuged, and the supernatant was collected to obtain plasma. The separated plasma samples were added to the reaction tubes provided in the kit. The reaction tubes were placed in a colorimeter, and the absorbance values ​​for each assay were read according to the kit instructions and converted into the corresponding detection results.

[0050] The results are as follows Figure 11 As shown, after intervention with enzymatic hydrolysis of water-soluble polysaccharides from Poria cocos, the serum ammonia, urea nitrogen, creatinine, and uric acid levels in CKD mice all decreased significantly. This indicates that enzymatic hydrolysis of water-soluble polysaccharides from Poria cocos can reduce the glomerular filtration burden and improve renal function.

[0051] ③Inflammatory factors The levels of IL-10, IL-6, TNF-α, and IL-1β were determined using an ELISA kit. Mouse plasma was first pretreated, and then the samples, along with controls and blanks, were added to microplates. After incubation with antibody at room temperature and washing, the plates were incubated with enzyme-labeled secondary antibody and washed again. Substrate was then added for color development, and the reaction was stopped with a stop solution. Finally, the absorbance was read, and the levels of IL-10, IL-6, TNF-α, and IL-1β were calculated.

[0052] The results are as follows Figure 12 As shown, enzymatic hydrolysis of Poria cocos water-soluble polysaccharides significantly reduced serum levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in CKD mice, while increasing the level of the anti-inflammatory factor IL-10. This suggests that enzymatic hydrolysis of Poria cocos water-soluble polysaccharides can improve inflammation in CKD mice and delay the progression of renal fibrosis.

[0053] ④ Antioxidant indicators of the kidneys The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in kidney tissue were determined using a biochemical reagent kit. Results are as follows: Figure 13 As shown, the addition of enzymatically hydrolyzed water-soluble polysaccharides from Poria cocos significantly increased the contents of SOD and GSH-PX, while significantly decreasing the content of malondialdehyde. This indicates that the sample can enhance the body's antioxidant capacity and has a mitigating effect on CKD.

[0054] Implementation Case 3 (1) Raw material pretreatment: The dried Poria cocos was pulverized and passed through a 100-mesh sieve to obtain Poria cocos powder; 200g of Poria cocos powder was weighed and mixed with 60% ethanol at a material-to-liquid ratio of 1:10g / mL, and extracted twice at room temperature for 2 hours each time; then it was mixed with distilled water at a material-to-liquid ratio of 1:8g / mL, and extracted twice at 90℃ for 2 hours each time; and then dried at 60℃. (2) Extraction of alkali-soluble polysaccharides from Poria cocos: The above-obtained system was mixed with sodium hydroxide solution at a material-to-liquid ratio of 1:16 g / mL, stirred at room temperature for 1 h, and then centrifuged to obtain supernatant; the pH of the supernatant was adjusted to 7 with 37% acetic acid solution, allowed to stand, washed repeatedly with distilled water 4 times, and then freeze-dried to obtain alkali-soluble polysaccharides from Poria cocos. (3) Enzymatic extraction of water-soluble polysaccharides from Poria cocos: The alkali-soluble polysaccharides from Poria cocos were dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. A compound enzyme was added, and the enzymatic hydrolysis reaction was carried out under the conditions of a compound enzyme ratio of 1:3, a substrate concentration of 1%, a hydrolysis pH of 4, a hydrolysis temperature of 30℃, a hydrolysis time of 1h, and an enzyme dosage of 9000U / mL. The hydrolysate was then subjected to enzyme inactivation, centrifugation, precipitation with four times the volume of ethanol, dialysis, and freeze-drying to obtain water-soluble polysaccharides from Poria cocos.

[0055] The polysaccharide extraction rate was found to be 14.07%.

[0056] Implementation Case 4 (1) Raw material pretreatment: The dried Poria cocos was pulverized and passed through a 100-mesh sieve to obtain Poria cocos powder; 200g of Poria cocos powder was weighed and mixed with 60% ethanol at a material-to-liquid ratio of 1:10g / mL, and extracted twice at room temperature for 2 hours each time; then it was mixed with distilled water at a material-to-liquid ratio of 1:8g / mL, and extracted twice at 90℃ for 2 hours each time; and then dried at 60℃. (2) Extraction of alkali-soluble polysaccharides from Poria cocos: The above-obtained system was mixed with sodium hydroxide solution at a material-to-liquid ratio of 1:16 g / mL, stirred at room temperature for 1 h, and then centrifuged to obtain supernatant; the pH of the supernatant was adjusted to 7 with 37% acetic acid solution, allowed to stand, washed repeatedly with distilled water 4 times, and then freeze-dried to obtain alkali-soluble polysaccharides from Poria cocos. (3) Enzymatic extraction of water-soluble polysaccharides from Poria cocos: The alkali-soluble polysaccharides from Poria cocos were dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. A compound enzyme was added, and the enzymatic hydrolysis reaction was carried out under the conditions of a compound enzyme ratio of 1:1, a substrate concentration of 3%, a hydrolysis pH of 5, a hydrolysis temperature of 40℃, a hydrolysis time of 1.5h, and an enzyme dosage of 11000U / mL. The hydrolysate was then subjected to enzyme inactivation, centrifugation, precipitation with four times the volume of ethanol, dialysis, and freeze-drying to obtain water-soluble polysaccharides from Poria cocos.

[0057] The polysaccharide extraction rate was found to be 20.01%.

[0058] Implementation Case 5 (1) Raw material pretreatment: The dried Poria cocos was pulverized and passed through a 100-mesh sieve to obtain Poria cocos powder; 200g of Poria cocos powder was weighed and mixed with 60% ethanol at a material-to-liquid ratio of 1:10g / mL, and extracted twice at room temperature for 2 hours each time; then it was mixed with distilled water at a material-to-liquid ratio of 1:8g / mL, and extracted twice at 90℃ for 2 hours each time; and then dried at 60℃. (2) Extraction of alkali-soluble polysaccharides from Poria cocos: The above-obtained system was mixed with sodium hydroxide solution at a material-to-liquid ratio of 1:16 g / mL, stirred at room temperature for 1 h, and then centrifuged to obtain supernatant; the pH of the supernatant was adjusted to 7 with 37% acetic acid solution, allowed to stand, washed repeatedly with distilled water 4 times, and then freeze-dried to obtain alkali-soluble polysaccharides from Poria cocos. (3) Enzymatic extraction of water-soluble polysaccharides from Poria cocos: The alkali-soluble polysaccharides from Poria cocos were dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. A compound enzyme was added, and the enzymatic hydrolysis reaction was carried out under the conditions of a compound enzyme ratio of 2:1, a substrate concentration of 4%, a hydrolysis pH of 6, a hydrolysis temperature of 50℃, a hydrolysis time of 2h, and an enzyme dosage of 12000U / mL. The hydrolysate was then subjected to enzyme inactivation, centrifugation, precipitation with four times the volume of ethanol, dialysis, and freeze-drying to obtain water-soluble polysaccharides from Poria cocos.

[0059] The polysaccharide extraction rate was found to be 27.24%.

[0060] Implementation Case 6 (1) Raw material pretreatment: The dried Poria cocos was pulverized and passed through a 100-mesh sieve to obtain Poria cocos powder; 200g of Poria cocos powder was weighed and mixed with 60% ethanol at a material-to-liquid ratio of 1:10g / mL, and extracted twice at room temperature for 2 hours each time; then it was mixed with distilled water at a material-to-liquid ratio of 1:8g / mL, and extracted twice at 90℃ for 2 hours each time; and then dried at 60℃. (2) Extraction of alkali-soluble polysaccharides from Poria cocos: The above-obtained system was mixed with sodium hydroxide solution at a material-to-liquid ratio of 1:16 g / mL, stirred at room temperature for 1 h, and then centrifuged to obtain supernatant; the pH of the supernatant was adjusted to 7 with 37% acetic acid solution, allowed to stand, washed repeatedly with distilled water 4 times, and then freeze-dried to obtain alkali-soluble polysaccharides from Poria cocos. (3) Enzymatic extraction of water-soluble polysaccharides from Poria cocos: The alkali-soluble polysaccharides from Poria cocos were dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. A compound enzyme was added, and the enzymatic hydrolysis reaction was carried out under the conditions of a compound enzyme ratio of 3:1, a substrate concentration of 5%, a hydrolysis pH of 7, a hydrolysis temperature of 35℃, a hydrolysis time of 2.5 h, and an enzyme dosage of 13000 U / mL. The hydrolysate was then subjected to enzyme inactivation, centrifugation, precipitation with four times the volume of ethanol, dialysis, and freeze-drying to obtain water-soluble polysaccharides from Poria cocos.

[0061] The polysaccharide extraction rate was found to be 33.63%.

[0062] Implementation Case 7 (1) Raw material pretreatment: The dried Poria cocos was pulverized and passed through a 100-mesh sieve to obtain Poria cocos powder; 200g of Poria cocos powder was weighed and mixed with 60% ethanol at a material-to-liquid ratio of 1:10g / mL, and extracted twice at room temperature for 2 hours each time; then it was mixed with distilled water at a material-to-liquid ratio of 1:8g / mL, and extracted twice at 90℃ for 2 hours each time; and then dried at 60℃. (2) Extraction of alkali-soluble polysaccharides from Poria cocos: The above-obtained system was mixed with sodium hydroxide solution at a material-to-liquid ratio of 1:16 g / mL, stirred at room temperature for 1 h, and then centrifuged to obtain supernatant; the pH of the supernatant was adjusted to 7 with 37% acetic acid solution, allowed to stand, washed repeatedly with distilled water 4 times, and then freeze-dried to obtain alkali-soluble polysaccharides from Poria cocos. (3) Enzymatic extraction of water-soluble polysaccharides from Poria cocos: The alkali-soluble polysaccharides from Poria cocos were dissolved in sodium hydroxide solution at a material-to-liquid ratio of 1:2 g / mL. A compound enzyme was added, and the enzymatic hydrolysis reaction was carried out under the conditions of a compound enzyme ratio of 3:1, a substrate concentration of 3%, a hydrolysis pH of 6, a hydrolysis temperature of 35℃, a hydrolysis time of 1.5h, and an enzyme dosage of 11000U / mL. The hydrolysate was then subjected to enzyme inactivation, centrifugation, precipitation with four times the volume of ethanol, dialysis, and freeze-drying to obtain water-soluble polysaccharides from Poria cocos.

[0063] The polysaccharide extraction rate was found to be 34.63%.

[0064] This invention provides a method for preparing water-soluble polysaccharides from Poria cocos based on enzyme-base synergy. Through a multi-step extraction process, water-soluble polysaccharides can be efficiently extracted from Poria cocos with high extraction efficiency and purity, while retaining the active components of the polysaccharides, providing high-quality raw materials for subsequent pharmacological research and applications.

[0065] The enzyme-alkali synergistic extraction method not only improves the extraction rate and purity of water-soluble polysaccharides from Poria cocos, but also improves the structure and functional properties of polysaccharides through enzymatic hydrolysis, making them more suitable for pharmacological research and development. By combining the specific catalytic effect of enzymes, the method achieves the goal of efficient, green and high-purity extraction, improves the activity of polysaccharides, thereby increasing the extraction rate, and is suitable for large-scale production of functional foods and pharmaceuticals.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing water-soluble polysaccharides from Poria cocos based on enzyme and alkali synergism, characterized in that, The method comprises the following steps: S1, raw material pretreatment: dry Poria cocos is crushed and passed through a 50-100 mesh sieve to obtain Poria cocos powder; the Poria cocos powder is mixed with 50-70% ethanol at a solid-liquid ratio of 1:5-1:10 g / mL, stirred at room temperature, and extracted for 2-3 times, each time for 1.5-2.5 h; then the Poria cocos powder is mixed with distilled water at a solid-liquid ratio of 1:6-1:8 g / mL, stirred at 85-95°C, and extracted for 2-3 times, each time for 1.5-2.5 h, and finally dried at a temperature of 55-65°C; S2, extraction of Poria cocos alkali-soluble polysaccharides: the system obtained above is mixed with a sodium hydroxide solution at a solid-liquid ratio of 1:15-1:17 g / mL, stirred at room temperature for 1-2 h, and then centrifuged to obtain supernatant; the supernatant is adjusted to a pH of 6.5-7.0 with a 36-40% acetic acid solution, allowed to stand, washed repeatedly with distilled water for 3-4 times, and freeze-dried to obtain Poria cocos alkali-soluble polysaccharides; S3, enzymatic extraction of Poria cocos water-soluble polysaccharides: the system obtained above is dissolved in a sodium hydroxide solution at a ratio of 1:1-1:3 g / mL, and a complex enzyme is added to perform enzymolysis under different ratios of the complex enzyme, substrate concentrations, enzymolysis pH, enzymolysis temperatures, enzymolysis times, and enzyme dosages; the enzymolysis liquid is subjected to enzyme inactivation, centrifugation, four-fold volume ethanol precipitation, dialysis, and freeze-drying to obtain Poria cocos water-soluble polysaccharides.

2. The method for preparing water-soluble polysaccharides from Poria cocos based on enzyme and alkali synergism according to claim 1, characterized in that, In step S3, the conditions for the enzymolysis are determined by single-factor and response surface methods.

3. The method for preparing water-soluble polysaccharides from Poria cocos based on enzyme and alkali synergism according to claim 1, characterized in that, In step S3, the complex enzyme is cellulase and β-glucanase.

4. The method for preparing water-soluble polysaccharides from Poria cocos based on enzyme and alkali synergism according to claim 1, characterized in that, The ratio of the cellulase to the β-glucanase is 1:3-3:1, the substrate concentration is 1-5%, the enzymolysis pH is 4-8, the enzymolysis temperature is 30-50°C, the enzymolysis time is 0.5-2.5 h, and the enzyme dosage is 9,000-13,000 U / mL.

5. The method for preparing water-soluble polysaccharides from Poria cocos based on enzyme and alkali synergism according to claim 1, characterized in that, On the basis of the single-factor experiment, a Box-Benhken experimental design in Design-Expert 13 statistical analysis software is used to take the extraction rate of Poria cocos water-soluble polysaccharides as a response value, take the three factors with the greatest impact in the single-factor experiment, i.e., enzyme dosage, substrate concentration, and enzymolysis time, as independent variables, and establish a three-factor three-level central composite design.

6. The method for preparing water-soluble polysaccharides from Poria cocos based on enzyme and alkali synergism according to claim 1, characterized in that, The extraction rate of the Poria cocos water-soluble polysaccharides can reach 40-42%.

7. Use of a water-soluble polysaccharide of Poria cocos, characterized in that, The Poria cocos water-soluble polysaccharides prepared according to any one of claims 1-6 are used for intervention in chronic kidney disease to delay the progression of renal fibrosis.

8. The use of a water-soluble polysaccharide of Poria cocos according to claim 7, wherein the water-soluble polysaccharide of Poria cocos is administered in an amount of 0.1 to 10 g per day. The chronic kidney disease includes one or more diseases such as renal function decline, inflammation-induced kidney lesions, and antioxidant capacity-induced kidney damage.

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