Low-ash edible mushroom polysaccharide and application thereof

Abstract

The invention relates to extraction of edible fungi, in particular to an extraction method of poria peel polysaccharide, which comprises the following steps: taking poria peel, adding an aqueous alkali solution, carrying out reflux extraction, filtering, taking filtrate, and further preparing the poria peel polysaccharide from the obtained aqueous alkali extracting solution through concentration, neutralization and deliming. The invention further protects the poria cocos peel polysaccharide prepared by the method and application of the poria cocos peel polysaccharide.

Description

Technical Field

[0001] The invention relates to an edible fungus extract, in particular to an extraction method of low-ash Poria peel polysaccharide and application thereof. Background Art

[0002] Poria cocos, the dried sclerotium of the Polyporaceae fungus Poria cocos (Schw.) Wolf, is an important edible fungus. Poria cocos peel is the brown to dark brown outer skin collected and shaved after processing and then dried in the shade. With the increasing demand for Poria cocos in pharmaceutical and food processing, despite containing a large number of functional ingredients, problems remain, such as insufficient processing and utilization, and significant waste. Reportedly, Poria cocos processing produces 10,000 tons of Poria cocos peel annually. Poria cocos peel has a complex composition, and some sugar extracts have low activity. Current extraction methods are still mostly focused on Poria cocos. Literature and patents primarily utilize alcohol extraction for Poria cocos peel, with alkaline extraction occasionally used. However, these methods often prioritize or even focus on alcohol-soluble active ingredients. The purity, quality, and activity of Poria cocos peel sugars remain understudied, and some processes even modify or denature sugars. Reports suggest that β-Poriacocos polysaccharides have limited functionality, failing to combat aging or enhance human immunity. Summary of the Invention

[0003] The applicant has discovered that the ash content of polysaccharides extracted from the peel of Poria cocos by alkali extraction and alcohol precipitation is high. Excessive ash content can cause dehydration, palpitations, and even cancer in the user. The present invention overcomes this technical deficiency and provides a new method for extracting and preparing polysaccharides from the peel of Poria cocos. It also finds that this low-ash polysaccharide has antioxidant and other effects.

[0004] In a first aspect, the extraction scheme adopted by the present invention comprises the following steps:

[0005] 1) Take Poria cocos peel, add alkaline aqueous solution with a pH value of 10-12 for extraction, filter, and obtain a filtrate (crude extract);

[0006] 2) Optionally, the filtrate obtained in step 1 is concentrated to obtain a concentrate;

[0007] 3) adjusting the pH of the alkaline aqueous extract to be purified to 7-8, centrifuging, and then adding ethanol to precipitate the polysaccharide to obtain a precipitated extract;

[0008] 4) The extract is redissolved with water, and the resulting redissolved solution is subjected to cation exchange resin chromatography, the lower column liquid is collected as the purified liquid, and the pH value is controlled to 7-8 to obtain low-ash Poria peel polysaccharide.

[0009] Optionally, in step 1, the material-liquid ratio of the Poria peel to the alkaline water is 1:10-30, such as 1:15-20. The material-liquid ratio is defined as the same as conventionally defined in the art, that is, the mass volume ratio of the raw material to the extract, in g / mL.

[0010] Optionally, in step 1, the pH of the alkaline aqueous solution is 11.

[0011] Optionally, the extraction temperature is above room temperature, and may be above reflux temperature, such as 90-110°C, preferably 100°C.

[0012] Optionally, the particle size of the Poria peel raw material in step 1 is controlled to be less than 3 cm, such as about 2 cm or less.

[0013] Optionally, step 1 is performed to extract Poria cocos 1-2 times, and the extraction object may include the filter residue obtained by filtration, and the filtrate obtained by extracting the filter residue is combined with the filtrate of the initial extraction of Poria cocos peel for subsequent separation of Poria cocos peel polysaccharide. Each time, the alkaline aqueous solution can be used for extraction, or the filter residue can be extracted with water (the ratio of water to the filter residue or Poria cocos peel raw material can be 10-30:1); the time for each extraction can be 2-4 hours. Optionally, in step (1), the extraction uses a jacketed reflux extraction tank.

[0014] Optionally, in step 2, the extract is concentrated to a relative density of 1.05 to 1.15, such as 1.1. Relative density herein refers to the density multiple of water.

[0015] Optionally, the pH value is adjusted to 7 in step 3.

[0016] Optionally, the centrifugal speed in step 3 is 1000-2000 r / min, such as 1200 r / min, and the centrifugation time can be 5-20 min, such as 10 min.

[0017] Optionally, the amount of alcohol added in step 3 is such that the final ethanol concentration is 75-80% (% in this field refers to volume percentage).

[0018] Optionally, the cation exchange resin in step 4 is a strong acid type or a weak acid type, such as a sulfonic acid group or a carboxylic acid group, such as 001*7 type, D113, D85 or D61 type.

[0019] Optionally, in step 4, the column volume is 400-500 mL, the sample concentration can be 20-25 mg / mL, the flow rate can be 1-2 BV / h (BV refers to the column volume), and the column liquid within 12 hours of the column can be collected as the purified product.

[0020] Optionally, after adjusting the pH in step 4, the low-ash Poria peel polysaccharide solid is obtained by concentration and drying, which can then be converted into powder. As an example, the method of the present invention includes:

[0021] (1) Cut the washed Poria peel into small pieces, add an alkaline aqueous solution with a pH value of 10 to 12, heat and extract in an extraction tank, filter and collect the filtrate, extract twice and combine the filtrates;

[0022] (2) taking the filtrate obtained in step (1), concentrating under reduced pressure to an extract density of 1.05 to 1.10 to obtain a concentrated solution;

[0023] (3) adjusting the pH value of the concentrate to 7.0-8.0, centrifuging, stirring, adding ethanol to precipitate the polysaccharide, and leaving it for 12-16 hours to obtain the precipitated extract, which was then re-dissolved in water;

[0024] (4) subjecting the reconstituted solution to cation exchange resin chromatography, collecting the purified liquid, adjusting the pH value to 7.0-8.0, concentrating, vacuum drying, and crushing to obtain a low-ash Poria peel polysaccharide extract;

[0025] Furthermore, in step (1), the extraction tank is a jacketed reflux extraction tank.

[0026] Furthermore, in the step (1), the extraction is performed twice, wherein a 0.1-0.3% NaOH solution is added in the first extraction, and the ratio of alkaline water volume L / Poria cocos peel weight kg is 15-30:1; and a 0.1-0.3% NaOH solution is added in the second extraction, and the ratio of alkaline water volume L / Poria cocos peel weight kg is 10-25:1; both extraction times are 2 hours, and the extraction temperature is 90-100°C;

[0027] Furthermore, the reduced pressure concentration equipment in step (2) is a hot reflux extraction and concentration unit, the vacuum degree during the reduced pressure concentration can be ≤0.08Mpa, and the temperature can be 30-60°C;

[0028] Furthermore, the centrifugal equipment in step (3) is a three-legged decanter centrifuge with a centrifugal speed of 1200 r / min and a centrifugal time of 10 min;

[0029] Furthermore, in step (3), the concentration of the alcohol precipitation ethanol solution is 93-95%, and the concentration of the alcohol precipitation ethanol solution after stirring is 75-80%;

[0030] Furthermore, in step (4), the column volume is 400-500 mL, the sample concentration is 20-25 mg / mL, the flow rate is 1-2 BV / h, and the column liquid within 12 hours of the column is collected; BV refers to the column volume;

[0031] Furthermore, in step (4), the cation exchange resin is 001*7 type;

[0032] Furthermore, the vacuum drying temperature in step (4) is ≤60°C.

[0033] This method extracts polysaccharides from Poria peel, and the extraction yield is significantly higher than that of water extraction, and the ash content of the polysaccharide is greatly reduced, which solves the problem of waste of Poria peel resources and greatly reduces the production cost of Poria polysaccharide. Moreover, the obtained low-ash Poria peel polysaccharide product has excellent activity, especially antioxidant activity.

[0034] In a second aspect, the present invention protects the Poria peel candy prepared by the method.

[0035] In a third aspect, the present invention further protects the application of the Poria peel candy.

[0036] The low-ash Poria peel polysaccharide obtained by the invention has anti-oxidation and anti-radiation effects, and can also be used as a raw material for further separation and purification to obtain Poria peel candy with higher purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The DPPH free radical scavenging ability of low-ash Poria peel polysaccharide

[0038] Figure 2 Low-ash Poria peel polysaccharide for scavenging ABTS + Free Radical Capacity

[0039] Figure 3 It is the total reducing power of low-ash Poria peel polysaccharide. DETAILED DESCRIPTION

[0040] The technical solutions of the present invention are further described in detail below with reference to specific embodiments, but the present invention is not limited to the following technical solutions.

[0041] A. Experimental Examples

[0042] Example 1

[0043] Cut the Poria peel into small pieces with a diameter of about 2 cm and use them as raw materials. Add alkaline water with a material-liquid ratio of 1:10 (g / L) and place them in a jacketed reflux extraction tank. The alkaline water concentration is prepared as a NaOH solution with a pH of 11. Heat and reflux extraction at 100°C for 2 hours. Filter and collect the filtrate ① and the residue ①. Add water 10 times the weight of the raw materials to the residue ① again in the jacketed reflux extraction tank. Heat and reflux extraction for 2 hours again. Filter to obtain filtrate ②. Combine the filtrate ① and the filtrate ②, and concentrate the filtrate at ≤60°C and 0.08Mpa vacuum conditions to a solution with a specific gravity greater than or equal to 1.10. The concentrated solution ① is prepared by adding 3.6% hydrochloric acid solution to the concentrated solution ① to neutralize it to pH = 7, the neutralized solution is centrifuged with a tripod sedimentation centrifuge to obtain a centrifuge, 93% ethanol solution is added to the centrifuge to a final ethanol concentration of 80%, and the solution is allowed to stand for 12 hours. The supernatant and the extract are separated, the extract is dried and redissolved, the redissolved solution is subjected to cation exchange resin 001*7, washed with water, the washing liquid is collected and adjusted to pH 7, and concentrated to a concentrated solution ② with a volume of 1 / 4 of the washing liquid, and the concentrated solution ② is vacuum dried at 60°C to obtain low-ash Poria peel polysaccharide with a polysaccharide yield of 0.82%.

[0044] Example 2

[0045] Cut the Poria peel into small pieces with a diameter of about 2 cm and use them as raw materials. Add alkaline water with a material-liquid ratio of 1:15 (g / L) and place them in a jacketed reflux extraction tank. The alkaline water concentration is prepared as a NaOH solution with a pH of 11. Heat and reflux extraction at 100°C for 2 hours. Filter and collect the filtrate ① and the residue ①. Add water 10 times the weight of the raw materials to the residue ① again in the jacketed reflux extraction tank. Heat and reflux extraction for 2 hours again. Filter to obtain filtrate ②. Combine filtrate ① and filtrate ②, and concentrate the filtrate at ≤60°C and 0.08Mpa vacuum conditions to a solution with a specific gravity greater than or equal to 1.10. Concentrate ①, add 3.6% hydrochloric acid solution to the concentrate ① to neutralize to pH = 7, centrifuge the neutralized solution with a tripod sedimentation centrifuge to obtain a centrifuge, stir and add 93% ethanol solution to the centrifuge to an ethanol concentration of 80%, let it stand for 12 hours, separate the supernatant and the extract, dry the extract and redissolve it, apply cation exchange resin 001*7 to the redissolved solution, wash it with water, collect the water washing liquid and adjust its pH to 7, concentrate it to a concentrate ② with a volume of 1 / 4 of the water washing liquid, and vacuum dry the concentrate ② at 60°C to obtain low-ash Poria peel polysaccharide with a polysaccharide yield of 9.04%.

[0046] Example 3

[0047] Cut the Poria peel into small pieces with a diameter of about 2 cm and use them as raw materials. Add alkaline water with a material-liquid ratio of 1:20 (g / L) and place them in a jacketed reflux extraction tank. The alkaline water concentration is configured as a NaOH solution with Ph=11. Heat and reflux extraction at 100°C for 2 hours. Filter to obtain filtrate ① and filter residue ①. Add water 10 times the weight of the raw materials to the filter residue ①, heat and reflux extraction again in the jacketed reflux extraction tank for 2 hours, and filter to obtain filtrate ②. Combine filtrate ① and filtrate ②, and concentrate the filtrate at ≤60°C and 0.08Mpa vacuum conditions to a solution with a specific gravity greater than or equal to 1.10. The concentrated solution ① was prepared by adding 3.6% hydrochloric acid solution to the concentrated solution ① to neutralize the pH value to 7, the neutralized solution was centrifuged with a tripod sedimentation centrifuge to obtain a centrifuge, 93% ethanol solution was added to the centrifuge to make the ethanol concentration 80%, and the solution was allowed to stand for 12 hours. The supernatant and the extract were separated, the extract was dried and redissolved, the redissolved solution was added with cation exchange resin 001*7, washed with water, the washing liquid was collected and the pH value was adjusted to 7, and the solution was concentrated to a concentrated solution ② with a volume of 1 / 4 of the washing liquid. The concentrated solution ② was vacuum dried at 60°C to obtain low-ash Poria peel polysaccharide with a polysaccharide yield of 5.90%.

[0048] Example 4

[0049] Cut the Poria peel into small pieces with a diameter of about 2 cm and use them as raw materials. Add alkaline water with a solid-liquid ratio of 1:30 (g / L) and place them in a jacketed reflux extraction tank. The alkaline water concentration is configured to be a NaOH solution with a pH of 11. Heat and reflux extraction at 100°C for 2 hours. Filter to obtain filtrate ① and filter residue ①. Add water 10 times the weight of the raw materials to the filter residue ①, heat and reflux extraction again in the jacketed reflux extraction tank for 2 hours, and filter to obtain filtrate ②. Combine filtrate ① and filtrate ②, and concentrate the filtrate at ≤60°C and 0.08Mpa vacuum conditions to a solution with a specific gravity greater than or equal to 1.10. The concentrated solution ① was prepared by adding 3.6% hydrochloric acid solution to the concentrated solution ① to neutralize the pH value to 7, the neutralized solution was centrifuged with a tripod sedimentation centrifuge to obtain a centrifuge, 93% ethanol solution was added to the centrifuge to make the ethanol concentration 80%, and the solution was allowed to stand for 12 hours. The supernatant and the extract were separated, the extract was dried and redissolved, the redissolved solution was added with cation exchange resin 001*7, washed with water, the washing liquid was collected and the pH value was adjusted to 7, and the concentrated solution was concentrated to 1 / 4 of the volume of the washing liquid ②, and the concentrated solution ② was vacuum dried at 60°C to obtain low-ash Poria peel polysaccharide with a polysaccharide yield of 4.06%.

[0050] Parallel Example:

[0051] The above four examples abandon the step of extracting the filter residue ①, and the other steps remain unchanged, that is, only the filtrate ① is used for subsequent concentration ... cation exchange, etc. to prepare Poria peel polysaccharide, which are recorded as Examples 1' to 4' in sequence. The polysaccharide yield of low-ash Poria peel polysaccharide prepared in each parallel example is shown in Table 1

[0052] Table 1

[0053] product Example 1 Example 2 Example 3 Example 4 Polysaccharide yield (%) 0.22 5.18 4.47 3.81

[0054] B. Analysis of polysaccharide content and ash content:

[0055] The polysaccharide content in the products of various embodiments of the present invention is relatively high, and the ash content is relatively low, such as:

[0056] product Polysaccharide content (%) ash content Example 2 27.60％ 6.11％ Example 3 40.61％ 7.35％ Example 4 46.74％ 5.33％

[0057] C. Activity Analysis

[0058] The Poria peel candy obtained by the present invention has antioxidant capacity. Taking the product of Example 1 as an example, the test data are as follows: Figure 1-3 The detection methods involved in this embodiment are all conventional methods in the field, and the key detection steps are as follows:

[0059] 1. Detection method of Poria peel polysaccharide content of the present invention (phenol-sulfuric acid method):

[0060] 1. Preparation of reference solution:

[0061] Accurately weigh an appropriate amount of glucose reference substance dried to constant weight at 105°C, add water to make a solution containing 100 μg per 1 ml.

[0062] 2. Preparation of standard curve:

[0063] Accurately pipette 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of the reference solution into 20mL glass test tubes, add water to 1.0mL, add 0.5mL of 5% phenol solution respectively, mix well, quickly add 5.0mL of sulfuric acid vertically, shake well, use the corresponding reagent as the blank, and measure the absorbance at a wavelength of 490nm by ultraviolet-visible spectrophotometry (Appendix VA of Part I of the Chinese Pharmacopoeia 2010 Edition). Draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0064] 3. Preparation of test solution:

[0065] Take approximately 10 mg of this product, accurately weigh it, and place it in a 10 mL volumetric flask. Add 5 mL of water and sonicate to dissolve it, then adjust the volume to 10 mL. Centrifuge the solution at 4000 r / min for 10 minutes. Accurately measure 1 mL of the above solution and follow the method under Preparation of Standard Curve, starting from "Add 0.5 mL of 5% phenol solution separately". Measure the absorbance according to the method. Read the weight (mg) of anhydrous glucose in the test solution from the standard curve and calculate the result.

[0066] 2. Total ash content of Poria peel polysaccharide

[0067] The test was carried out according to the ash content determination method in 2302 of the General Chapter 4 of the 2015 edition of the Chinese Pharmacopoeia.

[0068] Weigh 300 mg of the sample to be tested to the nearest 0.001 g in a crucible that has been previously calcined to constant weight. Gently shake the crucible to evenly distribute the sample in the ash dish. Open the furnace door, place the crucible in the chamber, and set the muffle furnace to 550°C. Close the furnace door and calcine at 550°C for 4 hours. Remove the crucible from the furnace, transfer it to a desiccator, cool it to room temperature, and then weigh it. Calculate the ash content of the sample after calcining to constant weight.

[0069] 3. In vitro antioxidant activity of Poria peel polysaccharide

[0070] (1) DPPH free radical scavenging ability test

[0071] Prepare a solution of appropriate concentration using the lyophilized sample powder. Mix 2 mL of the sample solution with 2 mL of a 0.1 mmol / L DPPH solution in anhydrous ethanol. After incubating in the dark at room temperature for 20 minutes, measure the absorbance of the mixture at 517 nm. Use a mixture of 2 mL of ultrapure water and 2 mL of anhydrous ethanol as a blank control.

[0072] The DPPH scavenging ability of the polysaccharide crude extract was calculated according to the following formula.

[0073] DPPH clearance rate (%) = [1-(A1-A2) / A0] × 100%

[0074] Where A0 is a mixture of 2 mL of ultrapure water and 2 mL of DPPH solution, A1 is a mixture of 2 mL of sample solution and 2 mL of DPPH solution, and A2 is a mixture of 2 mL of sample solution and 2 mL of anhydrous ethanol solution.

[0075] Preparation of 0.1mmol / L DPPH anhydrous ethanol solution: Dissolve 3.94mg of DPPH in 100mL of anhydrous ethanol and dissolve in the dark. (2) ABTS + Free radical scavenging ability test

[0076] Mix 7 mmol / L ABTS+ and 2.45 mmol / L potassium persulfate (final concentration) and let it stand overnight at room temperature in the dark. Dilute the resulting ABTS+ solution with anhydrous ethanol so that its absorbance at 30°C and 734 nm is 0.7 ± 0.02 to obtain the ABTS+ working solution. Add 100 μL of the sample solution of appropriate concentration to a test tube, then add 3.9 mL of ABTS+ solution. Replace the sample solution with anhydrous ethanol in the blank tube and replace the ABTS solution in the control tube. + The working solution was placed in the dark at room temperature for 6 minutes and its absorbance was measured at a wavelength of 734 nm. Each sample was run in parallel 3 times and the calculation formula was:

[0077] ABTS clearance (%) = (1-Ai / Ac) × 100%

[0078] Where Ai is the ratio of sample solution to ABTS + Absorbance of the mixed solution, Ac is the absorbance of anhydrous ethanol and ABTS + Absorbance of the mixture. 7mmol / L ABTS+ Preparation: Dissolve 19.2mg ABTS in 5mL deionized water.

[0079] Preparation of 140mmol / L potassium persulfate: Dissolve 37.84mg of potassium persulfate in 1mL of deionized water.

[0080] ABTS stock solution: Add 5 mL of ABTS solution to 89 μL of potassium persulfate solution and allow to react for 12-16 hours in the dark.

[0081] ABTS reaction solution: Take 1 mL of ABTS stock solution and add about 90 mL of anhydrous ethanol so that the absorbance of the mixture at 734 nm is between 0.680 and 0.720.

[0082] (3) Total reducing power test

[0083] Mix 2 mL of sample of varying concentrations, 2 mL of phosphate buffer (pH 6.6, 0.2 mol / L), and 2 mL of 1% potassium ferricyanide solution. The mixture is allowed to stand at 50°C for 20 minutes. 2 mL of 10% trifluoroacetic acid is added to terminate the reaction, and the mixture is centrifuged at 3000 rpm for 10 minutes. 2 mL of the supernatant is mixed with 2 mL of ultrapure water and 0.4 mL of 0.1% FeCl₃ solution. The mixture is allowed to stand at 50°C for 10 minutes, and the absorbance at 700 nm is measured. Ultrapure water serves as a blank control.

Claims

1. A method for extracting low-ash Poria peel polysaccharide, comprising the following steps 1) Take Poria cocos peel, add alkaline aqueous solution with a pH value of 10-12, extract, and filter to obtain a crude extract; 2) optionally concentrating the crude extract obtained in step 1 to obtain a concentrated solution; 3) The pH value of the Poria peel polysaccharide solution obtained in the previous step is adjusted to 7-8, and after centrifugation, ethanol is added to precipitate the polysaccharide to obtain a precipitated extract; 4) Redissolving the extract with water, subjecting the obtained reconstituted solution to cation exchange resin chromatography, collecting the purified liquid, and controlling the pH value to 7-8 to obtain low-ash Poria peel polysaccharide.

2. The method according to claim 1, wherein: In step 1, the material-liquid ratio of the Poria peel to the alkaline water is 1:10-30, such as 1:15-20.

3. The method according to claim 1, wherein: In step 1, the pH of the alkaline aqueous solution is 11, and the extraction temperature can be above the reflux temperature, such as 90-110°C, preferably 100°C.

4. The method according to claim 1, wherein: In step 2, the extract is concentrated to a relative density of 1.05 to 1.15, such as 1.

1.

5. The method according to claim 1, wherein: The pH value was adjusted to 7 as described in step 3.

6. The method according to claim 1, wherein: The centrifugal speed in step 3 is 1000-2000 r / min, such as 1200 r / min, and the centrifugal time can be 5-20 min, such as 10 min.

7. The method according to claim 1, wherein: The amount of alcohol added in step 3 is such that the final ethanol concentration is 75-80%.

8. The method according to claim 1, wherein the cation exchange resin in step 4 is a strong acid type or a weak acid type, such as 001*7 type, D113, D85 or D61 type.

9. Poria peel polysaccharide obtained by the method according to any one of claims 1 to 8.

10. Use of the Poria peel polysaccharide according to the preceding claim in the preparation of antioxidant products.

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