Method for extracting agaric polysaccharide from agaric

Abstract

The invention relates to the technical field of bioactive component extraction, in particular to a method for extracting auricularia auricula polysaccharide from auricularia auricula, which comprises the following steps: firstly, adding a specific synergist A into cleaned and crushed auricularia auricula, and carrying out ultrasonic-assisted pretreatment; adding a special compound enzyme and a regulating agent B, and carrying out microwave radiation enzymolysis; extracting by using hot water and adding a bifunctional reagent C, concentrating by using a nanofiltration membrane, and finally performing alcohol precipitation, washing and vacuum drying to obtain a product. The extraction method has a remarkable effect; the extraction efficiency can be greatly improved, and the polysaccharide purity and activity are improved; cost, raw material and energy consumption and enzyme consumption can be reduced, acid-base pollution is avoided, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive component extraction, in particular to a method for extracting wood ear polysaccharide from wood ear. Background Art

[0002] As a valuable bioactive ingredient, wood ear polysaccharides have attracted considerable attention. Their diverse benefits, including immunomodulatory, antioxidant, and lipid-lowering properties, hold great promise for the development of functional products. However, conventional methods for extracting polysaccharides from wood ear mushrooms present numerous challenges.

[0003] Although the traditional hot water extraction method is relatively simple to operate, the extraction efficiency is extremely low. Due to the compact structure of wood ear cells, polysaccharides are difficult to fully dissolve from the cells, which often requires the consumption of large amounts of raw materials and long-term high-temperature treatment. This not only leads to energy waste, but high temperatures may also destroy the biological activity of polysaccharides and reduce product quality. Although the enzymatic hydrolysis method can improve the extraction rate to a certain extent, the limited selection of enzyme types and the difficulty in accurately controlling the enzymatic hydrolysis conditions make the polysaccharide extraction effect unstable. Moreover, the cost of enzymes is high, and large-scale application will significantly increase production costs. In addition, the common acid-base extraction method has strict requirements on equipment, and acid and alkali residues are easy to pollute the environment. At the same time, it may cause changes in the polysaccharide structure and affect its functional properties.

[0004] With the advancement of science and technology and the growing market demand for high-quality wood ear polysaccharides, existing extraction technologies are no longer able to meet the needs of industrial development. There is an urgent need to develop an efficient, environmentally friendly, and cost-effective extraction method that maximizes polysaccharide activity. Against this backdrop, the present method was developed. Summary of the Invention

[0005] (1) Technical problems solved

[0006] In view of the shortcomings of the existing technology, the present invention provides a method for extracting fungus polysaccharides from fungus.

[0007] (2) Technical solution

[0008] An innovative method for extracting fungus polysaccharides from fungus, the method comprising the following key steps:

[0009] Pretreatment stage: fresh fungus is selected, washed and crushed; then a specially synthesized high-efficiency wall-breaking synergist A is added, which is composed of coumarin-3-carboxylic acid C 10 It is prepared by the reaction of H6O4 and mercaptoethylamine hydrochloride C2H7NS·HCl in the presence of an alkaline catalyst. The reaction formula is:

[0010]

[0011] The amount of synergist A added is 0.2%-0.8% of the mass of the fungus, and the fungus is stirred at 35-45°C and an ultrasonic power of 100-200W for 1.5-2.5 hours to significantly loosen the fungus cell structure and facilitate subsequent extraction;

[0012] Composite enzymatic hydrolysis: A special composite enzyme, consisting of β-glucanase, xylanase, and laccase in a mass ratio of 3:2:1, is added to the pretreated fungus. A metal ion regulator B with a unique structure is also added at a level of 0.08% to 0.25% of the fungus's mass. Enzymatic hydrolysis is carried out for 2.5 to 3.5 hours at a pH of 4.8 to 5.8, a temperature of 48 to 58°C, and a microwave radiation power of 50 to 100W, significantly promoting the release of fungus polysaccharides.

[0013] Hot water extraction step: The enzymatically hydrolyzed mixture is extracted in hot water at 85-95°C for 3.5-4.5 hours, with the solid-to-liquid ratio controlled at 1:18-1:22 g / mL; during this period, an antioxidant-solubilizing agent C is added at an amount of 0.05%-0.15% of the weight of the fungus to prevent polysaccharide oxidation and enhance its solubility in the extract;

[0014] Separation and concentration process: The extract is first centrifuged to remove the residue, and then concentrated through a nanofiltration membrane with a molecular weight cutoff of 2000-4000Da to 1 / 6-1 / 8 of the original volume. The operating pressure is 0.2-0.4MPa.

[0015] Alcohol precipitation and drying operation: slowly add 4-6 times the volume of anhydrous ethanol to the concentrated solution at a rate of 1.5-2.5 mL / min, and let it stand at 5-12° C. for 18-30 hours to precipitate the wood ear polysaccharide; collect the precipitate by centrifugation, wash it with anhydrous ethanol and acetone in sequence, and vacuum dry it at 42-52° C. and a vacuum degree of -0.09-0.1 MPa to constant weight to obtain a high-purity wood ear polysaccharide product.

[0016] Preferably, when the high-efficiency wall-breaking synergist A is synthesized, the molar ratio of coumarin-3-carboxylic acid to mercaptoethylamine hydrochloride is 1:1.2-1:1.6.

[0017] Preferably, the total added amount of the special complex enzyme is 0.8%-1.8% of the mass of the fungus.

[0018] Preferably, the metal ion regulator B can accurately complex specific metal ions, optimize the microenvironment of the enzyme active center, and improve the enzymatic efficiency by 20%-30%.

[0019] Preferably, the antioxidant-solubility-promoting dual-functional reagent C can increase the solubility of the fungus polysaccharide in the extract by 15%-25%, and effectively inhibit the oxidative degradation of the polysaccharide.

[0020] Preferably, during the nanofiltration membrane concentration process, the transmembrane pressure difference is maintained at 0.1-0.3 MPa and the temperature is controlled at 25-35°C.

[0021] Preferably, the pH value of the solution is adjusted to 6.5-7.5 during alcohol precipitation, which can improve the integrity and purity of the polysaccharide precipitation.

[0022] Preferably, the vacuum drying time is 4-6 hours, which can ensure that the fungus polysaccharide is fully dried without affecting its structure and activity.

[0023] Preferably, the wood ear polysaccharide extracted by the method for extracting wood ear polysaccharide from wood ear has a purity of 85%-98%, a molecular weight mainly distributed between , and has high biological activity.

[0024] Preferably, the fungus polysaccharide is used in the preparation of functional foods, biopharmaceutical preparations or high-end skin care products.

[0025] (3) Beneficial technical effects

[0026] Compared with the existing technology, the beneficial effects of the present invention are:

[0027] 1. By adding a self-synthesized, highly effective cell-breaking enhancer A, combined with ultrasound-assisted pretreatment, the dense cell structure of the fungus is rapidly broken down, creating favorable conditions for subsequent enzymatic hydrolysis and hot water extraction, significantly improving polysaccharide extraction compared to traditional methods. The specially formulated complex enzyme, in synergistic effect with the metal ion regulator B and microwave radiation, significantly boosts enzymatic hydrolysis efficiency, further promoting polysaccharide release.

[0028] 2. The antioxidant-solubility-promoting dual-functional reagent C not only effectively prevents polysaccharide oxidation but also improves its solubility in the extract, helping to obtain high-purity, highly active wood ear polysaccharides. Testing has shown that the wood ear polysaccharides extracted using this method are highly pure and retain their biologically active structures, performing well in functional tests such as immunomodulation and antioxidant testing.

[0029] 3. This method reduces raw material consumption and lowers energy costs. Compared with traditional enzymatic hydrolysis methods, the rational ratio of the specially formulated compound enzyme and the precise control of enzymatic hydrolysis conditions reduce the amount of enzyme used and save costs. At the same time, the entire process avoids the use of large amounts of acid and alkali, reducing environmental pollution. In addition, nanofiltration membrane concentration and precise alcohol precipitation and drying conditions improve the recovery rate of polysaccharides, further reducing production costs, providing a feasible solution for the large-scale industrial production of high-quality wood ear polysaccharides, with significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention is a flow chart of a method for extracting fungus polysaccharide from fungus;

[0031] Figure 2is a bar graph comparing the polysaccharide extraction rate and polysaccharide purity of the examples and comparative examples;

[0032] Figure 3 This is a line chart comparing the cumulative polysaccharide extraction amounts after pretreatment and hot water extraction in the embodiment and the comparative example;

[0033] Figure 4 H NMR spectrum of the high-efficiency wall-breaking synergist A proposed in the present invention. DETAILED DESCRIPTION

[0034] according to Figures 1 to 4 , the specific implementation of the present invention is as follows:

[0035] Example 1

[0036] Raw material preparation

[0037] Select fresh, high-quality wood ear mushrooms, remove impurities and rotten parts, and rinse them repeatedly with deionized water.

[0038] The raw materials include 500 grams of fresh and pest-free wood ear, 1.5 grams of a high-efficiency wall-breaking enhancer A synthesized from coumarin-3-carboxylic acid and mercaptoethylamine hydrochloride according to a specific process, 4 grams of a special complex enzyme (in which the mass ratio of β-glucanase, xylanase, and laccase is 3:2:1 and the enzyme activity meets the standard), 0.6 grams of an analytically pure metal ion regulator B, namely sodium ethylenediamine di-o-phenylacetate, 0.3 grams of a chemically pure antioxidant-solubilizing bifunctional reagent C, namely ferulic acid-polyethylene glycol ester, and an appropriate amount of analytically pure anhydrous ethanol and an appropriate amount of analytically pure acetone.

[0039] Extraction steps

[0040] Pretreatment: Crush the cleaned wood ear mushrooms into particles approximately 2-3 mm in size. Place them in a reactor, add 1.5 g of high-efficiency wall-breaking synergist A, and then add 1000 mL of deionized water. Adjust the temperature to 35°C, start the ultrasonic equipment at 100 W, and stir for 2.5 hours.

[0041] Composite enzymatic hydrolysis: add 4 g of special composite enzyme and 0.6 g of metal ion regulator B to the pretreated mixture, adjust the pH to 4.8 with dilute hydrochloric acid or sodium hydroxide solution, raise the temperature of the reactor to 48°C, turn on the microwave radiation equipment, set the power to 50 W, and perform enzymatic hydrolysis for 3.5 hours.

[0042] Hot water extraction: After the enzymatic hydrolysis is completed, the reactor temperature is raised to 85°C, 0.3 g of antioxidant-solubility-promoting dual-functional reagent C is added, and the extraction is continued for 4.5 hours while stirring. The solid-liquid ratio is 1:22 (g / mL).

[0043] Separation and concentration: The extract was centrifuged at 4000 rpm for 20 minutes to remove the residue. The supernatant was concentrated through a nanofiltration membrane with a molecular weight cutoff of 2000 Da and an operating pressure of 0.2 MPa to 1 / 8 of its original volume.

[0044] Alcohol precipitation and drying: Slowly add 4 times the volume of anhydrous ethanol to the concentrate at a rate of 1.5 mL / min, adjust the solution pH to 6.5, and let it stand at 5°C for 30 hours. Collect the precipitate by centrifugation, wash it with anhydrous ethanol and acetone 2-3 times in sequence, and then place the precipitate in a vacuum drying oven at 42°C and a vacuum degree of -0.09 MPa for 6 hours to obtain the fungus polysaccharide product.

[0045] Performance Testing

[0046] The polysaccharide extraction rate of the sample was 35%, the polysaccharide purity reached 86%, and its molecular weight distribution was mainly in the range of 1.5×10 4 -3×10 5 Within the scope of Da.

[0047] Example 2

[0048] Raw material preparation

[0049] The raw materials include 500 grams of fresh and pest-free wood ear mushrooms, 3 grams of a high-efficiency wall-breaking enhancer A synthesized from coumarin-3-carboxylic acid and mercaptoethylamine hydrochloride according to a specific process, 6 grams of a special complex enzyme (in which the mass ratio of β-glucanase, xylanase, and laccase is 3:2:1 and the enzyme activity meets the standards), 1 gram of an analytically pure metal ion regulator B, namely sodium ethylenediamine di-o-phenylacetate, 0.5 grams of a chemically pure antioxidant-solubilizing bifunctional reagent C, namely ferulic acid-polyethylene glycol ester, and an appropriate amount of analytically pure anhydrous ethanol and an appropriate amount of analytically pure acetone.

[0050] Extraction steps

[0051] Pretreatment: After the fungus was crushed, 3 g of high-efficiency wall-breaking synergist A and 1000 mL of deionized water were added, and stirred at 40°C and an ultrasonic power of 150 W for 2 hours.

[0052] Compound enzymatic hydrolysis: add 6g of special compound enzyme and 1g of metal ion regulator B, adjust the pH to 5.3, and perform enzymatic hydrolysis at 53°C and microwave power 75W for 3 hours.

[0053] Hot water extraction: Heat to 90°C, add 0.5 g of antioxidant-solubilizing agent C, and extract for 4 hours with a solid-liquid ratio of 1:20 (g / mL).

[0054] Separation and concentration: After centrifugation to remove the residue, the product was concentrated through a nanofiltration membrane with a molecular weight cut-off of 3000Da and an operating pressure of 0.3MPa to 1 / 7 of the original volume.

[0055] Alcohol precipitation and drying: add 5 times the volume of anhydrous ethanol at a rate of 2 mL / min, adjust the pH to 7, and let it stand at 8°C for 24 hours. After washing the precipitate, dry it at 47°C and vacuum degree -0.095 MPa for 5 hours.

[0056] Performance Testing

[0057] The polysaccharide extraction rate of the substance is 42%, the polysaccharide purity reaches 92%, and its molecular weight distribution is mainly in the range of 2×10 4 -5×10 5 Between.

[0058] Example 3

[0059] Raw material preparation

[0060] The raw materials and specifications required for the preparation are as follows: 500 grams of fresh and pest-free wood ear mushrooms; 4 grams of a high-efficiency wall-breaking enhancer A, synthesized from coumarin-3-carboxylic acid and mercaptoethylamine hydrochloride according to a specific process; 9 grams of a specially prepared complex enzyme, wherein the mass ratio of β-glucanase, xylanase, and laccase is 3:2:1, and the enzyme activity meets the standard; 1.25 grams of an analytically pure metal ion regulator B, i.e., sodium ethylenediamine di-o-phenylacetate; 0.75 grams of a chemically pure antioxidant-solubilizing bifunctional reagent C, i.e., ferulic acid-polyethylene glycol ester; and appropriate amounts of analytically pure anhydrous ethanol and analytically pure acetone.

[0061] Extraction steps

[0062] Pretreatment: After crushing the fungus, add 4g of high-efficiency wall-breaking synergist A and 1000mL of deionized water, and stir at 45°C and 200W ultrasonic power for 1.5 hours.

[0063] Compound enzymatic hydrolysis: add 9g of special compound enzyme and 1.25g of metal ion regulator B, adjust the pH to 5.8, and perform enzymatic hydrolysis at 58°C and microwave power of 100W for 2.5 hours.

[0064] Hot water extraction: Heat to 95°C, add 0.75 g of antioxidant-solubilizing agent C, and extract for 3.5 hours. The solid-liquid ratio is 1:18 (g / mL).

[0065] Separation and concentration: After centrifugation, the product was concentrated through a nanofiltration membrane with a molecular weight cut-off of 4000Da and an operating pressure of 0.4MPa to 1 / 6 of the original volume.

[0066] Alcohol precipitation and drying: add 6 times the volume of anhydrous ethanol at a rate of 2.5 mL / min, adjust the pH to 7.5, and let it stand at 12°C for 18 hours. After the precipitate is washed, dry it at 52°C and vacuum degree -0.1 MPa for 4 hours.

[0067] Performance Testing

[0068] The performance index test results of the sample showed that the polysaccharide extraction rate was 48%, the polysaccharide purity reached 96%, and the molecular weight distribution was mainly in the range of 3×10 4 -8×10 5 Between.

[0069] Comparative Example

[0070] Raw material preparation

[0071] Select 500g of fresh fungus the same as in the embodiment.

[0072] Extraction steps

[0073] Using a traditional hot water extraction method, the fungus was crushed and added to 10,000 mL of deionized water. Extraction was performed at 100°C for 6 hours with constant stirring. The extract was filtered and concentrated to 1 / 10 of its original volume. Three volumes of anhydrous ethanol were then added, and the mixture was allowed to stand at 4°C for 24 hours. The precipitate was collected by centrifugation, washed with anhydrous ethanol, and dried at 50°C to constant weight.

[0074] Performance Testing

[0075] The performance index test results of the substance showed that the polysaccharide extraction rate was 18%, the polysaccharide purity was 70%, and the molecular weight distribution was relatively dispersed without obvious concentrated range.

[0076] It can be seen from the above three embodiments and one comparative example that the method for extracting wood ear polysaccharide from wood ear of the present invention has significant advantages in terms of polysaccharide extraction rate, purity and molecular weight distribution control, and can effectively improve the extraction efficiency and product quality of wood ear polysaccharide.

[0077] The comprehensive performance comparison between the embodiment and the comparative example is shown in the following table:

[0078] Table 1

[0079]

[0080] Conclusion: This table visually demonstrates the differences in key performance indicators between the Examples and the Comparative Examples. The Examples excel in polysaccharide extraction efficiency, purity, and molecular weight distribution control, with performance improving significantly as the Example number increases. The Comparative Examples, on the other hand, perform far worse than the Examples in all respects, highlighting the advantages of the extraction method of the present invention.

[0081] The degradation rates of harmful gases in the examples and the comparative examples at different time points are compared in the following table:

[0082] Table 2

[0083]

[0084] Conclusion: This table shows the polysaccharide purity of each example at different drying temperatures. Different examples achieved high purities at their respective drying temperatures, demonstrating the significant influence of drying temperature on polysaccharide purity and the precise control achieved in the present method.

[0085] The comparison of the cumulative extraction amount of polysaccharides in different process stages is shown in the following table:

[0086] Table 3

[0087]

[0088] The technology presented here significantly improves polysaccharide extraction efficiency through phased process optimization. The pretreatment phase, with its wall-breaking synergist, increases the extraction yield of the examples to 10-15%, far exceeding the 2% achieved by the conventional comparative process. The combined enzymatic hydrolysis phase further contributes an additional 15-20% to the extraction yield. Finally, after hot water extraction, the total extraction yield of the examples reaches 35-48%, a 97-167% improvement over the comparative process. Data demonstrate that the synergistic effects of wall-breaking synergism, enhanced enzymatic hydrolysis, and low-temperature extraction can overcome the efficiency bottleneck of traditional high-temperature extraction methods, achieving simultaneous optimization of extraction yield and product quality.

[0089] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An innovative method for extracting fungus polysaccharides from fungus, characterized in that: The method includes the following key steps: Pretreatment stage: fresh fungus is selected, washed and crushed; then a specially synthesized high-efficiency wall-breaking synergist A is added, which is composed of coumarin-3-carboxylic acid C 10 It is prepared by the reaction of H6O4 and mercaptoethylamine hydrochloride C2H7NS·HCl in the presence of an alkaline catalyst. The reaction formula is: The amount of synergist A added is 0.2%-0.8% of the mass of the fungus, and the mixture is stirred at 35-45°C and an ultrasonic power of 100-200W for 1.5-2.5 hours; Composite enzymatic hydrolysis step: Add a composite enzyme composed of β-glucanase, xylanase and laccase in a mass ratio of 3:2:1 to the pretreated fungus; at the same time, add a metal ion regulator B in an amount of 0.08%-0.25% of the fungus mass, and perform enzymatic hydrolysis for 2.5-3.5 hours at a pH of 4.8-5.8, a temperature of 48-58°C, and a microwave radiation power of 50-100W; Hot water extraction step: extracting the enzymatically hydrolyzed mixture in hot water at 85-95°C for 3.5-4.5 hours, with the solid-liquid ratio controlled at 1:18-1:22 g / mL; during this period, adding an antioxidant-solubilizing agent C in an amount of 0.05%-0.15% of the weight of the fungus; Separation and concentration process: The extract is first centrifuged to remove the residue, and then concentrated through a nanofiltration membrane with a molecular weight cutoff of 2000-4000Da to 1 / 6-1 / 8 of the original volume. The operating pressure is 0.2-0.4MPa. Alcohol precipitation and drying operation: slowly add 4-6 times the volume of anhydrous ethanol to the concentrated solution at a rate of 1.5-2.5 mL / min, and let it stand at 5-12°C for 18-30 hours to precipitate the fungus polysaccharide; collect the precipitate by centrifugation, wash it with anhydrous ethanol and acetone in turn, and vacuum dry it at 42-52°C and a vacuum degree of -0.09 to -0.1 MPa to constant weight.

2. The method for extracting fungus polysaccharide from fungus according to claim 1, characterized in that: When the high-efficiency wall-breaking synergist A is synthesized, the molar ratio of coumarin-3-carboxylic acid to mercaptoethylamine hydrochloride is 1:1.2-1:1.

6.

3. The method for extracting fungus polysaccharide from fungus according to claim 1, characterized in that: The total added amount of the special compound enzyme is 0.8%-1.8% of the mass of the fungus.

4. The method for extracting fungus polysaccharide from fungus according to claim 1, characterized in that: Metal ion regulator B can accurately complex specific metal ions, optimize the microenvironment of the enzyme active center, and improve enzymatic hydrolysis efficiency by 20%-30%.

5. The method for extracting fungus polysaccharide from fungus according to claim 1, characterized in that: The antioxidant-solubility-promoting dual-function reagent C can increase the solubility of the fungus polysaccharide in the extract by 15%-25%.

6. The method for extracting fungus polysaccharide from fungus according to claim 1, characterized in that: During the nanofiltration membrane concentration process, the transmembrane pressure difference is maintained at 0.1-0.3 MPa and the temperature is controlled at 25-35°C.

7. The method for extracting fungus polysaccharide from fungus according to claim 1, characterized in that: During alcohol precipitation, the pH value of the solution was adjusted to 6.5-7.

5.

8. The method for extracting fungus polysaccharide from fungus according to claim 1, characterized in that: The vacuum drying time is 4-6 hours.

9. The method for extracting auricularia auricularia polysaccharide from auricularia auricularia according to claim 1, wherein: The purity of the wood ear polysaccharide reaches 85%-98%.

10. Use of the fungus polysaccharide according to claim 9 in the preparation of functional foods, biopharmaceutical preparations or high-end skin care products.

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