Process for extracting auricularia auricula polysaccharide by complex enzyme-assisted subcritical water extraction method

Abstract

The invention belongs to the technical field of extraction processes, and particularly relates to a process for extracting auricularia auricula polysaccharide by a compound enzyme-assisted subcritical water extraction method. The method specifically comprises the following steps: performing degreasing treatment on agaric to obtain degreased agaric powder; mixing the degreased auricularia auricula powder with water, then adding cellulose-pectin compound enzyme into the mixture, and performing enzymolysis to obtain an enzymolysis auricularia auricula solution; and carrying out subcritical extraction on the enzymolysis agaric solution to obtain the agaric polysaccharide. The compound enzyme-assisted subcritical water extraction method disclosed by the invention not only effectively shortens the extraction time, but also has the advantages of improving the polysaccharide yield and the polysaccharide biological activity.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction technology, and in particular relates to a process for extracting auricularia auricula polysaccharide by using a composite enzyme-assisted subcritical water extraction method. Background Art

[0002] Auricularia auricula, also known as tree fungus, is favored by people for its excellent flavor and nutritional value. The high protein and rich auricularia polysaccharides make it the "meat in plants", "the king of vegetarian food" and "the pearl of the fungus world". The auricularia polysaccharides in auricularia have multiple biological activities such as anti-tumor proliferation activity, anti-blood coagulation process, antioxidant properties, regulating blood cholesterol levels, delaying biological aging process and enhancing the body's immune response. Relevant studies mainly focusing on diabetes (a metabolic disease characterized by long-term high blood sugar levels) have shown that edible fungus auricularia polysaccharides are an effective way to treat diabetes. For this reason, auricularia polysaccharides have attracted much attention in the research of medicine and food, and its research results continue to emerge.

[0003] In recent years, the research on the extraction technology of polysaccharides from wood ear has been quite extensive, covering hot water extraction, enzymatic hydrolysis, microwave-assisted extraction and ultrasonic-assisted extraction. The efficiency of hot water extraction is low, it takes a long time, and consumes a lot of energy; the efficiency of enzymatic hydrolysis is low and the extraction cycle is long; although microwave-assisted extraction has good penetration ability, its mass transfer efficiency is not ideal; ultrasonic-assisted extraction has limitations in thermal effect and cavitation bubble formation. Summary of the invention

[0004] In view of the shortcomings of the above-mentioned prior art, the present invention provides a process for extracting black fungus polysaccharides by a composite enzyme-assisted subcritical water extraction method. The present invention first defattes the black fungus, then mixes it with water and a cellulose pectin composite enzyme and performs enzymolysis, and then performs subcritical extraction to obtain black fungus polysaccharides. The composite enzyme-assisted subcritical water extraction method of the present invention not only effectively shortens the extraction time, but also has the advantages of increasing the polysaccharide yield and the biological activity of the polysaccharide.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A process for extracting auricularia auricularia polysaccharide by a composite enzyme-assisted subcritical water extraction method comprises the following steps:

[0007] The fungus is defatted to obtain defatted fungus powder.

[0008] The defatted black fungus powder is mixed with water, and then cellulose pectin complex enzyme is added thereto and enzymolysis is performed to obtain an enzymolysis black fungus solution.

[0009] The enzymatically hydrolyzed fungus solution is subjected to subcritical extraction to obtain fungus polysaccharide.

[0010] Preferably, the mass volume ratio of defatted black fungus powder to water is 1g:80-160mL. The solid-liquid ratio affects the yield of black fungus polysaccharides extracted by subcritical water method. A larger liquid-solid ratio dilutes the polysaccharide concentration in the extract.

[0011] Preferably, the mass volume ratio of defatted wood ear powder to water is 1g:120mL.

[0012] Preferably, the subcritical extraction conditions are: extraction at 105-185°C and a rotation speed of 50-170r / min for 15-35min. Too high a temperature will accelerate the decomposition rate of polysaccharides and destroy the structural integrity of polysaccharides, which will cause a decrease in the yield of black fungus polysaccharides; if the extraction time is too long, black fungus polysaccharides will be degraded, thereby reducing the yield; if the rotation speed is too high, the disturbance of the liquid surface will increase, thereby reducing the yield of black fungus polysaccharides to a certain extent.

[0013] Preferably, the subcritical extraction conditions are: extraction at 165° C. and a rotation speed of 140 r / min for 25 min.

[0014] Preferably, during subcritical extraction, subcritical extraction is performed with the assistance of 1-2 MPa pressure. As the pressure increases, the solubility of the fungus polysaccharide will also increase.

[0015] Preferably, the mass ratio of defatted wood ear powder to cellulose pectin complex enzyme is 1:50.

[0016] Preferably, the enzymolysis time is 2 hours. If the enzymolysis time is too long, the production efficiency will be reduced, and if the enzymolysis time is too short, it will be unfavorable for the dissolution of polysaccharides.

[0017] Preferably, the petroleum ether and the fungus are mixed and then allowed to stand, and the fungus is defatted using the petroleum ether.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention first defattes the fungus, then mixes it with water and cellulose pectin complex enzyme and performs enzymolysis, and then performs subcritical extraction, extracts for 15-35min at 105-185°C and a rotation speed of 50-170r / min to obtain fungus polysaccharides. Degreasing is first performed to avoid the lipid components in the fungus from hindering the contact between the enzyme and the polysaccharide in the cell, thereby reducing the enzymolysis efficiency. At the same time, degreasing can effectively avoid the chemical reaction that may occur between the lipid and the polysaccharide, and protect the activity of the polysaccharide; in view of the large amount of colloid and cellulose in the fungus, the defatted fungus is enzymolyzed by using cellulose pectin complex enzyme to effectively improve the enzymolysis effect and the final polysaccharide yield; then subcritical extraction is performed, using water as an extractant, using subcritical water with a high solubility, and adjusting the temperature and rotation speed to complete efficient extraction in a short time, effectively reducing the difficulty of purification in the subsequent process of polysaccharide extraction, improving the polysaccharide yield, and significantly shortening the extraction time.

[0020] 2. The present invention studies the extraction of wood ear polysaccharides with certain antioxidant activity by using cellulose pectin complex enzyme-assisted subcritical water extraction, and optimizes the process. Taking the yield of wood ear polysaccharides, DPPH· scavenging rate and hydroxyl radical scavenging rate as evaluation indicators, the orthogonal test is used to optimize the extraction process of wood ear polysaccharides on the basis of single factors, and a comparative study is conducted with hot water extraction. The results show that when 2% cellulose pectin complex enzyme (1:1) is used for enzymatic hydrolysis for 2h, under the conditions of solid-liquid ratio of 1:120 (g / mL), extraction temperature of 165℃, extraction time of 25min, and rotation speed of 140r, the yield of wood ear polysaccharides extracted by subcritical water method is 24.45%, DPPH· scavenging rate is 21.29%, and hydroxyl radical scavenging rate is 81.62%, which has good antioxidant activity; and compared with hot water extraction method, it has obvious advantages of short extraction time, high yield, and significantly increased DPPH· free radical scavenging rate and hydroxyl free radical scavenging rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Glucose standard curve.

[0022] Figure 2 This is a graph showing the effect of the material-liquid ratio of Examples 1 to 4 and 6 on the yield of Auricularia auricula polysaccharide.

[0023] Figure 3 This is a graph showing the effect of the material-liquid ratio of Examples 1 to 5 on the DPPH·free radical scavenging rate of Auricularia auricula polysaccharide.

[0024] Figure 4 This is a graph showing the effect of the material-liquid ratio of Examples 7 to 11 on the scavenging rate of hydroxyl radicals of Auricularia auricula polysaccharide.

[0025] Figure 5 This is a graph showing the effect of the extraction temperature on the yield of Auricularia auricula polysaccharide in Examples 7 to 11.

[0026] Figure 6 This is a graph showing the effect of the extraction temperature of Example 7 to Example 11 on the DPPH· free radical scavenging rate of Auricularia auricularia polysaccharide.

[0027] Figure 7 This is a graph showing the effect of the extraction temperature of Example 7 to Example 11 on the scavenging rate of hydroxyl radicals of Auricularia auricularia polysaccharide.

[0028] Figure 8 This is a graph showing the effect of the extraction time on the yield of Auricularia auricula polysaccharides in Examples 12 to 16.

[0029] Fig. 9 This is a graph showing the effect of the extraction time of Example 12 to Example 16 on the DPPH· free radical scavenging rate of Auricularia auricularia polysaccharide.

[0030] Fig.10 This is a graph showing the effect of the extraction time of Example 12 to Example 16 on the scavenging rate of hydroxyl radicals of Auricularia auricularia polysaccharide.

[0031] Fig.11 This is a graph showing the effect of the rotation speed on the yield of wood ear polysaccharides in Examples 17 to 21.

[0032] Fig.12 This is a graph showing the effect of the rotation speed on the DPPH free radical scavenging rate of Auricularia auricularia polysaccharide in Examples 17 to 21.

[0033] Fig.13 This is a graph showing the effect of the rotation speed on the scavenging rate of hydroxyl radicals of Auricularia auricularia polysaccharide in Examples 17 to 21. DETAILED DESCRIPTION

[0034] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0035] Subcritical technology has attracted much attention in the extraction of polysaccharides from wood ear plants, due to its advantages of saving time and effectively improving the yield. In view of the practical application of subcritical water extraction technology in the field of wood ear polysaccharide extraction and its modification. The present invention uses a composite enzyme-assisted subcritical water extraction method to extract wood ear polysaccharides, with yield and biological activity (DPPH free radical scavenging rate and hydroxyl free radical scavenging rate) as the main evaluation indicators. By optimizing the process conditions to improve the yield and biological activity indicators of wood ear polysaccharides, it aims to provide a scientific basis for the development of functional foods for people with high blood sugar.

[0036] Compared with the hot water extraction method, the composite enzyme-assisted subcritical water extraction method of the present invention has a short extraction time and a high yield, overcomes the technical defects of the water extraction method such as low efficiency, long time consumption and high energy consumption, and has the advantages of significantly increasing the DPPH free radical scavenging rate and the hydroxyl free radical scavenging rate.

[0037] Compared with the enzymatic hydrolysis method, the composite enzyme-assisted subcritical water extraction method of the present invention can effectively improve the extraction efficiency and shorten the extraction cycle.

[0038] Compared with the microwave-assisted extraction method, the composite enzyme-assisted subcritical water extraction method of the present invention has significantly high mass transfer efficiency and can effectively improve the solubility of the substance in the aqueous solution.

[0039] Compared with the ultrasonic assisted extraction method, the composite enzyme assisted subcritical water extraction method of the present invention can effectively overcome the limitations of the ultrasonic assisted extraction method in terms of thermal effect and cavitation bubble formation. The technical scheme of the present invention is studied using embodiments and comparative examples, and the specific research methods and results are as follows:

[0040] Example 1

[0041] A process for extracting auricularia auricularia polysaccharide by a composite enzyme-assisted subcritical water extraction method comprises the following steps:

[0042] S1. Pretreatment of black fungus: rinse the black fungus, dry it in an oven, and grind it through an 80-mesh sieve, then add petroleum ether for degreasing, and place it in a fume hood at room temperature for 24 hours to obtain defatted black fungus powder.

[0043] S2. Extraction: Weigh 1.5 g of defatted black fungus powder, first mix it with distilled water at a solid-liquid ratio of 1 g: 80 mL, stir it thoroughly, add 2% cellulose pectin complex enzyme (1:1), and then wait for the defatted black fungus powder to be enzymatically hydrolyzed for 2 h; then transfer it into a subcritical reactor, extract black fungus polysaccharides for 25 min at a temperature of 120 ° C and a rotation speed of 120 r / min to obtain an extract.

[0044] S3. Evaporation and concentration: Take out the extract, centrifuge it in a centrifuge at 4000r / min for 20min, take out the supernatant, evaporate and concentrate it at 55°C to 1 / 5 of the original supernatant volume, add 95% ethanol by volume, let it stand for 24h, centrifuge it at 4000r / min for 20min, discard the supernatant, and obtain the crude product of Auricularia auricula polysaccharide.

[0045] Example 2

[0046] The preparation steps are the same as those in Example 1, except that the material-liquid ratio is changed from 1 g:80 mL to 1 g:100 mL.

[0047] Example 3

[0048] The preparation steps are the same as those in Example 1, except that the material-liquid ratio is changed from 1 g:80 mL to 1 g:120 mL.

[0049] Example 4

[0050] The preparation steps are the same as those in Example 1, except that the material-liquid ratio is changed from 1 g:80 mL to 1 g:140 mL.

[0051] Example 5

[0052] The preparation steps are the same as those in Example 1, except that the material-liquid ratio is changed from 1 g:80 mL to 1 g:150 mL.

[0053] Example 6

[0054] The preparation steps are the same as those in Example 1, except that the material-liquid ratio is changed from 1 g:80 mL to 1 g:160 mL.

[0055] Example 7

[0056] A process for extracting auricularia auricularia polysaccharide by a composite enzyme-assisted subcritical water extraction method comprises the following steps:

[0057] S1. Pretreatment of black fungus: rinse the black fungus, dry it in an oven, and grind it through an 80-mesh sieve, then add petroleum ether for degreasing, and place it in a fume hood at room temperature for 24 hours to obtain defatted black fungus powder.

[0058] S2. Extraction: Weigh 1.5 g defatted black fungus powder, first mix it with distilled water at a solid-liquid ratio of 1 g: 120 mL, stir it thoroughly, add 2% cellulose pectin complex enzyme (1:1), and then wait for the defatted black fungus powder to be enzymatically hydrolyzed for 2 h; then transfer it into a subcritical reactor, extract black fungus polysaccharides for 25 min at a temperature of 105 ° C and a rotation speed of 120 r / min to obtain an extract.

[0059] S3. Evaporation and concentration: Take out the extract, centrifuge it in a centrifuge at 4000r / min for 20min, take out the supernatant, evaporate and concentrate it at 55°C to 1 / 5 of the original supernatant volume, add 95% ethanol by volume, let it stand for 24h, centrifuge it at 4000r / min for 20min, discard the supernatant, and obtain the crude product of Auricularia auricula polysaccharide.

[0060] Example 8

[0061] The preparation steps are the same as those of Example 7, except that the temperature is replaced from 105°C to 125°C.

[0062] Example 9

[0063] The preparation steps are the same as those of Example 7, except that the temperature is replaced from 105°C to 145°C.

[0064] Example 10

[0065] The preparation steps are the same as those of Example 7, except that the temperature is replaced from 105°C to 165°C.

[0066] Embodiment 11

[0067] The preparation steps are the same as those of Example 7, except that the temperature is replaced from 105°C to 185°C.

[0068] Example 12

[0069] A process for extracting auricularia auricularia polysaccharide by a composite enzyme-assisted subcritical water extraction method comprises the following steps:

[0070] S1. Pretreatment of black fungus: rinse the black fungus, dry it in an oven, and grind it through an 80-mesh sieve, then add petroleum ether for degreasing, and place it in a fume hood at room temperature for 24 hours to obtain defatted black fungus powder.

[0071] S2. Extraction: Weigh 1.5 g of defatted black fungus powder, first mix it with distilled water at a solid-liquid ratio of 1 g: 120 mL, stir it thoroughly, add 2% cellulose pectin complex enzyme (1:1), and then wait for the defatted black fungus powder to be enzymatically hydrolyzed for 2 h; then transfer it into a subcritical reactor, extract black fungus polysaccharides for 25 min at a temperature of 150 ° C and a rotation speed of 120 r / min to obtain an extract.

[0072] S3. Evaporation and concentration: Take out the extract, centrifuge it in a centrifuge at 4000r / min for 20min, take out the supernatant, evaporate and concentrate it at 55°C to 1 / 5 of the original supernatant volume, add 95% ethanol by volume, let it stand for 24h, centrifuge it at 4000r / min for 20min, discard the supernatant, and obtain the crude product of Auricularia auricula polysaccharide.

[0073] Embodiment 13

[0074] The preparation steps are the same as those of Example 12, except that the time is replaced from 25 min to 15 min.

[0075] Embodiment 14

[0076] The preparation steps are the same as those of Example 12, except that the time is replaced from 25 min to 20 min.

[0077] Embodiment 15

[0078] The preparation steps are the same as those of Example 12, except that the time is replaced from 25 min to 30 min.

[0079] Example 16

[0080] The preparation steps are the same as those of Example 12, except that the time is replaced from 25 min to 35 min.

[0081] Embodiment 17

[0082] A process for extracting auricularia auricularia polysaccharide by a composite enzyme-assisted subcritical water extraction method comprises the following steps:

[0083] S1. Pretreatment of black fungus: rinse the black fungus, dry it in an oven, and grind it through an 80-mesh sieve, then add petroleum ether for degreasing, and place it in a fume hood at room temperature for 24 hours to obtain defatted black fungus powder.

[0084] S2. Extraction: Weigh 1.5 g of defatted black fungus powder, first mix it with distilled water at a solid-liquid ratio of 1 g: 120 mL, stir it thoroughly, add 2% cellulose pectin complex enzyme (1: 1), and then wait for the defatted black fungus powder to be enzymatically hydrolyzed for 2 h; then transfer it into a subcritical reactor, extract black fungus polysaccharides for 25 min at a temperature of 150 ° C and a rotation speed of 50 r / min to obtain an extract.

[0085] S3. Evaporation and concentration: Take out the extract, centrifuge it in a centrifuge at 4000r / min for 20min, take out the supernatant, evaporate and concentrate it at 55°C to 1 / 5 of the original supernatant volume, add 95% ethanol by volume, let it stand for 24h, centrifuge it at 4000r / min for 20min, discard the supernatant, and obtain the crude product of Auricularia auricula polysaccharide.

[0086] Embodiment 18

[0087] The preparation steps are the same as those in Example 17, except that the rotation speed is replaced from 50 r / min to 80 r / min.

[0088] Embodiment 19

[0089] The preparation steps are the same as those of Example 17, except that the rotation speed is replaced from 50 r / min to 110 r / min.

[0090] Embodiment 20

[0091] The preparation steps are the same as those in Example 17, except that the rotation speed is replaced from 50 r / min to 140 r / min.

[0092] Embodiment 21

[0093] The preparation steps are the same as those in Example 17, except that the rotation speed is replaced from 50 r / min to 170 r / min.

[0094] Embodiment 22

[0095] A process for extracting auricularia auricularia polysaccharide by a composite enzyme-assisted subcritical water extraction method comprises the following steps:

[0096] S1. Pretreatment of black fungus: rinse the black fungus, dry it in an oven, and grind it through an 80-mesh sieve, then add petroleum ether for degreasing, and place it in a fume hood at room temperature for 24 hours to obtain defatted black fungus powder.

[0097] S2. Extraction: Weigh 1.5 g of defatted black fungus powder, mix it with distilled water at a solid-liquid ratio of 1 g: 120 mL, stir it thoroughly, add 2% cellulose pectin complex enzyme (1:1), and then wait for the defatted black fungus powder to be enzymatically hydrolyzed for 2 h; then transfer it into a subcritical reactor, extract black fungus polysaccharides for 25 min at a temperature of 165 ° C and a rotation speed of 140 r / min to obtain an extract.

[0098] S3. Evaporation and concentration: Take out the extract, centrifuge it in a centrifuge at 4000r / min for 20min, take out the supernatant, evaporate and concentrate it at 55°C to 1 / 5 of the original supernatant volume, add 95% ethanol by volume, let it stand for 24h, centrifuge it at 4000r / min for 20min, discard the supernatant, and obtain the crude product of Auricularia auricula polysaccharide.

[0099] Embodiment 23

[0100] The preparation steps are the same as those in Example 21, except that the pressure in the subcritical reactor is increased by 2 MPa, and the yield of wood ear polysaccharide is 25.71%, the DPPH scavenging rate is 32.38%, and the hydroxyl radical scavenging rate is 85.44%. This is because the solubility of wood ear polysaccharide increases with the increase of pressure.

[0101] Comparative Example 1

[0102] A process for extracting fungus polysaccharides by water extraction comprises the following steps:

[0103] S1. Pretreatment of black fungus: rinse the black fungus, dry it in an oven, and grind it through an 80-mesh sieve, then add petroleum ether for degreasing, and place it in a fume hood at room temperature for 24 hours to obtain defatted black fungus powder.

[0104] S2. Weigh 1.5 g of defatted black fungus powder, first mix it with distilled water at a solid-liquid ratio of 1 g:40 mL, then soak it in a 100°C water bath for 4 h, centrifuge it, and continue to soak the precipitate in a 100°C water bath for 4 h, soak it twice, combine the supernatants, concentrate the supernatants to 1 / 5 of the volume of the original solution on a rotary evaporator, add 5 times the volume fraction of 95% ethanol, let it stand at 4°C for 24 h, centrifuge it at 4500 r / min for 30 min, discard the supernatant, and obtain a crude black fungus polysaccharide.

[0105] Based on the above single factor experiment, the yield of auricularia auricula polysaccharide, DPPH scavenging rate and hydroxyl radical scavenging rate were used as evaluation indicators, and four factors including solid-liquid ratio (mL / g), extraction temperature (℃), extraction time (min) and rotation speed (r / min) were selected. Three levels were selected for each factor to carry out orthogonal experiment. The factor level design is shown in Table 3.

[0106] Table 1 Orthogonal test factors

[0107]

[0108] Note: A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, and D3 in the table are the selected optimal parameters of each factor.

[0109] 1. Determination method of fungus polysaccharide yield, DPPH scavenging rate and hydroxyl free radical scavenging rate:

[0110] 1.1 Determination of the yield of fungus polysaccharides

[0111] (1) Preparation of standard curve

[0112] Using the phenol-sulfuric acid colorimetric method, take 7 clean test tubes, accurately draw 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of 100μg / mL glucose solution, and then add distilled water to 2.0mL. Add 1.0mL of 6% phenol to each test tube, and then quickly add 5.0mL of concentrated sulfuric acid, shake well, place in a boiling water bath for 15min, take out and cool for 10min, and measure the absorbance at 490nm. Use 2.0mL of distilled water instead of glucose solution and follow the same steps as a blank test. Draw a glucose standard curve and calculate the regression equation.

[0113] (2) Determination of the yield of fungus polysaccharides

[0114] Accurately weigh 50 mg of crude black fungus polysaccharide, put it into a 1L volumetric flask, and dilute it with distilled water to obtain a black fungus polysaccharide solution. Take 2 mL of the black fungus polysaccharide solution in a test tube, operate according to the steps of (1) above, measure its absorbance, calculate the concentration C of black fungus polysaccharide according to the absorbance value obtained, and calculate the yield Y of black fungus polysaccharide using the equation.

[0115] Y=[C×G / 50 / W]×100%

[0116] Wherein, Y is the yield of auricularia auriculariae polysaccharide, %; C is the mass concentration of auricularia auriculariae polysaccharide, mg / L; G is the weight of crude auricularia auriculariae polysaccharide, mg; and W is the mass of defatted auricularia auriculariae powder, mg.

[0117] 1.2 Determination of biological activity of Auricularia auricula polysaccharides

[0118] (1) Determination of DPPH free radical scavenging rate

[0119] Prepare 0.1mmol / L DPPH solution with anhydrous ethanol and store it in the dark. Fully mix 1mL of DPPH solution with equal amounts of wood ear polysaccharide solution of different gradients, react in the dark for 30min to obtain a reaction solution, and measure the absorbance of the reaction solution at 517nm. Use distilled water instead of wood ear polysaccharide solution as a blank control, measure 3 times in parallel and take the average value.

[0120]

[0121] Blank group: the absorbance value measured by replacing auricularia auricularia polysaccharide with anhydrous ethanol; sample group: the absorbance value of auricularia auricularia polysaccharide; control group: the absorbance value of DPPH with anhydrous ethanol.

[0122] (2) Determination of hydroxyl radical scavenging rate

[0123] Mix 1 mL of 9 mmol / L FeSO4 solution and an equal amount of 8.8 mmol / L H2O2 solution, add 1 mL of different gradients of wood ear polysaccharide sample solution and 1 mL of 9 mmol / L salicylic acid solution, mix thoroughly, and let stand for 1 hour to obtain a reaction solution. The absorbance of the reaction solution was measured at 517 nm. Distilled water was used as a blank group instead of the wood ear polysaccharide sample solution, and salicylic acid was used as a control group. The test was repeated 3 times and the average was taken.

[0124]

[0125] Blank group: H2O replaced the absorbance value of the Auricularia auriculariae polysaccharide sample; sample group: the absorbance value of the Auricularia auriculariae polysaccharide sample; control group: H2O replaced the absorbance value of H2O2.

[0126] 2. Results and Analysis

[0127] 2.1 Establishment of glucose standard curve

[0128] The glucose standard curve established by the phenol-sulfuric acid method is as follows: Figure 1 As shown, the regression equation y = 0.0094x + 0.0065, the equation's fit R 2 =0.9955, and the linear relationship is satisfied within 0-60 mg / L. The concentration of Auricularia auricula polysaccharide is obtained according to the linear regression equation, and then the yield of Auricularia auricula polysaccharide is obtained.

[0129] 2.2 Single factor test results and analysis

[0130] 2.2.1 Effect of solid-liquid ratio on yield and biological activity of fungus polysaccharides

[0131] like Figure 2 , Figure 3 , Figure 4 As shown. Figure 2 It can be seen that the solid-liquid ratio has an effect on the yield of auricularia auricula polysaccharides extracted by complex enzyme-assisted subcritical water extraction. When the solid-liquid ratio is between 1:80 (g / mL) and 1:120 (g / mL), the yield increases with the increase of the solid-liquid ratio; and reaches a peak value of 11.44% at 1:120 (mL / g). This may be because the contact area between the solvent and the defatted auricularia auricula powder increases with the increase of the solid-liquid ratio, the dissolution amount increases, and the yield is improved. When the solid-liquid ratio is greater than 1:120 (g / mL), the yield decreases, which may be because the larger liquid-to-solid ratio dilutes the concentration of auricularia auricula polysaccharides in the extract.

[0132] Depend on Figure 3 It can be seen that the DPPH· free radical scavenging rate of the auricularia auricula polysaccharide extracted with different solid-liquid ratios is different, and decreases with the increase of the dilution of the crude auricularia auricula polysaccharide. When the dilution factor is 1, the DPPH· free radical scavenging rate is the highest, all above 10%. Among them, at the level of 1:150 (g / mL), the scavenging rate of the auricularia auricula polysaccharide solution is much higher than other levels, reaching 20.24%. At 1:120 (g / mL), the scavenging rate of the auricularia auricula polysaccharide solution with a dilution factor of 8 is lower than other levels as a whole.

[0133] Depend on Figure 4 It can be seen that the scavenging rates of hydroxyl radicals of Auricularia auricula polysaccharides extracted with different solid-liquid ratios are different, and decrease with the increase of the dilution of crude Auricularia auricula polysaccharides. When the dilution ratio is 1, the scavenging rates of hydroxyl radicals are all high, all above 5%, among which the scavenging rate of Auricularia auricula polysaccharide solution with a solid-liquid ratio of 1:140 (g / mL) is higher than other levels, reaching 26.37%. In addition, when the solid-liquid ratio is 1:80 (g / mL), the scavenging rates of Auricularia auricula polysaccharide solutions with different dilution ratios are lower than other levels; when the solid-liquid ratio is 1:140 (g / mL), the scavenging rates of Auricularia auricula polysaccharide solutions with different dilution ratios are higher than other levels.

[0134] 2.2.2 Effect of extraction temperature on the yield and biological activity of Auricularia auricularia polysaccharides

[0135] like Figure 5 , Figure 6 , Figure 7 As shown by Figure 5 It can be seen that the extraction temperature has a certain effect on the yield of polysaccharides extracted from fungus by complex enzyme-assisted subcritical water extraction. When the temperature is between 105℃ and 145℃, the yield of polysaccharides from fungus increases with the increase of temperature and reaches a peak value of 12.03% at 145℃. This may be because the polarity of water decreases with the increase of temperature, making polysaccharides from fungus easier to extract, while reducing the viscosity of the fluid, making it easier for the fluid to penetrate into the extract, and increasing the yield of polysaccharides from fungus; when the temperature is higher than 145℃, the yield decreases, which may be because the increase in temperature causes the movement of water molecules to intensify, thereby strengthening its diffusion properties and significantly improving the permeability and solubility of polysaccharides from fungus, which is conducive to the precipitation of polysaccharides from fungus, thereby increasing the yield of polysaccharides from fungus. However, too high a temperature will accelerate the decomposition rate of polysaccharides from fungus and destroy the structural integrity of polysaccharides from fungus, which will cause a decrease in the yield of polysaccharides from fungus.

[0136] Depend on Figure 6 It can be seen that there are differences in the DPPH·radical scavenging rates of wood ear polysaccharides extracted at different extraction temperatures, and they decrease with the increase of the dilution of crude wood ear polysaccharides. When the dilution multiple is greater than 2 times, the DPPH·radical scavenging rate shows an obvious downward trend. When the dilution multiple is 1 times, the DPPH·radical scavenging rates are all high, all above 10%. Among them, at 185°C, the scavenging rate of wood ear polysaccharide solution is higher than other levels, reaching 50.16%. At 105°C, the scavenging rate of wood ear polysaccharide solution diluted 8 times is lower than other levels. At a temperature of 185°C, the scavenging rates of wood ear polysaccharide solutions of different dilution multiples are better than the other four groups as a whole.

[0137] Depend on Figure 7 It can be seen that the scavenging rate of hydroxyl radicals of Auricularia auricula polysaccharides extracted at different extraction temperatures is different, and decreases with the increase of the dilution of crude Auricularia auricula polysaccharides. When the dilution ratio is greater than 4 times, the scavenging rate of hydroxyl radicals decreases significantly. When the dilution ratio is 1 times, the scavenging rate of hydroxyl radicals is relatively high, all above 10%. When the extraction temperature is 105°C, the scavenging rate of Auricularia auricula polysaccharide solution is higher than other levels, reaching 33.26%. In addition, when the temperature is 185°C, the scavenging rates of Auricularia auricula polysaccharide solutions of different dilution ratios are lower than other levels.

[0138] 2.2.3 Effect of extraction time on the yield and biological activity of Auricularia auricularia polysaccharides

[0139] like Figure 8 , Fig. 9 , Fig.10 As shown by Figure 8 It can be seen that the extraction time has an effect on the yield of polysaccharides extracted from wood ear by complex enzyme-assisted subcritical water extraction. When the time is between 15 and 25 minutes, the yield of polysaccharides from wood ear increases with the increase of extraction time, reaching a peak at 25 minutes, with a yield of 18%. This may be because subcritical water has a strong solubility at high temperature and can complete the extraction in a short time. It can also be seen that when the extraction time is greater than 25 minutes, the yield shows a downward trend, indicating that too long a time will not increase the yield of polysaccharides from wood ear. The extraction environment may reach solid-liquid equilibrium at around 25 minutes of extraction. As time goes on, polysaccharides from wood ear degrade, resulting in a decrease in yield.

[0140] Depend on Fig. 9 It can be seen that the DPPH· free radical scavenging rate of the auricularia polysaccharide extracted at different extraction times is different, and decreases with the increase of the dilution of the crude auricularia polysaccharide. When the dilution multiple is 1, the DPPH· free radical scavenging rate is relatively high, all above 20%. Among them, at 20 minutes, the scavenging rate of the auricularia polysaccharide solution is higher than other levels, reaching 56.32%. At 15 minutes, the scavenging rates of the auricularia polysaccharide solutions at different dilution multiples are lower than other levels.

[0141] Depend on Fig.10 It can be seen that the scavenging rate of hydroxyl radicals of Auricularia auricula polysaccharides extracted at different extraction times is different, and decreases with the increase of the dilution of crude Auricularia auricula polysaccharides. When the dilution factor is 1, the scavenging rate of hydroxyl radicals is the highest, all above 20%. When the extraction time is 35min, the scavenging rate of Auricularia auricula polysaccharide solution is higher than other levels, reaching 37.04%; at 25min, the scavenging rate of Auricularia auricula polysaccharide solution with a dilution factor of 8 is lower than other levels as a whole.

[0142] 2.2.4 Effect of extraction speed on the yield and biological activity of Auricularia auricularia polysaccharides

[0143] like Fig.11 , Fig.12 , Fig.13 As shown by Fig.11 It can be seen that different rotation speeds have an effect on the yield of polysaccharides extracted from fungus by complex enzyme-assisted subcritical water extraction. When the rotation speed is between 50 and 140 r / min, the yield of polysaccharides from fungus increases with the increase of the rotation speed, reaching a peak value of 12.91% at a rotation speed of 140 r / min. This is because the appropriate rotation speed can accelerate the mixing and dissolution of the material, thereby increasing the yield of polysaccharides from fungus; when the rotation speed is greater than 140 r / min, a downward trend occurs, which is due to the increase in the disturbance of the liquid surface, which causes the yield of polysaccharides from fungus to decrease to a certain extent.

[0144] Depend on Fig.12It can be seen that the DPPH· free radical scavenging rate of the auricularia auricula polysaccharide extracted at different speeds is different, and decreases with the increase of the dilution of the crude auricularia auricula polysaccharide. When the dilution ratio is 1, the DPPH· free radical scavenging rate increases, all above 40%. When the speed is 170r / min, the scavenging rate of the auricularia auricula polysaccharide solution is higher than other levels, reaching 49.77%.

[0145] Depend on Fig.13 It can be seen that the scavenging rate of hydroxyl radicals of Auricularia auricula polysaccharides extracted at different speeds is different, and decreases with the increase of the dilution of crude Auricularia auricula polysaccharides. When the dilution ratio is 1, the scavenging rate of hydroxyl radicals increases, all above 15%. Among them, when the speed is 50r / min, the scavenging rate of Auricularia auricula polysaccharide solution is higher than other levels, reaching 34.69%; at 110r / min, the scavenging rates of Auricularia auricula polysaccharide solutions at different dilution ratios are lower than other levels.

[0146] 3. Orthogonal test to optimize the extraction process of fungus polysaccharides

[0147] In order to optimize the extraction process, an orthogonal experiment was designed. Three levels were selected for each of the four factors: solid-liquid ratio (A), extraction temperature (B), extraction time (C) and rotation speed (D). The orthogonal experiment was conducted with the yield of Auricularia auricula polysaccharide, DPPH scavenging rate and hydroxyl radical scavenging rate as indicators. The results are shown in Tables 2 and 3.

[0148] Table 2 Orthogonal test factor level table

[0149]

[0150] Table 3 Orthogonal test factor level table

[0151]

[0152]

[0153] In Tables 2 and 3, A1 is 1:120 (g / mL), A2 is 1:140 (g / mL), A3 is 1:160 (g / mL), B1 is 20 min, B2 is 25 min, B3 is 30 min, C1 is 145°C, C2 is 165°C, C3 is 185°C, D1 is 110 r / min, D2 is 140 r / min, and D3 is 170 r / min.

[0154] The orthogonal experimental design and results are shown in Table 2 and Table 3. As can be seen from the table, (1) the range of each factor's rate difference R is B>D>C>A, that is, B has the greatest influence, followed by D and C, and finally A, and the optimal combination obtained by the experiment is A1B3C2D3; (2) the range of each factor's DPPH clearance rate difference R is D>B>A>C, that is, D has the greatest influence, followed by B and A, and finally C, and the optimal combination obtained by the experiment is A2B3C1D3; (3) the range of each factor's hydroxyl radical clearance rate difference R is A>B>D>C, that is, A has the greatest influence, followed by B and D, and finally C, and the optimal combination obtained by the experiment is A2B2C1D2.

[0155] The comprehensive score is calculated by the yield accounting for 60%, DPPH· scavenging rate and hydroxyl radical scavenging rate accounting for 20% each. The range R of the comprehensive score of each factor is B>D>C>A, that is, B has the greatest influence, followed by D and C, and finally A. The optimal combination is A1B2C2D2, that is, the solid-liquid ratio is 1:120 (g / mL), the extraction time is 25min, the extraction temperature is 165℃, and the rotation speed is 140r / min. Under this condition, the verification experiment was carried out, and the yield of fungus polysaccharide was 24.45%, the DPPH· free radical scavenging rate was 21.29%, and the hydroxyl radical scavenging rate was 81.62%.

[0156] 4. Comparative study of compound enzyme-assisted subcritical water extraction and traditional hot water extraction

[0157] Auricularia auricularia polysaccharides were extracted according to the optimal process example 22 and compared with the comparative example 1. The results are shown in Table 4.

[0158] Table 4 Comparative study of compound enzyme-assisted subcritical water extraction and traditional hot water extraction

[0159]

[0160] As shown in Table 4, all the data measured under the optimal conditions of the complex enzyme-assisted subcritical water extraction method are higher than those of the hot water extraction method. Compared with the hot water extraction method, the extraction time was shortened from 4h to 20min, the yield increased from 16.09% to 24.45%, the DPPH· free radical scavenging rate increased from 9.97% to 21.29%, and the hydroxyl free radical scavenging rate increased from 14.26% to 81.62%. It can be seen that the subcritical water method for extracting Auricularia auricula polysaccharides has strong feasibility and practicality.

[0161] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and variations. The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and their protection scope is not limited thereto. Equivalent substitutions or changes made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention, and the protection scope of the present invention shall be subject to the claims.

Claims

1. A process for extracting fungus polysaccharides by using a complex enzyme-assisted subcritical water extraction method, characterized in that: The steps include: defatting the fungus powder to obtain defatted fungus powder; Mixing defatted wood ear powder with water, then adding cellulose pectin complex enzyme thereto and performing enzymolysis to obtain an enzymatically hydrolyzed wood ear solution; The enzymatically hydrolyzed fungus solution is subjected to subcritical extraction to obtain fungus polysaccharide; The subcritical extraction conditions are: extraction at 105-185° C. and a rotation speed of 50-170 r / min for 15-35 minutes.

2. The process for extracting fungus polysaccharides by a complex enzyme-assisted subcritical water extraction method according to claim 1, characterized in that: The subcritical extraction conditions were: extraction at 165°C and a rotation speed of 140 r / min for 25 min.

3. The process for extracting fungus polysaccharides by a complex enzyme-assisted subcritical water extraction method according to claim 1, characterized in that: During subcritical extraction, subcritical extraction is performed with the assistance of a pressure of 1-2 MPa.

4. The process for extracting fungus polysaccharides by a complex enzyme-assisted subcritical water extraction method according to claim 1, characterized in that: The mass volume ratio of defatted wood ear powder to water is 1g:80-160mL.

5. The process for extracting fungus polysaccharides by a complex enzyme-assisted subcritical water extraction method according to claim 4, characterized in that: The mass volume ratio of defatted wood ear powder to water is 1g:120mL.

6. The process for extracting fungus polysaccharides by a complex enzyme-assisted subcritical water extraction method according to claim 1, characterized in that: The mass ratio of defatted wood ear powder to cellulose pectin complex enzyme is 1:40-50.

7. The process for extracting fungus polysaccharides by a complex enzyme-assisted subcritical water extraction method according to claim 1, characterized in that: The enzymatic hydrolysis time is 1.5h-2h.

8. The process for extracting fungus polysaccharides by a complex enzyme-assisted subcritical water extraction method according to claim 1, characterized in that: petroleum ether and fungus powder are mixed and allowed to stand, and the fungus powder is defatted by using petroleum ether.

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