Staged extraction method of tremella aurantialba polysaccharide based on hot water extraction and ultrafiltration

Abstract

The invention relates to the technical field of extraction, and particularly discloses a tremella aurantialba polysaccharide segmented extraction method based on hot water extraction and ultrafiltration, which comprises the following steps: S1, after tremella aurantialba sporocarp is crushed, low-temperature plasma treatment is adopted, and a composite enzymatic hydrolysate containing 0.5-1.5% of cellulase and 0.3-1% of pectinase is added; s2, adding water into the material after enzymolysis according to the ratio of 1: 20-1: 40, and extracting in two stages; s3, leach liquor sequentially passes through a 100kDa ultrafiltration membrane system, a 30kDa ultrafiltration membrane system and a 10kDa ultrafiltration membrane system which are connected in series; s4, respectively dissolving the F1-F3 components, loading the dissolved F1-F3 components to a DEAE-SepharoseFastFlow anion column, carrying out gradient elution by adopting 00.5 M NaCl, and collecting four main peaks of P1-P4 according to the change of conductivity; s5, compounding the components and enhancing the effect; according to the method, through the synergistic effect of ultra-low-temperature plasma pretreatment and APG enhanced enzymolysis, the cell wall breaking efficiency is remarkably improved, efficient dissolution of tremella aurantialba polysaccharide is achieved under the mild condition, and the extraction period is greatly shortened.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of extraction, and particularly relates to a segmented extraction method of Auricularia auricula polysaccharide based on hot water extraction and ultrafiltration. BACKGROUND

[0002] The extraction of Auricularia auricula polysaccharide mainly depends on traditional physical and chemical methods and biological enzymatic hydrolysis technology. As the most commonly used method, hot water extraction usually adopts high-temperature treatment at 60-100 DEG C, combined with response surface optimization parameters, but the polysaccharide yield is generally low. In order to improve the efficiency, ultrasonic-assisted extraction is introduced, and the cell wall is broken through cavitation effect, so that the yield can be improved.

[0003] However, the hot water extraction method takes as long as 3-10 hours, and high temperature can easily damage the structure of heat-sensitive polysaccharide, resulting in a decrease in immune activity; although the ultrasonic method shortens the time, the mechanical shear force can degrade high molecular weight polysaccharide, affecting the probiotic activity. SUMMARY

[0004] The purpose of the present application is to provide a segmented extraction method of Auricularia auricula polysaccharide based on hot water extraction and ultrafiltration, so as to solve the problems in the background art.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0006] A segmented extraction method of Auricularia auricula polysaccharide based on hot water extraction and ultrafiltration, comprising the following steps:

[0007] S1, after crushing Auricularia auricula fruiting bodies, low-temperature plasma treatment (power 200-500 W, time 1-3 min) is adopted, and a complex enzymolysis solution containing 0.5-1.5% cellulase and 0.3-1% pectinase (w / w) is added, and enzymolysis is carried out at pH 4.5-5.5 and 45-55 DEG C for 1-2 h;

[0008] S2, after enzymolysis, the material is added with water at a ratio of 1:20-1:40 (g / mL), and is extracted in two stages:

[0009] First stage: 80-85 DEG C extraction for 1.5 h;

[0010] Second stage: 95-100 DEG C extraction for 2 h, and the filtrate is combined;

[0011] S3, the extraction solution is sequentially passed through a 100 kDa, 30 kDa and 10 kDa ultrafiltration membrane system in series, and >100 kDa component (F1), 30-100 kDa component (F2), 10-30 kDa component (F3) and <10 kDa permeate (reserved) are collected, respectively;

[0012] S4, after dissolving F1-F3 components respectively, loading into DEAE-Sepharose Fast Flow anion column, using 0.5M NaCl gradient elution, collecting P1-P4 four main peaks according to conductivity change;

[0013] S5, component compounding and synergism: compounding <10kDa permeate with P1-P4 peak according to mass ratio 1:5-1:10, adding 0.1% trehalose+0.05% mannitol (w / v) as freeze-drying protective agent, freeze-drying to obtain refined polysaccharide component.

[0014] Preferably, 0.1% surfactant alkyl glycoside is further added in the complex enzyme hydrolysate of step S1.

[0015] Preferably, the ultrafiltration membrane system of step S3 uses ceramic-organic hybrid membrane, and the operation pressure is 0.4-0.8MPa, and the temperature is 40-50℃.

[0016] Preferably, 0.02% nano-hydroxyapatite is further added in the freeze-drying protective agent of step S5.

[0017] Compared with the prior art, the beneficial effects of the present application are:

[0018] (1) Through the synergistic effect of low-temperature plasma pretreatment and APG intensified enzyme hydrolysis, the cell wall breaking efficiency is significantly improved, the efficient dissolution of Auricularia auricula polysaccharide is realized under mild conditions, the extraction period is greatly shortened, the multi-stage ultrafiltration membrane system separates polysaccharide components with different molecular weights, and the activity degradation caused by traditional hot reflux is avoided; the pH ladder elution strategy of ion column chromatography effectively improves the resolution of acidic polysaccharide.

[0019] (2) Through the small molecule polysaccharide reuse technology, the ultrafiltration permeate is converted into a freeze-drying protective matrix, which not only improves the product yield but also reduces the auxiliary material cost; the composite freeze-drying protective agent (containing nano-hydroxyapatite) maintains the polysaccharide space network through physical anchoring effect, ensuring long-term storage activity. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The flowchart of the present application. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0022] Example 1:

[0023] Please refer to Figure 1 A segmented extraction method of Chrysosporium merdarium polysaccharide based on hot water extraction and ultrafiltration is shown, comprising:

[0024] S1, raw material pretreatment: take 100g of dried Chrysosporium merdarium fruiting body, crush through a 60 mesh sieve, place in a low temperature plasma reaction chamber (power 300W, argon atmosphere), treat for 2min, add a complex enzymatic hydrolysis solution (containing 1% cellulase + 0.5% pectinase + 0.1% APG, pH 5.0), and enzymatically hydrolyze at 50°C for 50min;

[0025] S2, gradient hot water extraction: after enzymatic hydrolysis, the material is added with water at a ratio of 1:30 (g / mL) and extracted in two stages:

[0026] First stage: constant temperature stirring extraction at 82°C for 1.5h;

[0027] Second stage: reflux extraction at 98°C for 2h;

[0028] Combine the filtrate and centrifuge (8000rpm, 15min) to obtain 2.8L of clear extraction solution.

[0029] S3, multi-stage ultrafiltration fractionation: the extraction solution is subjected to a series connection of ultrafiltration systems (100kDa ceramic membrane→30kDa organic hybrid membrane→10kDa organic hybrid membrane), and the operating conditions are:

[0030] Membrane stage Pressure (MPa) Temperature (°C) Retained components 100 kDa 0.6 45 F1 component 30 kDa 0.5 45 F2 component 10 kDa 0.4 40 F3 component

[0031] Collect polysaccharides in each molecular weight range: F1 (>100kDa) 3.2g, F2 (30-100kDa) 4.1g, F3 (10-30kDa) 5.3g, and <10kDa permeate 1.8L;

[0032] S4, ion column chromatography purification: dissolve the F1 component in 0.01M Tris-HCl buffer (pH 7.0) and load a DEAE-Sepharose Fast Flow column (2.6×40cm);

[0033] Elution procedure:

[0034]

[0035]

[0036] Collect each main peak, dialyze and freeze-dry to obtain purified polysaccharides: P1 0.8g, P2 1.2g, P3 1.5g, and P4 0.9g;

[0037] S5, component compounding and freeze-drying: take <10 kDa permeate 200 mL, add P1-P4 components 4.4 g (mass ratio 1:8) altogether, add freeze-drying protectant (0.1% trehalose + 0.05% mannitol + 0.02% nano-hydroxyapatite), pre-freeze at -50°C for 12 h, freeze-dry to obtain compounded polysaccharide.

[0038] Experimental Example One:

[0039] Plasma-enzymolysis synergistic experiment, the experimental design is as follows:

[0040] Set 3 groups of parallel experiments (n=3), and the raw material is 100 g of dried golden ear powder:

[0041] Control group A: no pretreatment, direct enzymolysis (1% cellulase + 0.5% pectinase, pH 5.0, 50°C);

[0042] Control group B: only plasma treatment (300 W, 2 min), same enzymolysis condition;

[0043] Experimental group C: after plasma treatment (300 W, 2 min), add 0.1% APG in the enzymolysis solution;

[0044] After enzymolysis, add water at 1:30 (g / mL), extract at 82°C for 1.5 h, centrifuge (8000 rpm, 15 min) to take the supernatant, and determine the polysaccharide yield (%) by the phenol-sulfuric acid method (yield (%) = extracted polysaccharide mass / raw material mass x 100), and record the time required for enzymolysis to reach the maximum yield of 90%, and the specific data are as follows:

[0045] Group Polysaccharide yield (%) Enzymolysis time (min) Relative synergistic effect A: Untreated 8.7±0.3 120±5 - B: Plasma 12.1±0.5 90±4 +39.1％ C: Plasma + APG 15.3±0.6 50±3 +75.9％

[0046] As can be seen from the above, plasma treatment increases the cell wall permeability, the addition of APG further reduces the surface tension of the extraction system, the enzymolysis time is shortened, and the yield is higher than that of the control group A;

[0047] Compound freeze-drying protectant stability experiment, the experimental design is as follows:

[0048] Take 1.0 g of polysaccharide (30-100 kDa) of the same batch F2 component, and divide it into 3 groups:

[0049] Control group D: no protectant, direct freeze-drying;

[0050] Control group E: add 0.1% trehalose + 0.05% mannitol;

[0051] Experimental group F: add 0.1% trehalose + 0.05% mannitol + 0.02% nano-hydroxyapatite;

[0052] Each group was dissolved in 50 mL ultrapure water, pre-frozen at -50℃ for 12 h, freeze-dried (-55℃, 0.05 mbar, 24 h), and the recovery rate of polysaccharides was determined after re-dissolution: mass after freeze-drying / mass before freeze-drying x 100%;

[0053] Retention rate of β-glucan: reference Megazyme β-Glucan Assay Kit (GOPOD method);

[0054] The specific data are as follows:

[0055]

[0056] As can be seen from the above, the nano-hydroxyapatite anchors the polysaccharide chain through hydrogen bonds (FTIR shows that the O-H stretching vibration peak is enhanced), and the retention rate of β-glucan is increased to 96.3%, which is significantly higher than that of the control group D;

[0057] Structural stability characterization, detection method as follows:

[0058] Fourier infrared spectroscopy (FTIR): comparison of characteristic peak changes of polysaccharides before and after freeze-drying (3400 cm -1 O-H, 1650 cm -1 C=O);

[0059] Scanning electron microscopy (SEM): observation of the microstructure of freeze-dried products;

[0060] The specific data are as follows:

[0061] Group O-H / C=O peak area ratio SEM observation results Group D 2.1±0.2 Surface cracks, porous structure collapse Group F 3.4±0.3 Compact lamellar structure, uniform embedding of nanoparticles

[0062] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for segmental extraction of polysaccharides from Auricularia auricula based on hot water extraction and ultrafiltration, characterized in that, The method comprises the following steps: S1, after crushing the fruiting body of the gold ear, the fruiting body is treated by low-temperature plasma (power 200-500 W, time 1-3 min), and a complex enzymolysis solution containing 0.5-1.5% cellulase and 0.3-1% pectinase (w / w) is added, and the enzymolysis is carried out at pH 4.5-5.5 and 45-55 DEG C for 1-2 h; S2, after the enzymolysis, the material is added with water at a ratio of 1:20-1:40 (g / mL), and the extraction is carried out in two stages: the first stage: extraction at 80-85 DEG C for 1.5 h; the second stage: extraction at 95-100 DEG C for 2 h, and the filtrates are combined; S3, the extraction solution is sequentially passed through a 100 kDa, 30 kDa and 10 kDa ultrafiltration membrane system in series, and >100 kDa components (F1), 30-100 kDa components (F2), 10-30 kDa components (F3) and <10 kDa permeate (reserved) are collected respectively; S4, after the F1-F3 components are dissolved, they are loaded onto a DEAE-Sepharose Fast Flow anion column, and eluted by a 00.5M NaCl gradient, and four main peaks P1-P4 are collected according to the conductivity change; S5, component compounding and synergistic effect: the <10 kDa permeate and the P1-P4 peaks are compounded at a mass ratio of 1:5-1:10, 0.1% trehalose and 0.05% mannitol (w / v) are added as freeze-drying protectants, and refined polysaccharide components are obtained by freeze-drying.

2. The method according to claim 1, wherein the method is characterized by: The complex enzymolysis solution of step S1 further adds 0.1% surfactant alkyl glycoside.

3. The method according to claim 1, wherein the method is characterized by the following steps: The ultrafiltration membrane system of step S3 uses ceramic-organic hybrid membrane, and the operation pressure is 0.4-0.8 MPa and the temperature is 40-50 DEG C.

4. The method according to claim 1, wherein the method is characterized by: The freeze-drying protectant of step S5 further adds 0.02% nano-hydroxyapatite.

Images