Method for regulating and controlling polymerization degree of tannin in diaphragma juglandis based on composite enzymolysis technology

Abstract

The invention discloses a method for regulating and controlling the polymerization degree of tannin in diaphragma juglandis based on a composite enzymolysis technology, and relates to the technical field of plant active ingredient processing. Comprising the following steps: (1) raw material pretreatment: freeze-drying, crushing and sieving dried diaphragma juglandis, and adding a pretreatment solution for pretreatment; (2) step enzymolysis: sequentially carrying out cellulase treatment, tannase and beta-glucosidase compounding treatment and peroxidase treatment; and (3) inactivation and purification: carrying out heat treatment to terminate enzyme activity, and then carrying out gradient elution and purification by adopting macroporous resin. The invention provides a method for regulating and controlling the polymerization degree of walnut diaphragma juglandis tannin on the basis of a composite enzymolysis technology, which can reduce the polymerization degree under a mild condition and maintain relatively high biological activity at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant active ingredient processing, and particularly relates to a method for regulating the polymerization degree of tannins in walnut septum based on a compound enzymolysis technology. Background Art

[0002] As the main by-product in the walnut processing process, walnut septum has long been regarded as agricultural waste, and its utilization rate is less than 5%. According to statistics, more than 20 million tons of walnut septum are produced globally every year, and more than 90% of them are directly incinerated or landfilled, which not only causes waste of resources but also brings environmental pressure. However, recent studies have found that walnut septum contains up to 12 - 18% of condensed tannins, and such polyphenolic compounds have significant antioxidant, anti-inflammatory and hypoglycemic activities, showing important application values in the fields of functional foods, pharmaceutical excipients, etc. However, limited by its special structural characteristics, there are still major technical bottlenecks in its industrial application.

[0003] When the polymerization degree of condensed tannins exceeds 5, the absorption rate of tannins by intestinal epithelial cells drops sharply to less than 15%, which is mainly due to the following reasons: macromolecular tannins cannot passively diffuse through the intestinal mucosa; form irreversible complexes with digestive enzymes, inhibiting enzyme activity; combine with dietary proteins to form insoluble precipitates, etc. More seriously, high-polymerization-degree tannins (DP≥8) can stimulate the intestine to produce excessive mucin, resulting in adverse reactions such as abdominal distension and diarrhea. These characteristics seriously restrict its application in oral preparations, and most of the existing commercially available walnut septum extracts are used as industrial tanning agents, and the added value is less than 1 / 3 of that of traditional medicinal plant extracts. Although the traditional acid hydrolysis method can partially reduce the polymerization degree of tannins, strong acid conditions are likely to damage the active structure (such as the ring opening of flavan-3-ol units), resulting in the loss of antioxidant activity, and there is also a risk of acid solution residue.

[0004] Therefore, in order to solve the above technical problems, a new technical solution is needed to solve this technical problem, especially a method for regulating the polymerization degree of tannins in walnut septum based on a compound enzymolysis technology. Summary of the Invention

[0005] The purpose of the present invention is to solve the technical problems of the low utilization rate of existing walnut septum and the difficulty in absorbing and utilizing condensed tannins therein, and provides a method for regulating the polymerization degree of tannins in walnut septum based on a compound enzymolysis technology.

[0006] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a method for regulating the polymerization degree of tannins in walnut septum based on a compound enzymolysis technology, comprising the following steps:

[0008] (1) Raw material pretreatment: After harvesting walnut green fruits, remove the green peel, take the dried walnut septum, freeze-dry and pulverize it to 40 to 80 mesh, add a 35%-45% ethanol solution containing 1.5%-2% citric acid according to a solid-liquid ratio of 1:12 - 1:15, and perform ultrasonic treatment at 40 to 50 °C for 15 - 25 minutes. After solid-liquid separation, retain the filter residue. In this step, a citric acid / ethanol / water system (0.1 - 0.2M:35 - 45%:v / v, pH 4.2 - 4.8) is used for ultrasonic-assisted treatment at 40 - 50 °C (40 kHz, 200 W) to break the lignin-cellulose hydrogen bond network (verified by the peak shift of 2920 cm-1 in FTIR).

[0009] In the pretreatment, the pretreatment conditions are 1.5 - 2.5% citric acid and ultrasonic treatment at 40 kHz, which can chelate metal ions and destroy the lignin-cellulose cross-linked structure (shown by the peak shift of 2920 cm-1 in FTIR), while maintaining the system pH at 4.2 - 4.8 and an ultrasonic frequency not exceeding 45 kHz to protect the phenolic hydroxyl groups of tannins from oxidation.

[0010] (2) Stepwise enzymatic hydrolysis: First stage: Use cellulase from Trichoderma reesei with an activity of 0.4 - 0.6 U / g and stir at 50 ± 2 °C for 45 - 75 minutes; Second stage: Use tannase with an activity of 1.0 - 1.5 U / g and β-glucosidase with an activity of 0.7 - 0.9 U / g and compound them in a molar ratio of 1:0.6 - 0.8, and treat at 38 - 42 °C for 1.5 - 2.5 hours; Third stage: Add peroxidase with an activity of 0.5 - 0.8 U / g and 0.05 - 0.1 mM H2O2, and treat at 45 - 50 °C for 30 - 50 minutes.

[0011] (3) Inactivation and purification: Use AB-8 or XDA-6 type macroporous resin for gradient elution, and its elution gradient is 30% → 50% → 70% ethanol solution; control the elution flow rate at 1.0 - 1.5 BV / h.

[0012] Preferably, in step (2), pH adjustment is required between the cellulase treatment step and the enzyme compounding treatment: After the first stage, use 0.1M sodium citrate buffer to adjust the pH to 5.0 - 5.5. Since citric acid has been used in step (1), using sodium citrate to adjust the pH will not introduce new anions. At the same time, because citrate can chelate metal ions such as Fe2+ and Cu2+ in plant materials that catalyze phenolic oxidation reactions, it can prevent component oxidation; citric acid is a natural organic acid that is non-toxic and meets safety standards; sodium citrate, as an alkaline salt, is suitable for the operation of increasing the pH between the cellulase treatment step and the enzyme compounding treatment.

[0013] Preferably, in step (1), the pretreatment solution is an ethanol aqueous solution containing 2% citric acid, wherein the volume concentration of ethanol is 40%. Ultrasonic-assisted treatment is carried out at a material-liquid ratio of 1:12, and ultrasonic treatment is carried out at 45 °C for 20 minutes, and the ultrasonic frequency is controlled at 40 kHz.

[0014] Preferably, in step (2), the activities of tannase and β-glucosidase in the enzyme complex treatment are 1.2 U / g of raw material and 0.8 U / g of raw material respectively, and the treatment is carried out at 40 °C for 120 minutes. In the second stage of the stepwise enzymatic hydrolysis, the selection of tannase and β-glucosidase enables the cooperative hydrolysis of ester bonds and glycosidic bonds.

[0015] Preferably, in step (2), the activity of cellulase in the cellulase treatment is 0.5 U / g of raw material, and the treatment is carried out at 50 °C for 60 minutes. In the first stage of the stepwise enzymatic hydrolysis, cellulase has the optimal activity at 50 °C, and cell wall breaking is insufficient at lower or higher temperatures.

[0016] Preferably, in step (2), the activity of peroxidase in the peroxidase treatment step is 0.6 U / g, and it is treated with 0.08 mM H2O2 at 45-50 °C for 30-50 minutes. In the third stage, peroxidase needs to act synergistically with H2O2, and the concentration of H2O2 is lower than 0.1 mM to avoid overoxidation and damage to the tannin phenolic hydroxyl structure.

[0017] Preferably, in step (1), the conditions for freeze-drying treatment are -50 °C and 0.2 Mpa.

[0018] Preferably, in step (1), the particle size of the sieving is 55-65 mesh.

[0019] Preferably, in step (1), the particle size of the sieving is 60 mesh, which can enable the lignin-cellulose cross-linked structure of the persimmon calyx to contact the pretreatment solution more fully, and enable the persimmon calyx powder to contact the enzyme solution more fully, and can significantly improve the effect of subsequent steps. At the same time, the fineness of 60 mesh is not difficult to achieve, and higher mesh numbers will increase the grinding time.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) Through the synergistic effect of a composite enzyme system (cellulase breaks the matrix barrier + tannase / β-glucosidase targets and cleaves the tannin glycosidic bond to remove side chain groups, introducing peroxidase (Peroxidase), which generates hydroxyl radicals (·OH) under the mediation of H2O2, specifically attacking the C-C bond of condensed tannins, thereby significantly reducing its degree of polymerization), this step forms a three-level synergistic degradation system with cellulase and tannase / β-glucosidase, breaking through the limitations of the existing enzymatic hydrolysis process, achieving a reduction in the degree of polymerization under mild conditions while retaining high biological activity.

[0022] (2) The low-degree-of-polymerization persimmon calyx tannin prepared by the present invention, as a natural component of walnuts, has a lower impact on digestibility and can better exert its antioxidant and anti-inflammatory biological functions compared to condensed tannins with a larger degree of polymerization and a larger mass.

[0023] (3) As a by-product or even waste in the walnut industry, using persimmon calyx as a raw material for extracting bioactive substances can improve the utilization rate of the whole walnut fruit, expand the market application prospects, give full play to its resource value, and conform to the concept of modern sustainable development.

[0024] (4) Compared with the traditional acid hydrolysis method, the present invention has no problem of acid residue, improves the safety of the finished product, and is suitable for promotion to different product raw materials for further development. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 are the high-degree-of-polymerization tannin content and DPPH scavenging rate of the samples obtained in each example and comparative example of the present invention.

[0026] Figure 2 is the effect of different cellulase hydrolysis times of the present invention on the extraction rate and activity. DETAILED DESCRIPTION OF THE INVENTION

[0027] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following specific embodiments are used to describe the specific implementation manners of the present invention in detail.

[0028] Many specific details are set forth in the following description to facilitate a thorough understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0029] Second, the "one embodiment" or "embodiment" referred to herein means a specific feature, structure, or characteristic that may be included in at least one implementation manner of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that are mutually exclusive of other embodiments.

[0030] Example 1: A Method for Regulating the Polymerization Degree of Tannins in Walnut Septum by Composite Enzymolysis Technique I

[0031] Raw material treatment: Take dry walnut septum, crush it to 60 mesh, add a 40% ethanol solution containing 2% citric acid according to a solid-liquid ratio of 1:12, and ultrasonically treat it at 45 °C (40 kHz) for 20 minutes. After solid-liquid separation, retain the filter residue.

[0032] Enzymolysis process: Add 0.5 U / g of cellulase and react with shaking at 50 °C for 60 minutes; adjust the pH to 5.0 (0.1 M sodium citrate buffer), add 1.2 U / g of tannase and 0.8 U / g of β-glucosidase, and react at 40 °C for 120 minutes; add 0.6 U / g of peroxidase and 0.08 mM H2O2, and treat at 45 °C for 40 minutes.

[0033] Inactivation and purification: After maintaining at 80 °C for 10 minutes, load onto an AB-8 resin column, elute successively with 3 BV of 30% ethanol and 5 BV of 50% ethanol, collect the 50% ethanol eluate, freeze-dry it, and then detect the content of condensed tannins with different polymerization degrees and the DPPH radical scavenging efficiency.

[0034] Example 2: A Method for Regulating the Polymerization Degree of Tannins in Walnut Septum by Composite Enzymolysis Technique II

[0035] Raw material treatment: Take dry walnut septum, crush it to 80 mesh, add a 45% ethanol solution containing 1.5% citric acid according to a solid-liquid ratio of 1:10, and ultrasonically treat it at 50 °C (45 kHz) for 15 minutes. After solid-liquid separation, retain the filter residue.

[0036] Enzymolysis process: Add 0.6 U / g of cellulase and react with shaking at 50 °C for 45 minutes; adjust the pH to 5.0 (0.1 M sodium citrate buffer), add 1.5 U / g of tannase and 0.7 U / g of β-glucosidase, and react at 40 °C for 90 minutes; add 0.4 U / g of peroxidase and 0.05 mM H2O2, and treat at 45 °C for 40 minutes.

[0037] Inactivation and purification: After maintaining at 80 °C for 10 minutes, load onto an AB-8 resin column, elute successively with 3 BV of 30% ethanol and 5 BV of 50% ethanol, collect the 50% ethanol eluate, freeze-dry it, and then detect the content of condensed tannins with different polymerization degrees and the DPPH radical scavenging efficiency.

[0038] Example 3: Method 3 for Regulating the Polymerization Degree of Tannins in Walnut Septum by Composite Enzymolysis Technology

[0039] Raw material treatment: Take dry walnut septum, crush it to 40 mesh, add 35% ethanol solution containing 2.5% citric acid according to the solid-liquid ratio of 1:15, and treat it by ultrasonic wave (35 kHz) at 40 °C for 15 minutes. After solid-liquid separation, retain the filter residue.

[0040] Enzymolysis process: Add 0.4 U / g of cellulase and react with shaking at 50 °C for 75 minutes; adjust the pH to 5.0 (0.1 M sodium citrate buffer solution), add 1.0 U / g of tannase and 0.9 U / g of β-glucosidase, and react at 40 °C for 150 minutes; add 1.0 U / g of peroxidase and 0.12 mM H2O2, and treat at 45 °C for 40 minutes.

[0041] Inactivation and purification: After maintaining at 80 °C for 10 minutes, load the sample onto an AB-8 resin column, and elute it successively with 3 BV of 30% ethanol and 5 BV of 50% ethanol. Collect the 50% ethanol eluate, freeze-dry it, and then detect the content of condensed tannins with different polymerization degrees and the DPPH radical scavenging efficiency.

[0042] Among them, the determination method of the content of condensed tannins with different polymerization degrees is as follows:

[0043] Filter the inactivated and purified sample through a 0.45 μm organic filter membrane for HPLC analysis. Use tannin standard products (DP2-DP10) for polyphenol analysis, and calibrate the curve with procyanidin B2 standard products for quantification. Chromatographic conditions: Phenomenex Luna HILIC (150 mm × 4.6 mm, 3 μm); mobile phase A: acetonitrile: acetic acid (98:2, v / v), mobile phase B: methanol: water: acetic acid (95:3:2, v / v / v); gradient elution: 0-25 min, 100% A; 25-30 min, 55% A; 30-34 min, 100% A; flow rate: 1 mL / min; injection volume: detection wavelength: 316 nm; column temperature 30 °C. After obtaining the chromatographic analysis data of different samples, integrate the chromatographic peaks, and the content of the polymer is expressed in terms of procyanidin B2 equivalent (mg PE / g).

[0044] Among them, the determination method of the DPPH radical scavenging rate is as follows:

[0045] Take the purified freeze-dried sample and prepare a test solution with absolute ethanol at a concentration of 0.1 mg / mL. Accurately weigh 2.5 mg of DPPH reagent (1,1-diphenyl-2-picrylhydrazyl), and make up the volume to 50 mL with absolute ethanol to prepare a 0.1 mM DPPH radical solution, which is stored in the dark. Take 2 mL of the sample test solution and mix it with 2 mL of the DPPH solution, vortex for 10 seconds, and then react in the dark at 25 °C for 30 minutes. Use an equal volume of absolute ethanol to replace the sample solution as a blank control, and mix an equal volume of the DPPH solution with absolute ethanol as a background control. After the reaction, measure the absorbance value at a wavelength of 517 nm (using a UV-1800 type ultraviolet-visible spectrophotometer and a 1 cm quartz cuvette). Each group of experiments is measured in parallel 3 times, and then calculate the radical scavenging rate using this formula: Scavenging rate = (1 - (A_sample - A_background) / A_blank) × 100%.

[0046] Comparative Example 1: The necessity of citric acid in the pretreatment

[0047] This Comparative Example 1 is based on Example 1, and the difference from Example 1 is that: cancel the addition of citric acid in the pretreatment step, and only use a 40% ethanol solution for ultrasonic treatment.

[0048] Comparative Example 2: The necessity of ultrasonic treatment in the pretreatment

[0049] This Comparative Example 2 is based on Example 1, and the difference from Example 1 is that: cancel the ultrasonic treatment in the pretreatment step.

[0050] Comparative Example 3: The irreplaceability of cellulase in breaking the mechanism barrier

[0051] This Comparative Example 3 is based on Example 1, and the difference from Example 1 is that: cancel the cellulase treatment step.

[0052] Comparative Example 4: The irreplaceability of tannase in the specific cleavage of tannins

[0053] This Comparative Example 4 is based on Example 1, and the difference from Example 1 is that: cancel the addition of tannase during the enzymatic hydrolysis process.

[0054] Comparative Example 5: The irreplaceability of β-glucosidase in the specific cleavage of tannins

[0055] This Comparative Example 5 is based on Example 1, and the difference from Example 1 is that: cancel the addition of β-glucosidase during the enzymatic hydrolysis process.

[0056] Comparative Example 6: The logical necessity of the stepwise enzymatic hydrolysis sequence for matrix breaking and tannin release

[0057] This Comparative Example 6 is based on Example 1, and the difference from Example 1 is that: cancel the addition of β-glucosidase during the enzymatic hydrolysis process.

[0058] Comparative Example 7: Necessity of peroxidase

[0059] Based on Example 4, the peroxidase treatment in the third stage was cancelled, and the other conditions were the same.

[0060] Comparative Example 8: Effects of different cellulase hydrolysis times on the degree of polymerization and activity of tannins

[0061] This Comparative Example 7 is based on Example 1. The difference from Example 1 is that the cellulase hydrolysis times were set to 30, 40, 50, 70, 80, and 90 minutes respectively.

[0062] Comparative Example 9: Effects of different compound enzyme hydrolysis temperatures on the degree of polymerization and activity of tannins

[0063] This Comparative Example 8 is based on Example 1. The difference from Example 1 is that the cellulase hydrolysis times were set to 35, 38, 42, 45, and 48 minutes respectively.

[0064] Comparative Example 10: Effects of different ultrasonic pretreatment times on tannin release and activity

[0065] This Comparative Example 9 is based on Example 1. The difference from Example 1 is that the ultrasonic treatment times were set to 5, 10, 15, 25, and 30 minutes respectively.

[0066] Table 1 High-polymerization-degree tannin content and DPPH scavenging rate of samples obtained in each example and comparative example

[0067]

[0068] Table 2 Effects of different cellulase hydrolysis times on tannin extraction rate and activity

[0069]

[0070]

[0071] Table 3 Effects of different compound enzyme hydrolysis temperatures on the degree of polymerization and activity of tannins

[0072]

[0073] Table 4 Effects of different ultrasonic pretreatment times on tannin release and activity

[0074]

[0075]

[0076] According to the data analysis in Table 1, it can be seen that:

[0077] (1) In Comparative Example 1, citric acid was missing in the pretreatment step, resulting in insufficient pretreatment and a decrease in the tannin release efficiency.

[0078] (2) In Comparative Example 2, ultrasonic treatment was missing in the pretreatment step, resulting in insufficient pretreatment and a decrease in the tannin release efficiency.

[0079] (3) In Comparative Example 3, cellulase treatment was missing in the stepwise enzymatic hydrolysis process, and the cell wall breaking could not be well completed, resulting in a decrease in the tannin release rate.

[0080] (4) In Comparative Example 4, tannase was missing in the complex enzyme in the stepwise enzymatic hydrolysis process, and the hydrolyzable tannins in the mixed components were not degraded, greatly affecting the accessibility to the condensed tannin substrate in the subsequent steps.

[0081] (5) In Comparative Example 5, β-glucosidase was missing in the complex enzyme in the stepwise enzymatic hydrolysis process, and it did not form a cellulolytic enzyme system with cellulase in the first stage to further break the cell wall, resulting in incomplete tannin release.

[0082] (6) In Comparative Example 6, the order of the stepwise enzymatic hydrolysis process was reversed, the release rate of condensed tannins in the tissue was low, and the accessibility of subsequent enzymes was greatly reduced, resulting in the inability to effectively reduce the degree of polymerization.

[0083] (7) In the series of Comparative Example 7, as the enzymatic hydrolysis time increased, peroxidase was missing, and it could not react with H2O2 to oxidize the resorcinol ring of the flavan-3-ol unit in the condensed tannins to decrease the degree of polymerization.

[0084] It can be seen from the data analysis in Table 2 that:

[0085] (8) In the series of Comparative Example 8, as the cellulase hydrolysis time increased, the tannin release efficiency also increased. However, after the enzymatic hydrolysis time was greater than 60 min, the extraction rate of tannins tended to be stable, and the tannin release was roughly completed.

[0086] It can be seen from the data analysis in Table 3 that:

[0087] (9) In the series of Comparative Example 9, as the complex enzyme hydrolysis temperature increased, the decomposition efficiency of condensed tannins also increased. However, after the enzymatic hydrolysis temperature was greater than 40 min, the activity of the complex enzyme decreased with the increase in temperature.

[0088] It can be seen from the data analysis in Table 4 that:

[0089] (10) In the series of Comparative Example 10, as the ultrasonic time increased, the energy of the cavitation effect increased, and the tannin extraction rate increased. However, excessive ultrasonic time would cause the hydroxyl radicals generated by ultrasound to oxidize the tannins.

[0090] In Tables 1 to 4, it is obtained that Example 1 has the optimal experimental parameters. Among them, the enzymatic hydrolysis temperature and the concentration of H2O2 in the three steps of gradient enzymatic hydrolysis are key parameters. The optimal enzymatic hydrolysis temperatures (50 °C, 40 °C, 45 °C) in the three steps ensure that each of the four enzymes plays its role: tannase can degrade the hydrolyzable tannin component as a pre-step; cellulase and β-glucosidase form a cellulolytic enzyme system to further break the cell wall, and at the same time also degrade some of the glycosyl groups in the side chain and the sugar components in the mixture as a pre-step, improving the accessibility of peroxidase to the substrate condensed tannin; peroxidase catalyzes H2O2 to release active oxygen, oxidizing the resorcinol ring of the flavan-3-ol unit in condensed tannin to reduce its degree of polymerization. The concentration of H2O2 is selected as 0.08 mM. If the concentration is too low, it is difficult to provide sufficient active oxygen to break the inter-unit linkage bonds of condensed tannin. If the concentration is too high, it will damage the groups outside the target group in the substrate and reduce its biological activity.

[0091] The present invention provides a method for obtaining condensed tannins with a low degree of polymerization from walnut septum powder by subjecting the walnut septum to pulverization as a raw material, pretreatment, gradient enzymatic hydrolysis, and dynamic purification. The method for reducing the degree of polymerization of tannins in walnut septum of the present invention is green without reagent residue and does not damage the antioxidant active components of walnut septum, providing a new technology for the utilization rate of walnut septum and having a good market prospect.

[0092] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Those of ordinary skill in the relevant technical fields can also make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention. The patent protection scope of the present invention shall be defined by the claims.

Claims

1. A method for regulating the polymerization degree of tannins in walnut septum based on a composite enzymatic hydrolysis technology, characterized in that, It includes the following steps: (1) Raw material pretreatment: Take dry walnut septum, freeze-dry and crush it to 40 to 80 mesh, add an ethanol solution containing 1.5%-2% citric acid with a solid-liquid ratio of 1:12 - 1:15, and perform ultrasonic treatment at 40 to 50 °C for 15 - 25 minutes. After solid-liquid separation, retain the filter residue; (2) Stepwise enzymatic hydrolysis: First stage: Use cellulase with an activity of 0.4 - 0.6 U / g and stir at 50 ± 2 °C for 45 - 75 minutes; Second stage: Use tannase with an activity of 1.0 - 1.5 U / g and β-glucosidase with an activity of 0.7 - 0.9 U / g and compound them at a molar ratio of 1:0.6 - 0.8, and treat at 38 - 42 °C for 1.5 - 2.5 hours; Third stage: Add peroxidase with an activity of 0.5 - 0.8 U / g and 0.05 - 0.1 mM H2O2, and treat at 45 - 50 °C for 30 - 50 minutes; (3) Inactivation and purification: Use macroporous resin of type AB-8 or XDA-6 for gradient elution, and its elution gradient is 30% → 50% → 70% ethanol solution; control the elution flow rate at 1.0 - 1.5 BV / h.

2. The method according to claim 1, wherein In step (2), pH adjustment is required between the cellulase treatment step and the enzyme compounding treatment: After the first stage, use 0.1M sodium citrate buffer to adjust the pH to 5.0 - 5.

5.

3. In the method according to claim 1, wherein In step (1), the pretreatment solution is: an ethanol aqueous solution containing 2% citric acid, where the volume concentration of ethanol is 40%, perform ultrasonic-assisted treatment at a solid-liquid ratio of 1:12, and perform ultrasonic treatment at 45 °C for 20 minutes, and control the ultrasonic frequency at 40 kHz.

4. In the method according to claim 1, characterized in that, In step (2), the activities of tannase and β-glucosidase in the enzyme compounding treatment are 1.2 U / g of raw material and 0.8 U / g of raw material respectively, and treat at 40 °C for 120 minutes.

5. In the method according to claim 1, wherein In step (2), the activity of cellulase in the cellulase treatment is 0.5 U / g of raw material, and treat at 50 °C for 60 minutes.

6. In the method according to claim 1, wherein In step (2), the activity of peroxidase in the peroxidase treatment step is 0.6 U / g, and treat with 0.08 mM H2O2 at 45 - 50 °C for 30 - 50 minutes.

7. In the method according to claim 1, it is characterized in that, In step (1), the conditions for freeze-drying treatment are -50 °C and 0.2 Mpa.

8. In the method according to claim 1, wherein In step (1), the particle size of sieving is 55 - 65 mesh.

9. In the method according to claim 2, wherein, In step (1), the particle size of sieving is 60 mesh.

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