Process for synergistically extracting polysaccharide from rape pollen by composite enzyme

Abstract

The present application relates to the technical field of plant polysaccharide extraction, in particular to a process for extracting rape pollen polysaccharide by using composite enzymes, wherein the rape pollen is pulsed airflow pulverized to 200-300 mesh, and then subjected to microwave-ultrasonic degreasing with 85% ethanol solution; after being mixed with a pH 4.8 acetic acid-sodium acetate buffer solution, a composite enzyme system immobilized by Fe3O4@SiO2 nanoparticles at a mass ratio of 2:1:1:0.5 of cellulase, pectinase, protease and beta-glucosidase is added; the enzymolysis is carried out at 50 DEG C constant temperature water bath and 15V / cm pulse electric field for 4 hours; after the enzymolysis solution is inactivated, gradient ethanol precipitation is carried out, and the polysaccharide is obtained by ultrafiltration deproteinization and vacuum pulsating freeze drying. The present application improves the cell wall breakage rate and enzymolysis efficiency by using pulsed airflow pulverization, magnetic nano-immobilized enzyme and pulse electric field assistance, and the polysaccharide yield reaches 7.32%, the purity is 91%, the beta-glycosidic bond proportion and antioxidant activity are significantly improved, the enzyme can be recycled and reused, the drying time is shortened, and high-efficiency and low-consumption extraction is realized.

Description

Technical Field

[0001] This invention relates to the field of plant polysaccharide extraction technology, specifically a process for the synergistic extraction of rapeseed pollen polysaccharides using a compound enzyme. Background Technology

[0002] Rapeseed pollen, a natural resource rich in polysaccharides, proteins, flavonoids, and other active ingredients, has attracted much attention due to its polysaccharide components' immunomodulatory, antioxidant, and antitumor bioactivities. However, traditional rapeseed pollen polysaccharide extraction processes face significant technical bottlenecks. Early hot water extraction methods relied on high temperatures to destroy cell walls, leading not only to polysaccharide structural denaturation but also yields generally below 4%, failing to meet the demands of industrial production. While subsequent enzymatic hydrolysis methods utilize cellulases and pectins to disrupt cell walls, their free enzyme systems suffer from low catalytic efficiency and limited enzyme-substrate interaction sites. Especially when dealing with the complex cell wall structure of rapeseed pollen, polysaccharide release is insufficient, and the enzymes cannot be recovered and reused, resulting in high production costs.

[0003] With increasing demands for efficiency and environmental friendliness in the extraction of natural products, existing technologies such as ultrasound-assisted and microwave-assisted extraction, while enhancing mass transfer, lack selectivity in cell wall destruction due to their purely physical nature. This often results in degradation of active ingredients and significantly increases energy costs. For example, while traditional ultrasound extraction can shorten extraction time, polysaccharide purity is easily affected by impurities such as lipids and proteins, complicating subsequent purification processes. Furthermore, existing complex enzyme systems are mostly limited to simple combinations of cellulase and pectinase, lacking design for the synergistic degradation of proteins and glycosidic bonds in rapeseed pollen cell walls. This leads to a low proportion of β-glycosidic bonds, hindering the full realization of polysaccharide bioactivity.

[0004] In current industrial production, the extraction of rapeseed pollen polysaccharides faces multiple challenges, including low yield, poor purity, weak activity, and high cost. There is an urgent need to develop an extraction process that combines efficient cell wall disruption, selective enzymatic hydrolysis, low energy consumption, and recyclability to overcome the bottlenecks of traditional technologies in polysaccharide yield, structure preservation, and industrial application. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a process for the synergistic extraction of rapeseed pollen polysaccharides using a compound enzyme.

[0007] (II) Technical Solution

[0008] A process for synergistic extraction of rapeseed pollen polysaccharides using a compound enzyme includes the following steps:

[0009] S1. The rapeseed pollen was pulverized to 200-300 mesh by pulsed airflow. During the pulverization process, nitrogen gas at -5℃ was introduced to control the temperature to ≤30℃. Then, microwave-ultrasound combined degreasing was performed with 85% ethanol solution: microwave power 400W and ultrasonic frequency 45kHz were used simultaneously for 25 minutes. After centrifugation, the precipitate was collected.

[0010] S2. The precipitate was mixed with an acetate-sodium acetate buffer solution at pH 4.8, and a magnetic nano-immobilized complex enzyme system was added. The enzyme system consisted of cellulase, pectinase, protease, and β-glucosidase immobilized by Fe3O4@SiO2 nanoparticles. The amount of enzyme added was 2% of the pollen mass. Enzymatic hydrolysis was carried out in a constant temperature water bath at 50°C with a pulsed electric field. During the enzymatic hydrolysis, 0.1M acetic acid was automatically added dropwise using an online pH sensor to maintain the stability of the system.

[0011] S3. After the enzyme hydrolysate is inactivated at 85℃, a gradient ethanol precipitation method is used: first, add 2 times the volume of 95% ethanol and let stand for 8 hours. After centrifugation, the precipitate is then precipitated again with 4 times the volume of 70% ethanol. After standing at 4℃ for 12 hours, the precipitate is collected by centrifugation.

[0012] S4. Precipitation was carried out using a hollow fiber ultrafiltration membrane for dynamic deproteinization at an operating pressure of 0.15 MPa and a flow rate of 20 L / h. Subsequently, it was pre-frozen at -40℃ for 2 hours and then freeze-dried under vacuum for 30 hours to obtain rapeseed pollen polysaccharides.

[0013] Preferably, during the microwave-ultrasound synergistic degreasing process, 0.5% wt of citric acid is added to the ethanol solution as a penetration enhancer.

[0014] Preferably, the magnetic nano-immobilized composite enzyme system achieves enzyme recycling and reuse through an external magnet with a magnetic field strength of 0.8T.

[0015] Preferably, during the pulsed electric field enzymatic hydrolysis process, a pulsed electric field lasting 5 minutes is applied every 30 minutes.

[0016] Preferably, in the gradient ethanol precipitation method, the initial precipitation temperature is controlled at 25°C, and the secondary precipitation temperature is reduced to 4°C.

[0017] Preferably, during the deproteinization process of the hollow fiber ultrafiltration membrane, a 0.05% wt chitosan solution is simultaneously introduced as a filter aid.

[0018] Preferably, the pulsating pressure range of the vacuum pulsating freeze-drying is 5-10 Pa, the drying time is shortened by 20% compared with traditional freeze-drying, and the polysaccharide activity retention rate is ≥95%.

[0019] Preferably, the process integrates a near-infrared spectroscopy online monitoring system to measure the polysaccharide concentration in the enzymatic hydrolysate in real time, and automatically triggers the enzyme inactivation program when the concentration reaches 12 mg / mL.

[0020] Preferably, the pulse airflow pulverizer operates at a pulverizing pressure of 0.6 MPa and pulverizes 3 times.

[0021] Preferably, the yield of rapeseed pollen polysaccharides extracted by this process is ≥7.1%, the purity is ≥91%, and the proportion of β-glycosidic bonds is increased by 35% compared with the traditional enzymatic hydrolysis method.

[0022] (iii) Beneficial technical effects

[0023] Compared with existing technologies, the beneficial effects of this invention are:

[0024] 1. The combination of pulsed airflow pulverization and microwave-ultrasound synergistic degreasing technology achieves a pollen cell wall fragmentation rate of over 95%, increasing the degreasing rate and creating sufficient contact conditions for subsequent enzymatic hydrolysis. Compared with traditional pulverization and degreasing processes, this increases the polysaccharide yield, achieving a purity of over 92%. 2. The magnetic nano-immobilized composite enzyme system, through the immobilization of Fe3O4@SiO2 nanoparticles, maintains an enzyme activity retention rate of over 80% even after 5 reuses, reducing costs compared to free enzyme systems. Simultaneously, pulsed electric field-assisted enzymatic hydrolysis promotes increased cell wall porosity and a higher proportion of β-glycosidic bonds, significantly enhancing the antioxidant activity of polysaccharides, with a DPPH scavenging rate exceeding 86%.

[0025] 2. The integration of gradient ethanol precipitation and vacuum pulsed freeze-drying processes solves the problems of polysaccharide loss and activity degradation during traditional precipitation, resulting in higher yield, shorter drying time, and polysaccharide activity retention exceeding 95% compared to single-concentration precipitation. This process achieves dynamic control of the enzymatic hydrolysis process through online near-infrared spectroscopy monitoring, automatically triggering enzyme inactivation when the polysaccharide concentration reaches 12 mg / mL, ensuring process stability and repeatability. Attached Figure Description

[0026] Figure 1 This is a process flow diagram of a compound enzyme synergistic extraction process for rapeseed pollen polysaccharides proposed in this invention;

[0027] Figure 2 This is a comparison chart of polysaccharide yield and purity between the examples and the comparative examples;

[0028] Figure 3 This is a line graph comparing the DPPH scavenging rates of the examples and the comparative examples;

[0029] Figure 4 This is a radar comparison chart created by standardizing the dimensions of the data comparing the effects of the embodiments and the comparative examples. Detailed Implementation

[0030] according to Figures 1 to 4 The specific embodiments of the present invention are as follows:

[0031] I. Process Principles and System Composition

[0032] This process utilizes pulsed airflow to disrupt the cell walls of rapeseed pollen, combined with microwave-ultrasound synergistic defatting to remove lipid interference. A magnetically immobilized complex enzyme system (cellulase, pectinase, protease, and β-glucosidase) is then used to enzymatically release polysaccharides under the assistance of a pulsed electric field. The polysaccharides are then extracted through gradient ethanol precipitation, ultrafiltration to remove proteins, and vacuum pulsed freeze-drying, achieving high-purity extraction of rapeseed pollen polysaccharides. The Fe3O4@SiO2 nanoparticle-immobilized enzymes are recovered via a magnetic field.

[0033] II. Example 1: Extraction of Standard Process Parameters

[0034] S1. Raw material pretreatment

[0035] Take 100g of rapeseed pollen during its peak flowering period and place it in a pulse jet mill. The milling pressure is 0.6MPa, and nitrogen gas at -5℃ is introduced to control the temperature. Mill the pollen three times until it reaches a mesh size of 200, with a cell wall breakage rate of 95%. Add 1000mL of 85% ethanol solution and 0.5% wt citric acid to the milled pollen. Degrease the pollen for 25 minutes under microwave power of 400W and ultrasonic frequency of 45kHz. Keep the pollen in a 45℃ water bath and then centrifuge at 4000r / min for 15 minutes. Discard the supernatant and keep the precipitate.

[0036] S2. Magnetic nano-immobilized enzymatic hydrolysis

[0037] Prepare 1200 mL of acetic acid-sodium acetate buffer solution (pH 4.8) and add the precipitate to the buffer. Prepare the Fe3O4@SiO2 nanoparticle immobilized complex enzyme system: Weigh 2 g of cellulase, 1 g of pectinase, 1 g of protease, and 0.5 g of β-glucosidase according to the mass ratio of cellulase:pectinase:protease:β-glucosidase = 2:1:1:0.5, mix with 100 mg of Fe3O4@SiO2 nanoparticles, and immobilize using a cross-linking agent. Add the immobilized enzyme system to the reaction system at 2% of the pollen mass. In a 50℃ constant temperature water bath, apply a pulsed electric field with a strength of 15 V / cm and a pulse frequency of 10 Hz, and stir at 200 r / min for 4 hours. Monitor the pH in real time with an online pH sensor, and add 0.1 M acetic acid dropwise to maintain pH stability.

[0038] S3. Gradient ethanol precipitation

[0039] The enzyme hydrolysate was inactivated at 85°C for 18 minutes, centrifuged at 4000 rpm for 15 minutes, and the supernatant was collected. Two volumes of 95% ethanol were added to the supernatant, and the mixture was allowed to stand at 25°C for 8 hours. The precipitate was then collected by centrifugation. Four volumes of 70% ethanol were added to the precipitate, and the mixture was allowed to stand at 4°C for 12 hours. The polysaccharide precipitate was collected by centrifugation at 6000 rpm for 10 minutes.

[0040] S4. Purification and Drying

[0041] The precipitate was deproteinized using a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 5000 Da at an operating pressure of 0.15 MPa and a flow rate of 20 L / h, while a 0.05% wt chitosan solution was simultaneously introduced. The ultrafiltered solution was pre-frozen at -40°C for 2 hours, then transferred to a vacuum pulse freeze dryer at a vacuum degree of 5 Pa and a pulse frequency of 0.5 Hz for 30 hours, yielding 7.32 g of rapeseed pollen polysaccharide.

[0042] III. Example 2: Optimization of Pulse Electric Field Parameters

[0043] S1. Raw material pretreatment

[0044] Take 100g of rapeseed pollen during its peak flowering period and place it in a pulse jet mill. The milling pressure is 0.6MPa, and nitrogen gas at -5℃ is introduced to control the temperature. Mill the pollen three times until it reaches a mesh size of 200, with a cell wall breakage rate of 95%. Add 1000mL of 85% ethanol solution and 0.5% wt citric acid to the milled pollen. Degrease the pollen for 25 minutes under microwave power of 400W and ultrasonic frequency of 45kHz. Keep the pollen in a 45℃ water bath and then centrifuge at 4000r / min for 15 minutes. Discard the supernatant and keep the precipitate.

[0045] S2. Magnetic nano-immobilized enzymatic hydrolysis

[0046] Prepare 1200 mL of acetic acid-sodium acetate buffer solution (pH 4.8) and add the precipitate to the buffer. Prepare the Fe3O4@SiO2 nanoparticle-immobilized complex enzyme system: Weigh 2 g of cellulase, 1 g of pectinase, 1 g of protease, and 0.5 g of β-glucosidase according to the mass ratio of cellulase:pectinase:protease:β-glucosidase = 2:1:1:0.5, mix with 100 mg of Fe3O4@SiO2 nanoparticles, and immobilize using a cross-linking agent. Add the immobilized enzyme system to the reaction system at 2% of the pollen mass. In a 50℃ constant temperature water bath, apply a pulsed electric field with a strength of 20 V / cm and a pulse frequency of 15 Hz, applying the pulsed electric field for 5 minutes every 20 minutes, stirring at 200 r / min for 4 hours. Monitor the pH in real time with an online pH sensor, and add 0.1 M acetic acid dropwise to maintain pH stability.

[0047] S3. Gradient ethanol precipitation

[0048] The enzyme hydrolysate was inactivated at 85°C for 18 minutes, centrifuged at 4000 rpm for 15 minutes, and the supernatant was collected. Two volumes of 95% ethanol were added to the supernatant, and the mixture was allowed to stand at 25°C for 8 hours. The precipitate was then collected by centrifugation. Four volumes of 70% ethanol were added to the precipitate, and the mixture was allowed to stand at 4°C for 12 hours. The polysaccharide precipitate was collected by centrifugation at 6000 rpm for 10 minutes.

[0049] S4. Purification and Drying

[0050] The precipitate was deproteinized using a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 5000 Da at an operating pressure of 0.15 MPa and a flow rate of 20 L / h, while a 0.05% wt chitosan solution was simultaneously introduced. The ultrafiltered solution was pre-frozen at -40°C for 2 hours, then transferred to a vacuum pulse freeze dryer at a vacuum degree of 8 Pa and a pulse frequency of 0.5 Hz for 28 hours to obtain 7.51 g of rapeseed pollen polysaccharide.

[0051] IV. Example 3: Adjustment of Compound Enzyme Ratio

[0052] S1. Raw material pretreatment

[0053] Take 100g of rapeseed pollen during its peak flowering period and place it in a pulse jet mill. The milling pressure is 0.6MPa, and nitrogen gas at -5℃ is introduced to control the temperature. Mill the pollen three times until it reaches a mesh size of 200, with a cell wall breakage rate of 95%. Add 1000mL of 85% ethanol solution and 0.5% wt citric acid to the milled pollen. Degrease the pollen for 25 minutes under microwave power of 400W and ultrasonic frequency of 45kHz. Keep the pollen in a 45℃ water bath and then centrifuge at 4000r / min for 15 minutes. Discard the supernatant and keep the precipitate.

[0054] S2. Magnetic nano-immobilized enzymatic hydrolysis

[0055] Prepare 1200 mL of acetate-sodium acetate buffer solution at pH 4.8, and add the precipitate to the buffer solution. Prepare the Fe3O4@SiO2 nanoparticle immobilized complex enzyme system: Weigh 1.5 g of cellulase, 1 g of pectinase, 1 g of protease, and 0.5 g of β-glucosidase according to the mass ratio of cellulase:pectinase:protease:β-glucosidase = 1.5:1:1:0.5, mix with 100 mg of Fe3O4@SiO2 nanoparticles, and immobilize using a cross-linking agent. Add the immobilized enzyme system to the reaction system at 2% of the pollen mass. In a 50℃ constant temperature water bath, apply a pulsed electric field with a strength of 15 V / cm and a pulse frequency of 10 Hz, and stir at 200 r / min for 4 hours. Monitor the pH in real time with an online pH sensor, and add 0.1 M acetic acid dropwise to maintain pH stability.

[0056] S3. Gradient ethanol precipitation

[0057] The enzyme hydrolysate was inactivated at 85°C for 18 minutes, centrifuged at 4000 rpm for 15 minutes, and the supernatant was collected. Two volumes of 95% ethanol were added to the supernatant, and the mixture was allowed to stand at 25°C for 8 hours. The precipitate was then collected by centrifugation. Four volumes of 70% ethanol were added to the precipitate, and the mixture was allowed to stand at 4°C for 12 hours. The polysaccharide precipitate was collected by centrifugation at 6000 rpm for 10 minutes.

[0058] S4. Purification and Drying

[0059] The precipitate was deproteinized using a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 5000 Da at an operating pressure of 0.15 MPa and a flow rate of 20 L / h, while a 0.05% wt chitosan solution was simultaneously introduced. The ultrafiltered solution was pre-frozen at -40°C for 2 hours, then transferred to a vacuum pulse freeze dryer at a vacuum degree of 5 Pa and a pulse frequency of 0.5 Hz for 30 hours, yielding 7.15 g of rapeseed pollen polysaccharide.

[0060] V. Comparative Example: Traditional Hot Water Extraction Method

[0061] S1. Preprocessing

[0062] 100g of rapeseed pollen was pulverized to 80 mesh using a regular grinder, defatted with 85% ethanol solution at room temperature for 30 minutes by stirring, and then centrifuged to collect the precipitate.

[0063] S2. Hot water extraction

[0064] The precipitate was mixed with water at a ratio of 1:20, extracted in a 90°C water bath for 3 hours, and the supernatant was collected by filtration.

[0065] S3. Precipitation and Purification

[0066] Four times the volume of 95% ethanol was added directly to the supernatant to precipitate the precipitate. After standing for 24 hours, the protein was removed five times by the Sevag method after centrifugation and dialyzed for 48 hours. After normal freeze drying, 3.21g of polysaccharide was obtained.

[0067] The effects of the examples and comparative examples are compared in the table below:

[0068] Table 1

[0069] Group Example 1 Example 2 Example 3 Comparative Example Polysaccharide yield (%) 7.32 7.51 7.15 3.21 purity(%) 92.5 93.2 91.8 68.3 β-glycosidic bond ratio (%) 48.7 51.2 47.3 32.5 DPPH clearance rate (%) 86.3 88.5 85.1 52.1 Enzyme activity retention rate (after 5 reuses) 82% 80% 81% 0

[0070] The yields of Examples 1-3 were 128%-134% higher than the comparative example, indicating that the synergistic effect of the composite enzyme and pulsed electric field significantly improved the polysaccharide release efficiency. Example 2, by optimizing the pulsed electric field parameters, increased the β-glycosidic bond ratio to 51.2%, confirming the promoting effect of electric field strength and frequency on cell wall porosity. The magnetically immobilized enzyme, after five reuses, achieved an enzyme activity retention rate ≥80%, significantly higher than that of the free enzyme, thus reducing costs. Vacuum pulsed freeze-drying shortened the drying time by 20% compared to traditional drying, achieving a polysaccharide activity retention rate of 95%, while the DPPH removal rate of the comparative example was only 52.1%, verifying the protective effect of the process on the active ingredients.

[0071] The table below compares the response of electric field strength and pulse frequency to yield:

[0072] Table 2

[0073] Electric field strength (V / cm) Pulse frequency (Hz) Polysaccharide yield (%) 15 10 7.32 15 15 7.45 20 10 7.48 20 15 7.51

[0074] The pulsed electric field parameters significantly affect the polysaccharide yield. When the electric field strength is 20V / cm and the pulse frequency is 15Hz, the yield reaches the maximum value of 7.51%, which is 2.6% higher than the basic parameters (15V / cm, 10Hz), indicating that there is an optimal range for the synergistic effect of the electric field.

[0075] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A process for synergistic extraction of rapeseed pollen polysaccharides using a compound enzyme, characterized in that, Includes the following steps: S1. The rapeseed pollen was pulverized to 200-300 mesh by pulsed airflow. During the pulverization process, nitrogen gas at -5℃ was introduced to control the temperature to ≤30℃. Then, microwave-ultrasound combined degreasing was performed with 85% ethanol solution: microwave power 400W and ultrasonic frequency 45kHz were used simultaneously for 25 minutes. After centrifugation, the precipitate was collected. S2. The precipitate was mixed with an acetate-sodium acetate buffer solution at pH 4.8, and a magnetic nano-immobilized complex enzyme system was added. The enzyme system consisted of cellulase, pectinase, protease, and β-glucosidase immobilized using Fe3O4@SiO2 nanoparticles. The amount of enzyme added was 2% of the pollen mass. Enzymatic hydrolysis was performed in a constant temperature water bath at 50°C using a pulsed electric field. During the pulsed electric field hydrolysis, the electric field strength was 15-20 V / cm and the pulse frequency was 10-15 Hz. During the hydrolysis, 0.1 M acetic acid was automatically added dropwise using an online pH sensor to maintain the stability of the system. S3. After the enzyme hydrolysate is inactivated at 85℃, a gradient ethanol precipitation method is used: first, add 2 times the volume of 95% ethanol and let stand for 8 hours. After centrifugation, the precipitate is then precipitated again with 4 times the volume of 70% ethanol. After standing at 4℃ for 12 hours, the precipitate is collected by centrifugation. S4. Precipitation was carried out using a hollow fiber ultrafiltration membrane for dynamic deproteinization at an operating pressure of 0.15 MPa and a flow rate of 20 L / h. Subsequently, it was pre-frozen at -40℃ for 2 hours and then freeze-dried under vacuum for 30 hours to obtain rapeseed pollen polysaccharides. During the pulsed electric field enzymatic hydrolysis process, a pulsed electric field lasting 5 minutes is applied every 30 minutes. The vacuum pulse freeze-drying process has a pulse pressure range of 5-10 Pa, which shortens the drying time by 20% compared to traditional freeze-drying, and the polysaccharide activity retention rate is ≥95%. The process integrates a near-infrared spectroscopy online monitoring system to measure the polysaccharide concentration in the enzymatic hydrolysate in real time, and automatically triggers the enzyme inactivation program when the concentration reaches 12 mg / mL. The pulse airflow pulverizer operates at a pulverizing pressure of 0.6 MPa and pulverizes three times.

2. The process for synergistic extraction of rapeseed pollen polysaccharides using a compound enzyme according to claim 1, characterized in that, During the microwave-ultrasound synergistic degreasing process, 0.5% wt of citric acid is added to the ethanol solution as a penetration enhancer.

3. The process for synergistic extraction of rapeseed pollen polysaccharides using a compound enzyme according to claim 1, characterized in that, The magnetic nano-immobilized composite enzyme system achieves enzyme recycling and reuse through an external magnet with a magnetic field strength of 0.8T.

4. The process for synergistic extraction of rapeseed pollen polysaccharides using a compound enzyme according to claim 1, characterized in that, In the gradient ethanol precipitation method, the initial precipitation temperature is controlled at 25°C, and the secondary precipitation temperature is reduced to 4°C.

5. The process for synergistic extraction of rapeseed pollen polysaccharides using a compound enzyme according to claim 1, characterized in that, During the deproteinization process of the hollow fiber ultrafiltration membrane, a 0.05% wt chitosan solution is simultaneously introduced as a filter aid.

6. The process for synergistic extraction of rapeseed pollen polysaccharides using a compound enzyme according to claim 1, characterized in that, The yield of rapeseed pollen polysaccharides extracted by this process is ≥7.1%, the purity is ≥91%, and the proportion of β-glycosidic bonds is increased by 35% compared with the traditional enzymatic hydrolysis method.

Images