Segmented enzymolysis-cascade membrane separation extraction method of cyclocarya paliurus combined-state selenium polysaccharide and combined-state selenium polysaccharide prepared by same

Abstract

The invention belongs to the technical field of plant active ingredient extraction, and relates to a segmented enzymolysis-cascade membrane separation extraction method of cyclocarya paliurus combined-state selenium polysaccharide and the combined-state selenium polysaccharide prepared by the method. The method comprises the following steps: firstly, constructing a first extraction system for a cyclocarya paliurus leaf raw material, carrying out first-stage enzymolysis, inactivation and solid-liquid separation, separating a first liquid phase through a nanofiltration membrane to form a low-molecular selenium-containing component flow, and excluding the low-molecular selenium-containing component flow in a subsequent alcohol precipitation and recovery step; and constructing a second extraction system from the first solid phase, carrying out second-stage enzymolysis, inactivation and solid-liquid separation, sequentially carrying out microfiltration, first ultrafiltration, second ultrafiltration and nanofiltration washing on the second liquid phase, and carrying out ethanol precipitation to obtain the combined-state selenium polysaccharide. The proportion of target molecular weight interval components in the obtained product is increased, the mass fraction of polysaccharide is increased, and the proportion of inorganic selenium, the mass fraction of protein and the ash content are reduced.

Description

A segmented enzymatic hydrolysis-stepwise membrane separation extraction method for bound selenium polysaccharides from *Cyclocarya paliurus* and the resulting bound selenium polysaccharides. Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction technology, and relates to a segmented enzymatic hydrolysis-step membrane separation extraction method for bound selenium polysaccharides from Eucommia ulmoides and the obtained bound selenium polysaccharides. Background Technology

[0002] Cyclocarya paliurus is a plant belonging to the genus Cyclocarya in the family Juglandaceae. Its leaves contain various components, including polysaccharides. With the continuous advancement of research on functional components derived from plants, numerous studies have been conducted on the extraction, separation, and application of polysaccharides from Cyclocarya paliurus leaves.

[0003] In existing technologies, the extraction routes for *Cyclocarya paliurus* polysaccharides or selenium-containing polysaccharides typically include steps such as raw material drying and pulverization, water extraction or enzymatic extraction, filtration or centrifugation, concentration, alcohol precipitation, and drying. Some technical solutions also introduce unit operations such as ultrafiltration membrane, nanofiltration membrane, or resin separation after enzymatic extraction to further enrich and purify the components in the extract. Other technical routes involve further extraction and recovery of the residue after preliminary enzymatic hydrolysis, or uniform concentration and precipitation of the supernatant after enzymatic hydrolysis. Although the above methods can obtain polysaccharides or selenium-containing components from *Cyclocarya paliurus*, existing technologies generally use the primary extract as the direct recovery target, or integrate the liquid phases obtained from different stages into the subsequent concentration and alcohol precipitation path. There is a lack of clear process division regarding whether the low-molecular-weight selenium-containing components in the initial liquid phase should be treated separately from the target polysaccharide components in the later stage, how the liquid and solid phases at different stages correspond in the subsequent recovery path, and the molecular weight range that the final target component should fall into. Therefore, in actual processing, existing technologies often result in the low molecular weight components in the front stage and the target components in the back stage entering the final product together. This leads to a wide molecular weight distribution of the obtained product, with more low molecular weight components being introduced. At the same time, non-target components such as inorganic selenium, protein, and ash are also more likely to enter the final precipitate. As a result, although the obtained product has a certain total selenium content, the proportion of inorganic selenium is high, and there are more impurities such as protein and ash, resulting in a dispersed composition. This is not conducive to obtaining selenium-containing products that are mainly in the polysaccharide bound state, and the absorption and utilization rate by the human body is low.

[0004] Therefore, it is necessary to provide a new method for extracting selenium-containing polysaccharides from Eucommia ulmoides. Based on the existing enzymatic extraction and membrane separation processes, a clearer separation and recovery relationship should be established for the liquid and solid phase components obtained at different stages. The source range and molecular weight boundary of the final target component should be further defined, and the obtained product should achieve more favorable results in terms of molecular weight distribution, polysaccharide mass fraction, inorganic selenium ratio, and impurity control. Summary of the Invention

[0005] The purpose of this invention is to provide a segmented enzymatic hydrolysis-step membrane separation extraction method for Cyclocarya paliurus bound selenium polysaccharides, as well as the Cyclocarya paliurus bound selenium polysaccharides obtained by this method and their applications, so as to solve the problems existing in the extraction process of Cyclocarya paliurus polysaccharides or selenium-containing polysaccharides.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The present invention proposes a segmented enzymatic hydrolysis-stepwise membrane separation extraction method for *Cyclocarya paliurus* bound selenium polysaccharides, comprising the following steps: S1, drying and pulverizing *Cyclocarya paliurus* leaf raw material to obtain *Cyclocarya paliurus* leaf powder, and mixing the *Cyclocarya paliurus* leaf powder with water or buffer solution to form a first extraction system; S2, performing a first-stage enzymatic hydrolysis on the first extraction system, and sequentially performing a first-stage inactivation and a first-stage solid-liquid separation on the first-stage hydrolysate to obtain a first liquid phase and a first solid phase; S3, separating the first liquid phase through a nanofiltration membrane, collecting its permeate as a low-molecular-weight selenium-containing component stream, wherein the low-molecular-weight selenium-containing component stream is a liquid with an apparent molecular weight not exceeding 3000 Da as measured by gel permeation chromatography. S4. The first solid phase is mixed with water or buffer to form a second extraction system. The second extraction system is subjected to a second-stage enzymatic hydrolysis, and the second-stage enzymatic hydrolysate is subjected to a second-stage inactivation and a second-stage solid-liquid separation to obtain a second liquid phase. S5. The second liquid phase is subjected to microfiltration, a first ultrafiltration and a second ultrafiltration. The liquid phase obtained by the first ultrafiltration permeate and the second ultrafiltration is collected as the target component liquid. The target component liquid is a liquid phase component with an apparent molecular weight of 5000~80000 Da as determined by gel permeation chromatography. S6. The target component liquid is washed by nanofiltration and then ethanol is added. After standing, the precipitate is collected and dried to obtain the *Cyclocarya paliurus* bound selenium polysaccharide.

[0007] Further, in step S2, the pH of the first stage of enzymatic hydrolysis is 3.5~5.5, the temperature is 35~50℃, and the time is 0.5~3h. The first stage of enzymatic hydrolysis uses pectinase, hemicellulase, or a first composite enzyme composed of pectinase and hemicellulase. The total amount of enzyme added is 0.1%~3.0% of the mass of Eucommia ulmoides leaf powder. The inactivation conditions of the first stage are 80~95℃ for 5~20min.

[0008] Further, in step S3, the molecular weight cutoff of the nanofiltration membrane is 150~1000 Da, and the low molecular weight selenium-containing component stream is the permeate separated by the nanofiltration membrane, or it is a combined solution of the permeate and the washing permeate obtained when a constant volume washing method is used in the nanofiltration membrane separation process.

[0009] Furthermore, between step S3 and step S4, a first solid phase washing step is included, in which the first solid phase is washed 1 to 5 times with water or an aqueous ethanol solution with a volume fraction of 5% to 30%. The washing liquid is incorporated into the low molecular weight selenium-containing component stream, and the washing liquid does not enter the subsequent target component liquid recovery step.

[0010] Further, in step S4, the pH of the second stage of enzymatic hydrolysis is 4.5~7.5, the temperature is 45~60℃, and the time is 1~5h. The second stage of enzymatic hydrolysis uses cellulase, protease, or a second complex enzyme composed of cellulase and protease. The total amount of enzyme added is 0.2%~5.0% of the mass of the first solid phase. The inactivation conditions of the second stage are 85~100℃ for 5~30min.

[0011] Furthermore, the pore size of the microfiltration in step S5 is 0.05~0.45μm, the molecular weight cutoff of the first ultrafiltration is 30~80kDa, the molecular weight cutoff of the second ultrafiltration is 8~20kDa, and the components with an apparent molecular weight of 8000~50000Da in the target component solution account for more than 60% of the total polysaccharide components in the target component solution.

[0012] Furthermore, in step S6, the nanofiltration washing is performed using a nanofiltration membrane with a molecular weight cutoff of 150~500 Da, and a constant volume washing method is adopted. The volume of water used for washing is 1~8 times the volume of the target component liquid. After the ethanol is added, the final ethanol concentration of the system is 60%~90%. The standing temperature is 2~10℃, and the standing time is 4~24h. The drying is performed by freeze drying or vacuum drying.

[0013] On the other hand, the present invention proposes a Cyclocarya paliurus-bound selenium polysaccharide, which is prepared by the method described in the present invention, and has an apparent molecular weight of 5000~80000 Da as measured by gel permeation chromatography, a total selenium content of 0.02%~1.00%, a polysaccharide mass fraction of 55%~95% as measured by the anthrone-sulfuric acid method, and an inorganic selenium content accounting for no more than 20% of the total selenium content.

[0014] Furthermore, in the *Cyclocarya paliurus*-bound selenium polysaccharide, the component with an apparent molecular weight of 8000~50000 Da accounts for more than 60% of the total polysaccharide component, the protein content is 0.1%~8.0%, and the ash content is not higher than 5.0%.

[0015] Furthermore, this invention also proposes the application of *Cyclocarya paliurus*-bound selenium polysaccharide in the preparation of an encapsulated selenium-containing food composition. In the encapsulated selenium-containing food composition, the *Cyclocarya paliurus*-bound selenium polysaccharide serves as the core material, and the wall material is selected from one or at least two of maltodextrin, gum arabic, resistant dextrin, and pectin. The mass ratio of the core material to the wall material is 1:1 to 1:20.

[0016] This invention offers the following advantages: First, the first liquid phase obtained after the first-stage enzymatic hydrolysis is separated separately using a nanofiltration membrane to form a low-molecular-weight selenium-containing component stream, which is explicitly excluded from the subsequent ethanol precipitation and recovery step. Simultaneously, the first solid phase obtained from the first-stage solid-liquid separation is used for the second-stage enzymatic hydrolysis, ensuring that the final product entering the precipitation and recovery step is no longer the entire extract after the first enzymatic hydrolysis, but rather a specific component derived from the second liquid phase after further release from the first solid phase. This reduces the carryover of low-molecular-weight selenium-containing components in the final product. This invention limits the low-molecular-weight selenium-containing component stream to liquid phase components with an apparent molecular weight not exceeding 3000 Da, and limits the target component liquid to liquid phase components with an apparent molecular weight of 5000-80000 Da. The target component liquid is formed by sequentially passing the second liquid phase through microfiltration, first ultrafiltration, and second ultrafiltration. Test results show that, using the process of this invention, the proportion of components with an apparent molecular weight in the 8000-50000 Da range in the obtained product significantly increases compared to the total polysaccharide components.

[0017] This invention involves further nanofiltration and ethanol precipitation after obtaining the target component solution. This ensures that the final recovery path is limited to the specific liquid phase component after molecular weight sieving, while the low-molecular-weight selenium-containing components and some soluble accompanying components from the initial stage no longer precipitate with the target component. The resulting product maintains a high total selenium content and a high polysaccharide mass fraction, while significantly reducing the proportion of inorganic selenium, protein mass fraction, and ash content. Because the total selenium content remains at a high level while the proportion of inorganic selenium decreases, the proportion of selenium components existing in organically bound form in the resulting product is correspondingly increased, and impurities are reduced. This results in a more concentrated composition of *Cyclocarya paliurus*-bound selenium polysaccharides, providing a more favorable material basis for the human body's absorption and utilization of selenium. Attached Figure Description

[0018] Figure 1 is a flowchart of the overall process for extracting selenium polysaccharides from Cyclocarya paliurus. Detailed Implementation

[0019] To make the technical solution, technical features, and effects of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Equivalent substitutions, conventional adjustments, or corresponding modifications made by those skilled in the art based on this specification without departing from the concept of the present invention should all fall within the scope of protection of the present invention.

[0020] The segmented enzymatic hydrolysis-stepwise membrane separation extraction method for *Cyclocarya paliurus* bound selenium polysaccharides described in this invention uses *Cyclocarya paliurus* leaves as the starting material. Through a first extraction system, a low-molecular-weight selenium-containing component stream is formed by first-stage enzymatic hydrolysis, first-stage inactivation, first-stage solid-liquid separation, and nanofiltration of the first liquid phase. Based on the first solid phase obtained from the first-stage solid-liquid separation, a second extraction system is constructed, undergoing second-stage enzymatic hydrolysis, second-stage inactivation, second-stage solid-liquid separation, and microfiltration, first ultrafiltration, second ultrafiltration, and nanofiltration washing of the second liquid phase to obtain a concentrated bound selenium polysaccharide solution. This solution is then precipitated with ethanol and dried to obtain the *Cyclocarya paliurus* bound selenium polysaccharide. Thus, the low-molecular-weight selenium-containing component stream and the target component solution are distinguished in terms of source, processing path, and final recovery method.

[0021] In this invention: "low molecular weight selenium-containing component stream" refers to the permeate obtained after the first liquid phase is separated by nanofiltration membrane, or the combined liquid of the permeate and the washing permeate obtained when constant volume washing filtration is used in the nanofiltration membrane separation process. This low molecular weight selenium-containing component stream does not enter the subsequent alcohol precipitation recovery step; "target component liquid" refers to the liquid phase component obtained after the second liquid phase is separated by microfiltration, first ultrafiltration and second ultrafiltration in sequence, which is formed by the retention of the first ultrafiltration permeate by the second ultrafiltration; "Qingqianliu bound selenium polysaccharide" refers to the product obtained by the method of this invention that meets the requirements of apparent molecular weight, total selenium content, polysaccharide mass fraction and inorganic selenium ratio.

[0022] In the following specific embodiments, unless otherwise specified, the raw materials of *Cyclocarya paliurus* leaves, pectinase, hemicellulase, cellulase, protease, buffer solution, nanofiltration membrane, microfiltration membrane, ultrafiltration membrane, ethanol, and various conventional chemical reagents can all be conventional products in the field; the filtration, centrifugation, temperature control, membrane separation, drying, and detection equipment used can also all be conventional equipment in the field.

[0023] In the following specific embodiments, unless otherwise specified, all percentage contents are mass percentages, temperature is in °C, time is in h or min, molecular weight cutoff is in Da or kDa, and pore size is in μm. Apparent molecular weight was determined by gel permeation chromatography; total selenium content was determined by inductively coupled plasma mass spectrometry; inorganic selenium content was determined by conventional inorganic selenium detection methods in the art; polysaccharide mass fraction was determined by the anthrone-sulfuric acid method; protein mass fraction was determined by the Coomassie brilliant blue method; and ash content was determined by the high-temperature ignition method. For the proportion of components with an apparent molecular weight in the range of 8000~50000 Da in the total polysaccharide components, the proportion of the peak area of ​​the corresponding retention time range in gel permeation chromatography to the relevant peak area of ​​the total polysaccharide was calculated.

[0024] The raw material for *Cyclocarya paliurus* leaves can be artificially cultivated or naturally grown. Preferably, the raw material is selected after harvesting and removing obvious impurities. The leaves are first rinsed with clean water to remove surface residue, then dried with hot air at 50-70℃, and subsequently pulverized using a pulverizing device and sieved through a 20-80 mesh screen to obtain *Cyclocarya paliurus* leaf powder with relatively uniform particle size.

[0025] Enzyme preparations used for the first stage of enzymatic hydrolysis include pectinase, hemicellulase, or a first complex enzyme composed of pectinase and hemicellulase; enzyme preparations used for the second stage of enzymatic hydrolysis include cellulase, protease, or a second complex enzyme composed of cellulase and protease. All of the above enzyme preparations can be commercially available enzyme preparations conventional in the art.

[0026] The nanofiltration membrane separation of the first liquid phase uses a nanofiltration membrane with a molecular weight cutoff of 150-1000 Da. The stepwise membrane separation of the second liquid phase includes microfiltration, first ultrafiltration, second ultrafiltration, and nanofiltration washing. The microfiltration uses a microfiltration membrane with a pore size of 0.05-0.45 μm; the first ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 30-80 kDa; the second ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 8-20 kDa; and the nanofiltration washing uses a nanofiltration membrane with a molecular weight cutoff of 150-500 Da.

[0027] Unless otherwise specified in subsequent embodiments, the material-liquid ratio, stirring speed, circulation flow rate, transmembrane pressure difference, centrifugal speed, filtration time, number of washing cycles, drying time, and other conditions involved in this invention can be conventionally adjusted by those skilled in the art based on equipment specifications, raw material conditions, and processing scale.

[0028] Example 1 This example provides a segmented enzymatic hydrolysis-step membrane separation and extraction method for bound selenium polysaccharides from Eucommia ulmoides.

[0029] S1. Constructing the first extraction system: Weigh 1000g of cleaned, dried, pulverized and passed through a 40-mesh sieve of Eucommia ulmoides leaf powder, add 15000mL of citric acid-sodium citrate buffer solution, adjust the pH of the system to 4.6, stir and mix for 20min to obtain the first extraction system.

[0030] S2. Perform the first stage of enzymatic hydrolysis, the first stage of inactivation, and the first stage of solid-liquid separation: Add 8g of pectinase and 12g of hemicellulase to the first extraction system, and enzymatically hydrolyze at 45℃ for 2h to obtain the first stage of enzymatic hydrolysate; then heat the first stage of enzymatic hydrolysate to 88℃ and maintain it for 10min for the first stage of inactivation. After inactivation, cool it to below 35℃, filter it through a filter cloth, and then centrifuge it at 4500r / min for 15min. Collect the supernatant as the first liquid phase and collect the precipitated solid as the first solid phase.

[0031] S3. Perform nanofiltration membrane separation on the first liquid phase to form a low molecular weight selenium-containing component stream: Input the first liquid phase into a nanofiltration membrane system with a molecular weight cutoff of 300 Da for nanofiltration membrane separation, control the operating temperature at 25~30℃, and control the transmembrane pressure difference at 1.0~1.5 MPa; perform nanofiltration concentration first, and then add deionized water using a constant volume washing filter method, with the total volume of washing water being twice the volume of the first liquid phase; collect the permeate obtained from nanofiltration membrane separation and the washing permeate, and combine them as the low molecular weight selenium-containing component stream; detect the low molecular weight selenium-containing component stream using gel permeation chromatography, and its main distribution range is below 3000 Da.

[0032] S4. Wash the first solid phase and construct the second extraction system: Add 4000 mL of a 10% (v / v) ethanol aqueous solution to the first solid phase, stir for 15 min, filter, and collect the washing liquid; wash once more in the same manner. Combine the washing liquids obtained from the two washings and add them to the low molecular weight selenium-containing component stream. Add 12000 mL of deionized water to the washed first solid phase and adjust the pH of the system to 5.8 to obtain the second extraction system.

[0033] S5. Perform the second-stage enzymatic hydrolysis, second-stage inactivation, and second-stage solid-liquid separation: Add 15g of cellulase and 10g of protease to the second extraction system, and hydrolyze at 52℃ for 3h to obtain the second-stage enzymatic hydrolysate. Then, heat the second-stage enzymatic hydrolysate to 92℃ and maintain it for 12min for the second-stage inactivation. After inactivation, cool it to below 40℃, filter it through a filter cloth, and then centrifuge it at 5000r / min for 15min. Collect the supernatant as the second liquid phase.

[0034] S6. The second liquid phase is subjected to stepwise membrane separation, nanofiltration washing, ethanol precipitation, and drying to obtain *Cyclocarya paliurus*-bound selenium polysaccharide. The second liquid phase is first treated with a microfiltration membrane with a pore size of 0.22 μm to obtain a microfiltration permeate. The microfiltration permeate is then fed into a first ultrafiltration membrane system with a molecular weight cutoff of 50 kDa, and the first ultrafiltration permeate is collected. The first ultrafiltration permeate is then fed into a second ultrafiltration membrane system with a molecular weight cutoff of 10 kDa, and the second ultrafiltration retentate is collected as the target component solution. The target component solution is detected by gel permeation chromatography, and its main distribution range is in the range of 5000~80000 Da.

[0035] The target component solution was fed into a nanofiltration membrane system with a molecular weight cutoff of 300 Da for nanofiltration washing. The washing method was constant volume washing, and the volume of washing water was 4 times the volume of the target component solution. The nanofiltration membrane retentate was collected to obtain a concentrated solution of bound selenium polysaccharides. Anhydrous ethanol was slowly added to the concentrated solution of bound selenium polysaccharides to bring the final ethanol concentration of the system to 75%, and then the solution was allowed to stand at 4°C for 12 hours. After standing, the precipitate was collected, washed twice with 95% ethanol, and then freeze-dried to constant weight to obtain 121.6 g of bound selenium polysaccharides from *Cyclocarya paliurus*.

[0036] Example 2 This example provides a segmented enzymatic hydrolysis-step membrane separation extraction method for bound selenium polysaccharides from *Cyclocarya paliurus*. Except for the following parameters and conditions, all other steps are performed according to Example 1.

[0037] S1. Constructing the first extraction system: Weigh 1000g of cleaned, dried, pulverized and passed through a 40-mesh sieve of Eucommia ulmoides leaf powder, add 15000mL of citric acid-sodium citrate buffer solution, adjust the pH of the system to 4.4, stir and mix for 20min to obtain the first extraction system.

[0038] S2. Perform the first stage of enzymatic hydrolysis, the first stage of inactivation, and the first stage of solid-liquid separation: Add 10g of pectinase and 10g of hemicellulase to the first extraction system, and perform enzymatic hydrolysis at 46℃ for 2.5h to obtain the first stage enzymatic hydrolysate. Then, heat the first stage enzymatic hydrolysate to 90℃ and maintain it for 8min to perform the first stage of inactivation. After inactivation, perform the first stage of solid-liquid separation as described in Example 1 to obtain the first liquid phase and the first solid phase.

[0039] S3. The first liquid phase is separated using nanofiltration to form a low-molecular-weight selenium-containing component stream: The first liquid phase is fed into a nanofiltration membrane system with a molecular weight cutoff of 500 Da for nanofiltration separation. A constant-volume washing method is used, with the total volume of washing water being three times the volume of the first liquid phase. The permeate obtained from nanofiltration and the washing permeate are collected and combined to form the low-molecular-weight selenium-containing component stream. The low-molecular-weight selenium-containing component stream is detected using gel permeation chromatography, and its main distribution range is below 3000 Da.

[0040] S4. Wash the first solid phase and construct the second extraction system: Add 3500 mL of 8% (v / v) ethanol aqueous solution to the first solid phase, stir for 15 min, filter, and collect the washing liquid; wash twice more in the same manner. Combine the washing liquids obtained from the three washes and add them to the low molecular weight selenium-containing component stream. Add 11000 mL of deionized water to the washed first solid phase and adjust the pH of the system to 5.6 to obtain the second extraction system.

[0041] S5. Perform the second stage of enzymatic hydrolysis, second stage of inactivation, and second stage of solid-liquid separation: Add 16g of cellulase and 8g of protease to the second extraction system, and hydrolyze at 50°C for 3.5h to obtain the second stage enzymatic hydrolysate. Then, heat the second stage enzymatic hydrolysate to 90°C and maintain it for 15min to perform the second stage of inactivation. After inactivation, perform the second stage of solid-liquid separation as described in Example 1 to obtain the second liquid phase.

[0042] S6. The second liquid phase was subjected to stepwise membrane separation, nanofiltration washing, ethanol precipitation, and drying to obtain *Cyclocarya paliurus*-bound selenium polysaccharide: The second liquid phase was subjected to microfiltration and first ultrafiltration as described in Example 1. The permeate from the first ultrafiltration was then fed into a second ultrafiltration membrane system with a molecular weight cutoff of 8 kDa, and the second ultrafiltration retentate was collected as the target component solution. The target component solution was fed into a nanofiltration membrane system with a molecular weight cutoff of 300 Da for nanofiltration washing using a constant volume washing method, with the washing water volume being 5 times the volume of the target component solution. Anhydrous ethanol was added to the obtained bound selenium polysaccharide concentrate to bring the final ethanol concentration of the system to 72%, and then the mixture was allowed to stand at 4°C for 10 hours. After standing, the precipitate was collected, washed twice with 95% ethanol, and then freeze-dried to constant weight to obtain 118.4 g of *Cyclocarya paliurus*-bound selenium polysaccharide.

[0043] Example 3 This example provides a segmented enzymatic hydrolysis-step-membrane separation extraction method for bound selenium polysaccharides from *Cyclocarya paliurus*. Except for the step-membrane separation window in the second stage and its corresponding subsequent processing parameters, the remaining steps are performed according to Example 1.

[0044] The obtained second liquid phase was processed according to the following steps: S6, the second liquid phase was subjected to stepwise membrane separation, nanofiltration washing, ethanol precipitation and drying to obtain *Cyclocarya paliurus*-bound selenium polysaccharide: the second liquid phase was first treated with a microfiltration membrane with a pore size of 0.22 μm to obtain microfiltration permeate; then the microfiltration permeate was fed into a first ultrafiltration membrane system with a molecular weight cutoff of 80 kDa, and the first ultrafiltration permeate was collected; then the first ultrafiltration permeate was fed into a second ultrafiltration membrane system with a molecular weight cutoff of 15 kDa, and the second ultrafiltration retentate was collected as the target component solution. The target component solution was detected by gel permeation chromatography, and its main distribution range was in the range of 15000~80000 Da, with a relatively high proportion of components in the range of 8000~50000 Da.

[0045] The target component solution was fed into a nanofiltration membrane system with a molecular weight cutoff of 300 Da for nanofiltration washing. The washing method was constant volume washing, and the volume of washing water was three times the volume of the target component solution. The nanofiltration membrane retentate was collected to obtain a concentrated bound selenium polysaccharide solution. Anhydrous ethanol was slowly added to the concentrated bound selenium polysaccharide solution to bring the final ethanol concentration of the system to 78%, and then the solution was allowed to stand at 4°C for 12 hours. After standing, the precipitate was collected, washed twice with 95% ethanol, and then freeze-dried to constant weight to obtain 124.8 g of *Cyclocarya paliurus* bound selenium polysaccharide.

[0046] Example 4 This example provides a method for preparing an encapsulated selenium-containing food composition, wherein the selenium polysaccharide bound to Eucommia ulmoides is used as the core material, and the wall material is maltodextrin and gum arabic.

[0047] S1. Preparation of wall material solution: Weigh 400g of maltodextrin and 200g of gum arabic, add 5400mL of deionized water, and stir to dissolve for 30min at 40℃ to obtain the wall material solution. The mass ratio of maltodextrin to gum arabic is 2:1.

[0048] S2. Preparation of core material dispersion and formation of pre-embedding mixture: Weigh 120g of the *Cyclocarya paliurus*-bound selenium polysaccharide obtained in Example 1, add 600mL of deionized water, and stir and disperse at room temperature for 20min to obtain the core material dispersion. Slowly add the core material dispersion to the wall material solution, and continue stirring at 45℃ for 30min to obtain the pre-embedding mixture. The *Cyclocarya paliurus*-bound selenium polysaccharide serves as the core material, and the maltodextrin and gum arabic serve as the wall material, with a core material to wall material mass ratio of 1:5.

[0049] S3. Homogenization: The pre-embedding mixture is fed into a high-shear homogenizer and homogenized at 8000 r / min for 8 min to obtain a uniform dispersion. After homogenization, the uniform dispersion is allowed to stand for 15 min to degas.

[0050] S4. Drying into powder to obtain an encapsulated selenium-containing food composition: The defoamed uniform dispersion is fed into a spray drying device, the inlet air temperature is controlled at 165℃ and the outlet air temperature is controlled at 82℃, and spray drying is performed. The resulting powder is collected and sieved through a 60-mesh sieve to obtain an encapsulated selenium-containing food composition.

[0051] The obtained encapsulated selenium-containing food composition was tested. The product was a light yellow to light brownish-yellow powder with no obvious agglomeration. The total selenium content was determined to be 0.031% by inductively coupled plasma mass spectrometry; the polysaccharide mass fraction was determined to be 18.6% by the anthrone-sulfuric acid method. The obtained powder was redissolved in deionized water, and after stirring, the system was evenly dispersed, and no obvious insoluble precipitate visible to the naked eye was observed.

[0052] Comparative Example 1: This comparative example provides a method for extracting selenium-containing polysaccharides from Eucommia ulmoides. Except that the first liquid phase does not form a separate low-molecular-weight selenium-containing component stream, but is combined with the washing liquid and the second liquid phase before entering the subsequent recovery step, the other steps are carried out according to Example 1.

[0053] S1 and S2 are performed according to Example 1.

[0054] S3. Processing the first liquid phase: In this comparative example, the first liquid phase is not separated by nanofiltration membrane to avoid forming a low-molecular-weight selenium-containing component stream. The first liquid phase is temporarily stored for subsequent combined processing with the washing liquid and the second liquid phase.

[0055] S4. Wash the first solid phase and construct the second extraction system: Add 4000 mL of a 10% (v / v) ethanol aqueous solution to the first solid phase, stir for 15 min, filter, and collect the washing liquid; wash once more in the same manner. Combine the washing liquids obtained from the two washings and add them to the first liquid phase. Add 12000 mL of deionized water to the washed first solid phase and adjust the pH of the system to 5.8 to obtain the second extraction system.

[0056] S5. Proceed according to Example 1 to obtain the second liquid phase.

[0057] S6. Perform stepwise membrane separation, nanofiltration washing, ethanol precipitation, and drying on the combined liquid: Combine the first liquid phase, washing liquid, and second liquid phase, and first treat with a microfiltration membrane with a pore size of 0.22 μm to obtain a microfiltration permeate; then input the microfiltration permeate into a first ultrafiltration membrane system with a molecular weight cutoff of 50 kDa, and collect the first ultrafiltration permeate; then input the first ultrafiltration permeate into a second ultrafiltration membrane system with a molecular weight cutoff of 10 kDa, and collect the second ultrafiltration retentate. Input the obtained second ultrafiltration retentate into a nanofiltration membrane system with a molecular weight cutoff of 300 Da for nanofiltration washing using a constant volume washing method, with the washing water volume being 4 times the volume of the second ultrafiltration retentate, and collect the nanofiltration membrane retentate. Slowly add anhydrous ethanol to the obtained concentrate to achieve a final ethanol concentration of 75%, and then let it stand at 4°C for 12 h. After standing, collect the precipitate, wash twice with 95% ethanol, and then freeze-dry to constant weight to obtain 135.2 g of product.

[0058] Comparative Example 2 provides a method for extracting selenium-containing polysaccharides from Eucommia ulmoides. Except for the second liquid phase, which is not separated by first and second ultrafiltration, all other steps are performed according to Example 1.

[0059] S1 to S5 were all performed according to Example 1.

[0060] S6. Microfiltration, nanofiltration, ethanol precipitation, and drying of the second liquid phase: The second liquid phase is first treated with a microfiltration membrane with a pore size of 0.22 μm to obtain the microfiltration permeate. The microfiltration permeate is then directly fed into a nanofiltration membrane system with a molecular weight cutoff of 300 Da for nanofiltration washing. The washing method is constant volume washing, and the volume of washing water is 4 times the volume of the microfiltration permeate. The nanofiltration membrane retentate is collected. Anhydrous ethanol is slowly added to the resulting concentrate to bring the final ethanol concentration of the system to 75%, and then the mixture is allowed to stand at 4°C for 12 hours. After standing, the precipitate is collected, washed twice with 95% ethanol, and then freeze-dried to constant weight to obtain 128.7 g of product.

[0061] Comparative Example 3 provides a method for extracting selenium-containing polysaccharides from *Cyclocarya paliurus*. Except for the low-molecular-weight selenium-containing component, which may be included in the subsequent ethanol precipitation and recovery step, all other steps were performed according to Example 1.

[0062] S1 to S5 are all carried out according to Example 1, wherein the first liquid phase is separated by nanofiltration membrane to form a low molecular weight selenium-containing component stream.

[0063] S6. The second liquid phase is subjected to stepwise membrane separation, combined with the low-molecular-weight selenium-containing component stream, followed by nanofiltration washing, ethanol precipitation, and drying: The second liquid phase is separated by microfiltration, first ultrafiltration, and second ultrafiltration as described in Example 1, and the second ultrafiltration retentate is collected. The second ultrafiltration retentate is combined with the low-molecular-weight selenium-containing component stream and fed into a nanofiltration membrane system with a molecular weight cutoff of 300 Da for nanofiltration washing. The washing method is constant volume washing, and the volume of washing water is 4 times the combined liquid volume. The nanofiltration membrane retentate is collected. Anhydrous ethanol is slowly added to the obtained concentrate to achieve a final ethanol concentration of 75%, and then the mixture is allowed to stand at 4°C for 12 hours. After standing, the precipitate is collected, washed twice with 95% ethanol, and then freeze-dried to constant weight to obtain 139.5 g of product.

[0064] Comparative Example 4 provides a method for extracting selenium-containing polysaccharides from Eucommia ulmoides. Except for omitting the segmented enzymatic hydrolysis and the first solid phase entering the second stage of enzymatic hydrolysis, the subsequent separation and recovery conditions are kept as consistent as possible with those in Example 1.

[0065] S1. Constructing the extraction system: Weigh 1000g of cleaned, dried, pulverized and passed through a 40-mesh sieve of Eucommia ulmoides leaf powder, add 15000mL of citric acid-sodium citrate buffer solution, adjust the pH of the system to 5.0, stir and mix for 20min to obtain the extraction system.

[0066] S2. Perform single-stage enzymatic hydrolysis, inactivation, and solid-liquid separation: Add 8g of pectinase, 12g of hemicellulase, 15g of cellulase, and 10g of protease to the extraction system, and hydrolyze at 50℃ for 4 hours to obtain the hydrolysate. Then, heat the hydrolysate to 90℃ and maintain it for 12 minutes for inactivation. After inactivation, cool it to below 40℃, filter it through a filter cloth, and then centrifuge it at 5000r / min for 15 minutes. Collect the supernatant as the extract.

[0067] S3. The extract was subjected to microfiltration, ultrafiltration, nanofiltration washing, ethanol precipitation, and drying: The extract was first treated with a microfiltration membrane with a pore size of 0.22 μm to obtain the microfiltration permeate; then, the microfiltration permeate was fed into an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa, and the ultrafiltration retentate was collected; then, the ultrafiltration retentate was fed into a nanofiltration membrane system with a molecular weight cutoff of 300 Da for nanofiltration washing using a constant volume washing method, with the volume of washing water being 4 times the volume of the ultrafiltration retentate, and the nanofiltration retentate was collected. Anhydrous ethanol was slowly added to the resulting concentrate to bring the final ethanol concentration of the system to 75%, and then the mixture was allowed to stand at 4°C for 12 h. After standing, the precipitate was collected, washed twice with 95% ethanol, and then freeze-dried to constant weight to obtain 130.4 g of product.

[0068] The products obtained in Examples 1-3 and Comparative Examples 1-4 were analyzed. The analysis included the main distribution range of apparent molecular weight, the proportion of components with an apparent molecular weight of 8000-50000 Da in the total polysaccharide composition, total selenium content, the proportion of inorganic selenium content in the total selenium content, polysaccharide mass fraction, protein mass fraction, and ash content. Simultaneously, gel permeation chromatography was performed on the low-molecular-weight selenium-containing component streams from Examples 1-3 and the finally obtained *Cyclocarya paliurus*-bound selenium polysaccharide to compare the compositional differences between the initial effluent and the target recovered component. The results are shown in Tables 1 and 2.

[0069] Table 1. Detection results of the products obtained in the examples and comparative examples.

[0070] Table 2 Comparison of molecular weight distribution of low molecular weight selenium-containing components and final products

[0071] As shown in Table 1, the main distribution ranges of the apparent molecular weight of the products obtained in Examples 1 to 3 are 5000~80000 Da, 5000~80000 Da, and 15000~80000 Da, respectively. The proportions of the components with an apparent molecular weight of 8000~50000 Da in the total polysaccharide components are 68.7%, 72.9%, and 74.1%, respectively, which are significantly higher than those in Comparative Examples 1 to 4. Meanwhile, the polysaccharide mass fractions of the products obtained in Examples 1-3 were 78.4%, 81.6%, and 82.1%, respectively; the protein mass fractions were 3.2%, 2.7%, and 2.5%, respectively; and the ash content was 2.8%, 2.3%, and 2.1%, respectively. In contrast, the polysaccharide mass fractions of the products obtained in Comparative Examples 1-4 were 69.2%, 70.5%, 66.8%, and 64.9%, respectively; the protein mass fractions were 4.8%, 4.1%, 4.6%, and 5.1%, respectively; and the ash content was 4.4%, 3.9%, 4.7%, and 4.9%, respectively. These results indicate that by employing the segmented enzymatic hydrolysis-ladder membrane separation extraction pathway described in this invention, the proportion of components within the target molecular weight range in the obtained products is increased, the polysaccharide mass fraction is increased, and the protein mass fraction and ash content are decreased, resulting in a more concentrated product composition.

[0072] As shown in Table 1, the total selenium content of the products obtained in Examples 1 to 3 was 0.18%, 0.21%, and 0.20%, respectively, and the proportion of inorganic selenium content to the total selenium content was 11.6%, 9.8%, and 10.3%, respectively. In contrast, the total selenium content of the products obtained in Comparative Examples 1 to 4 was 0.17%, 0.16%, 0.17%, and 0.14%, respectively, and the proportion of inorganic selenium content to the total selenium content was 18.9%, 16.8%, 19.2%, and 17.6%, respectively. These results indicate that under the conditions of forming a low-molecular-weight selenium-containing component stream in the first liquid phase and preventing it from entering the subsequent ethanol precipitation and recovery step, while simultaneously allowing the second liquid phase to undergo first and second ultrafiltration to form the target component liquid, the proportion of inorganic selenium content to the total selenium content in the obtained products decreases, and the total selenium content remains at a high level.

[0073] As shown in Table 2, the main distribution range of the low molecular weight selenium-containing component streams in Examples 1 to 3 is all below 3000 Da, while the main distribution range of the finally obtained *Cyclocarya paliurus*-bound selenium polysaccharide is located at 5000~80000 Da, 5000~80000 Da, and 15000~80000 Da, respectively. This result indicates that, under the technical solution described in this invention, the low molecular weight selenium-containing component stream formed by nanofiltration membrane separation of the first liquid phase and the target component liquid formed by step-membrane separation of the second liquid phase have significant differences in apparent molecular weight distribution, and the components discharged in the first stage and the target recovered components in the second stage can be distinguished.

[0074] Based on the results of Comparative Example 1 in Table 1, when the first liquid phase does not form a low-molecular-weight selenium-containing component stream separately, but instead enters the subsequent recovery path together with the washing liquid and the second liquid phase, the proportion of the 8000~50000 Da range component in the obtained product decreases, while the proportion of inorganic selenium content in the total selenium content, protein mass fraction, and ash content all increase. Based on the results of Comparative Example 2, when the second liquid phase undergoes nanofiltration washing and ethanol precipitation directly without first and second ultrafiltration separation, the apparent molecular weight distribution of the obtained product becomes significantly wider, and the proportion of the target molecular weight range component decreases. Based on the results of Comparative Example 3, even if a low-molecular-weight selenium-containing component stream has already been formed in the initial stage, if it is reintegrated into the subsequent recovery path, the low-molecular-weight segment component in the obtained product increases, while the polysaccharide mass fraction decreases and the inorganic selenium proportion increases. Based on the results of Comparative Example 4, if the overall process is changed to single-stage enzymatic hydrolysis, followed by direct subsequent membrane separation and recovery, the obtained product is lower than that of Examples 1-3 in terms of molecular weight concentration, polysaccharide mass fraction, and inorganic selenium proportion.

[0075] Further comparison of Examples 1 to 3 shows that, within the scope of the present invention, after adjusting the first-stage diversion conditions or the second-stage step-membrane separation window, the obtained product still maintains a high concentration of the target components. Specifically, Example 2, by adjusting the first-stage enzymatic hydrolysis parameters, the first liquid-phase nanofiltration membrane separation conditions, and the second-stage enzymatic hydrolysis parameters, increased the proportion of components in the 8000-50000 Da range in the obtained product to 72.9%, increased the total selenium content to 0.21%, decreased the proportion of inorganic selenium content to 9.8% of the total selenium content, and increased the polysaccharide mass fraction to 81.6%. Example 3, by adjusting the second-stage step-membrane separation window, further concentrated the main distribution range of the obtained product in the 15000-80000 Da range, increased the proportion of components in the 8000-50000 Da range to 74.1%, increased the polysaccharide mass fraction to 82.1%, and decreased the protein mass fraction and ash content to 2.5% and 2.1%, respectively. This indicates that, under the premise that the overall process path described in this invention remains unchanged, by adjusting the upstream diversion conditions or the downstream membrane separation window, the compositional characteristics of the target component solution can be further improved, and the final obtained Cyclocarya paliurus-bound selenium polysaccharide can achieve more favorable detection results in terms of molecular weight distribution, polysaccharide mass fraction, and impurity control.

[0076] In summary, this invention, by forming a low-molecular-weight selenium-containing component stream in the first liquid phase and excluding it from the subsequent ethanol precipitation and recovery steps, while allowing the first solid phase to continue into the second stage of enzymatic hydrolysis, and by subjecting the second liquid phase to first and second ultrafiltration to form the target component solution, followed by nanofiltration and ethanol precipitation to obtain the final product, enables the final obtained *Cyclocarya paliurus* bound selenium polysaccharide to form a more concentrated molecular weight distribution within the target molecular weight range, increasing the proportion of components in the 8000~50000 Da range, increasing the polysaccharide mass fraction, reducing the proportion of inorganic selenium content in the total selenium content, and reducing protein mass fraction and ash content.

[0077] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for segmented enzymatic hydrolysis-step-membrane separation and extraction of *Cyclocarya paliurus*-bound selenium polysaccharides, characterized in that, Includes the following steps: S1. Dry and pulverize the raw material of *Cyclocarya paliurus* leaves to obtain *Cyclocarya paliurus* leaf powder, and mix the *Cyclocarya paliurus* leaf powder with water or buffer solution to form a first extraction system; S2. Perform a first-stage enzymatic hydrolysis on the first extraction system, and sequentially perform a first-stage inactivation and a first-stage solid-liquid separation on the first-stage hydrolysate to obtain a first liquid phase and a first solid phase; S3. Separate the first liquid phase through a nanofiltration membrane, and collect its permeate as a low-molecular-weight selenium-containing component stream. The low-molecular-weight selenium-containing component stream is a liquid phase component with an apparent molecular weight of no more than 3000 Da as measured by gel permeation chromatography, and does not enter the subsequent alcohol precipitation recovery step; S4. The first solid phase is mixed with water or buffer solution to form a second extraction system. The second extraction system is subjected to a second-stage enzymatic hydrolysis, and the second-stage enzymatic hydrolysate is subjected to a second-stage inactivation and a second-stage solid-liquid separation to obtain a second liquid phase. S5. The second liquid phase is subjected to microfiltration, a first ultrafiltration, and a second ultrafiltration. The liquid phase obtained by the first ultrafiltration permeate and the second ultrafiltration is collected as the target component liquid. The target component liquid is a liquid phase component with an apparent molecular weight of 5000~80000 Da as determined by gel permeation chromatography. S6. The target component liquid is washed by nanofiltration, ethanol is added, and after standing, the precipitate is collected and dried to obtain Cyclocarya paliurus-bound selenium polysaccharide.

2. The extraction method according to claim 1, characterized in that, In step S2, the pH of the first stage of enzymatic hydrolysis is 3.5~5.5, the temperature is 35~50℃, and the time is 0.5~3h. The first stage of enzymatic hydrolysis uses pectinase, hemicellulase, or a first composite enzyme composed of pectinase and hemicellulase. The total amount of enzyme added is 0.1%~3.0% of the mass of Eucommia ulmoides leaf powder. The inactivation conditions of the first stage are 80~95℃ for 5~20min.

3. The extraction method according to claim 1, characterized in that, The nanofiltration membrane in step S3 has a molecular weight cutoff of 150~1000 Da, and the low molecular weight selenium-containing component stream is the permeate separated by the nanofiltration membrane, or it is a combined liquid of the permeate and the washing permeate obtained when a constant volume washing method is used in the nanofiltration membrane separation process.

4. The extraction method according to claim 1, characterized in that, Between steps S3 and S4, there is also a first solid phase washing step, in which the first solid phase is washed 1 to 5 times with water or an aqueous solution of ethanol with a volume fraction of 5% to 30%. The washing liquid is incorporated into the low molecular weight selenium-containing component stream, and the washing liquid does not enter the subsequent target component liquid recovery step.

5. The extraction method according to claim 1, characterized in that, In step S4, the pH of the second stage of enzymatic hydrolysis is 4.5~7.5, the temperature is 45~60℃, and the time is 1~5h. The second stage of enzymatic hydrolysis uses cellulase, protease, or a second complex enzyme composed of cellulase and protease. The total amount of enzyme added is 0.2%~5.0% of the mass of the first solid phase. The inactivation conditions of the second stage are 85~100℃ for 5~30min.

6. The extraction method according to claim 1, characterized in that, The microfiltration in step S5 has a pore size of 0.05~0.45μm, the first ultrafiltration has a molecular weight cutoff of 30~80kDa, the second ultrafiltration has a molecular weight cutoff of 8~20kDa, and the components with an apparent molecular weight of 8000~50000Da in the target component solution account for more than 60% of the total polysaccharide components in the target component solution.

7. The extraction method according to claim 1, characterized in that, The nanofiltration washing in step S6 is performed using a nanofiltration membrane with a molecular weight cutoff of 150~500 Da, and a constant volume washing method is adopted. The volume of washing water is 1~8 times the volume of the target component liquid. After the ethanol is added, the final ethanol concentration of the system is 60%~90%. The standing temperature is 2~10℃ and the standing time is 4~24h. The drying is performed by freeze drying or vacuum drying.

8. A selenium polysaccharide bound to Eucommia ulmoides, characterized in that, The *Cyclocarya paliurus*-bound selenium polysaccharide is prepared by the method described in any one of claims 1 to 7, and the apparent molecular weight measured by gel permeation chromatography is 5000 to 80000 Da, the total selenium content is 0.02% to 1.00%, the polysaccharide mass fraction measured by the anthrone-sulfuric acid method is 55% to 95%, and the inorganic selenium content accounts for no more than 20% of the total selenium content.

9. The *Cyclocarya paliurus*-bound selenium polysaccharide according to claim 8, characterized in that, In the *Cyclocarya paliurus*-bound selenium polysaccharide, the component with an apparent molecular weight of 8000~50000 Da accounts for more than 60% of the total polysaccharide component, the protein content is 0.1%~8.0%, and the ash content is not higher than 5.0%.

10. The application of the *Cyclocarya paliurus*-bound selenium polysaccharide according to claim 8 or 9 in the preparation of an encapsulated selenium-containing food composition, characterized in that... In the encapsulated selenium-containing food composition, the *Cyclocarya paliurus* bound selenium polysaccharide serves as the core material, and the wall material is selected from one or at least two of maltodextrin, gum arabic, resistant dextrin, and pectin. The mass ratio of the core material to the wall material is 1:1 to 1:20.

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