High-activity Chinese chive polysaccharide extraction method based on gradient enzymolysis-membrane separation combination
By using a gradient enzymatic hydrolysis-membrane separation method, the problems of low extraction efficiency and activity loss of leek polysaccharides were solved, achieving efficient and low-cost extraction of highly active polysaccharides, which is suitable for functional foods and pharmaceuticals.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-19
- Publication Date
- 2026-04-07
AI Technical Summary
In existing technologies, the extraction efficiency of leek polysaccharides is low, the purification process is complex, the activity is not well preserved, and high temperature or chemical reagent treatment leads to serious loss of antioxidant activity, making it difficult to meet industrial needs.
A gradient enzymatic hydrolysis-membrane separation method was adopted, including freeze-drying and pulverization, low- and high-intensity ultrasonic cell disruption, gradient enzymatic hydrolysis, and microfiltration and ultrafiltration treatment, to control the molecular weight range of 5~30kDa and retain highly active polysaccharide components.
It significantly improved the yield and purity of leek polysaccharides, shortened the extraction cycle, reduced energy consumption and reagent costs, and increased antioxidant activity by more than 40%, making it suitable for industrial production.
Abstract
Description
Technical Field
[0001] This invention relates to the fields of natural product extraction and biomedicine, specifically to a method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation. Background Technology
[0002] Leek polysaccharides are a class of natural active ingredients extracted from leeks. They are high-molecular-weight carbohydrates composed of multiple monosaccharide molecules. Studies have shown that they have the potential to activate immunity and have antioxidant properties, and have broad application prospects in the fields of functional foods, health products and medicines.
[0003] Existing methods for extracting polysaccharides from leeks suffer from low extraction efficiency, complex purification processes, and insufficient activity retention. For example, traditional water extraction and alcohol precipitation methods take 12-24 hours, yielding only 3-5% polysaccharide, and are prone to degradation of heat-sensitive polysaccharides. Single-column chromatography (such as DEAE-cellulose column) has a long purification cycle of 3-5 days, with purity less than 60%, which is insufficient to meet the industrialization needs of high-activity polysaccharides. High-temperature extraction (such as reflux extraction) or chemical reagent-assisted extraction (such as acid / alkali) can destroy the glycosidic bond structure of polysaccharides, resulting in a loss of more than 30% of antioxidant activity, thus limiting industrial applications.
[0004] Therefore, this invention proposes a method for extracting leek polysaccharides. Through this extraction method, not only can the yield and purity of the obtained leek polysaccharides be effectively improved, but the activity of the leek polysaccharides is also significantly preserved, which has broad application prospects. Summary of the Invention
[0005] The purpose of this invention is to provide a highly active leek polysaccharide extraction method based on gradient enzymatic hydrolysis-membrane separation, in order to solve the problems of long extraction time and severe loss of antioxidant activity of leek polysaccharides obtained by existing water extraction and alcohol precipitation methods, as well as high temperature and acid-base treatment.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation, comprising the following steps: S1. Take fresh, clean chives, freeze-dry them, then grind and sift them to obtain dried chive powder; S2. The dried chive powder from S1 is pulverized and then subjected to cell wall breaking treatment to obtain cell wall breaking liquid; the cell wall breaking liquid is subjected to enzymatic hydrolysis to obtain enzymatic hydrolysate. The cell wall disruption described herein is a primary cell wall disruption process using low-intensity ultrasound combined with cellulase, and a secondary cell wall disruption process using high-intensity ultrasound. The enzymatic hydrolysis is a process of gradient enzymatic hydrolysis of the cell wall disruption solution; the gradient enzymatic hydrolysis is a first enzymatic hydrolysis process at a first temperature and a first degrading enzyme, and a second enzymatic hydrolysis process at a second temperature and a second degrading enzyme. S3. The S2 enzymatic hydrolysate is subjected to membrane treatment and freeze-drying to obtain highly active leek polysaccharide; The membrane treatment described is a combined process of microfiltration and ultrafiltration.
[0007] Furthermore, in S1, the freeze-drying temperature is -30℃ to -50℃, the vacuum degree is 0.05Mpa to 0.1Mpa, and the freeze-drying time is 12h to 24h.
[0008] Furthermore, in S1, the material is pulverized and then passed through an 80-100 mesh sieve.
[0009] Furthermore, in step S2, the dried leek powder is mixed with distilled water to form a slurry, and the mass-volume ratio of the dried leek powder to the distilled water is 1:10~30, g / mL.
[0010] Furthermore, in S2, during the first cell wall breaking process, the temperature is 50±5℃, the amount of cellulase added is 0.1%~0.3% of the mass of the dried leek powder, the power of low-intensity ultrasound is 40W~60W, the frequency is 20kHz~25kHz, and the processing time is 40min~60min; during the second cell wall breaking process, the intensity of high-intensity ultrasound is 200W~400W, the power is 30kHz~50kHz, and the processing time is 20min~30min; during both the first and second cell wall breaking processes, the stirring intensity is 200~400rpm.
[0011] Furthermore, in S2, the first temperature is 45±2℃, and the first degrading enzyme is a complex enzyme of cellulase, pectinase, and protease; the second temperature is 50±2℃, and the second degrading enzyme is protease.
[0012] Furthermore, in the first enzymatic hydrolysis process, the amount of compound enzyme added is 1-3% of the mass of dried leek powder, the mass ratio of cellulase:pectinase:protease in the compound enzyme is 3:2:1, the pH is 5.0±0.2, and the hydrolysis time is 80min-120min; in the second enzymatic hydrolysis process, the amount of protease added is 0.5%-1% of the mass of dried leek powder, the pH is 6.0±0.2, and the hydrolysis time is 40min-80min.
[0013] Furthermore, in step S2, after the secondary enzymatic hydrolysis process is completed, the solution is heated in a water bath at 70℃~80℃ for 10min~30min to obtain the enzymatic hydrolysate.
[0014] Furthermore, in S3, the microfiltration process uses an alumina ceramic membrane with a diameter of 0.22 μm, an operating pressure of 0.3 ± 0.2 MPa, and an operating temperature of 40 ± 2 °C; the ultrafiltration process uses polyethersulfone membranes with molecular weight cutoffs of 30 kDa and 5 kDa, respectively, with an operating pressure of 0.2 ± 0.2 MPa and an operating temperature of 35 ± 2 °C.
[0015] Furthermore, in S3, the freeze-drying process involves pre-freezing the membrane-treated liquid at -50℃±5℃ for 2~4 hours, followed by sublimation drying at a vacuum of 0.01~0.05MPa for 12~24 hours.
[0016] The beneficial effects of this invention are: 1. The method of the present invention has significantly improved efficiency, with the yield of leek polysaccharide reaching more than 8% and the purity reaching more than 90%. Membrane separation technology replaces column chromatography, shortening the extraction cycle by about 60%, avoiding organic solvent pollution, reducing reagent costs by 50%, and reducing energy consumption by 25%, making it suitable for industrial continuous production. 2. By employing a dual-cell wall breaking and dual-enzyme hydrolysis strategy, we achieve efficient cell wall breaking and targeted degradation of cellulose, pectin, and protein, thus solving the problems of low cell wall breaking rate, low efficiency of single-enzyme hydrolysis, and high impurity residue in traditional methods. 3. By controlling the molecular weight range of ultrafiltration to 5~30kDa, highly active polysaccharide components are retained in a targeted manner, and the scavenging rate of DPPH free radicals and ABTS free radicals reaches 90~95%, and its antioxidant activity is improved by more than 40% compared with the existing technology (p<0.01). Detailed Implementation
[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
[0018] The principle of this invention is as follows: This invention employs freeze-drying, pulverization, cell wall disruption, enzymatic hydrolysis, and membrane treatment to achieve highly active extraction of leek polysaccharides from leeks.
[0019] Fresh chives are washed, freeze-dried, and then pulverized. The particle size of the pulverized chives is controlled to be 80-100 mesh, which helps in the subsequent cell wall breaking and enzymatic hydrolysis processes.
[0020] In the method of this invention, the cell wall breaking adopts a two-stage breaking process, which can improve the cell wall breaking rate to over 95%. The first breaking process is a pretreatment process of low-intensity ultrasound combined with cellulase, with an ultrasound intensity of 40-60W. The second breaking process is a rapid breaking process under high-intensity ultrasound, with an ultrasound intensity of 200-400W. After the breaking process, the cell wall breaking rate can reach over 95%. The enzymatic hydrolysis adopts a two-stage enzymatic hydrolysis process. By controlling different types of enzymes and different temperatures, the efficiency of enzymatic hydrolysis is further improved. The first enzymatic hydrolysis uses cellulase, pectinase, and protease, while the second enzymatic hydrolysis uses protease. The two-stage enzymatic hydrolysis further removes proteins and can effectively prevent proteins from forming complexes with polysaccharides, thereby improving the product yield.
[0021] In the method of this invention, the enzymatic hydrolysate is treated by microfiltration and ultrafiltration, which can effectively obtain leek polysaccharides with a specific molecular weight range. It has been verified that leek polysaccharides with a molecular weight range of 5~30kDa have strong antioxidant activity and have broad application prospects.
[0022] Example 1 In this embodiment, the process of the present invention is used to extract leek polysaccharides, as detailed below: 1. Raw material pretreatment Take 1 kg of fresh chives, wash them with clean water, freeze-dry them at -40℃ and 0.08 MPa vacuum for 24 hours, pulverize them and pass them through a 100-mesh sieve to obtain dried chive powder with a moisture content of 3.2%.
[0023] 2. Cell wall disruption and enzymatic hydrolysis treatment Take 100g of dried leek powder, mix the powder with distilled water at a ratio of 1:20 (g / mL), heat to 45℃ while stirring at 200rpm, then add 0.1g of cellulase, and place in an ultrasonic instrument. First, treat at 50W and 20kHz for 60min, then continue to treat at 300W and 40kHz for 20min to obtain a cell wall disruption solution. The cell wall disruption rate in the solution reached 97.2%.
[0024] Add a complex enzyme of cellulase, pectinase, and protease to the above cell wall-breaking solution, with a cellulase:pectinase:protease ratio of 3:2:1 (mass ratio), and a total complex enzyme addition of 1.5g. Perform enzymatic hydrolysis at 45℃ and pH 5.0 for 90min. Then, raise the temperature to 50℃, adjust the pH to 6.0, and add another 0.5g of protease, continuing enzymatic hydrolysis for another 60min. After hydrolysis, heat in a 70℃ water bath for 15min to ensure an enzyme inactivation rate >99%, obtaining the enzymatic hydrolysate.
[0025] In this embodiment, cellulase (5000 U / g), pectinase (3000 U / g), and protease (2000 U / g) were all commercially available (Beijing Solarbio Science & Technology Co., Ltd.). The cellulase mainly consists of exo-β-glucanase, endo-β-glucanase, and β-glucosidase, thus solving the problem of incomplete removal of non-cellulose impurities (such as proteins and pectins) by single enzymatic hydrolysis.
[0026] 3. Membrane separation The above enzymatic hydrolysate was microfiltered through a 0.22 μm ceramic membrane (alumina material, operating pressure 0.3 MPa, temperature 40 °C) to obtain a microfiltration permeate with a transmittance greater than 95%. The microfiltration permeate was then subjected to ultrafiltration through 30 kDa and 5 kDa polyethersulfone (PES) membranes (the operating pressure for both ultrafiltration stages was 0.2 MPa, temperature 35 °C). The ultrafiltration permeate obtained after treatment with the 30 kDa PES membrane was then treated with a 5 kDa PES membrane to obtain the ultrafiltration retentate.
[0027] 4. Freeze-drying The ultrafiltration retentate was pre-frozen at -50℃ for 2 hours, and then sublimated and dried under a vacuum of 0.01 MPa for 12 hours to obtain a white flocculent polysaccharide powder. After freeze-drying, 10.8 g of leek polysaccharide powder was obtained, with a water content of 2.2%, an oven-dry purity of 92.5%, a pure product yield of 9.77%, a DPPH free radical scavenging rate of 94.7% (1 mL of 0.1 mmol / L DPPH free radical solution + 1 mL of 1.0 mg / mL leek polysaccharide solution), and an ABTS free radical scavenging rate of 92.4% (1 mL of 0.1 mmol / L ABTS free radical solution + 1 mL of 0.5 mg / mL leek polysaccharide solution). Accelerated experiments (40℃, RH 75%) showed that the polysaccharide powder retained >90% of its activity within 6 months, meeting the requirements for drug stability. In terms of immunomodulation, this polysaccharide can significantly increase the spleen index of cyclophosphamide-induced immunosuppressed mice (by 1.8 times).
[0028] 5. Membrane cleaning and regeneration Flux recovery rate of membrane is >90% when circulated and flushed with 0.5% NaOH solution for 30 minutes, extending membrane life to more than 50 cycles.
[0029] Comparative Example 1 In this comparative example, the traditional water extraction and alcohol precipitation method was used to extract leek polysaccharides.
[0030] Using 100g of dried leek powder from Example 1, the extract was obtained by water bath extraction at 80℃ for 2 hours. Ethanol was added to the extract to a concentration of 70% to precipitate polysaccharides. The crude product yield was 4.1%, the purity was 68.3%, and the DPPH free radical scavenging rate was 52%, which was significantly lower than that of the process of this invention.
[0031] Testing revealed that the leek polysaccharide obtained in Example 1 of this invention contained 20% glucuronic acid. Compared with the leek polysaccharide obtained in the comparative example, in vitro experiments showed that it increased the NO release of RAW264.7 macrophages by 3.2 times, confirming that its immunomodulatory activity was significantly enhanced.
[0032] Comparative Example 2 In this comparative example, the steps of "heating to 45°C under stirring at 200 rpm, then adding 0.1 g of cellulase, and placing it in an ultrasonic instrument for treatment at 50 W and 20 kHz for 60 min" in Example 1 are omitted. Instead, the treatment is carried out directly at 300 W and 40 kHz for 20 min, and other operations are the same as in Example 1.
[0033] In this comparative example, 9.1 g of leek polysaccharide was obtained, with a moisture content of 2.5%, an oven-dry purity of 92.3%, a pure product yield of 8.20%, and a DPPH free radical scavenging rate of 86.4%.
[0034] It can be seen that by omitting the corresponding steps in Example 1, the yield of leek polysaccharide and the DPPH free radical scavenging rate both decreased significantly.
[0035] Comparative Example 3 In this comparative example, the gradient enzymatic hydrolysis step in Example 1 is omitted, that is, the step of "then raising the temperature to 50°C, adjusting the pH to 6.0, adding 0.5g of protease, and continuing enzymatic hydrolysis for 60min" is omitted. Other operations are the same as in Example 1.
[0036] In this comparative example, 6.5g of leek polysaccharide was obtained, with a moisture content of 2.4%, an oven-dry purity of 92.6%, a pure product yield of 5.87%, and a DPPH free radical scavenging rate of 64.8%.
[0037] Comparative Example 4 In this comparative example, the activity of the obtained leek polysaccharide was investigated when ultrafiltration was used with different molecular weight cutoffs; other steps were the same as in Example 1.
[0038] When the molecular weight cutoff of ultrafiltration is less than 5 kDa (the first ultrafiltration treatment with a molecular weight cutoff of 5 kDa, and the permeate is collected), the DPPH radical scavenging rate is 45%; when the molecular weight cutoff of ultrafiltration is 30~100 kDa (same as the secondary ultrafiltration treatment in Example 1, the first ultrafiltration treatment with a molecular weight cutoff of 100 kDa, and the permeate is collected, the second ultrafiltration treatment with a molecular weight cutoff of 30 kDa, and the tack liquid is collected), the DPPH radical scavenging rate is 62%; when the molecular weight cutoff of ultrafiltration is greater than 100 kDa (same as the secondary ultrafiltration treatment in Example 1, the first ultrafiltration treatment with a molecular weight cutoff greater than 100 kDa, and the permeate is collected, the second ultrafiltration treatment with a molecular weight cutoff of 100 kDa, and the tack liquid is collected), the DPPH radical scavenging rate is 38%.
[0039] It is evident that the leek polysaccharides within the molecular weight range of this invention possess excellent free radical scavenging capabilities.
[0040] This invention is not limited to the preferred embodiments described above. Anyone can derive other forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.
Claims
1. A method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation, characterized in that, Includes the following steps: S1. Take fresh, clean chives, freeze-dry them, then grind and sift them to obtain dried chive powder; S2. The dried chive powder from S1 is pulverized and then subjected to cell wall breaking treatment to obtain cell wall breaking liquid; the cell wall breaking liquid is subjected to enzymatic hydrolysis to obtain enzymatic hydrolysate. The cell wall disruption described herein is a primary cell wall disruption process using low-intensity ultrasound combined with cellulase, and a secondary cell wall disruption process using high-intensity ultrasound. The enzymatic hydrolysis is a process of gradient enzymatic hydrolysis of the cell wall disruption solution; the gradient enzymatic hydrolysis is a first enzymatic hydrolysis process at a first temperature and a first degrading enzyme, and a second enzymatic hydrolysis process at a second temperature and a second degrading enzyme. S3. The S2 enzymatic hydrolysate is subjected to membrane treatment and freeze-drying to obtain highly active leek polysaccharide; The membrane treatment described is a combined process of microfiltration and ultrafiltration.
2. The method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation according to claim 1, characterized in that: In S1, the freeze-drying temperature is -30℃ to -50℃, the vacuum degree is 0.05Mpa to 0.1Mpa, and the freeze-drying time is 12h to 24h.
3. The method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation according to claim 1, characterized in that: In S1, the material is pulverized and then passed through an 80-100 mesh sieve.
4. The method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation according to claim 1, characterized in that: In step S2, dried leek powder is mixed with distilled water to make a slurry. The mass-volume ratio of dried leek powder to distilled water is 1:10~30, g / mL.
5. The method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation according to claim 1, characterized in that: In S2, during the first cell wall breaking process, the temperature is 50±5℃, the amount of cellulase added is 0.1%~0.3% of the mass of the dried leek powder, the power of low-intensity ultrasound is 40W~60W, the frequency is 20kHz~25kHz, and the processing time is 40min~60min; during the second cell wall breaking process, the intensity of high-intensity ultrasound is 200W~400W, the power is 30kHz~50kHz, and the processing time is 20min~30min; during both the first and second cell wall breaking processes, the stirring intensity is 200~400rpm.
6. The method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation according to claim 1, characterized in that: In S2, the first temperature is 45±2℃, and the first degrading enzyme is a complex enzyme of cellulase, pectinase and protease; the second temperature is 50±2℃, and the second degrading enzyme is protease.
7. The method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation according to claim 6, characterized in that: In the first enzymatic hydrolysis process, the amount of compound enzyme added is 1-3% of the mass of dried leek powder, the mass ratio of cellulase:pectinase:protease in the compound enzyme is 3:2:1, the pH is 5.0±0.2, and the hydrolysis time is 80min-120min; in the second enzymatic hydrolysis process, the amount of protease added is 0.5%-1% of the mass of dried leek powder, the pH is 6.0±0.2, and the hydrolysis time is 40min-80min.
8. The method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation according to claim 1, characterized in that: In step S2, after the secondary enzymatic hydrolysis process is completed, the solution is heated in a water bath at 70℃~80℃ for 10min~30min to obtain the enzymatic hydrolysate.
9. The method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation according to claim 1, characterized in that: In S3, the microfiltration process uses an alumina ceramic membrane with a diameter of 0.22 μm, an operating pressure of 0.3 ± 0.2 MPa, and an operating temperature of 40 ± 2 °C; the ultrafiltration process uses polyethersulfone membranes with molecular weight cutoffs of 30 kDa and 5 kDa, respectively, with an operating pressure of 0.2 ± 0.2 MPa and an operating temperature of 35 ± 2 °C.
10. The method for extracting highly active leek polysaccharides based on gradient enzymatic hydrolysis-membrane separation according to claim 1, characterized in that: In S3, the freeze-drying process involves pre-freezing the membrane-treated liquid at -50℃±5℃ for 2-4 hours, followed by sublimation drying at a vacuum of 0.01-0.05MPa for 12-24 hours.