A method for refining active polysaccharides of fomes fomentarius based on simulated moving bed chromatography

Abstract

A method for purifying active polysaccharides from *Fodium argentea* based on simulated moving bed chromatography is disclosed, relating to the food industry. The method includes the following steps: (1) extracting *Fodium argentea* to obtain a polysaccharide extract, and removing small-molecule sugars using membrane filtration; (2) fractionating and purifying the polysaccharide extract using simulated moving bed chromatography to obtain a purified solution; (3) concentrating and freeze-drying the purified solution after purification and impurity removal to obtain the purified active polysaccharides from *Fodium argentea*. The active polysaccharides from *Fodium argentea* obtained using this method exhibit a purity of 80-90% and a recovery rate of 70-80%, respectively, and possess high activity. This method provides high product activity, high yield, high separation efficiency, and large throughput, significantly improving raw material utilization, enabling continuous operation, and reducing production costs.

Description

Technical Field

[0001] This invention belongs to the field of food industry, specifically relating to a simulated moving bed chromatography purification technology, and particularly to a technology for separating and purifying polysaccharides from *Phellinus truncatus* using simulated moving bed chromatography. Background Technology

[0002] *Fodium lauricularis*, also known as *Fodium fimbriatum*, belongs to the Basidiomycetes class and is a higher medicinal fungus belonging to the Polyporaceae family. It primarily parasitizes broad-leaved trees such as birch and poplar. It is abundant in Northeast, North, Southwest China, as well as Shaanxi, Xinjiang, Henan, and Guangxi provinces. Traditional pharmacopoeias record that due to its hard texture, it is often used as an acupuncture tool and can also be used to stop bleeding; a decoction can be used to dispel stagnation and blood stasis, or it can be boiled into a hemostatic ointment. Both the fruiting body and mycelium of *Fodium lauricularis* contain many bioactive substances, the most representative being *Fodium lauricularis* polysaccharide. In China and many other Asian countries, a decoction of *Fodium lauricularis* fruiting body is widely used to treat esophageal cancer, gastric cancer, oral ulcers, gastrointestinal disorders, inflammation, and various colds. Domestic and international research on *Fodium lauricularis* indicates that its polysaccharide has significant anti-fatigue, tumor cell growth inhibition, immune function enhancement, and antioxidant effects. Currently, there is limited research on the isolation and purification of polysaccharides from *Pheretima aspergillum*, and there is a lack of advanced extraction techniques, resulting in insufficient utilization of the polysaccharides and a waste of resources.

[0003] Chinese Patent 202211055989.7 discloses a method for extracting polysaccharides from *Lactarius deliciosus*, which involves adding cellulase, followed by ultrasonication and water bath protein removal. While the extraction method is simple and convenient, it fails to effectively remove impurities, resulting in a low polysaccharide extraction rate. Chinese Patent 201810086539.1 discloses a method for separating flavonoids from *Ginkgo biloba* leaf extract using a simulated moving bed chromatography. This patent employs an old-style simulated moving bed with eight columns, which not only wastes a large amount of eluent but also yields low purity and low yield of flavonoids. Chinese Patent 201811420403.6 discloses a method for purifying flavonoids from buckwheat hulls using simulated moving bed chromatography. This patent utilizes 20 columns divided into five zones for separation. Although the purity and yield of the obtained flavonoids are high, it uses a large amount of ethanol regenerant, and the excessively long columns affect the efficiency of separation and purification.

[0004] Currently, there is considerable research on polysaccharide extraction and purification methods, with commonly used methods including hot water extraction, ultrasonic extraction, surfactant-ultrasonic synergistic extraction, supercritical CO2 extraction, macroporous resin column chromatography, and high-speed countercurrent chromatography. However, existing methods suffer from high costs, low efficiency, unstable purity, and complex operation, making them unsuitable for industrial-scale production. There is an urgent need for efficient industrial-scale purification technology for *Fomitopsis pinnatifida* polysaccharides.

[0005] Simulated moving bed chromatography (SMB) is a highly efficient purification method. Compared to single-column chromatography, SMB offers higher resin utilization, larger feed rates, lower eluent usage, and continuous production capability. Building upon the advantages of moving bed chromatography, SMB retains the continuous operation and excellent separation performance while avoiding the difficulties associated with solid countercurrent. It features good separation efficiency and high production efficiency. This technology also boasts compact equipment, low investment costs, minimal environmental pollution, and ease of automation. Currently, there are no reports on research into the purification of *Polyporus cirrhosa* polysaccharides using simulated moving bed chromatography.

[0006] Against this backdrop, the inventors researched a technique for purifying polysaccharides from *Phellinus truncatula* using simulated moving bed chromatography, simplifying the operation steps, reducing production losses, achieving continuous production, improving production efficiency, and establishing an industrial method for purifying polysaccharides from *Phellinus truncatula*. Summary of the Invention

[0007] The purpose of this invention is to solve the problem of inefficient and continuous purification of *Phellinus cirrhosa* polysaccharides. A method for purifying *Phellinus cirrhosa* polysaccharides using simulated moving bed chromatography has been developed, comprising the following steps:

[0008] (1) Extraction of polysaccharides: Polysaccharides were extracted from *Fodium tumefaciens* using hot water extraction. 1000g of wild *Fodium tumefaciens* was accurately weighed and added to 70% ethanol at a material-to-liquid ratio of 1:15 (g / mL). The mixture was incubated in a water bath at 80℃ for 2 hours. After cooling, it was centrifuged at 4000r / min for 10 minutes, and the precipitate was dried. Ten times the volume of distilled water was added, and the mixture was heated and stirred at 85℃ for 3 hours. After cooling, it was centrifuged at 4000r / min for 10 minutes, and the supernatant was collected. The precipitate was extracted once more. The two extracts were combined after centrifugation, concentrated by rotary evaporation, and ethanol was added to bring the final ethanol concentration to 80%. Protein was removed by ethanol precipitation to obtain the *Fodium tumefaciens* polysaccharide extract.

[0009] (2) Primary purification of polysaccharides: The polysaccharide extract of *Fomitopsis fusiforme* was concentrated under reduced pressure, and after standing, concentrated precipitate a and concentrated liquid a were obtained. Concentrated liquid a was concentrated under reduced pressure again, and after standing, concentrated precipitate b and concentrated liquid b were obtained. Concentrated precipitate a and concentrated precipitate b were mixed to obtain concentrated precipitate c. Concentrated precipitate c was added to sodium hydroxide solution and stirred evenly. The precipitate was filtered to remove the precipitate. The product was combined with concentrated liquid a and concentrated liquid b. Then, protein acid K solution was added to it, and enzymatic hydrolysis was performed at 50-60℃ for 1-2 hours. Precipitation was added to sorbitol solution, and the supernatant was removed by centrifugation. The product was dried to obtain crude *Fomitopsis fusiforme* polysaccharide. Water was added to the crude *Fomitopsis fusiforme* polysaccharide, and ultrafiltration was performed using an ultrafiltration membrane. During the ultrafiltration process, the membrane was sealed to prevent oxygen from entering the permeate and the retentate. The final retentate was collected and concentrated under reduced pressure to obtain *Fomitopsis fusiforme* polysaccharide extract with small molecule sugars removed.

[0010] (3) Fractional purification of active polysaccharides: The polysaccharide extract of *Porphyra yezoensis* was fractionated and purified by simulated moving bed chromatography to obtain a purified solution;

[0011] The simulated mobile chromatography system comprises 24 columns, divided into five zones: adsorption, purification, desorption, regeneration, and washing. The number of columns in each zone is 10, 3, 3, 5, and 3 respectively. Each zone has one inlet and one outlet. The inlet of the adsorption and desorption zones is the first column in that zone, while the inlets of the purification, regeneration, and washing zones are the last columns in that zone. All zones are connected in series; the outlet of the washing zone is connected to the inlet of the desorption zone, and the outlet of the desorption zone is connected to the inlet of the purification zone. During operation, all zones work simultaneously. When the running time reaches the switching time, each column moves clockwise by one column position.

[0012] (4) Freeze-drying: After desalting, the refined liquid is concentrated and freeze-dried to obtain refined active polysaccharides of *Porphyra yezoensis*.

[0013] Furthermore, the feed solutions for the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are respectively the primary purified *Fomitopsis pinicola* active polysaccharide solution, 0.5 mol / L NaCl, 1.0 mol / L NaCl, 2.0 mol / L NaCl, and deionized water;

[0014] Furthermore, the flow rates of the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are 2-5 BV / h, 5-8 BV / h, 2-5 BV / h, 5-8 BV / h, and 7-10 BV / h, respectively.

[0015] Furthermore, the switching time is 1000s-1500s;

[0016] Furthermore, the stationary phase of the chromatographic column is DEAE-52 cellulose anion exchange resin, DEAE-agarose gel FF, etc.;

[0017] Furthermore, the operating temperature during the storage step is 25℃-35℃.

[0018] The present invention also provides a polysaccharide of *Pterocarpus styracifolius* prepared according to the above method.

[0019] The method for purifying *Fodium styracifolium* polysaccharides provided by this invention has low operating costs, high feed concentration, and yields *Fodium styracifolium* active polysaccharides with a purity of 80%–90% and a yield of 75%–85%. The immunomodulatory activity of RAW264.7 macrophages is increased by more than 30% compared to before fractionation. This method is pollution-free, reduces subsequent concentration costs, simplifies operation steps, enables continuous production, and improves production efficiency. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a flowchart illustrating the simulated moving bed chromatography purification process of the present invention. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] The simulated moving chromatography device used in this invention consists of 24 chromatographic columns, and the feed and discharge are controlled by a rotary valve.

[0028] The following examples illustrate the preparation method of the extract: Accurately weigh 1000g of wild *Fomitopsis pilosula*, add 70% ethanol at a material-to-liquid ratio of 1:15 (g / mL), incubate in a water bath at 80℃ for 2 hours, cool, centrifuge at 4000 rpm for 10 minutes, and dry the precipitate. Add ten times the volume of distilled water, heat and stir at 85℃ for 3 hours. After cooling, centrifuge at 4000 rpm for 10 minutes, collect the supernatant, and repeat the extraction once more. Combine the two extracts after centrifugation, concentrate by rotary evaporation, add ethanol to achieve a final ethanol concentration of 80%, and precipitate to remove proteins to obtain the *Fomitopsis pilosula* polysaccharide extract.

[0029] The primary purification method used in the following examples is as follows: The extract of *Fomitopsis pilosula* polysaccharide was concentrated under reduced pressure, and after standing, concentrated precipitate a and concentrated liquid a were obtained. Concentrated liquid a was concentrated again under reduced pressure, and after standing, concentrated precipitate b and concentrated liquid b were obtained. Concentrated precipitate a and concentrated precipitate b were mixed to obtain concentrated precipitate c. Concentrated precipitate c was added to sodium hydroxide solution and stirred evenly, filtered, and the precipitate was removed. The product was combined with concentrated liquid a and concentrated liquid b. Then, protein acid K solution was added, and enzymatic hydrolysis was performed at 50-60℃ for 1-2 hours. Precipitation was then performed in sorbitol solution, the supernatant was removed by centrifugation, and the product was dried to obtain crude *Fomitopsis pilosula* polysaccharide. Water was added to the crude *Fomitopsis pilosula* polysaccharide, and ultrafiltration was performed using an ultrafiltration membrane. During ultrafiltration, the membrane was sealed to prevent oxygen absorption, and permeate and retentate were obtained. The final retentate was collected and concentrated under reduced pressure to obtain *Fomitopsis pilosula* polysaccharide extract with small molecule sugars removed.

[0030] Example 1

[0031] (1) Extraction of polysaccharides: Polysaccharide extract of Porphyra yezoensis was obtained;

[0032] (2) Primary purification of polysaccharides: The polysaccharide extract of *Porphyra yezoensis* with small molecule sugars removed was obtained.

[0033] (3) Fractional purification of active polysaccharides: The polysaccharide extract of *Porphyra yezoensis* was fractionated and purified by simulated moving bed chromatography to obtain a purified solution;

[0034] The simulated mobile chromatography system comprises 24 columns, divided into five zones: adsorption, purification, desorption, regeneration, and washing. The number of columns in each zone is 10, 3, 3, 5, and 3 respectively. Each zone has one inlet and one outlet. The inlet of the adsorption and desorption zones is the first column in that zone, while the inlets of the purification, regeneration, and washing zones are the last columns in that zone. All zones are connected in series; the outlet of the washing zone is connected to the inlet of the desorption zone, and the outlet of the desorption zone is connected to the inlet of the purification zone. During operation, all zones work simultaneously. When the running time reaches the switching time, each column moves clockwise by one column position.

[0035] The feed solutions for the adsorption, purification, desorption, regeneration, and washing zones were respectively the primary purified *Fomitopsis pilosula* active polysaccharide solution, 0.5 mol / L NaCl, 1.0 mol / L NaCl, 2.0 mol / L NaCl, and deionized water; the flow rates for the adsorption, purification, desorption, regeneration, and washing zones were 2 BV / h, 5 BV / h, 2 BV / h, 5 BV / h, and 7 BV / h, respectively; the switching time was 1000 s; the stationary phase of the chromatographic column was DEAE-52 cellulose anion exchange resin; and the operating temperature was 25℃.

[0036] (4) Freeze-drying: After desalting, the refined liquid is concentrated and freeze-dried to obtain refined active polysaccharides of *Porphyra yezoensis*.

[0037] The active polysaccharide of *Pheretima aspergillum* prepared in this embodiment has a purity of 81% and a yield of 76%, and the immunomodulatory activity of RAW264.7 macrophages is increased by 35% compared with that before grading.

[0038] Example 2

[0039] (1) Extraction of polysaccharides: Polysaccharide extract of Porphyra yezoensis was obtained;

[0040] (2) Primary purification of polysaccharides: The polysaccharide extract of *Porphyra yezoensis* with small molecule sugars removed was obtained.

[0041] (3) Fractional purification of active polysaccharides: The polysaccharide extract of *Porphyra yezoensis* was fractionated and purified by simulated moving bed chromatography to obtain a purified solution;

[0042] The simulated mobile chromatography system comprises 24 columns, divided into five zones: adsorption, purification, desorption, regeneration, and washing. The number of columns in each zone is 10, 3, 3, 5, and 3 respectively. Each zone has one inlet and one outlet. The inlet of the adsorption and desorption zones is the first column in that zone, while the inlets of the purification, regeneration, and washing zones are the last columns in that zone. All zones are connected in series; the outlet of the washing zone is connected to the inlet of the desorption zone, and the outlet of the desorption zone is connected to the inlet of the purification zone. During operation, all zones work simultaneously. When the running time reaches the switching time, each column moves clockwise by one column position.

[0043] The feed solutions for the adsorption, purification, desorption, regeneration, and washing zones were respectively primary purified *Fomitopsis pilosula* active polysaccharide solution, 0.5 mol / L NaCl, 1.0 mol / L NaCl, 2.0 mol / L NaCl, and deionized water; the flow rates for the adsorption, purification, desorption, regeneration, and washing zones were 3 BV / h, 6 BV / h, 3 BV / h, 6 BV / h, and 8 BV / h, respectively; the switching time was 1200 s; the stationary phase of the chromatographic column was DEAE-52 cellulose anion exchange resin; and the operating temperature was 30℃.

[0044] (4) Freeze-drying: After desalting, the refined liquid is concentrated and freeze-dried to obtain refined active polysaccharides of *Porphyra yezoensis*.

[0045] The active polysaccharide of *Pheretima aspergillum* prepared in this embodiment has a purity of 85% and a yield of 80%, and the immunomodulatory activity of RAW264.7 macrophages is increased by 38% compared with that before grading.

[0046] Example 3

[0047] (1) Extraction of polysaccharides: Polysaccharide extract of Porphyra yezoensis was obtained;

[0048] (2) Primary purification of polysaccharides: The polysaccharide extract of *Porphyra yezoensis* with small molecule sugars removed was obtained.

[0049] (3) Fractional purification of active polysaccharides: The polysaccharide extract of *Porphyra yezoensis* was fractionated and purified by simulated moving bed chromatography to obtain a purified solution;

[0050] The simulated mobile chromatography system comprises 24 columns, divided into five zones: adsorption, purification, desorption, regeneration, and washing. The number of columns in each zone is 10, 3, 3, 5, and 3 respectively. Each zone has one inlet and one outlet. The inlet of the adsorption and desorption zones is the first column in that zone, while the inlets of the purification, regeneration, and washing zones are the last columns in that zone. All zones are connected in series; the outlet of the washing zone is connected to the inlet of the desorption zone, and the outlet of the desorption zone is connected to the inlet of the purification zone. During operation, all zones work simultaneously. When the running time reaches the switching time, each column moves clockwise by one column position.

[0051] The feed solutions for the adsorption, purification, desorption, regeneration, and washing zones were respectively primary purified *Fomitopsis pilosula* active polysaccharide solution, 0.5 mol / L NaCl, 1.0 mol / L NaCl, 2.0 mol / L NaCl, and deionized water; the flow rates for the adsorption, purification, desorption, regeneration, and washing zones were 5 BV / h, 8 BV / h, 5 BV / h, 8 BV / h, and 10 BV / h, respectively; the switching time was 1500 s; the stationary phase of the chromatographic column was DEAE-52 cellulose anion exchange resin; and the operating temperature was 35℃.

[0052] (4) Freeze-drying: After desalting, the refined liquid is concentrated and freeze-dried to obtain refined active polysaccharides of *Porphyra yezoensis*.

[0053] The active polysaccharide of *Pheretima aspergillum* prepared in this embodiment has a purity of 90% and a yield of 85%, and the immunomodulatory activity of RAW264.7 macrophages is increased by 45% compared with that before grading.

[0054] The core technology of this invention is the technique of purifying polysaccharides from *Potamogeton crispus* using simulated moving bed chromatography, which yields polysaccharides with high purity and yield. This process not only ensures high purity and high yield of polysaccharides, but also has distinctive features in terms of cost savings, simplified procedures, and continuous production.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for purifying active polysaccharides from *Pheretima aspergillum* based on simulated moving bed chromatography, comprising the following steps: (1) Extraction of polysaccharides: Polysaccharides were extracted from *Fodium tumefaciens* using hot water extraction. 1000g of wild *Fodium tumefaciens* was accurately weighed and added to 70% ethanol at a material-to-liquid ratio of 1:15 (g / mL). The mixture was incubated in a water bath at 80℃ for 2 hours. After cooling, it was centrifuged at 4000r / min for 10 minutes, and the precipitate was dried. Ten times the volume of distilled water was added, and the mixture was heated and stirred at 85℃ for 3 hours. After cooling, it was centrifuged at 4000r / min for 10 minutes, and the supernatant was collected. The precipitate was extracted once more. The two extracts were combined after centrifugation, concentrated by rotary evaporation, and ethanol was added to bring the final ethanol concentration to 80%. Protein was removed by ethanol precipitation to obtain the *Fodium tumefaciens* polysaccharide extract. (2) Primary purification of polysaccharides: The polysaccharide extract of *Porphyra yezoensis* was concentrated under reduced pressure, and after standing, concentrated precipitate a and concentrated liquid a were separated. Concentrate a again under reduced pressure, and after standing, separate the pulp to obtain concentrated precipitate b and concentrated solution b. Mix concentrated precipitate a and concentrated precipitate b to obtain concentrated precipitate c. Add concentrated precipitate c to sodium hydroxide solution and stir evenly. Filter to remove the precipitate, and combine the product with concentrated solution a and concentrated solution b. Then add protein acid K solution and enzymatically hydrolyze at 50-60℃ for 1-2 hours. Add sorbitol solution to precipitate, centrifuge to remove the supernatant, and dry to obtain crude *Polygonum cuspidatum* polysaccharide. Add water to the crude *Polygonum cuspidatum* polysaccharide and use an ultrafiltration membrane for ultrafiltration. During the ultrafiltration process, seal and isolate oxygen to obtain permeate and retentate. Collect the final retentate and concentrate under reduced pressure to obtain *Polygonum cuspidatum* polysaccharide extract with small molecule sugars removed. (3) Fractional purification of active polysaccharides: The polysaccharide extract of *Porphyra yezoensis* was fractionated and purified by simulated moving bed chromatography to obtain a purified solution; The simulated mobile chromatography system comprises 24 columns, divided into five zones: adsorption, purification, desorption, regeneration, and washing. The number of columns in each zone is 10, 3, 3, 5, and 3 respectively. Each zone has one inlet and one outlet. The inlet of the adsorption and desorption zones is the first column in that zone, while the inlets of the purification, regeneration, and washing zones are the last columns in that zone. All zones are connected in series; the outlet of the washing zone is connected to the inlet of the desorption zone, and the outlet of the desorption zone is connected to the inlet of the purification zone. During operation, all zones work simultaneously. When the running time reaches the switching time, each column moves clockwise by one column position. (4) Freeze-drying: After desalting, the refined liquid is concentrated and freeze-dried to obtain refined active polysaccharides of *Porphyra yezoensis*.

2. The method for purifying active polysaccharides from *Pheretima aspergillum* based on simulated moving bed chromatography according to claim 1, characterized in that... The feed solutions for the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are respectively the primary purified *Fomitopsis pinicola* active polysaccharide solution, 0.5 mol / L NaCl, 1.0 mol / L NaCl, 2.0 mol / L NaCl, and deionized water.

3. The method for purifying active polysaccharides from *Pheretima aspergillum* based on simulated moving bed chromatography according to claim 1, characterized in that... The flow rates in the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are 2-5 BV / h, 5-8 BV / h, 2-5 BV / h, 5-8 BV / h, and 7-10 BV / h, respectively.

4. The method for purifying active polysaccharides from *Pheretima aspergillum* based on simulated moving bed chromatography according to claim 1, characterized in that... The switching time is 1000s-1500s.

5. The method for purifying active polysaccharides from *Pheretima aspergillum* based on simulated moving bed chromatography according to claim 1, characterized in that... The purified active polysaccharide from *Pheretima aspergillum* had a purity of 80%–90% and a yield of 75%–85%. The immunomodulatory activity of RAW264.7 macrophages was increased by more than 30% compared with that before grading.

6. The method for purifying active polysaccharides from *Pheretima aspergillum* based on simulated moving bed chromatography according to claim 1, characterized in that... The stationary phase of the chromatographic column is DEAE-52 cellulose anion exchange resin, DEAE-agarose gel FF, etc.

7. The method for purifying active polysaccharides from *Pheretima aspergillum* based on simulated moving bed chromatography according to claim 1, characterized in that... The operating temperature during the storage and processing step is 25℃-35℃.

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