A method for purifying active polysaccharides of comatricha lignosa based on continuous chromatography

Abstract

A method for purifying active polysaccharides from *Trichoderma harzianum* based on continuous chromatography is disclosed, relating to the food industry. The method includes the following steps: (1) extracting *Trichoderma harzianum* to obtain a polysaccharide extract; (2) fractionating and refining the polysaccharide extract using continuous chromatography to obtain a refined liquid; (3) removing salts from the refined liquid using membrane separation technology, concentrating the refined liquid, and freeze-drying it to obtain the refined active polysaccharides from *Trichoderma harzianum*. The active polysaccharides from *Trichoderma harzianum* obtained using this method achieve a purity of 80%-90% and a recovery rate of 75%-85%, respectively, and exhibit high activity. This method provides high product activity, high yield, high separation efficiency, and large processing capacity, significantly improving the utilization rate of raw materials, enabling continuous operation, and reducing labor costs.

Description

Technical Field

[0001] This invention belongs to the field of food industry, specifically relating to a continuous chromatography purification technique, and particularly to a technique for continuous chromatography purification of polysaccharide active substances from Trichophyton spp. Background Technology

[0002] Hexagona, also known as Longan Comb, belongs to the phylum Basidiomycota, order Aphyllophorales, family Polyporaceae, and genus Hexagona. It is a rare medicinal fungus that grows on the trunks of broad-leaved trees in tropical and subtropical regions. According to the "Chinese Materia Medica," Hexagona is slightly bitter, astringent, and slightly warm in nature, and has the effects of promoting bowel movement, regulating qi and relieving pain, and strengthening the stomach. In traditional Chinese medicine, 10-12 grams of dried Hexagona fruiting bodies are often decocted and taken warm to treat stomach ailments. It is also sometimes mixed with Crested Mushroom, Inonotus obliquus, and Ganoderma lucidum, processed into ultrafine powder, and taken with warm water as an adjunct treatment for stomach cancer. In traditional Chinese medicine, Hexagona fruiting bodies are also decocted and taken to treat chronic nephritis. In Guangdong, there have been cases of kidney stone patients experiencing stone expulsion after decocting and taking Hexagona fruiting bodies.

[0003] Current research indicates that *Trichoderma harzianum* has broad prospects for development and utilization in medicinal and health care applications. However, in nature, due to limitations such as growth environment and season, wild resources are relatively rare and difficult to obtain, with high harvesting costs and inconsistent quality. Furthermore, it is common to mistakenly identify other woody fungi such as *Ganoderma lucidum*, *Sanghuang*, and *Phellinus thunbergii* as *Trichoderma harzianum*, which hinders its development and utilization as a pharmaceutical and health product. Existing methods for extracting and purifying polysaccharide active substances from *Trichoderma harzianum* are numerous, 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, preventing industrial-scale production.

[0004] Chinese Patent 201110268344.7 discloses a method for preparing fucoidan. This method uses brown algae as raw material to obtain crude Panax notoginseng polysaccharide through filtration and ethanol precipitation, then purifies the fucoidan using ultrafiltration and desaltes the polysaccharide solution using electrodialysis. However, the fucoidan content prepared by this method is low, only about 35%, and the patent does not clearly describe the purity of the final polysaccharide product.

[0005] Chinese Patent 201911021251.7 discloses a method for extracting and applying *Hypericum hygroscopicum* mycelial oil. The extraction method includes: taking *Hypericum hygroscopicum* fruiting bodies, drying and pulverizing them, adding solvent and continuously refluxing and percolating at 50°C for extraction, collecting the extract, and recovering the solvent by water bath distillation at 50°C to obtain crude mycelial oil; passing the crude mycelial oil through an adsorption chromatography column, eluting with an eluent, collecting the eluent, and recovering the solvent by vacuum rotary distillation at 50°C to obtain the *Hypericum hygroscopicum* mycelial oil; the method is simple to operate, has a high oil yield, does not damage the structure of its effective components, and the solvent can be recycled.

[0006] Literature review reveals that large-scale research and production of extraction and purification techniques for the active substances in *Trichoderma hyacinthum* polysaccharides have not yet been conducted. However, with further research, significant progress will be made in the study of *Trichoderma hyacinthum* polysaccharides, leading to a substantial increase in demand and production, and a promising market prospect.

[0007] Continuous preparative chromatography is a highly efficient purification method that enables continuous production and provides excellent separation results. With increasingly fierce market competition, existing technologies for removing impurities from *Trichoderma* polysaccharides can no longer meet current technological demands, and there is an urgent need for efficient industrial-scale polysaccharide removal technologies.

[0008] Currently, there are no research reports on purification techniques for polysaccharide active substances from *Trichoderma hyacinthumbum*.

[0009] Against this backdrop, the inventors researched a technique for the continuous preparation of chromatographic and membrane separation purification of active polysaccharides from *Trichoderma gracilis*, simplifying operational steps, reducing production losses, achieving continuous production, improving production efficiency, and establishing an industrial method for purifying active polysaccharides from *Trichoderma gracilis*. Summary of the Invention

[0010] The purpose of this invention is to provide a method for continuous chromatography purification of polysaccharide active substances from *Trichoderma harzianum*, thereby solving the problems existing in the prior art. The method of this invention can obtain *Trichoderma harzianum* polysaccharide active substances with high purity and activity. Furthermore, the method of this invention has the advantages of cost savings, simple process steps, and continuous production capability, and includes the following steps:

[0011] (1) Polysaccharide extraction: Polysaccharides were extracted from the raw material of *Trichoderma gracilis* by hot water extraction. First, the raw material was selected and impurities were removed. It was then placed in a tray and dried in an oven at 60°C until dry. After pulverization, it was stored at -20°C until use. Then, 1000g of *Trichoderma gracilis* powder was accurately weighed and added to 70% anhydrous ethanol at a material-to-liquid ratio of 1:15 (g / mL). The mixture was then heated in a water bath at 80°C for 2 hours. After cooling, it was centrifuged at 4000r / min for 15 minutes and the precipitate was dried. Ten times the volume of distilled water was added, and the mixture was heated and stirred at 85°C 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. After centrifugation, the two extracts were combined, concentrated by rotary evaporation, and anhydrous ethanol was added to make the final ethanol concentration 80%. The protein was removed by ethanol precipitation to obtain the *Trichoderma gracilis* polysaccharide extract.

[0012] (2) Fractional purification of active polysaccharides: The polysaccharide extract of Trichoderma hyacinthus was fractionated and purified by continuous chromatography to obtain a purified solution;

[0013] The continuous chromatography system comprises one column. At the top of the column, five feed tanks are controlled by solenoid valves and a fluid pump: a feed tank, a purification tank, a desorption tank, a regeneration tank 1, and a regeneration tank 2. At the bottom of the column, two outlet tanks are controlled by solenoid valves: an impurity tank and a purification tank. The feed solution, purification solution, desorption solution, regeneration solution 1, and regeneration solution 2 are sequentially and precisely pumped into the column via a flow meter control system. Similarly, the impurity tank and purification tank are sequentially and precisely pumped into the column via a flow meter control system. After one cycle, this process is automatically repeated until the production task is completed.

[0014] (3) Membrane concentration and desalination: A 3000Da nanofiltration membrane is used to remove salt substances during the fractionation process, thereby achieving the purpose of desalination of the purified liquid.

[0015] (4) Freeze-drying: Concentrate and freeze-dry to obtain refined active polysaccharides of Trichoderma hyacinthus.

[0016] Furthermore, the raw material liquid, refined liquid, desorption liquid, regeneration liquid 1 and regeneration liquid 2 are respectively the extract of Trichoderma hyacinthumb polysaccharide, 0.5 mol / L NaCl, 1.0-1.5 mol / L NaCl, 2.5 mol / L NaCl and deionized water;

[0017] Furthermore, the flow rates of the raw material, purified solution, desorption solution, regenerated solution 1, and regenerated solution 2 at the upper end of the chromatographic column are 1-1.5 BV, 1.5-2 BV / h, 1-2 BV / h, 2-3 BV / h, and 3-5 BV / h, respectively.

[0018] Furthermore, the lower end of the chromatographic column is directly connected to the impurity tank. Only after the desorption solution is pumped in at 0.5-0.8 BV is it pumped into the purification tank. After the regeneration solution 1 is pumped in at 0.5 BV, it is switched back to the impurity tank.

[0019] Furthermore, the purified Trichoderma hyacinth active polysaccharide has a purity of 80%-90% and a yield of 75%-85%, and the immunomodulatory activity of RAW264.7 macrophages is increased by more than 50% compared with that before grading.

[0020] Furthermore, the stationary phase of the chromatographic column is DEAE-52 cellulose anion exchange resin, DEAE-agarose gel FF, etc.;

[0021] Furthermore, the working temperature in the purification step is 25℃-35℃.

[0022] The present invention also provides a polysaccharide active substance of *Trichoderma harzianum* prepared according to the above method.

[0023] This invention develops a technique for the continuous preparation and chromatographic purification of active polysaccharides from *Trichoderma gracilis*, and establishes a highly efficient method for purifying these active polysaccharides. This method not only ensures the high purity and high activity of the active polysaccharides from *Trichoderma gracilis*, but also achieves cost savings in the entire process, with simple steps and the ability to be continuously produced. Attached Figure Description

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0025] Figure 1 This is a process flow diagram of the continuous chromatographic purification of polysaccharide active substances from Trichoderma harzianum according to the present invention. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] The following examples illustrate the preparation method of the extract: Accurately weigh 1000g of *Hypericum cristatum* powder and add 70% anhydrous ethanol at a material-to-liquid ratio of 1:15 (g / mL). Incubate in a water bath at 80℃ for 2 hours. After cooling, centrifuge at 4000 rpm for 15 minutes and dry the precipitate. Add 10 times the volume of distilled water and 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. Combine the two extracts after centrifugation, perform rotary evaporation, and concentrate. Add anhydrous ethanol to achieve a final ethanol concentration of 80%. Eliminate proteins by ethanol precipitation to obtain the *Hypericum cristatum* polysaccharide extract.

[0032] The polysaccharide purification method used in the following examples is as follows: A continuous chromatography process is used to fractionate and purify the polysaccharide extract of *Trichoderma gracilis* to obtain a purified solution. The continuous chromatography column comprises one column. The upper end of the column is controlled by a flow meter to sequentially and precisely pump in the *Trichoderma gracilis* polysaccharide extract, 0.5 mol / L NaCl, 1.0-1.5 mol / L NaCl, 2.5 mol / L NaCl, and deionized water. The lower end of the column is controlled by a flow meter to sequentially and precisely pump in the impurity tank and the purification tank. After one cycle, this process is automatically repeated until the production task is completed. A 3000 Da nanofiltration membrane is used to remove salts from the fractionation process, achieving desalination of the purified solution. The solution is then concentrated and freeze-dried to obtain the purified active polysaccharide from *Trichoderma gracilis*.

[0033] Example 1:

[0034] (1) Extraction of polysaccharides: Polysaccharide extract of Trichophyton spp. was obtained;

[0035] (2) Fractional purification of active polysaccharides: The polysaccharide extract of Trichoderma hyacinthus was fractionated and purified by continuous chromatography to obtain a purified solution;

[0036] The continuous chromatography system comprises one column. At the top of the column, five feed tanks are controlled by solenoid valves and a fluid pump: a feed tank, a purification tank, a desorption tank, a regeneration tank 1, and a regeneration tank 2. At the bottom of the column, two outlet tanks are controlled by solenoid valves: an impurity tank and a purification tank. The feed solution, purification solution, desorption solution, regeneration solution 1, and regeneration solution 2 are sequentially and precisely pumped into the column via a flow meter control system. Similarly, the impurity tank and purification tank are sequentially and precisely pumped into the column via a flow meter control system. After one cycle, this process is automatically repeated until the production task is completed.

[0037] The feed solution, purified solution, desorption solution, regenerated solution 1, and regenerated solution 2 are respectively: *Trichoderma hyacinthii* polysaccharide extract, 0.5 mol / L NaCl, 1.0-1.5 mol / L NaCl, 2.5 mol / L NaCl, and deionized water; the flow rates of the feed solution, purified solution, desorption solution, regenerated solution 1, and regenerated solution 2 are 1 BV, 1.5 BV / h, 1 BV / h, 2 BV / h, and 3 BV / h, respectively; after the desorption solution is pumped in at 0.5 BV, it is pumped into the purified tank, and after the regenerated solution 1 is pumped in at 0.5 BV, it is switched to the impurity tank; the stationary phase of the chromatographic column is DEAE-52 cellulose anion exchange resin; the operating temperature is 25℃.

[0038] (3) Membrane concentration and desalination: A 3000Da nanofiltration membrane is used to remove salt substances during the fractionation process, thereby achieving the purpose of desalination of the purified liquid.

[0039] (4) Freeze-drying: Concentrate and freeze-dry to obtain refined active polysaccharides of Trichoderma hyacinthus.

[0040] The active polysaccharide prepared in this embodiment has a purity of 82% and a yield of 78%, and the immunomodulatory activity of RAW264.7 macrophages is increased by 45% compared with that before grading.

[0041] Example 2:

[0042] (1) Extraction of polysaccharides: Polysaccharide extract of Trichophyton spp. was obtained;

[0043] (2) Fractional purification of active polysaccharides: The polysaccharide extract of Trichoderma hyacinthus was fractionated and purified by continuous chromatography to obtain a purified solution;

[0044] The continuous chromatography system comprises one column. At the top of the column, five feed tanks are controlled by solenoid valves and a fluid pump: a feed tank, a purification tank, a desorption tank, a regeneration tank 1, and a regeneration tank 2. At the bottom of the column, two outlet tanks are controlled by solenoid valves: an impurity tank and a purification tank. The feed solution, purification solution, desorption solution, regeneration solution 1, and regeneration solution 2 are sequentially and precisely pumped into the column via a flow meter control system. Similarly, the impurity tank and purification tank are sequentially and precisely pumped into the column via a flow meter control system. After one cycle, this process is automatically repeated until the production task is completed.

[0045] The feed solution, purified solution, desorption solution, regenerated solution 1, and regenerated solution 2 are respectively: *Trichoderma hyacinthus* polysaccharide extract, 0.5 mol / L NaCl, 1.0-1.5 mol / L NaCl, 2.5 mol / L NaCl, and deionized water; the flow rates of the feed solution, purified solution, desorption solution, regenerated solution 1, and regenerated solution 2 are 1.2 BV, 1.7 BV / h, 1.5 BV / h, 2.5 BV / h, and 4 BV / h, respectively; after the desorption solution is pumped in at 0.6 BV, it is pumped into the purified tank, and after the regenerated solution 1 is pumped in at 0.5 BV, it is switched to the impurity tank; the stationary phase of the chromatographic column is DEAE-52 cellulose anion exchange resin; the operating temperature is 30℃.

[0046] (3) Membrane concentration and desalination: A 3000Da nanofiltration membrane is used to remove salt substances during the fractionation process, thereby achieving the purpose of desalination of the purified liquid.

[0047] (4) Freeze-drying: Concentrate and freeze-dry to obtain refined active polysaccharides of Trichoderma hyacinthus.

[0048] The active polysaccharide of Trichoderma harzianum prepared in this embodiment has a purity of 85.4% and a yield of 80.6%, and the immunomodulatory activity of RAW264.7 macrophages is increased by 50.3% compared with that before grading.

[0049] Example 3:

[0050] (1) Extraction of polysaccharides: Polysaccharide extract of Trichophyton spp. was obtained;

[0051] (2) Fractional purification of active polysaccharides: The polysaccharide extract of Trichoderma hyacinthus was fractionated and purified by continuous chromatography to obtain a purified solution;

[0052] The continuous chromatography system comprises one column. At the top of the column, five feed tanks are controlled by solenoid valves and a fluid pump: a feed tank, a purification tank, a desorption tank, a regeneration tank 1, and a regeneration tank 2. At the bottom of the column, two outlet tanks are controlled by solenoid valves: an impurity tank and a purification tank. The feed solution, purification solution, desorption solution, regeneration solution 1, and regeneration solution 2 are sequentially and precisely pumped into the column via a flow meter control system. Similarly, the impurity tank and purification tank are sequentially and precisely pumped into the column via a flow meter control system. After one cycle, this process is automatically repeated until the production task is completed.

[0053] The feed solution, purified solution, desorption solution, regenerated solution 1, and regenerated solution 2 are respectively: *Trichoderma hyacinthus* polysaccharide extract, 0.5 mol / L NaCl, 1.0-1.5 mol / L NaCl, 2.5 mol / L NaCl, and deionized water; the flow rates of the feed solution, purified solution, desorption solution, regenerated solution 1, and regenerated solution 2 are 1.5 BV, 2 BV / h, 2 BV / h, 3 BV / h, and 5 BV / h, respectively; after the desorption solution is pumped in at 0.8 BV, it is pumped into the purified tank, and after the regenerated solution 1 is pumped in at 0.5 BV, it is switched to the impurity tank; the stationary phase of the chromatographic column is DEAE-52 cellulose anion exchange resin; the operating temperature is 35℃.

[0054] (3) Membrane concentration and desalination: A 3000Da nanofiltration membrane is used to remove salt substances during the fractionation process, thereby achieving the purpose of desalination of the purified liquid.

[0055] (4) Freeze-drying: Concentrate and freeze-dry to obtain refined active polysaccharides of Trichoderma hyacinthus.

[0056] The active polysaccharide of Trichoderma harzianum 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 55.4% compared with that before grading.

[0057] The core technology of this invention is the technique of continuous chromatography purification of Trichoderma hyacinthumb polysaccharide, which yields Trichoderma hyacinthumb polysaccharide with high purity and yield. This process not only ensures high purity, high yield and high activity of Trichoderma hyacinthumb polysaccharide, but also has distinctive features in terms of cost saving, simple process steps and continuous production.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A method for purifying active polysaccharides from *Trichoderma harzianum* based on continuous chromatography, comprising the following steps: (1) Polysaccharide extraction: Polysaccharides were extracted from the raw material of *Trichoderma gracilis* by hot water extraction. First, the raw material was selected and impurities were removed. It was then placed in a tray and dried in an oven at 60°C until dry. After pulverization, it was stored at -20°C until use. Then, 1000g of *Trichoderma gracilis* powder was accurately weighed and added to 70% anhydrous ethanol at a material-to-liquid ratio of 1:15 (g / mL). The mixture was then heated in a water bath at 80°C for 2 hours. After cooling, it was centrifuged at 4000r / min for 15 minutes and the precipitate was dried. Ten times the volume of distilled water was added, and the mixture was heated and stirred at 85°C 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. After centrifugation, the two extracts were combined, concentrated by rotary evaporation, and anhydrous ethanol was added to make the final ethanol concentration 80%. The protein was removed by ethanol precipitation to obtain the *Trichoderma gracilis* polysaccharide extract. (2) Fractional purification of active polysaccharides: The polysaccharide extract of Trichoderma hyacinthus was fractionated and purified by continuous chromatography to obtain a purified solution; The continuous chromatography system comprises one column. At the top of the column, five feed tanks are controlled by solenoid valves and a fluid pump: a feed tank, a purification tank, a desorption tank, a regeneration tank 1, and a regeneration tank 2. At the bottom of the column, two outlet tanks are controlled by solenoid valves: an impurity tank and a purification tank. The feed solution, purification solution, desorption solution, regeneration solution 1, and regeneration solution 2 are sequentially and precisely pumped into the column via a flow meter control system. Similarly, the impurity tank and purification tank are sequentially and precisely pumped into the column via a flow meter control system. After one cycle, this process is automatically repeated until the production task is completed. (3) Membrane concentration and desalination: A 3000Da nanofiltration membrane is used to remove salt substances during the fractionation process, thereby achieving the purpose of desalination of the purified liquid. (4) Freeze-drying: Concentrate and freeze-dry to obtain refined active polysaccharides of Trichoderma hyacinthus.

2. The method for purifying active polysaccharides from *Trichoderma harzianum* based on continuous chromatography according to claim 1, characterized in that... The raw material solution, purified solution, desorption solution, regeneration solution 1 and regeneration solution 2 used are respectively the extract of Trichoderma hyacinthumb polysaccharide, 0.5 mol / L NaCl, 1.0-1.5 mol / L NaCl, 2.5 mol / L NaCl and deionized water.

3. The method for purifying active polysaccharides from Trichoderma harzianum based on continuous chromatography according to claim 1, characterized in that... The flow rates of the feed solution, purified solution, desorption solution, regenerated solution 1, and regenerated solution 2 at the top of the chromatographic column are 1-1.5 BV, 1.5-2 BV / h, 1-2 BV / h, 2-3 BV / h, and 3-5 BV / h, respectively.

4. The method for purifying active polysaccharides from Trichoderma harzianum based on continuous chromatography according to claim 1, characterized in that... The lower end of the chromatographic column is directly connected to the impurity tank. Only after the desorption solution is pumped in at 0.5-0.8 BV is it pumped into the purification tank. After the regeneration solution 1 is pumped in at 0.5 BV, it is switched back to the impurity tank.

5. The method for purifying active polysaccharides from *Trichoderma harzianum* based on continuous chromatography according to claim 1, characterized in that... The purified active polysaccharide from Trichoderma hygroscopicum had a purity of 80%-90% and a yield of 75%-85%. The immunomodulatory activity of RAW264.7 macrophages was increased by more than 50% compared with that before grading.

6. The method for purifying active polysaccharides from Trichoderma harzianum based on continuous 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 Trichoderma harzianum based on continuous chromatography according to claim 1, characterized in that... The working temperature during the purification step is 25℃-35℃.

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