A method for fractionally separating and purifying tremella polysaccharide and a polysaccharide membrane fractionation equipment

Abstract

The application discloses a method for grading separation and purification of tremella polysaccharide and a polysaccharide membrane grading refining equipment, and comprises the following steps: S1, clarifying the crude separation liquid of the tremella polysaccharide by tangential flow filtration through a ceramic membrane assembly; S2, adopting a first ultrafiltration membrane assembly with a molecular weight cut-off of 800-1200 kDa for primary ultrafiltration, and collecting the >800 kDa macromolecular weight component in the concentrated liquid; S3, adopting a second ultrafiltration membrane assembly with a molecular weight cut-off of 80-120 kDa for secondary ultrafiltration, and collecting the 100-1000 kDa medium molecular weight component in the concentrated liquid; and S4, adopting a nanofiltration membrane assembly with a molecular weight cut-off of 2-4 kDa for nanofiltration, collecting the 3-100 kDa small molecular weight component in the concentrated liquid, and discharging the permeate. The equipment comprises a feeding unit, a pretreatment clarification unit, a primary ultrafiltration unit, a secondary ultrafiltration unit and a nanofiltration unit which are sequentially connected. The application solves the problems of the existing tremella polysaccharide membrane grading, such as fuzzy boundary and difficulty in synchronously obtaining three high-purity products.

Description

Technical Field

[0001] This invention relates to a method for fractionation, separation, and purification of Tremella fuciformis polysaccharides and a polysaccharide membrane fractionation and purification device, belonging to the field of Tremella fuciformis polysaccharide extraction technology. Background Technology

[0002] Tremella polysaccharides are important active ingredients in Tremella fuciformis, possessing various biological activities such as immunomodulation, antioxidation, moisturizing, and antitumor effects. They have wide applications in the food, health products, cosmetics, and biopharmaceutical fields.

[0003] Currently, the separation and purification of Tremella fuciformis polysaccharides mainly employs methods such as water extraction and alcohol precipitation, ion exchange chromatography, or gel column chromatography. Water extraction and alcohol precipitation can only yield total polysaccharides and cannot achieve fractionation of components with different molecular weights. Although chromatography and column chromatography can achieve a certain degree of molecular weight fractionation, they have low throughput, high cost, and long operation cycles, making them difficult to implement for industrial production.

[0004] Tremella fuciformis polysaccharide, as a macromolecular acidic heteropolysaccharide, typically has a main peak molecular weight in the millions of Daltons, reaching approximately 3 million Daltons or even higher, obtained naturally or through mild extraction. Its aqueous solution exhibits both high viscosity and viscoelasticity at high concentrations, far exceeding the viscosity levels of common plant polysaccharides (such as soybean polysaccharide and shiitake mushroom polysaccharide). Therefore, using tens of thousands of Daltons as the boundary between large, medium, and small molecular weights would categorize most Tremella fuciformis polysaccharides as "large molecular weight," failing to accurately reflect the inherent molecular weight distribution differences within the polysaccharide itself. This invention uses components with molecular weights above 1 million Daltons as large molecular weight components, 100,000-1 million Daltons as medium molecular weight components, and below 100,000 Daltons as small molecular weight components. This better aligns with the million-level molecular weight background of Tremella fuciformis polysaccharides and facilitates addressing different application needs such as thickening and film formation, moisturizing and repairing, and absorption and utilization. Meanwhile, the high viscosity of Tremella polysaccharide solution easily forms concentration polarization layer and gel layer on the membrane surface during membrane separation, causing serious membrane fouling and flux decline. This is the core industry pain point that restricts the application of membrane separation technology to the fractionation and purification of Tremella polysaccharide.

[0005] While existing multi-stage membrane separation technology has been applied to the separation of other polysaccharides, proteins, or plant extracts, the membrane separation of Tremella fuciformis polysaccharide, due to its large molecular weight (millions of molecules), strong hydration capacity, and significantly high viscosity, cannot be simply equated with the bulk replacement of other materials. Existing solutions for Tremella fuciformis polysaccharide often focus on cell disruption, extraction, or degradation, with membrane fractionation often considered as a subsequent optional step. These solutions fail to address the combined challenges of Tremella fuciformis polysaccharide's high molecular weight, high viscosity, susceptibility to gelation contamination, and easy loss of small molecular fragments with the permeate, and do not establish a systematic process for fractionation boundaries, viscosity control, and progressively reducing the load at each stage. Therefore, the technical problem this invention aims to solve is not simply using multi-stage membranes, but rather establishing a 1 million / 100,000 Dalton fractionation boundary suitable for a million-molecule molecular weight distribution under high-viscosity Tremella fuciformis polysaccharide feed conditions. This is achieved through a combination of ceramic membrane pretreatment, tangential high-shear flow, low-concentration feeding, segmented retention, and constant-volume dialysis, enabling stable operation of multi-stage membrane separation and obtaining clearly defined large, medium, and small molecular weight components of Tremella fuciformis polysaccharide. Summary of the Invention

[0006] To address the aforementioned problems in existing technologies, this invention provides a method for the graded separation and purification of Tremella fuciformis polysaccharides and a polysaccharide membrane graded purification device, which can simultaneously obtain high, medium, and low molecular weight Tremella fuciformis polysaccharide products with clear boundaries and stable purity in the same batch.

[0007] The technical solution of the present invention is as follows: A method for fractional separation and purification of Tremella fuciformis polysaccharides, comprising the following steps: S1: The crude polysaccharide separation liquid of Tremella fuciformis is filtered tangentially through a ceramic membrane module with a pore size of 0.05μm-0.50μm to remove particles, colloids and suspended impurities, and obtain a clear solution; S2: The obtained clarified solution is tangentially filtered through a first ultrafiltration membrane module with a molecular weight cutoff of 800-1200kDa, preferably about 1000kDa. Large molecular weight components with a molecular weight ≥1000kDa are collected on the concentrate side to obtain the permeate. S3: The permeate is tangentially filtered through a second ultrafiltration membrane module with a molecular weight cutoff of 80-120kDa, preferably about 100kDa. Medium molecular weight Tremella polysaccharide fraction with a molecular weight cutoff of 100-1000kDa is collected on the concentrate side, and the permeate is then introduced into the next step. S4: The permeate is tangentially filtered through a nanofiltration membrane module or a compact ultrafiltration membrane module with a molecular weight cutoff of 3-10kDa. Small molecular weight components with a molecular weight of 3-100kDa are collected on the concentrate side, and the permeate is discharged. Three molecular weight ranges of Tremella polysaccharides are obtained simultaneously in the same batch.

[0008] In step S2, the molecular weight cutoff of the first ultrafiltration membrane is preferably 900-1100 kDa, and the operating pressure is 0.1 MPa-0.8 MPa; in step S3, the molecular weight cutoff of the second ultrafiltration membrane is preferably 90-110 kDa, and the operating pressure is 0.2 MPa-1.0 MPa; in step S4, the molecular weight cutoff of the nanofiltration membrane or compact ultrafiltration membrane is preferably 3-10 kDa, and the operating pressure is 0.5 MPa-2.0 MPa; the operating temperature in the fractionation and purification method is 10℃-60℃.

[0009] In step S2 and / or step S3, pure water is added to the ultrafiltration concentrate side for constant volume dialysis to improve the purity of components in the corresponding molecular weight range.

[0010] The crude polysaccharide separation solution is the liquid obtained after preliminary solid-liquid separation of the water extract or enzymatic hydrolysate of the fruiting body of Tremella fuciformis.

[0011] A membrane grading and refining device for trellised polysaccharides used in implementing the method includes a feeding unit, a pretreatment clarification unit, a primary ultrafiltration unit, a secondary ultrafiltration unit, and a nanofiltration unit connected in sequence. The pretreatment clarification unit includes a ceramic membrane module. The primary ultrafiltration unit includes a first ultrafiltration membrane module, whose inlet is connected to the permeate outlet of the pretreatment clarification unit. The concentrate outlet of the primary ultrafiltration unit is connected to a high molecular weight product tank. The secondary ultrafiltration unit includes a second ultrafiltration membrane module, whose inlet is connected to the permeate outlet of the primary ultrafiltration unit. The concentrate outlet of the secondary ultrafiltration unit is connected to a medium molecular weight product tank. The nanofiltration unit includes a nanofiltration membrane module, whose inlet is connected to the permeate outlet of the secondary ultrafiltration unit. The concentrate outlet of the nanofiltration unit is connected to a low molecular weight product tank, and the permeate outlet is connected to a permeate discharge pipeline.

[0012] The system also includes a reflux pipeline, one end of which is connected to the concentrate side of the primary ultrafiltration unit and / or the secondary ultrafiltration unit, and the other end returns to the inlet of the pretreatment clarification unit. A reflux pump is installed on the reflux pipeline.

[0013] At least one of the outlets of the pretreatment clarification unit, the primary ultrafiltration unit, the secondary ultrafiltration unit, or the nanofiltration unit is provided with a sampling interface for online detection of turbidity, conductivity, sugar content, viscosity, or molecular weight distribution.

[0014] The pretreatment clarification unit includes a ceramic membrane module with a pore size of 0.05-0.50 μm, a first ultrafiltration membrane module with a molecular weight cutoff of 800-1200 kDa, a second ultrafiltration membrane module with a molecular weight cutoff of 80-120 kDa, and a nanofiltration membrane module or compact ultrafiltration membrane module in the low molecular weight component concentration unit with a molecular weight cutoff of 3-10 kDa.

[0015] The ceramic membrane module incorporates a tubular or plate-type ceramic membrane, and the first ultrafiltration membrane module, the second ultrafiltration membrane module, and the nanofiltration membrane module are each independently selected from spiral wound membranes, hollow fiber membranes, or tubular membranes.

[0016] The present invention has the following beneficial effects: This invention utilizes a core process of ceramic membrane pretreatment, two-stage ultrafiltration at the million-level boundary, and a series of small molecular weight component concentration membranes to simultaneously obtain Tremella fuciformis polysaccharide products with three molecular weight ranges: ≥1000kDa, 100-1000kDa, and 3-100kDa, in the same batch operation. This achieves full utilization of the raw materials and significantly improves product diversity and comprehensive utilization rate.

[0017] This invention employs a membrane cascade design with progressively decreasing molecular weight cutoffs, resulting in clear boundaries between each molecular weight range, stable product purity and yield, and the ability of the three products to be tailored to different high-value application scenarios.

[0018] This invention uses nanofiltration or compact ultrafiltration membrane modules to retain 3-100kDa low molecular weight Tremella fuciformis polysaccharide components, while simultaneously achieving the retention and recovery of the target product and the online removal of small molecule impurities and salts. It combines the functions of fractionation and purification, solving the technical problem that low molecular weight Tremella fuciformis polysaccharides are easily lost with the permeate and are difficult to recover as an independent product.

[0019] This invention effectively removes particles, colloids and suspended impurities from the crude separation liquid of Tremella polysaccharide through ceramic membrane pretreatment, significantly reducing the pollution of subsequent membrane modules, maintaining stable membrane flux and extending membrane service life. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the Tremella polysaccharide membrane grading and refining equipment of the present invention. The reference numerals in the figure are as follows: 1. Feeding unit; 2. Pretreatment clarification unit; 3. Primary ultrafiltration unit; 4. Secondary ultrafiltration unit; 5. Nanofiltration unit; 61. High molecular weight product tank; 62. Medium molecular weight product tank; 63. Low molecular weight product tank; 7. Permeate discharge pipeline; 8. Return pump. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0022] Please see Figure 1 The invention provides a technical solution: The core technological concept of this invention lies in employing a four-stage cascaded membrane separation architecture: a ceramic membrane module, a first ultrafiltration membrane module, a second ultrafiltration membrane module, and a low molecular weight component concentration membrane module. The first ultrafiltration membrane module has a molecular weight cutoff of 800-1200 kDa (preferably 900-1100 kDa), the second ultrafiltration membrane module has a molecular weight cutoff of 80-120 kDa (preferably 90-110 kDa), and the low molecular weight component concentration membrane module has a molecular weight cutoff of 3-10 kDa. These three molecular weight cutoffs form a decreasing gradient from high to low, which aligns with the million-level distribution characteristic of natural Tremella fuciformis polysaccharide with a molecular weight of approximately 3 million Daltons, namely, a high molecular weight range ≥1000 kDa, a medium molecular weight range of 100-1000 kDa, and a low molecular weight range of 3-100 kDa. Using 1 million Daltons and 100,000 Daltons as dividing lines, the grading results have greater product differentiation and application targeting.

[0023] In the existing technology, although multi-stage membrane separation can be used for some polysaccharides or plant extracts, direct membrane fractionation of Tremella fuciformis polysaccharide has three unique technical difficulties: (1) The main molecular weight of Tremella fuciformis polysaccharide is in the millions of Daltons, with long molecular chains and large hydration radii. The viscosity and viscoelasticity of the feed solution are significantly higher than those of general plant polysaccharides, and the membrane surface is very prone to the formation of concentration polarization layer, gel layer and irreversible fouling; (2) If the conventional ultrafiltration boundary of tens of thousands of Daltons is directly applied, it cannot effectively distinguish between the macromolecular and medium molecular components of Tremella fuciformis polysaccharide, resulting in distortion of the fractionation product boundary; (3) Low molecular weight Tremella fuciformis polysaccharide is easily lost with the permeate when removing salt and oligosaccharide impurities, and the high viscosity feed solution will cause rapid flux decay when entering the small pore membrane. Therefore, the existing multi-stage membrane process for other materials cannot be directly transferred to the Tremella fuciformis polysaccharide system.

[0024] The reason why this solution can overcome the above-mentioned technical bottlenecks is that, firstly, before the crude extract enters ultrafiltration and nanofiltration, a ceramic membrane module is set up for pretreatment clarification, and the concentration and viscosity of the feed solids are controlled to remove large particles, mycelial residues, colloids and suspended impurities that are prone to forming gel layers. At the same time, tangential flow filtration is used to maintain membrane shear, and if necessary, dilution, batch feeding or constant volume dialysis are used to reduce the local concentration on the membrane surface, thereby slowing down concentration polarization and gel layer formation and maintaining stable membrane flux.

[0025] Secondly, this scheme raises the first-level boundary to about 1000 kDa and sets the second-level boundary at about 100 kDa, so that the grading boundary matches the natural molecular weight background of about 3 million Daltons of Tremella polysaccharide. This allows for the separate acquisition of high molecular weight components and medium molecular weight components, rather than simply classifying all components with a molecular weight greater than tens of thousands of Daltons into the same macromolecular segment.

[0026] Thirdly, because this solution reduces the turbidity and apparent viscosity of the feed solution through pretreatment with ceramic membrane modules, and removes high molecular weight (≥1000kDa) and medium molecular weight (100-1000kDa) components through the first and second ultrafiltration membrane modules in stages, the feed solution entering the low molecular weight component concentration membrane module is relatively clean, with lower viscosity and fewer impurities. Therefore, nanofiltration or compact ultrafiltration membrane modules can retain low molecular weight target products of 3-100kDa at a stable flux, and remove small molecule impurities and salts with a molecular weight less than 3kDa online with the permeate. This allows low molecular weight products to provide a basis for subsequent food, cosmetic, or biomaterial applications without the need for a separate complex desalination process.

[0027] A membrane fractionation and purification device for implementing the above-mentioned method for fractionation and purification of Tremella polysaccharides comprises, in its overall structure, a feeding unit 1, a pretreatment clarification unit 2, a primary ultrafiltration unit 3, a secondary ultrafiltration unit 4, and a nanofiltration unit 5 connected in sequence.

[0028] Feeding unit 1 receives the crude separated liquid of Tremella fuciformis polysaccharide and conveys it to pretreatment clarification unit 2. Pretreatment clarification unit 2 is equipped with a ceramic membrane module with a pore size of 0.05 to 0.50 micrometers. This module can be tubular or plate-type and is used for tangential flow filtration of the crude separated liquid to remove particles, colloids, and suspended impurities, thereby obtaining a clarified solution and reducing turbidity and apparent viscosity. This provides pre-treatment reduction for the subsequent stable membrane grading of the high-viscosity Tremella fuciformis polysaccharide solution.

[0029] The permeate outlet of the pretreatment clarification unit 2 is connected to the feed inlet of the primary ultrafiltration unit 3. The primary ultrafiltration unit 3 is equipped with a first ultrafiltration membrane module with a molecular weight cutoff of 800 to 1200 kDa. The membrane material is polyethersulfone, regenerated cellulose or other fouling-resistant ultrafiltration membrane material, and the configuration can be one of spiral wound, hollow fiber, tubular or plate and frame.

[0030] The first ultrafiltration membrane module performs tangential flow filtration on the clarified solution, enriching the high molecular weight Tremella polysaccharide component with a molecular weight ≥1000kDa on its concentrate side, and collecting it into the high molecular weight product tank 61 through pipeline; the permeate then enters the secondary ultrafiltration unit 4.

[0031] The secondary ultrafiltration unit 4 houses a second ultrafiltration membrane module with a molecular weight cutoff of 80 to 120 kDa. The membrane material and configuration are similar to those of the first ultrafiltration membrane module. The second ultrafiltration membrane module performs secondary tangential flow filtration on the permeate from the primary ultrafiltration unit. The concentrate side enriches the medium molecular weight Tremella fuciformis polysaccharide component with a molecular weight between 100 and 1000 kDa and collects it in the medium molecular weight product tank 62. The permeate then enters the nanofiltration unit 5.

[0032] Nanofiltration unit 5 is equipped with a nanofiltration membrane module or a compact ultrafiltration membrane module with a molecular weight cutoff of 3 to 10 kDa. The membrane material is a polyamide composite membrane, a polyethersulfone membrane, or a regenerated cellulose membrane, and the configuration can be spiral wound, hollow fiber, tubular, or plate and frame. The nanofiltration membrane module or compact ultrafiltration membrane module performs final concentration and desalination of the permeate from the secondary ultrafiltration. The concentrate side is enriched with low molecular weight Tremella fuciformis polysaccharide components with a molecular weight between 3 and 100 kDa and collected in the low molecular weight product tank 63. The permeate is discharged from the system through the permeate discharge pipeline 7. This permeate mainly contains salts and small molecule impurities with a molecular weight of less than 3 kDa.

[0033] To further improve the overall yield of the target components, a reflux pipeline is also installed in the equipment. One end of this reflux pipeline is connected to the concentrate side of the primary ultrafiltration unit 3 and / or the secondary ultrafiltration unit 4, while the other end returns to the inlet of the pretreatment clarification unit 2. A reflux pump 8 is installed on the reflux pipeline. When the concentrate of the primary ultrafiltration unit 3 or the secondary ultrafiltration unit 4 has not reached the ideal concentration or purity, it can be sent back to the pretreatment clarification unit 2 by the reflux pump 8, mixed with fresh feed, and then re-enter the membrane separation process. This achieves multiple cyclic filtration, effectively improving the recovery rate of high and medium molecular weight components and reducing losses.

[0034] In addition, to monitor the operational status and product quality of each separation stage in real time, the equipment is equipped with sampling interfaces at least at one of the following locations: the outlet of the pretreatment clarification unit 2, the outlet of the primary ultrafiltration unit 3, the outlet of the secondary ultrafiltration unit 4, and the outlet of the nanofiltration unit 5. Through these sampling interfaces, operators can periodically or online collect samples of the feed liquid to detect key indicators such as turbidity, conductivity, sugar content, viscosity, or molecular weight distribution, thereby adjusting operating parameters in a timely manner to ensure the stability of the purity and yield of each stage of the product.

[0035] To address the difficulties in membrane separation caused by the high viscosity of Tremella fuciformis polysaccharides, the present invention can also adopt the following operational control measures: control the total sugar concentration of the feed solution entering the first-stage ultrafiltration unit to 1-20 mg / mL, preferably 3-10 mg / mL; use tangential flow circulation to maintain the membrane surface flow velocity at 0.5-5.0 m / s; when the transmembrane pressure difference increases or the flux decreases beyond the set value, reduce the apparent viscosity of the feed solution by adding water and constant volume dialysis, reducing the concentration factor, discharging the high-viscosity concentrate in stages, or using low-temperature to medium-temperature conditions; and preferentially select tubular, plate-and-frame, or fouling-resistant hollow fiber membrane modules to reduce the problem of easy clogging of spiral wound membranes in high-viscosity polysaccharide systems.

[0036] The specific selection of each membrane module can be flexibly determined according to the processing scale and feed characteristics: the ceramic membrane module can be a tubular ceramic membrane or a plate ceramic membrane; the first ultrafiltration membrane module, the second ultrafiltration membrane module and the nanofiltration membrane module can each be selected independently from spiral wound membranes, hollow fiber membranes or tubular membranes.

[0037] Example 1: Take 10L of water extract of Tremella fruiting body, centrifuge to remove large particles of residue, and use it as the crude separation liquid of Tremella polysaccharide (total polysaccharide content is about 5.2mg / mL, molecular weight distribution range is about 50 kDa-3000 kDa, and the main peak is about 1800 kDa).

[0038] Step S1: Pretreatment clarification: The above crude separation liquid is passed through a ceramic membrane module with a membrane pore size of 0.20 μm and subjected to tangential flow filtration under the conditions of operating pressure of 0.2 MPa and operating temperature of 25 °C. The permeate is collected to obtain a clear Tremella fuciformis polysaccharide solution of about 9.5 L. The turbidity is reduced from 85.3 NTU at the feed to 1.2 NTU. The polysaccharide recovery rate is about 96.5 ± 1.2%.

[0039] Step S2: Primary Ultrafiltration – Retention of High Molecular Weight Components: The clarified solution obtained in Step S1 is passed through a first ultrafiltration membrane module (polyethersulfone) with a molecular weight cutoff of 1000 kDa. Tangential flow filtration is performed at an operating pressure of 0.3 MPa and an operating temperature of 25°C. When the volume of the concentrate is reduced to approximately 1.0 L, 2.0 L of pure water is added to the concentrate side for constant volume dialysis. The concentrate side components are finally collected and freeze-dried to obtain approximately 14.2 g of high molecular weight Tremella fuciformis polysaccharide product (≥1000 kDa), with a purity of 91.3 ± 1.5% and a yield of 86.7 ± 2.4%. The permeate proceeds to Step S3.

[0040] Step S3: Secondary Ultrafiltration – Retention of Medium Molecular Weight Components: The permeate obtained in Step S2 is passed through a second ultrafiltration membrane module (polyethersulfone) with a molecular weight cutoff of 100 kDa. Tangential flow filtration is performed at an operating pressure of 0.5 MPa and an operating temperature of 25°C. When the volume of the concentrate is reduced to approximately 1.2 L, 2.0 L of pure water is added to the concentrate side for constant volume dialysis. The concentrate side components are collected and freeze-dried to obtain approximately 16.8 g of medium molecular weight Tremella fuciformis polysaccharide product (100-1000 kDa), with a purity of 89.5 ± 1.1% and a yield of 84.2 ± 3.1%. The permeate proceeds to Step S4.

[0041] Step S4: Concentration of low molecular weight components: The permeate obtained in Step S3 is passed through a nanofiltration membrane module or a compact ultrafiltration membrane module with a molecular weight cutoff of 5 kDa for tangential flow filtration at an operating pressure of 1.2 MPa and an operating temperature of 25°C. The concentrate side components are collected and freeze-dried to obtain approximately 9.5 g of low molecular weight Tremella fuciformis polysaccharide product (3-100 kDa), with a purity of 92.1 ± 1.5% and a yield of 81.3 ± 2.7%. The permeate (containing salt and small molecule impurities with a molecular weight <3 kDa) is discharged.

[0042] In this embodiment, three different molecular weight ranges of Tremella polysaccharide products were obtained simultaneously from 10L of crude Tremella polysaccharide separation solution in the same batch operation. The total product recovery rate was approximately 84.0±1.8%, and the molecular weight distribution ranges of the three products were clear and the purity was high.

[0043] Example 2 Take 10L of enzymatic hydrolysate of Tremella fruiting body (treated with a combination of cellulase and pectinase), remove the residue by plate and frame filtration, and use it as the crude separation liquid of Tremella polysaccharide (total polysaccharide content is about 6.8mg / mL, molecular weight distribution range is about 30kDa-1200kDa, and the main peak is about 900 kDa).

[0044] Step S1: Pretreatment clarification: The crude separation liquid was passed through a ceramic membrane module with a membrane pore size of 0.15 μm and subjected to tangential flow filtration at an operating pressure of 0.3 MPa and an operating temperature of 40 °C to obtain approximately 9.3 L of clarified solution with a polysaccharide recovery rate of approximately 95.8 ± 0.8%.

[0045] Step S2: First-stage ultrafiltration: The clarified solution was passed through a first-stage ultrafiltration membrane module with a molecular weight cutoff of 1000 kDa, and subjected to tangential flow filtration at an operating pressure of 0.3 MPa and an operating temperature of 40°C. The concentrate was collected after constant-volume dialysis to obtain approximately 17.5 g of high molecular weight Tremella fuciformis polysaccharide product (≥1000 kDa) with a purity of 90.6 ± 1.2%.

[0046] Step S3: Secondary Ultrafiltration: The permeate from Step S2 is passed through a second ultrafiltration membrane module with a molecular weight cutoff of 100 kDa, and subjected to tangential flow filtration at an operating pressure of 0.5 MPa and an operating temperature of 40°C. The concentrate is collected after constant-volume dialysis to obtain approximately 21.3 g of medium molecular weight Tremella fuciformis polysaccharide product (100-1000 kDa) with a purity of 88.7 ± 1.1%.

[0047] Step S4: Concentration of low molecular weight components: The permeate from step S3 is passed through a nanofiltration membrane module or a compact ultrafiltration membrane module with a molecular weight cutoff of 5 kDa for tangential flow filtration at an operating pressure of 1.5 MPa and an operating temperature of 40°C. The concentrate is collected to obtain approximately 12.1 g of low molecular weight Tremella fuciformis polysaccharide product (3-100 kDa) with a purity of 91.5 ± 1.3%.

[0048] This embodiment uses enzymatic pretreatment, which increases the total polysaccharide extraction rate by about 30% compared with water extraction, and the purity of the products in each molecular weight range remains at a high level.

[0049] Example 3 (Scale-up Verification) The process of Example 1 was scaled up to a scale of 500L. The aqueous extract of *Tremella fuciformis* fruiting bodies was centrifuged and used as the crude separation liquid. For ceramic membrane pretreatment, a tubular ceramic membrane with a pore size of 0.20 μm (membrane area 2.5 m²) was used. For primary ultrafiltration, a spiral wound or tubular ultrafiltration membrane with a molecular weight cutoff of 1000 kDa (membrane area 8.0 m²) was used. For secondary ultrafiltration, a spiral wound ultrafiltration membrane with a molecular weight cutoff of 100 kDa (membrane area 6.5 m²) was used. For concentration of low molecular weight components, a nanofiltration membrane or compact ultrafiltration membrane with a molecular weight cutoff of 5 kDa (membrane area 5.0 m²) was used. The operating temperature for each stage was 25°C, and the operating pressures were 0.2 MPa (ceramic membrane), 0.3 MPa (primary ultrafiltration), 0.5 MPa (secondary ultrafiltration), and 1.2 MPa (low molecular weight component concentration), respectively. During operation, the membrane flux remained stable, and the molecular weight distribution of the products at each stage was consistent with the laboratory-scale test results. The product purity was all above 88%, verifying the industrial scale-up feasibility of the method of this invention.

[0050] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for fractional separation and purification of Tremella fuciformis polysaccharides, characterized in that, Includes the following steps: S1: The crude polysaccharide separation liquid of Tremella fuciformis is filtered tangentially through a ceramic membrane module with a pore size of 0.05μm-0.50μm to remove particles, colloids and suspended impurities, and obtain a clear solution; S2: The obtained clarified solution is tangentially filtered through a first ultrafiltration membrane module with a molecular weight cutoff of 800-1200kDa. Large molecular weight components with a molecular weight >1000kDa are collected on the concentrate side to obtain the permeate. S3: The permeate is tangentially filtered through a second ultrafiltration membrane module with a molecular weight cutoff of 80-120kDa. Medium molecular weight Tremella polysaccharide fraction with a molecular weight of 100-1000kDa is collected on the concentrate side, and the permeate proceeds to the next step. S4: The permeate is tangentially filtered through a nanofiltration membrane module with a molecular weight cutoff of 3-10kDa. Small molecular weight components with a molecular weight of 3-100kDa are collected on the concentrate side, while small molecular weight impurities and salts with a molecular weight of less than 3kDa are discharged with the permeate. Three molecular weight ranges of Tremella polysaccharides are obtained simultaneously in the same batch. The molecular weight of the crude tremella polysaccharide separation solution is not less than 1000 kDa. In step S2 and / or step S3, pure water is added to the ultrafiltration concentrate side for constant volume dialysis to maintain a constant liquid volume during the concentration process, thereby reducing membrane surface concentration polarization and improving the purity of components in the corresponding molecular weight range.

2. The method for fractionation, separation, and purification of Tremella fuciformis polysaccharides as described in claim 1, characterized in that: In step S1, the pore size of the ceramic membrane module is preferably 0.15-0.30 μm, and the operating pressure is 0.1-0.5 MPa; in step S2, the molecular weight cutoff of the first ultrafiltration membrane is preferably 900-1100 kDa, and the operating pressure is 0.1 MPa-0.8 MPa; in step S3, the molecular weight cutoff of the second ultrafiltration membrane is preferably 90-110 kDa, and the operating pressure is 0.2 MPa-1.0 MPa; in step S4, the molecular weight cutoff of the nanofiltration membrane or compact ultrafiltration membrane is preferably 3-10 kDa, and the operating pressure is 0.5 MPa-2.0 MPa; the operating temperature in the fractionation and purification method is 10℃-60℃.

3. The method for fractional separation and purification of Tremella polysaccharides as described in claim 1, characterized in that: The crude polysaccharide separation solution of Tremella fuciformis is the liquid obtained after preliminary solid-liquid separation from the aqueous extract or enzymatic hydrolysate of Tremella fuciformis fruiting body.

4. A device for grading and refining Tremella fuciformis polysaccharide membranes for implementing the method according to any one of claims 1-3, characterized in that: The system includes a feed unit (1), a pretreatment clarification unit (2), a primary ultrafiltration unit (3), a secondary ultrafiltration unit (4), and a nanofiltration unit (5) connected in sequence. The pretreatment clarification unit (2) includes a ceramic membrane module. The primary ultrafiltration unit (3) includes a first ultrafiltration membrane module, whose feed inlet is connected to the permeate outlet of the pretreatment clarification unit (2). The concentrate outlet of the primary ultrafiltration unit (3) is connected to a high molecular weight product tank (61). The secondary ultrafiltration unit (4) includes a second ultrafiltration membrane module, whose feed inlet is connected to the permeate outlet of the primary ultrafiltration unit (3). The concentrate outlet of the secondary ultrafiltration unit (4) is connected to a medium molecular weight product tank (62). The nanofiltration unit (5) includes a nanofiltration membrane module, whose feed inlet is connected to the permeate outlet of the secondary ultrafiltration unit (4). The concentrate outlet of the nanofiltration unit (5) is connected to a low molecular weight product tank (63), and the permeate outlet is connected to a permeate discharge pipeline (7).

5. The Tremella polysaccharide membrane grading and refining equipment as described in claim 4, characterized in that: It is also provided with a reflux pipeline, one end of which is connected to the concentrate side of the primary ultrafiltration unit (3) and / or the secondary ultrafiltration unit (4), and the other end returns to the inlet of the pretreatment clarification unit (2). A reflux pump (8) is provided on the reflux pipeline.

6. The Tremella polysaccharide membrane grading and refining equipment as described in claim 4, characterized in that: At least one of the outlets of the pretreatment clarification unit (2), the primary ultrafiltration unit (3), the secondary ultrafiltration unit (4), or the nanofiltration unit (5) is provided with a sampling interface for online detection of turbidity, conductivity, sugar content, viscosity, or molecular weight distribution.

7. The Tremella polysaccharide membrane grading and refining equipment as described in claim 4, characterized in that: The pretreatment clarification unit (2) includes a ceramic membrane module with a pore size of 0.05-0.50 μm, a first ultrafiltration membrane module with a molecular weight cutoff of 800-1200 kDa, a second ultrafiltration membrane module with a molecular weight cutoff of 80-120 kDa, and a nanofiltration membrane module or compact ultrafiltration membrane module in the small molecular weight component concentration unit with a molecular weight cutoff of 3-10 kDa.

8. The Tremella polysaccharide membrane grading and refining equipment as described in claim 4, characterized in that: The ceramic membrane module incorporates a tubular or plate-type ceramic membrane, and the first ultrafiltration membrane module, the second ultrafiltration membrane module, and the nanofiltration membrane module are each independently selected from spiral wound membranes, hollow fiber membranes, or tubular membranes.

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