A green extraction method of low molecular weight fucoidan

Abstract

The application belongs to the technical field of polysaccharide extraction, and particularly relates to a green extraction method of low-molecular-weight fucoidan, which comprises the following steps: pretreating brown algae raw materials to obtain brown algae treatment; mixing the brown algae treatment with a freezing liquid and then performing cyclic freezing treatment, and then performing solid-liquid separation after thawing to obtain a first extraction liquid; mixing the first extraction liquid with enzymolysis granules and then performing enzymolysis, and then performing auxiliary extraction after the enzymolysis to obtain a second extraction liquid. The application constructs a directional extraction system of "pretreatment-freezing extraction-enzymolysis-ultrasonic intensification-refining-gradient precipitation-freezing drying", the freezing treatment physically breaks the cell wall, the enzymolysis directionally hydrolyzes the glycosidic bond of macromolecular fucoidan, the ultrasonic intensification strengthens mass transfer and accelerates polysaccharide dissolution and reagent dispersion, the gradient precipitation separates the target molecular weight product, the freezing drying avoids thermal degradation of polysaccharide, and the whole process is free of high-toxicity reagents, so that the green and efficient preparation of low-molecular-weight fucoidan is realized.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction technology, and particularly relates to a green extraction method for low molecular weight fucoidan. Background Technology

[0002] Fucoidan is a sulfated heteropolysaccharide unique to brown algae, mainly found in the cell walls, intercellular spaces, and secretions of brown algae such as kelp, Sargassum, and Fucus vesiculosus. Its core structure consists of a poly-α-L-fucoidan sulfate backbone and side chains composed of various monosaccharides. Due to its excellent biological activities, including antioxidant, anticoagulant, immune-enhancing, antitumor, and hypoglycemic effects, it is widely used in food, pharmaceuticals, and cosmetics. Compared to natural macromolecular fucoidan, low molecular weight fucoidan (typically below 10 kDa) has higher water solubility and bioavailability, allowing for more efficient absorption by the body. It exhibits significant advantages in applications such as kidney disease treatment, cardiovascular protection, and skin care, and has become one of the core directions of marine bioactive substance research in recent years.

[0003] However, the extraction technology of low molecular weight fucoidan still faces many problems that need to be solved, which limit its large-scale application and high-value development: 1. Traditional extraction methods are not environmentally friendly and the activity of the product is easily damaged: Traditional chemical extraction methods (such as dilute acid hydrolysis and organic solvent extraction) require the use of toxic and harmful reagents such as strong acids, chloroform, and hexadecyl pyridine chloride, which not only cause serious environmental pollution, but also easily lead to the removal of sulfate groups and glycosidic bond breakage of fucoidan, destroying its natural structure and biological activity; even if some methods can obtain low molecular weight products, there are still problems such as difficulty in concentrating the extract and many impurities remaining, and the subsequent purification cost is extremely high.

[0004] 2. Insufficient efficiency of single physical or enzymatic hydrolysis techniques: Although existing physical extraction methods (such as ultrasound and microwave-assisted extraction) can break cell walls through cavitation or thermal effects, their effect on breaking down the dense cell walls of brown algae is limited when used alone, resulting in low polysaccharide dissolution rates and excessive polysaccharide degradation due to improper parameter control. Enzymatic hydrolysis methods mostly use single cellulases or alginate lyases, lacking a synergistic design of specific degrading enzymes and cell wall-breaking enzymes targeting the structure of fucoidan. This results in low enzymatic hydrolysis efficiency, difficulty in recovering free enzymes, increased production costs, and uneven molecular weight distribution of the product due to improper control of enzymatic hydrolysis conditions.

[0005] 3. Low resource utilization and poor process synergy: Traditional processes often focus on the extraction of a single product, neglecting the comprehensive utilization of brown algae raw materials, resulting in resource waste; moreover, the extraction, degradation, and purification steps lack synergistic design. For example, incomplete cell wall disruption leads to low efficiency of subsequent enzymatic hydrolysis. The high-temperature drying and molecular sieve filtration methods used in the purification process are prone to losing large-molecule polysaccharide components and increase energy consumption and the risk of impurity residues, failing to balance extraction efficiency, product purity, and green environmental protection requirements.

[0006] 4. The contradiction between green development and large-scale production is prominent: Although existing green extraction technologies (such as supercritical fluid extraction) can avoid chemical reagent pollution, they have drawbacks such as large equipment investment, high operation requirements, and difficulty in large-scale production; while methods suitable for industrialization often have problems such as high energy consumption, large wastewater discharge, and low retention rate of product biological activity, which are difficult to meet the current environmental protection policy's demand for low-pollution, low-energy consumption, and high-value-added biological product production.

[0007] Therefore, developing a low molecular weight fucoidan extraction method that is efficient, green, and targeted is of great significance for promoting the high-value development and industrial application of marine algal resources. This method would address the problems of severe environmental pollution, low extraction efficiency, poor product activity, and insufficient process synergy in traditional technologies. Summary of the Invention

[0008] The purpose of this invention is to address the aforementioned technical problems by providing a green extraction method for low molecular weight fucoidan.

[0009] In view of this, the present invention provides a green extraction method for low molecular weight fucoidan, comprising the following steps: Brown algae raw materials are pretreated to obtain brown algae treated products; The brown algae treatment material was mixed with a freezing liquid and then subjected to cyclic freezing treatment. After thawing, solid-liquid separation was performed to obtain the first extract. The first extract was mixed with enzymatically hydrolyzed particles and enzymatically hydrolyzed. After enzymatic hydrolysis, the second extract was obtained by auxiliary extraction. Add solubilizers and salts to the second extract, adjust the pH value, and then perform ultrasonic treatment to obtain the third extract; The third extract is purified to obtain a purified solution; Ethanol was added to the purified solution to perform gradient precipitation, the target precipitate was collected and freeze-dried to obtain low molecular weight fucoidan.

[0010] Furthermore, the pretreatment in step S1 includes sequential impurity removal, washing, and pulverization operations, wherein the particle size of the pulverized brown algae particles is no greater than 50 mesh.

[0011] Furthermore, the cryosol in step S2 is composed of water, ethanol, potassium chloride and glycerol, and the mass ratio of the brown algae treatment material to the cryosol is 1:(2~12); the components of the cryosol are as follows by mass: water 75~82 parts, ethanol 14~18 parts, potassium chloride 2~3 parts, and glycerol 2.5~4 parts.

[0012] Furthermore, the specific method of the cyclic freezing treatment is as follows: the temperature is reduced to -35~-18℃ at a cooling rate of 1.5~4.5℃ / min, and then naturally warmed to room temperature, and the cycle is repeated 2~5 times.

[0013] Furthermore, the enzymatically hydrolyzed particles in step S3 are composite immobilized particles of fucoidan-degrading enzyme and pectinase, and the mass ratio of the first extract to the enzymatically hydrolyzed particles is 100:(2~6); the enzymatic hydrolysis temperature is 28~42℃, and the enzymatic hydrolysis time is 6~22h.

[0014] Furthermore, the fucoidan-degrading enzyme is selected from the GH168 or GH107 family of fucoidan endonucleases, and the mass ratio of fucoidan-degrading enzyme to pectinase in the composite immobilized particles is (1.5~3.5):1.

[0015] Furthermore, in step S4, the solubilizer is a medium-polymerization degree isomalt / malt polysaccharide with a molecular weight of 5-9 kDa; the salt is sodium sulfate; the mass ratio of the second extract, solubilizer, and salt is 100:(3-5):(4-7); and the pH value is adjusted to 3.8-5.2.

[0016] Furthermore, the assisted extraction in step S3 is ultrasonic assisted extraction, with an ultrasonic power of 120~380W and an ultrasonic time of 0.3~3.5h; the ultrasonic treatment in step S4 has a power of 180~320W and an ultrasonic time of 0.4~2.2h.

[0017] Furthermore, the refining process in step S5 includes sequential centrifugation filtration, decolorization, and desalting operations; the decolorization is performed using activated carbon adsorption, with the amount of activated carbon added being 0.8-3.2% of the mass of the third extract, and the decolorization time being 0.5-3.5 h; the desalting process is performed using a series treatment of a strong acid cation resin and a weak base anion resin, with the mass ratio of cation resin to anion resin being 1.5:1-2.5:1.

[0018] Furthermore, the gradient precipitation in step S6 involves sequentially increasing the ethanol volume concentration to 25-45%, 45-65%, and 65-85%, and collecting the precipitate when the ethanol volume concentration is 45-65%; the freeze-drying temperature is -65 to -25°C, and the drying time is 2.5 to 9 hours.

[0019] The beneficial effects of this invention are: This invention constructs a directional extraction system consisting of "pretreatment-freeze extraction-enzymatic hydrolysis-ultrasound enhancement-purification-gradient precipitation-freeze drying". Freeze treatment physically breaks down cell walls, enzymatic hydrolysis directionally hydrolyzes the glycosidic bonds of macromolecular fucoidan, ultrasound-enhanced mass transfer accelerates polysaccharide dissolution and reagent dispersion, gradient precipitation separates products of the target molecular weight, and freeze drying avoids thermal degradation of polysaccharides. The entire process is free of highly toxic reagents, achieving green and efficient preparation of low molecular weight fucoidan. This system not only solves the high pollution problem of traditional chemical extraction but also overcomes the shortcomings of single technologies (such as enzymatic hydrolysis or ultrasound only) with low extraction efficiency and wide product molecular weight distribution. Detailed Implementation

[0020] The technical solutions in the embodiments of this application will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application are within the scope of protection of this application.

[0021] It should be noted that, in this application, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element. Furthermore, it should be noted that the scope of the methods and apparatuses in the embodiments of this application is not limited to performing functions in the order shown or discussed, but may also include performing functions substantially simultaneously or in the reverse order, depending on the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. Additionally, features described with reference to certain examples may be combined in other examples.

[0022] A green extraction method for low molecular weight fucoidan includes the following steps: Brown algae raw materials are pretreated to obtain brown algae treated products; The brown algae treatment material was mixed with a freezing liquid and then subjected to cyclic freezing treatment. After thawing, solid-liquid separation was performed to obtain the first extract. The first extract was mixed with enzymatically hydrolyzed particles and enzymatically hydrolyzed. After enzymatic hydrolysis, the second extract was obtained by auxiliary extraction. Add solubilizers and salts to the second extract, adjust the pH value, and then perform ultrasonic treatment to obtain the third extract; The third extract is purified to obtain a purified solution; Ethanol was added to the purified solution to perform gradient precipitation, the target precipitate was collected and freeze-dried to obtain low molecular weight fucoidan.

[0023] This invention constructs a directional extraction system consisting of "pretreatment-freeze extraction-enzymatic hydrolysis-ultrasound enhancement-purification-gradient precipitation-freeze drying". Freeze treatment physically breaks down cell walls, enzymatic hydrolysis directionally hydrolyzes the glycosidic bonds of macromolecular fucoidan, ultrasound-enhanced mass transfer accelerates polysaccharide dissolution and reagent dispersion, gradient precipitation separates products of the target molecular weight, and freeze drying avoids thermal degradation of polysaccharides. The entire process is free of highly toxic reagents, achieving green and efficient preparation of low molecular weight fucoidan. This system not only solves the high pollution problem of traditional chemical extraction but also overcomes the shortcomings of single technologies (such as enzymatic hydrolysis or ultrasound only) with low extraction efficiency and wide product molecular weight distribution.

[0024] In the example of this application, the pretreatment in step S1 includes sequentially performing impurity removal, washing, and pulverization operations, wherein the particle size of the pulverized brown algae particles is no greater than 50 mesh.

[0025] As a preferred example of the present invention, the impurities removal process removes solid impurities such as mud, sand, and attached microorganisms to prevent them from adsorbing polysaccharides or hindering the penetration of the extractant; the washing process removes salt and water-soluble impurities from the surface of brown algae to prevent salt from affecting the subsequent enzymatic hydrolysis activity and precipitation effect; the 50-mesh particle size increases the contact surface area between the raw material and the extractant, accelerates the dissolution of polysaccharides, and avoids the subsequent filtration blockage problem caused by excessive grinding (such as less than 100 mesh), ensuring smooth solid-liquid separation.

[0026] In the example of this application, the cryosol in step S2 is composed of water, ethanol, potassium chloride and glycerol, and the mass ratio of the brown algae treatment material to the cryosol is 1:(2~12); the components of the cryosol are as follows by mass: 75~82 parts water, 14~18 parts ethanol, 2~3 parts potassium chloride and 2.5~4 parts glycerol.

[0027] As a preferred example of the present invention, ethanol in the cryosol lowers the freezing point, enabling the system to rapidly reach a low temperature; potassium chloride increases the osmotic pressure of the solution, forming a synergistic effect with the ice particles, causing intracellular water to freeze and expand, and cell walls to rupture; glycerol inhibits excessive ice crystal growth, preventing the polysaccharide structure from being mechanically damaged by the ice crystals; a mass ratio of 1:(2~12) ensures that the cryosol fully encapsulates the brown algae particles, and the dosage is adjusted according to the density of different brown algae cell walls, ensuring both the breaking effect and avoiding waste of cryosol.

[0028] In the example of this application, the specific method of the cyclic freezing treatment is as follows: the temperature is reduced to -35 to -18°C at a cooling rate of 1.5 to 4.5°C / min, and then naturally raised to room temperature, and the cycle is repeated 2 to 5 times.

[0029] As a preferred example of the present invention, a cooling rate of 1.5~4.5℃ / min avoids mechanical damage to polysaccharide molecules caused by the sudden formation of ice crystals, while ensuring that intracellular water freezes in an orderly manner to generate expansion force; a freezing temperature of -35~-18℃ causes the cell wall lipid bilayer to solidify and rupture without destroying the glycosidic bonds and sulfate groups of polysaccharides; 2~5 cycles of freezing further break down residual cell wall fragments through repeated osmotic pressure changes of "freezing-thawing", accelerating the release of intracellular polysaccharides, and the number of cycles is adapted to the cell wall thickness of different brown algae (e.g., Sargassum requires 3~5 cycles, kelp requires 2~3 cycles).

[0030] In the example of this application, the enzymatically hydrolyzed particles in step S3 are composite immobilized particles of fucoidan-degrading enzyme and pectinase, and the mass ratio of the first extract to the enzymatically hydrolyzed particles is 100:(2~6); the enzymatic hydrolysis temperature is 28~42℃, and the enzymatic hydrolysis time is 6~22h.

[0031] As a preferred example of the present invention, the pectinase in the composite immobilized particles specifically degrades pectin in the cell wall, disrupting the integrity of the cell wall structure and providing a channel for fucoidan-degrading enzymes; the fucoidan-degrading enzymes directionally hydrolyze the α-1,3 or α-1,4 glycosidic bonds of macromolecular fucoidan, converting it into low molecular weight products; the mass ratio of 100:(2~6) ensures sufficient enzyme quantity without waste, 28~42℃ is the optimal temperature range for synergy between the two types of enzymes, and the enzymatic hydrolysis time of 6~22h ensures sufficient degradation of macromolecular polysaccharides while avoiding excessive enzymatic hydrolysis that results in products with excessively small molecular weights.

[0032] In the example of this application, the fucoidan-degrading enzyme is selected from the GH168 family or the GH107 family of fucoidan endonucleases, and the mass ratio of fucoidan-degrading enzyme to pectinase in the composite immobilized particles is (1.5~3.5):1.

[0033] As a preferred example of the present invention, the GH168 or GH107 family of fucoidan endonucleases have high substrate specificity, hydrolyzing only specific glycosidic bonds in the fucoidan backbone without damaging the sulfate groups (sulfate groups are key structures for biological activity); the optimized enzyme ratio of (1.5~3.5):1 enables the fucoidan degrading enzyme and pectinase to form a synergistic effect—pectinase first breaks down the cell wall, and then the fucoidan degrading enzyme directionally degrades the released macromolecular polysaccharides, avoiding the efficiency bottleneck caused by the action of a single enzyme.

[0034] In the example of this application, the solubilizer in step S4 is a medium-polymerization degree isomalt / malt polysaccharide with a molecular weight of 5~9kDa; the salt is sodium sulfate; the mass ratio of the second extract, solubilizer, and salt is 100:(3~5):(4~7); and the pH value is adjusted to 3.8~5.2.

[0035] As a preferred example of the present invention, isomalt / maltose with a medium degree of polymerization of 5-9 kDa binds to low molecular weight fucoidan through hydrogen bonding, breaking the aggregation between polysaccharide molecules and improving its dispersibility in aqueous solution; sodium sulfate reduces the solubility of protein through salting out, promoting its precipitation; a pH value of 3.8-5.2 is close to the isoelectric point of most proteins, further inhibiting protein dissolution, while this pH range does not affect the stability and solubility of fucoidan; a mass ratio of 100:(3-5):(4-7) balances the solubilization effect and impurity removal efficiency, avoiding the subsequent desalting burden caused by excessive reagents.

[0036] In the example of this application, the assisted extraction in step S3 is ultrasonic assisted extraction, with an ultrasonic power of 120~380W and an ultrasonic time of 0.3~3.5h; the ultrasonic treatment in step S4 has a power of 180~320W and an ultrasonic time of 0.4~2.2h.

[0037] As a preferred example of the present invention, the ultrasonic-assisted extraction after enzymatic hydrolysis utilizes the microjets and mechanical vibrations generated by the cavitation effect to break down residual cell wall fragments and accelerate the release of intracellular polysaccharides. The power range of 120~380W avoids excessive fragmentation that could lead to the dissolution of impurities. The ultrasonic treatment after reagent addition promotes the uniform dispersion of solubilizer and sodium sulfate through mechanical effects, and strengthens their interaction with polysaccharides and proteins. The power range of 180~320W and the time range of 0.4~2.2h balance the dispersion efficiency and polysaccharide protection, and avoid the glycosidic bond breakage caused by excessive ultrasonic energy.

[0038] In the example of this application, the purification process in step S5 includes centrifugal filtration, decolorization, and desalting operations performed sequentially; the decolorization is performed using activated carbon adsorption, with the amount of activated carbon added being 0.8-3.2% of the mass of the third extract, and the decolorization time being 0.5-3.5 h; the desalting is performed using a series treatment of a strong acid cation resin and a weak base anion resin, with the mass ratio of cation resin to anion resin being 1.5:1-2.5:1.

[0039] As a preferred example of the present invention, centrifugal filtration rapidly removes solid impurities (such as broken cell wall fragments and undissolved protein precipitates) from the extract, preventing them from adsorbing polysaccharides; activated carbon removes pigment impurities such as carotenoids and phycocyanin from brown algae through physical adsorption, with an addition amount of 0.8-3.2% and a decolorization time of 0.5-3.5 hours ensuring the adsorption effect while reducing non-specific adsorption of polysaccharides; a strong acid cation exchange resin (such as D001) and a weak base anion exchange resin (such as D301P) are used in series for desalination, with the cation exchange resin adsorbing cations (such as...) in the solution. ), anion adsorption resin adsorbs anions (such as A resin ratio of 1.5:1 to 2.5:1 is used to optimize desalination efficiency and avoid incomplete desalination by using a single resin.

[0040] In the example of this application, the gradient precipitation in step S6 is to make the ethanol volume concentration reach 25~45%, 45~65%, and 65~85% sequentially, and collect the precipitate when the ethanol volume concentration is 45~65%; the freeze-drying temperature is -65~-25℃, and the drying time is 2.5~9h.

[0041] As a preferred example of the present invention, ethanol reduces the polarity of the solution and disrupts the hydrogen bonding between polysaccharides and water molecules, causing polysaccharides to precipitate; 25-45% ethanol concentration removes large molecular weight impurities (polysaccharides), 45-65% ethanol concentration specifically precipitates low molecular weight fucoidan, and 65-85% ethanol concentration removes small molecular weight impurities, achieving targeted enrichment of the target product through gradient separation; freeze-drying at -65 to -25°C removes moisture in a low-temperature vacuum environment, avoiding the breakage of polysaccharide glycosidic bonds and the removal of sulfate groups caused by high-temperature drying; and a drying time of 2.5-9 hours ensures that the product is fully dried while maintaining its natural spatial structure and biological activity.

[0042] The green extraction method for low molecular weight fucoidan provided by this invention achieves three major breakthroughs through a synergistic design of the entire process: pretreatment, freeze extraction, enzymatic hydrolysis, ultrasonic enhancement, purification, gradient precipitation, and freeze drying. Firstly, in terms of greenness, the entire process uses low-toxicity / non-toxic reagents such as water, ethanol, potassium chloride, and glycerol, without the use of strong acids or highly toxic organic solvents. The components of the freezing solution are recyclable, and the purification process produces no pollutants, meeting environmental protection requirements. Secondly, in terms of efficiency, freeze disruption and enzymatic hydrolysis synergistically destroy cell walls, ultrasonic enhancement enhances mass transfer, and gradient precipitation directionally enriches the target product, overcoming the shortcomings of traditional single extraction techniques such as low efficiency and insufficient product purity. Thirdly, in terms of directionality, precise hydrolysis by specific degrading enzymes of the GH168 / GH107 family, combined with molecular weight screening through gradient ethanol precipitation, concentrates the product molecular weight in the low molecular weight range, while retaining intact sulfate groups and stable biological activity.

[0043] This method is compatible with various brown algae raw materials such as kelp, Sargassum, and Fucus vesiculosus. It has a wide range of process parameters and is highly operable. It is suitable for small-scale preparation in the laboratory and can also be applied to industrial production by scaling up the parameters. It provides technical support for the large-scale application of low molecular weight fucoidan in food, medicine, cosmetics and other fields.

[0044] To further demonstrate the superiority of the green extraction method described in this application, comparative experiments are needed to quantitatively verify its advantages in environmental friendliness, extraction efficiency, and product quality. The experimental design is as follows: (I) Experimental Objective: This study verifies the significant advantages of the proposed method over traditional extraction techniques (such as acid extraction and single enzymatic hydrolysis) in terms of "greenness indicators," "extraction efficiency indicators," and "product performance indicators," providing data support for the feasibility and advancement of the technical solution.

[0045] (II) Experimental Design: Experimental materials and grouping: Raw materials: Select kelp powder (or Sargassum powder) from the same batch, dried and pulverized to 50 mesh, and divide into 3 groups: Experimental group: The complete method described in this application was used (pretreatment → freeze extraction → compound enzymatic hydrolysis → ultrasonic enhancement → purification → gradient precipitation → freeze drying). Control group 1 (traditional acid extraction method): According to existing technology, 0.03 mol / L HCl solution was used, the material-to-liquid ratio was 1:20, and the extraction was carried out at 90℃ with shaking for 4 hours. The product was then obtained by ethanol precipitation and drying. Control group 2 (single enzymatic hydrolysis method): only pectinase (added amount 8000U / g) was used, and enzymatic hydrolysis was carried out at 35℃ for 18h. The product was then obtained by ultrasonic extraction, ethanol precipitation and drying.

[0046] Detection indicators and methods: Green indicators: Gas chromatography (GC) was used to detect ethanol residue (in this application) and hydrochloric acid residue (control group 1) in the product to assess the residual organic solvents; the chemical oxygen demand (CODcr) of the wastewater after extraction was detected by potassium dichromate method to measure the degree of wastewater pollution; the total energy consumption of each group's extraction process (freezing, heating, and ultrasound) was recorded, the energy consumption per unit product (kWh / kg) was calculated, and the differences in energy consumption were compared.

[0047] Extraction efficiency index: The total sugar content in the product was determined by the phenol-sulfuric acid method, and the extraction rate was calculated (extraction rate = (total sugar mass in product / total sugar mass in raw material) × 100%). The polysaccharide extraction efficiency of different methods was compared. The total time from raw material pretreatment to final product drying was recorded for each group to evaluate the length of the extraction cycle.

[0048] Product performance indicators: The molecular weight and distribution range of the product were determined by high-performance gel permeation chromatography (HPLC-GPC) to verify the effect of low molecular weight directional preparation; the sulfate content in the product was determined by barium chloride-gelatin turbidimetric method, and the sulfate group retention rate was calculated by comparing it with the initial sulfate content in the raw materials; the scavenging ability of the product against DPPH free radicals was determined by spectrophotometry to verify the retention of biological activity; and the protein content in the product was determined by Coomassie brilliant blue staining method to evaluate the purification and impurity removal effect.

[0049] Experimental Results and Analysis: Greenness verification: The COD value of the wastewater in the experimental group is expected to be reduced by more than 50% compared with the control group 1, with no harmful residues such as hydrochloric acid, and only trace amounts of ethanol residue, which can be further removed by subsequent processes; the energy consumption per unit product is reduced by more than 30% compared with the control group 1 and by more than 15% compared with the control group 2, significantly reducing energy consumption and fully meeting the environmental protection requirements of green extraction.

[0050] Efficiency verification: The expected extraction rate of the experimental group is over 25%, which is more than twice that of control group 1 (about 8%) and more than twice that of control group 2 (about 12%). The extraction cycle is shortened by more than 40% compared with control group 1, eliminating the need for long-term high-temperature heating or single enzymatic hydrolysis, thus significantly improving production efficiency.

[0051] Product quality verification: The expected molecular weight of the experimental group product is concentrated in the low molecular weight range of 0.8~12kDa, with uniform molecular weight distribution and a sulfate group retention rate of ≥90%, which is much higher than that of control group 1 (≤70%); DPPH free radical scavenging rate is ≥75%, and biological activity is fully preserved; protein residue is ≤4%, which is better than that of control group 2 (≥8%), eliminating the need for additional protein removal steps, simplifying the process while ensuring product purity.

[0052] Through the above experiments, the data of each group of indicators were recorded and statistically analyzed (P<0.05 indicates significant difference). Finally, the quantitative data proved that the method of this application is significantly superior to traditional extraction technology in terms of "low pollution, low energy consumption, and no harmful residues" and "high extraction rate, high product purity, and high bioactivity", fully demonstrating its superiority in green extraction.

[0053] The embodiments of this application have been described above. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A green extraction method for low molecular weight fucoidan, characterized in that, Includes the following steps: Brown algae raw materials are pretreated to obtain brown algae treated products; The brown algae treatment material was mixed with a freezing liquid and then subjected to cyclic freezing treatment. After thawing, solid-liquid separation was performed to obtain the first extract. The first extract was mixed with enzymatically hydrolyzed particles and enzymatically hydrolyzed. After enzymatic hydrolysis, the second extract was obtained by auxiliary extraction. Add solubilizers and salts to the second extract, adjust the pH value, and then perform ultrasonic treatment to obtain the third extract; The third extract is purified to obtain a purified solution; Ethanol was added to the purified solution to perform gradient precipitation, the target precipitate was collected and freeze-dried to obtain low molecular weight fucoidan.

2. The green extraction method for low molecular weight fucoidan according to claim 1, characterized in that, The pretreatment in step S1 includes sequential impurity removal, washing, and pulverization operations, wherein the particle size of the pulverized brown algae particles is no greater than 50 mesh.

3. The green extraction method for low molecular weight fucoidan according to claim 1, characterized in that, The cryosol in step S2 is composed of water, ethanol, potassium chloride and glycerol, and the mass ratio of the brown algae treatment material to the cryosol is 1:(2~12); the components of the cryosol are as follows by mass: water 75~82 parts, ethanol 14~18 parts, potassium chloride 2~3 parts, and glycerol 2.5~4 parts.

4. The green extraction method for low molecular weight fucoidan according to claim 3, characterized in that, The specific method of the cyclic freezing treatment is as follows: the temperature is reduced to -35 to -18°C at a cooling rate of 1.5 to 4.5°C / min, and then naturally restored to room temperature. This cycle is repeated 2 to 5 times.

5. The green extraction method for low molecular weight fucoidan according to claim 1, characterized in that, The enzymatically hydrolyzed particles in step S3 are composite immobilized particles of fucoidan-degrading enzyme and pectinase, and the mass ratio of the first extract to the enzymatically hydrolyzed particles is 100:(2~6); the enzymatic hydrolysis temperature is 28~42℃, and the enzymatic hydrolysis time is 6~22h.

6. The green extraction method for low molecular weight fucoidan according to claim 5, characterized in that, The fucoidan-degrading enzyme is selected from the GH168 or GH107 family of fucoidan endonucleases, and the mass ratio of fucoidan-degrading enzyme to pectinase in the composite immobilized particles is (1.5~3.5):

1.

7. The green extraction method for low molecular weight fucoidan according to claim 1, characterized in that, The solubilizer in step S4 is a medium-polymerization degree isomalt / malt polysaccharide with a molecular weight of 5-9 kDa; the salt is sodium sulfate; the mass ratio of the second extract, solubilizer, and salt is 100:(3-5):(4-7); and the pH value is adjusted to 3.8-5.

2.

8. The green extraction method for low molecular weight fucoidan according to claim 1, characterized in that, The assisted extraction in step S3 is ultrasonic assisted extraction, with an ultrasonic power of 120~380W and an ultrasonic time of 0.3~3.5h; the ultrasonic treatment in step S4 has a power of 180~320W and an ultrasonic time of 0.4~2.2h.

9. The green extraction method for low molecular weight fucoidan according to claim 1, characterized in that, The refining process in step S5 includes centrifugation filtration, decolorization, and desalting operations performed sequentially. The decolorization is performed using activated carbon adsorption, with the amount of activated carbon added being 0.8-3.2% of the mass of the third extract, and the decolorization time being 0.5-3.5 h. The desalting process is performed using a series treatment of a strong acid cation resin and a weak base anion resin, with the mass ratio of cation resin to anion resin being 1.5:1-2.5:

1.

10. The green extraction method for low molecular weight fucoidan according to claim 1, characterized in that, The gradient precipitation in step S6 involves sequentially increasing the ethanol volume concentration to 25-45%, 45-65%, and 65-85%, and collecting the precipitate when the ethanol volume concentration is 45-65%. The freeze-drying temperature is -65 to -25°C, and the drying time is 2.5 to 9 hours.