Method for increasing polysaccharide yield

Abstract

The invention provides a method for improving polysaccharide yield, which comprises the following steps: mixing raw material powder with deionized water to form a suspension, and adding cellulase, pectinase and polyethylene glycol 4000 according to a preset proportion; carrying out stepped temperature control enzymolysis on the constant-volume enzymolysis system to obtain an enzymolysis product; heating the enzymolysis product to 80-90 DEG C, and keeping the temperature for 5-10 minutes to inactivate the enzyme; carrying out pigment adsorption on the solution subjected to enzyme deactivation, and taking supernate; eluting the pigment-adsorbed solution with a low-concentration ethanol aqueous solution and a medium-concentration ethanol aqueous solution in sequence, and collecting a polysaccharide-containing ethanol aqueous solution obtained by eluting a high-concentration ethanol aqueous solution; and carrying out rotary evaporation concentration and freeze drying to obtain the target polysaccharide. Cellulase and pectinase are added to synergistically decompose a raw material structure, polyethylene glycol 4000 optimizes an enzymolysis environment, and polysaccharide is dissolved out to the maximum extent in combination with stepped temperature control enzymolysis; pigments and small molecular impurities are directionally removed through pigment adsorption so as to improve the purity of the polysaccharide, and finally, the target polysaccharide is separated and collected through gradient elution, so that the yield of the polysaccharide is improved.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction technology, and more specifically, to a method for improving polysaccharide yield. Background Technology

[0002] Polysaccharides, as an important class of biological macromolecules, are widely found in plants, animals, and microorganisms, and have shown great application value in many fields such as food, medicine, and cosmetics. In the food industry, polysaccharides can be used as thickeners, stabilizers, and preservatives to improve the texture, taste, and shelf life of food. They can also be developed into functional foods, such as polysaccharide beverages and oral liquids, providing health benefits to consumers. In the pharmaceutical industry, polysaccharides have various biological activities such as immunomodulation, anti-tumor activity, and hypoglycemia. They can be used as raw materials for the preparation of immunomodulators, anti-tumor drugs, and hypoglycemic drugs, and some polysaccharide products have already been used clinically. In the cosmetics industry, due to their moisturizing and antioxidant properties, polysaccharides are added to skin care products such as creams, lotions, and masks to achieve moisturizing and anti-aging effects. However, polysaccharide extraction currently faces numerous challenges, the most prominent being low yield. Traditional polysaccharide extraction methods, such as water extraction and alcohol precipitation, while simple and low-cost, suffer from low extraction efficiency and long processing times, limiting polysaccharide yield. Furthermore, the high polarity of water easily extracts water-soluble components such as proteins and glycosides, making the extract prone to spoilage during storage and hindering subsequent separation. While acid extraction can improve polysaccharide extraction rates to some extent—for example, some polysaccharides containing acidic groups like glucuronic acid are difficult to dissolve at low pH values—acetic acid or hydrochloric acid can be used to acidify the extract before adding ethanol to precipitate the polysaccharide. Alternatively, copper salts can be added to form insoluble complexes or salt precipitates. However, acidic conditions may cause the breakage of glycosidic bonds in the polysaccharide, and acids can corrode containers; therefore, these methods are generally unsuitable except for weak acids. In the alkaline extraction method, polysaccharides are stable in alkaline solutions. Alkali is beneficial for the leaching of acidic polysaccharides, which can increase the yield of polysaccharides and shorten the extraction time. However, the extract contains other impurities, which makes the viscosity too high, making filtration difficult. In addition, the extract has a strong alkaline taste and the solution is yellow, which will affect the flavor and color of the finished product. Organic solvent extraction methods suffer from solvent residue issues, and the use of organic solvents poses certain hazards to the environment and operators. While supercritical fluid extraction offers advantages such as preserving the activity of active ingredients and eliminating solvent residue, its high equipment investment limits its large-scale application. These limitations of existing methods severely restrict the large-scale production and application of polysaccharides, failing to meet the growing market demand. With the increasing global awareness of health, the market demand for plant polysaccharides, as a natural bioactive substance, is showing a continuous upward trend. Therefore, improving polysaccharide yield is particularly important and urgent. This can not only reduce production costs and improve production efficiency but also promote the large-scale and industrialized development of polysaccharide-related industries, meeting the market's growing demand for polysaccharides. Summary of the Invention

[0003] The main objective of this invention is to provide a method for improving polysaccharide yield, so as to at least solve the problems of low polysaccharide extraction efficiency and limited yield in the prior art.

[0004] To achieve the above objectives, the present invention provides a method for increasing polysaccharide yield, comprising the following steps: Step 1: Pre-treat the raw materials used for polysaccharide extraction. The pre-treatment includes washing, drying, and pulverizing the raw materials to obtain raw material powder. Step 2: Mix the raw material powder with deionized water to form a suspension, add cellulase, pectinase and polyethylene glycol 4000 according to the preset ratio, stir to dissolve, and obtain the enzymatic hydrolysis system, and make up the volume. Step 3: Perform step-temperature controlled enzymatic hydrolysis on the enzymatic hydrolysis system after adjusting the volume. The step-temperature controlled enzymatic hydrolysis is performed sequentially at 30~35℃ for 1~2 hours, 40~45℃ for 1~2 hours, and 50~55℃ for 0.5~1 hours to obtain the enzymatic hydrolysis product. Step 4: Heat the enzymatic hydrolysate to 80-90°C and maintain for 5-10 minutes to inactivate the enzyme; Step 5: Adsorb pigments from the enzyme-inactivated solution and collect the supernatant; Step 6: Perform gradient elution separation on the solution after adsorption of pigments, successively eluting with low-concentration and medium-concentration ethanol aqueous solutions, and collect the polysaccharide-containing ethanol aqueous solution obtained by high-concentration ethanol aqueous solution elution; concentrate the polysaccharide-containing ethanol aqueous solution by rotary evaporation and freeze-dry to obtain the target polysaccharide.

[0005] Optionally, in step 1, the drying is performed at 60~70℃ until the moisture content of the raw material is ≤8%.

[0006] Optionally, in step 2, the initial mixing mass-to-volume ratio of the raw material powder to deionized water is 1:10 (g / mL), and the concentration of the raw material powder in the enzymatic hydrolysis system is 80 g / L.

[0007] Optionally, the reagent used to adjust the pH in step 2 is a 0.1 mol / L hydrochloric acid solution or a 0.1 mol / L sodium hydroxide solution.

[0008] Optionally, in step 2, the cellulase activity is 4000~6000 U / g and the pectinase activity is 2000~4000 U / g.

[0009] Optionally, the mass ratio of cellulase to pectinase is 1:(0.9~1.5); The amount of polyethylene glycol 4000 added is 0.3% to 0.8% of the mass of the raw material powder in the suspension.

[0010] Optionally, in step 2, before making up the volume, the pH of the enzymatic hydrolysis system is adjusted to 4.5-5.0.

[0011] Optionally, the pigment adsorption conditions in step 5 include: using LX-8 resin, with the amount of LX-8 resin being 10%~15% of the volume of the solution after enzyme inactivation, a temperature of 25~30℃, a stirring speed of 40~60 r / min, and an adsorption time of 2~3 hours.

[0012] Optionally, before use, the LX-8 resin described in step 5 is soaked in 95% ethanol, washed with deionized water until there is no alcohol odor, then soaked in 5% hydrochloric acid solution, washed with deionized water until neutral, and finally soaked in 5% sodium hydroxide solution, washed with deionized water until neutral.

[0013] Optionally, the gradient elution conditions include: controlling the elution rate to 2~3 BV / h; and performing elution in the following order: (1) Use a 10% low-concentration ethanol aqueous solution with an elution volume of 1.0~1.5 BV; (2) Replace with a 30% medium-concentration ethanol aqueous solution, with an elution volume of 1.3~2.0 BV; (3) Replace with a 50% high-concentration ethanol aqueous solution, with an elution volume of 1.7~2.5 BV; Collect the effluent generated during the elution process of 50% ethanol aqueous solution in step (3).

[0014] This invention provides a method for improving polysaccharide yield, comprising pretreatment of the raw materials used for polysaccharide extraction, wherein the pretreatment includes washing, drying, and pulverizing the raw materials to obtain raw material powder; mixing the raw material powder with deionized water to form a suspension, adding cellulase, pectinase, and polyethylene glycol 4000 in a preset ratio, stirring to dissolve, obtaining an enzymatic hydrolysis system, and adjusting the volume; subjecting the adjusted-volume enzymatic hydrolysis system to stepwise temperature-controlled enzymatic hydrolysis, wherein the stepwise temperature-controlled enzymatic hydrolysis is performed sequentially at 30-35°C for 1-2 hours, 40-4... Enzymatic hydrolysis was performed at 5℃ for 1-2 hours and at 50-55℃ for 0.5-1 hours to obtain the enzymatic hydrolysis product. The enzymatic hydrolysis product was heated to 80-90℃ and held for 5-10 minutes to inactivate the enzyme. The solution after enzyme inactivation was subjected to pigment adsorption, and the supernatant was collected. The solution after pigment adsorption was subjected to gradient elution separation, successively eluted with low-concentration and medium-concentration ethanol aqueous solutions, and the polysaccharide-containing ethanol aqueous solution obtained by high-concentration ethanol aqueous solution was collected. The polysaccharide-containing ethanol aqueous solution was concentrated by rotary evaporation and freeze-dried to obtain the target polysaccharide. Thus, by pre-treating and pulverizing the raw materials, adding cellulase and pectinase to synergistically decompose the raw material structure, optimizing the enzymatic hydrolysis environment with polyethylene glycol 4000, and combining step-temperature controlled enzymatic hydrolysis to maximize the dissolution of polysaccharides, pigment adsorption was used to directionally remove pigments and small molecule impurities to improve the purity of polysaccharides, and finally the target polysaccharide was collected by gradient elution separation, the extraction efficiency and recovery of polysaccharides were improved, and the final yield of the target polysaccharide was effectively increased. Attached Figure Description

[0015] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a flowchart of an optional method for improving polysaccharide yield according to an embodiment of the present invention. Detailed Implementation

[0016] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0017] like Figure 1 As shown, this application provides a method for improving polysaccharide yield, comprising the following steps: Step 1: Pre-treat the raw materials used for polysaccharide extraction. The pre-treatment includes washing, drying, and pulverizing the raw materials to obtain raw material powder. Step 2: Mix the raw material powder with deionized water to form a suspension, add cellulase, pectinase and polyethylene glycol 4000 according to the preset ratio, stir to dissolve, and obtain the enzymatic hydrolysis system, and make up the volume. Step 3: Perform step-temperature controlled enzymatic hydrolysis on the enzymatic hydrolysis system after adjusting the volume. The step-temperature controlled enzymatic hydrolysis is performed sequentially at 30~35℃ for 1~2 hours, 40~45℃ for 1~2 hours, and 50~55℃ for 0.5~1 hours to obtain the enzymatic hydrolysis product. Step 4: Heat the enzymatic hydrolysate to 80-90°C and maintain for 5-10 minutes to inactivate the enzyme; Step 5: Adsorb pigments from the enzyme-inactivated solution and collect the supernatant; Step 6: Perform gradient elution separation on the solution after adsorption of pigments, successively eluting with low-concentration and medium-concentration ethanol aqueous solutions, and collect the polysaccharide-containing ethanol aqueous solution obtained by high-concentration ethanol aqueous solution elution; concentrate the polysaccharide-containing ethanol aqueous solution by rotary evaporation and freeze-dry to obtain the target polysaccharide.

[0018] Specifically, in step 1, the raw materials used for polysaccharide extraction are first cleaned by rinsing with clean water or soaking followed by rinsing to remove impurities such as mud, dust, and residual contaminants adhering to the surface of the raw materials, preventing impurities from entering the subsequent extraction system and affecting the purity of polysaccharides. After cleaning, the raw materials are dried by air drying or baking to remove free moisture, which not only prevents the raw materials from becoming moldy or deteriorating during storage or pulverization, but also makes the raw materials more uniform in texture, facilitating subsequent pulverization operations. Finally, the dried raw materials are placed in a pulverizer and pulverized to a particle size of 40-70 mesh. This particle size increases the specific surface area of ​​the raw materials, allowing for more thorough contact between the raw materials and enzymes and water in subsequent steps, creating conditions for improving enzymatic hydrolysis efficiency and promoting polysaccharide release, ultimately yielding raw material powder.

[0019] Step 2: Take the raw material powder obtained in Step 1 and mix it with deionized water in a certain proportion. Stir to evenly disperse the raw material particles in the deionized water to form a suspension, providing a carrier for the interaction between the enzyme and the raw material. Then, add cellulase, pectinase, and polyethylene glycol 4000 to the suspension in a preset proportion. Cellulase and pectinase are used to decompose the structural components such as cellulose and pectin in the raw material, helping to release the encapsulated polysaccharides. Polyethylene glycol 4000 helps optimize the system environment. Then, continue stirring for 10-30 minutes (the specific time can be adjusted according to the dissolution of the system) to ensure that the added enzymes and additives are fully dissolved and that the components are evenly distributed in the system, avoiding local concentrations that are too high or too low, which would affect enzyme activity, thus forming a preliminary enzymatic hydrolysis system. Finally, use a volumetric flask or designated container to bring the enzymatic hydrolysis system to a fixed volume by adding deionized water to determine the final concentration of the system, ensuring that the subsequent enzymatic hydrolysis reaction is carried out under stable and controllable conditions. Polyethylene glycol 4000 is food grade.

[0020] Step 3: Transfer the finalized enzymatic hydrolysis system to a reaction vessel with precise temperature control (such as a constant temperature water bath, constant temperature shaker, or bioreactor) to begin stepwise temperature-controlled enzymatic hydrolysis. First, set the temperature to 30-35°C and maintain this temperature for 1-2 hours. This temperature range is suitable for the initial activity of the enzymes, allowing them to gradually adapt to the system environment and slowly initiate the catalytic reaction, initially decomposing the cellulose and pectin on the surface of the raw materials, avoiding a sudden drop in enzyme activity due to a rapid temperature increase. Next, raise the temperature to 40-45°C and continue enzymatic hydrolysis for 1-2 hours. This temperature is closer to the optimal activity range of cellulase and pectinase, significantly increasing the catalytic rate and accelerating the process. The disruption of the cell wall structure of the raw material promotes the release of polysaccharides from the cells into the solution. Finally, the temperature is adjusted to 50-55℃ and incubated for 0.5-1 hour. During this stage, the catalytic effect of the enzyme is further enhanced by moderately increasing the temperature, ensuring that the difficult-to-decompose structural components in the raw material are fully degraded and the polysaccharides are released as fully as possible. At the same time, the short-term high temperature will not cause a large amount of degradation of the released polysaccharides. Throughout the enzymatic hydrolysis process, low-speed stirring (e.g., 20-50 r / min) can be maintained according to the system conditions to promote the contact between the substrate and the enzyme. After three-stage temperature control operations of heating and holding, an enzymatic hydrolysis product containing polysaccharides, enzymes, and incompletely degraded impurities is obtained.

[0021] Step 4: After Step 3, active cellulase and pectinase remain in the enzymatic hydrolysis product. If left untreated, these enzymes may continue to decompose polysaccharides or other components during subsequent transfer and settling, leading to polysaccharide chain breakage, reduced yield, or the generation of more small molecule impurities affecting purity. Therefore, the enzymatic hydrolysis product is transferred to a corrosion-resistant heating container (such as a glass beaker or stainless steel reactor). The temperature is slowly increased by water bath heating, oil bath heating, or electric heating to a stable temperature of 80-90°C (avoiding local overheating that could lead to polysaccharide carbonization). This temperature is maintained for 5-10 minutes. High temperatures will destroy the spatial structure of the enzyme (such as the breakage of hydrogen bonds and disulfide bonds), permanently eliminating its catalytic activity and thus completely terminating the enzymatic hydrolysis reaction. After enzyme inactivation, the container can be transferred to room temperature or a cold water bath for cooling to reduce the system temperature to room temperature or near room temperature. This prevents the high-temperature system from affecting the performance of the adsorbent in subsequent adsorption steps, ultimately yielding the enzyme-inactivated crude polysaccharide extract.

[0022] Step 5: The solution after enzyme inactivation contains impurities such as pigments from the raw materials (e.g., chlorophyll and flavonoids from plants, carotenoids from fungi) and small amounts of protein, in addition to the target polysaccharide. These pigments can cause the polysaccharide product to appear dark, affecting its appearance, and may also bind to the polysaccharide, affecting subsequent separation. Add the adsorbent to the enzyme-inactivated solution, transfer it to a container with a stirring function, and stir continuously at 40-60 rpm for 2-3 hours at 25-30°C. Alternatively, allow the adsorbent to fully contact the solution through static adsorption. Pigment molecules are captured by the adsorbent through physical adsorption (such as van der Waals forces, pore trapping) or chemical adsorption (such as hydrogen bonding). After adsorption, the adsorbent containing the pigment is separated from the solution by filtration (using qualitative filter paper, microporous filter membrane, or suction filtration with the help of Buchner funnel) or centrifugation (such as centrifugation at 3000~5000 r / min for 10~15 minutes). The clear solution without obvious pigment is taken as the supernatant. The supernatant mainly contains polysaccharide components, thus completing the pigment removal and reducing impurity interference for subsequent further separation and purification.

[0023] Step 6: Slowly load the supernatant after pigment adsorption into a pre-packed chromatography column containing separation media using a peristaltic pump or gravity dripping. Control the loading flow rate, ensuring the loading volume does not exceed 5% of the column bed volume to guarantee sufficient adsorption of the polysaccharide component onto the separation media surface. Simultaneously, some of the smaller molecular weight polyethylene glycol 4000 may elute with the unadsorbed liquid. After loading, use an ethanol-water solution as the eluent for gradient elution separation, with the gradient being low-concentration ethanol-water solution, medium-concentration ethanol-water solution, and high-concentration ethanol-water solution. The low-concentration ethanol-water solution preferentially elutes polyethylene glycol 4000 (which is readily soluble in the polar solvent of low-concentration ethanol), and collecting the eluent at this stage removes most of the polyethylene glycol 4000. The medium-concentration ethanol-water solution further elutes residual polyethylene glycol 4000 and small molecule impurities, ensuring no polyethylene glycol 4000 residue remains on the separation media surface. Only the polysaccharide ethanol-water solution obtained from the high-concentration ethanol-water solution is collected; at this point, the component is essentially free of polyethylene glycol 4000.

[0024] The collected polysaccharide-containing ethanol-water solution was transferred to the evaporation flask of a rotary evaporator and concentrated by rotary evaporation under reduced pressure (0.06~0.08MPa) and low temperature (40~50℃). Temperature control was required during this process to avoid polysaccharide degradation due to high temperature. Ethanol was recovered through a condenser to achieve solvent recycling. If trace amounts of polyethylene glycol 4000 remained during the concentration process, they would be further carried out with the evaporation of ethanol and water, reducing their content in the concentrate.

[0025] After concentrating the solution to a viscous consistency (solid content 20%~30%), the concentrate is transferred to the freeze-drying tray of a freeze dryer. It is first frozen at ~40 to ~50°C for 2~4 hours to completely freeze the concentrate into a solid. Then, the vacuum system is activated to maintain a vacuum level (e.g., 1~10 Pa), allowing the ice to sublimate directly into water vapor at low temperature to remove moisture. After freeze-drying, the resulting loose polysaccharide solid is pulverized (e.g., using a mortar and pestle or a small grinder), and sieved (80 mesh sieve) to obtain a uniformly sized dried target polysaccharide product. Finally, it can be packaged, sealed, and stored to prevent moisture absorption.

[0026] This application lays the foundation for polysaccharide release by pre-treating and pulverizing the raw materials. Then, it optimizes the enzymatic hydrolysis environment by adding cellulase and pectinase to synergistically decompose the raw material structure and by using polyethylene glycol 4000 to optimize the enzymatic hydrolysis environment. Combined with step-temperature controlled enzymatic hydrolysis, it precisely matches enzyme activity to maximize polysaccharide dissolution. Subsequently, it terminates enzymatic hydrolysis by enzyme inactivation to avoid polysaccharide degradation and removes pigments and small molecule impurities by adsorption to improve polysaccharide purity. Finally, it collects the target polysaccharide by gradient elution and then concentrates and dries it to preserve the integrity of the polysaccharide. Ultimately, it significantly improves the extraction efficiency and recovery amount of polysaccharides and effectively increases the final yield of the target polysaccharide.

[0027] In one possible implementation, in step 1, the drying is performed at 60-70°C until the moisture content of the raw material is ≤8%.

[0028] Specifically, from a temperature control perspective, 60-70℃ falls within the medium-low temperature range. This range allows for the gradual removal of free moisture from the raw materials through continuous heating, while also preventing damage to the polysaccharide structure due to excessively high temperatures (such as exceeding 80℃). Glycosidic bonds in polysaccharide molecules are sensitive to high temperatures; excessively high temperatures can easily cause them to break, leading to polysaccharide degradation or denaturation. Temperatures of 60-70℃ maximize the preservation of polysaccharide integrity, laying the foundation for subsequent extraction of high-quality polysaccharides. From a moisture content control perspective, limiting the final moisture content of the raw materials to ≤8% significantly inhibits the reproductive activity of microorganisms (such as molds and yeasts). Microbial growth requires certain moisture conditions. A dry environment with a moisture content of ≤8% can cut off its moisture supply, effectively preventing the raw materials from becoming moldy and deteriorating during subsequent storage or pulverization, and ensuring the stability of the raw material quality. On the other hand, raw materials with a moisture content of ≤8% will become dry and brittle, avoiding the clumping caused by excessive moisture content. This not only facilitates subsequent pulverization operations (easily pulverizing into uniform particle size powder, reducing clogging problems in pulverization equipment), but also allows the pulverized raw material powder to have more complete contact with the water and enzymes in the enzymatic hydrolysis system, reducing enzymatic hydrolysis dead zones caused by the agglomeration of raw material particles due to moisture, creating favorable conditions for improving subsequent enzymatic hydrolysis efficiency and promoting the full dissolution of polysaccharides.

[0029] In one possible implementation, the initial mixing mass-to-volume ratio of the raw material powder to deionized water in step 2 is [value missing], and the concentration of the raw material powder in the enzymatic hydrolysis system at a constant volume is 80 g / L.

[0030] Specifically, the initial mixing mass-to-volume ratio is 1:10 (g / mL), meaning 10 mL of deionized water is added for every 1 g of raw material powder. This ratio allows the raw material powder to initially disperse and form a uniform suspension, avoiding agglomeration due to insufficient water (agglomeration makes it difficult for subsequent enzymes to contact the polysaccharides inside the raw material), or excessive water leading to a low raw material concentration and wasted solvent during subsequent volume adjustment. After volume adjustment, the raw material powder concentration in the enzymatic hydrolysis system is 80 g / L, a final concentration achieved by adding deionized water based on the initial mixing. This concentration ensures that there is sufficient raw material in the system for enzymatic hydrolysis (avoiding insufficient total polysaccharide extraction due to low concentration), and also provides suitable space for cellulase and pectinase to function. The enzyme molecules can diffuse fully in the suspension at this concentration, uniformly contact the surface of the raw material particles and penetrate into the interior, efficiently decompose the cell wall structure to release polysaccharides, and will not cause the system viscosity to increase or the contact between the enzyme and the raw material to be hindered due to the raw material concentration being too high (above 120g / L), or the enzymatic hydrolysis efficiency to decrease or the energy consumption and time cost to increase due to the concentration being too low (such as below 50g / L).

[0031] On the one hand, the initial mixing ratio of 1:10 and the final concentration of 80 g / L can reduce the ineffective consumption of enzymes (avoiding waste due to enzymes not being able to contact the raw materials) and promote the full dissolution of polysaccharides, laying the foundation for improving the final yield. On the other hand, the clear ratio and concentration settings make the process more repeatable, and the state of the enzymatic hydrolysis system can be stably controlled during different batches of operation, avoiding differences in polysaccharide yield caused by uneven dispersion of raw materials or concentration fluctuations. At the same time, reasonable water control also reduces the amount of solvent to be processed in the subsequent concentration steps, reducing energy consumption and production costs.

[0032] In one possible implementation, the reagent used to adjust the pH in step 2 is a 0.1 mol / L hydrochloric acid solution or a 0.1 mol / L sodium hydroxide solution.

[0033] Specifically, 0.1 mol / L hydrochloric acid solution or 0.1 mol / L sodium hydroxide solution is selected to adjust the pH of the enzymatic hydrolysis system. The catalytic activity of cellulase and pectinase depends on a specific pH environment. The pH of the system must be adjusted to this range using acid-base reagents to ensure efficient enzyme function. A concentration of 0.1 mol / L satisfies the precision requirements of pH adjustment; the low concentration allows for a slow change in the system pH, avoiding sudden pH rises and falls due to excessive concentration (e.g., high concentrations of acids and bases can easily cause the pH to exceed the enzyme's optimal range, leading to enzyme denaturation and inactivation). It also reduces additional interference from the reagents, preventing excessive addition of Cl- from increasing the concentration in the system. - Na +Excessive plasma concentration can inhibit enzyme activity or affect subsequent polysaccharide purification steps. Meanwhile, hydrochloric acid and sodium hydroxide, as common chemical reagents, are stable, have easily controllable purity, and are relatively inexpensive. They are suitable for standardized pH adjustment in process operations, ensuring consistent pH conditions across different batches of enzymatic hydrolysis systems, thus providing a foundation for stable enzymatic hydrolysis efficiency.

[0034] In one possible implementation, in step 2, the cellulase activity is 4000~6000 U / g and the pectinase activity is 2000~4000 U / g.

[0035] Specifically, the cell walls of plant raw materials are mainly composed of cellulose, pectin, and other components. Polysaccharides are mostly encapsulated within the cell walls. Cellulase and pectinase are needed to break down the cell wall structure and allow the polysaccharides to fully dissolve. Cellulase, with an activity of 4000-6000 U / g (U being the enzyme activity unit, representing the enzyme's ability to catalyze substrates), can efficiently catalyze the hydrolysis of cellulose into small-molecule sugars. This avoids incomplete cellulose decomposition due to insufficient enzyme activity (cell walls cannot be fully broken down, hindering polysaccharide release) or waste of enzyme resources due to excessive enzyme activity (exceeding the amount of enzyme required to decompose cellulose in the raw material). Pectinase, with an activity of 2000-4000 U / g, can effectively decompose pectin components in the cell walls, assisting cellulase in breaking down the cell wall barrier, and forms a reasonable ratio with cellulase activity, avoiding an imbalance in enzymatic hydrolysis efficiency caused by excessive or insufficient use of a single enzyme. The amount of cellulase used is 4.0% to 6.0% of the raw material powder mass, and the amount of pectinase used is 5.0% to 7.0% of the raw material powder mass.

[0036] In one possible implementation, the mass ratio of the cellulase to the pectinase is 1:(0.9~1.5); The amount of polyethylene glycol 4000 added is 0.3% to 0.8% of the mass of the raw material powder in the suspension.

[0037] Specifically, regarding the enzyme ratio, cellulase is used to decompose the cellulose skeleton of plant cell walls, while pectinase is used to degrade the pectin-binding components in the cell walls. A mass ratio of 1:(0.9~1.5) avoids both excessive cellulase (leading to incomplete pectin decomposition, remaining cell wall adhesion, and hindered polysaccharide release) and excessive pectinase (resulting in wasted enzyme resources, and excess pectinase may bind with polysaccharides, interfering with subsequent purification). This ratio is suitable for the conventional cellulose to pectin content ratio in most plant raw materials, maximizing cell wall decomposition efficiency and opening channels for polysaccharide dissolution. The preferred mass ratio of cellulase to pectinase is 1:1.2.

[0038] Polyethylene glycol 4000 is used to improve the environment of the enzymatic hydrolysis system (such as reducing enzyme molecule aggregation and improving enzyme stability). If the addition amount is below 0.3%, it cannot effectively optimize the environment, and enzyme activity is easily affected by impurities in the system, leading to a decrease. If the addition amount is above 0.8%, it not only increases costs but may also cause an increase in system viscosity due to excessive polyethylene glycol 4000, which in turn affects the contact efficiency between the enzyme and the raw materials. Furthermore, it increases the difficulty of subsequent separation and removal of polyethylene glycol 4000. Preferably, the addition amount of polyethylene glycol 4000 is 0.5% of the mass of the raw material powder in the suspension.

[0039] In one possible implementation, in step 2, before final volume determination, the pH of the enzymatic hydrolysis system is adjusted to 4.5-5.0.

[0040] Specifically, cellulase activity is pH-sensitive. The optimal pH range for most industrial-grade cellulases is 4.0–5.0, within which the enzyme molecule's spatial structure is stable, resulting in the highest efficiency in catalyzing cellulose hydrolysis. While the optimal pH for pectinase is slightly lower (3.5–4.5), a pH range of 4.5–5.0 still ensures high activity, effectively degrading pectin components in cell walls. Adjusting the pH before volume adjustment prevents pH fluctuations caused by solvent dilution, ensuring the final enzymatic hydrolysis system's pH falls precisely within the enzyme's highly active range. This prevents enzyme denaturation and inactivation due to pH deviations, thus guaranteeing efficient cell wall decomposition and polysaccharide dissolution. The preferred pH value for this adjustment is 4.8.

[0041] In one possible implementation, the pigment adsorption conditions in step 5 include: using LX~8 resin, with the amount of LX~8 resin being 10%~15% of the volume of the solution after enzyme inactivation, a temperature of 25~30℃, a stirring speed of 40~60 r / min, and an adsorption time of 2~3 hours.

[0042] Specifically, LX~8 resin exhibits strong selective adsorption capacity for pigments such as polyphenols and flavonoids in plant enzymatic hydrolysates, while showing extremely low adsorption rate for polysaccharides (reducing polysaccharide loss along with pigments). It can remove impurities while protecting the target components. The resin dosage is 10%~15% of the solution volume after enzyme inactivation. This ensures sufficient adsorption sites for complete pigment adsorption (avoiding insufficient dosage leading to pigment residue that affects the color and purity of subsequent polysaccharide products), while also preventing excessive dosage from wasting resin and increasing subsequent solid-liquid separation costs. An adsorption temperature of 25~30℃ can balance... Regarding resin adsorption efficiency and stability, LX-8 resin exhibits high adsorption activity at this temperature, avoiding structural changes or pigment desorption caused by high temperatures, while also preventing excessively slow adsorption rates and prolonged process time due to low temperatures. A stirring speed of 40-60 r / min ensures sufficient contact between the resin and the solution (avoiding resin sedimentation leading to incomplete local adsorption) without causing resin particle wear or polysaccharide molecule shearing damage due to excessively high speed. An adsorption time of 2-3 hours promotes complete binding between pigments and resin, avoiding incomplete adsorption due to too short a time or increased energy consumption and process cycle due to too long a time.

[0043] The optimal conditions for pigment adsorption are as follows: LX-8 resin dosage is 12% of the solution volume after enzyme inactivation, temperature is 28℃, stirring speed is 50r / min, and adsorption time is 2.5 hours.

[0044] In one possible implementation, the LX-8 resin described in step 5 is soaked in 95% ethanol and washed with deionized water until there is no alcohol odor before use, then soaked in 5% hydrochloric acid solution and washed with deionized water until neutral, and finally soaked in 5% sodium hydroxide solution and washed with deionized water until neutral.

[0045] Specifically, LX-8 is first soaked in 95% ethanol. The solubility of ethanol can remove organic impurities (such as monomers, oligomers, and oil stains) remaining during the resin production or storage process. If these impurities remain, they will occupy the resin adsorption sites and reduce the adsorption capacity for pigments. The subsequent water washing until there is no alcohol odor prevents ethanol from entering the enzymatic hydrolysate and preventing it from having a potential impact on the polysaccharide structure. Next, it is soaked in 5% hydrochloric acid. The acidic environment can dissolve metal ion impurities (such as calcium and magnesium ions) on the resin surface and in the pores, while adjusting the surface charge state of the resin to create suitable conditions for the subsequent adsorption of polar pigments. The water washing until neutral can eliminate the interference of the acidic environment on the pH of the enzymatic hydrolysate. Finally, it is soaked in 5% sodium hydroxide. On the one hand, it can further remove residual alkaline impurities. On the other hand, it can moderately activate the functional groups of the resin (such as hydroxyl and amino groups) to enhance its adsorption affinity for pigment molecules. The water washing until neutral can prevent alkaline substances from entering the system and causing polysaccharide degradation or abnormal pH of the enzymatic hydrolysate. The entire pretreatment process is progressive, which not only ensures the cleanliness of the resin, but also optimizes its adsorption performance through acid and alkali treatment, laying the foundation for efficient and precise removal of pigments and impurities while reducing polysaccharide loss.

[0046] In one possible implementation, the gradient elution conditions include: controlling the elution rate at 2~3 BV / h; and performing elution in the following order: (1) Use a 10% ethanol aqueous solution with an elution volume of 1.0~1.5 BV; (2) Replace with 30% ethanol aqueous solution, with an elution volume of 1.3~2.0 BV; (3) Replace with 50% ethanol aqueous solution, with an elution volume of 1.7~2.5 BV; Collect the effluent generated during the elution process of 50% ethanol aqueous solution in step (3).

[0047] Specifically, controlling the elution rate to 2~3 BV / h (BV refers to the resin column volume, which is the total volume of LX-8 resin packed in the chromatography column) ensures sufficient contact between the eluent and the resin particles, allowing different components enough time to desorb gradually according to their adsorption strength. This avoids impurities and polysaccharides from co-eluting due to excessively high flow rates, while also preventing excessively slow flow rates from prolonging the process and increasing energy consumption. Eluting is performed sequentially with "low concentration → medium concentration → high concentration" ethanol. Low-concentration (10%) ethanol is particularly effective for removing small molecule impurities with weak resin adsorption (such as residual monosaccharides from enzymatic hydrolysis, low...). Polysaccharides and small amounts of water-soluble pigments have stronger desorption capabilities, and elution volumes of 1.0–1.5 BV can completely remove these impurities. Medium-concentration (30%) ethanol can specifically desorb impurities with moderate adsorption capacity (such as some polar pigments and oligosaccharides), and elution volumes of 1.3–2.0 BV can further purify the system. High-concentration (50%) ethanol has the strongest desorption capacity and can effectively displace target polysaccharides with strong binding to the resin from the adsorption sites. Volumes of 1.7–2.5 BV can ensure sufficient elution of polysaccharides and reduce yield loss due to incomplete elution. Collecting the eluted fraction from 50% ethanol can minimize the loss of polysaccharides during the initial low- and medium-concentration ethanol elution and eliminate interference from impurities.

[0048] In this application, the elution rate is preferably 2.5 BV / h; the elution volume of 10% low-concentration ethanol aqueous solution is preferably 1.2 BV, the elution volume of 30% medium-concentration ethanol aqueous solution is preferably 1.6 BV, and the elution volume of 50% high-concentration ethanol aqueous solution is preferably 2.2 BV.

[0049] The following examples further illustrate this.

[0050] I. Experimental Preparation Raw materials: Dried Ningxia wolfberries (moisture content 12.3%, initial polysaccharide content 10.5% as determined by high performance liquid chromatography (HPLC), 100.0g / set, discarding moldy and broken particles). Reagents: Cellulase (brand: Novozymes, model: Celluclast 1.5L, enzyme activity 5000U / g, purity 98.5%), pectinase (brand: Genentech, model: Pectinex UltraSP-L, enzyme activity 3000U / g, purity 97.8%), LX-8 macroporous adsorption resin (particle size 0.3~1.25mm, specific surface area 800~1000m² / g), 95% ethanol (analytical grade, purity 95.2%), 5% hydrochloric acid solution (accurate concentration 5.02%), 5% sodium hydroxide solution (concentration 5.05%), 0.1mol / L hydrochloric acid solution (concentration 0.102mol / L), deionized water.

[0051] Instruments: High-speed universal grinder (model FW100, sieve aperture 0.25mm, i.e., 60 mesh), constant temperature water bath (model HH-S4, temperature control accuracy ±0.1℃), high-speed centrifuge (model TGL-16M, maximum speed 16000r / min), rotary evaporator (model RE-52AA, vacuum degree 0.07MPa, temperature control accuracy ±0.5℃), freeze dryer (model FD-1A-50, cold trap temperature -50℃, vacuum degree 1Pa), high performance liquid chromatograph (model Agilent1260, chromatographic column ZORBAXSB-C18, 4.6×250mm).

[0052] II. Polysaccharide Preparation (Experimental Group) Take 100.0g of Ningxia wolfberries, rinse them 3 times with deionized water (500mL of water each time, 30 seconds each time), put them in an oven at 65℃ and dry for 4.5h. The moisture content was measured to be 7.0% by a moisture analyzer. The wolfberry powder was then pulverized for 30 seconds using an FW100 pulverizer and passed through a 60-mesh sieve. Add 100.0g of wolfberry powder and 1000.0mL of deionized water to a 2000mL beaker. Stir with a magnetic stirrer (300r / min) for 15 minutes to form a uniform suspension. Then add 5.0g of cellulase, 6.0g of pectinase (mass ratio 1:1.2), and 0.5g of polyethylene glycol (accurate to 0.001g). Continue stirring for 20 minutes. Adjust the pH of the system to 4.8 with 0.102mol / L hydrochloric acid solution (monitor in real time with a pH meter, error ±0.01). Transfer the solution to a 1250mL volumetric flask and add deionized water to the mark. At this point, the concentration of the raw material powder in the system is accurate to 80.0g / L. The entire enzymatic hydrolysis system after being brought to a constant volume was transferred to a 5000mL three-necked flask, placed in a constant temperature water bath, and kept at 32℃ for 1.5h (stirring speed 30r / min); then the temperature was increased to 43℃ and kept at 1.5h; finally the temperature was increased to 52℃ and kept at 0.8h, with continuous stirring during the process, to obtain the enzymatic hydrolysis product. Transfer the three-necked flask to an electric heating mantle and heat the enzymatic hydrolysis product to 85°C (monitor in real time with a thermometer, error ±0.5°C), and maintain for 8 minutes; then place the flask in a 20°C cold water bath and cool to 25°C to obtain the enzyme-inactivated solution, the volume of which is 1248 mL as measured by a graduated cylinder. Take 149.8 mL of pretreated LX-8 resin (accurately calculated based on 12% of the solution volume after enzyme inactivation) and add it to the enzyme-inactivated solution; transfer the mixture to a constant temperature stirring tank, set the temperature to 28℃, the stirring speed to 50 r / min, and continue stirring for 2.5 h; then transfer the mixture to centrifuge tubes, centrifuge at 4000 r / min for 12 minutes using a TGL-16M centrifuge, and take 1200 mL of the supernatant; Take a 20×300mm chromatography column and pack it with LX-8 resin to a column volume of 100mL (BV=100mL). Load the supernatant using a peristaltic pump (5mL sample, i.e., 0.05BV), controlling the elution rate at 2.5BV / h (i.e., 250mL / h). First, elute with 120mL (1.2BV) of 10% ethanol aqueous solution and collect the eluent. Then, replace the eluent with 160mL (1.6BV) of 30% ethanol aqueous solution and collect the eluent. Finally, replace the eluent with 22mL (1.6BV) of 50% ethanol aqueous solution. Elute with 0 mL (2.2 BV) and collect 218 mL of eluent from this stage; transfer the collected 50% ethanol eluent to a rotary evaporator, set the temperature to 45℃ and the vacuum degree to 0.07 MPa, and concentrate for 1.5 h to a solid content of 25.0% (measured by a refractometer); transfer the concentrate to a freeze-drying tray, place it in a freeze dryer, freeze at -45℃ for 3.0 h, and then freeze-dry at a vacuum degree of 5 Pa for 8.0 h; grind the freeze-dried polysaccharide solid with a mortar and pestle, pass it through an 80-mesh sieve, and weigh to obtain 12.30 g of polysaccharide. III. Polysaccharide Preparation (Control Group) Control group 1: No enzymatic hydrolysis, no resin adsorption, no gradient elution, extraction was completed only by water extraction at 80℃ (2h+1h re-extraction), concentration, precipitation with 3 times ethanol, and drying. Control group 2: The steps of “construction of enzymatic hydrolysis system + step temperature-controlled enzymatic hydrolysis” were removed. The raw material suspension was directly heated at 80℃ for 2.5h for extraction. The remaining steps (enzyme inactivation, adsorption, elution, etc.) were the same as those of the experimental group. Control group 3: The enzymatic hydrolysis stage was changed to 43℃ constant temperature enzymatic hydrolysis for 3.3h (the same as the total enzymatic hydrolysis time of the new scheme), and the remaining steps were the same as the experimental group. Control group 4: The "pigment adsorption" step was removed, and gradient elution was performed directly after enzyme inactivation. The remaining steps were the same as those in the experimental group.

[0053] Control group 5: Only cellulase was added (11.0g, the same as the total mass of cellulase + pectinase in the experimental group), and the enzymatic hydrolysis temperature, time and other steps (pH adjustment, adsorption, elution, etc.) were the same as the experimental group.

[0054] IV. Experimental Data Table 1. Polysaccharide yield and purity data prepared from the experimental and control groups.

[0055] As shown in Table 1, the experimental group was significantly better than the control group in both the polysaccharide yield and purity. The experimental group used a combination of "compound enzyme synergy + step-temperature controlled enzymatic hydrolysis + resin adsorption" to efficiently promote polysaccharide dissolution to increase yield and remove impurities to ensure purity. IV. Advantages and Applications of the Experimental Group The experimental group improved the yield and purity of polysaccharides through a triple optimization design: synergistic dissolution with compound enzymes, stepwise temperature control for increased efficiency, and resin adsorption for impurity removal. This process can be directly applied to the large-scale extraction of polysaccharides from plants such as wolfberry, poria cocos, and ganoderma lucidum. In the functional food industry, it can increase the content of effective ingredients and enhance product competitiveness; in the pharmaceutical industry, it can provide high-purity raw materials for polysaccharide drugs, reducing the potential interference of impurities on efficacy; and in the cosmetics industry, it can reduce the risk of skin irritation from pigments and small molecule impurities, broadening its application scenarios.

[0056] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for increasing polysaccharide yield, characterized in that, Includes the following steps: Step 1: Pre-treat the raw materials used for polysaccharide extraction. The pre-treatment includes washing, drying, and pulverizing the raw materials to obtain raw material powder. Step 2: Mix the raw material powder with deionized water to form a suspension, add cellulase, pectinase and polyethylene glycol 4000 according to the preset ratio, stir to dissolve, and obtain the enzymatic hydrolysis system, and make up the volume. Step 3: Perform step-temperature controlled enzymatic hydrolysis on the enzymatic hydrolysis system after adjusting the volume. The step-temperature controlled enzymatic hydrolysis is performed sequentially at 30~35℃ for 1~2 hours, 40~45℃ for 1~2 hours, and 50~55℃ for 0.5~1 hours to obtain the enzymatic hydrolysis product. Step 4: Heat the enzymatic hydrolysate to 80-90°C and maintain for 5-10 minutes to inactivate the enzyme; Step 5: Adsorb pigments from the enzyme-inactivated solution and collect the supernatant; Step 6: Perform gradient elution separation on the solution after adsorption of pigments, successively eluting with low-concentration and medium-concentration ethanol aqueous solutions, and collect the polysaccharide-containing ethanol aqueous solution obtained by high-concentration ethanol aqueous solution elution. The polysaccharide-containing ethanol aqueous solution was concentrated by rotary evaporation and freeze-dried to obtain the target polysaccharide.

2. The method for improving polysaccharide yield according to claim 1, characterized in that, In step 1, the drying process involves drying the raw material at 60-70°C until the moisture content is ≤8%.

3. The method for improving polysaccharide yield according to claim 1, characterized in that, In step 2, the initial mixing mass-to-volume ratio of the raw material powder to deionized water is 1:10 (g / mL), and the concentration of the raw material powder in the enzymatic hydrolysis system is 80 g / L.

4. The method for improving polysaccharide yield according to claim 1, characterized in that, The reagents used to adjust the pH in step 2 are 0.1 mol / L hydrochloric acid solution or 0.1 mol / L sodium hydroxide solution.

5. The method for improving polysaccharide yield according to claim 1, characterized in that, In step 2, the cellulase activity is 4000~6000 U / g and the pectinase activity is 2000~4000 U / g.

6. The method for improving polysaccharide yield according to claim 5, characterized in that, The mass ratio of cellulase to pectinase is 1:(0.9~1.5); The amount of polyethylene glycol 4000 added is 0.3% to 0.8% of the mass of the raw material powder in the suspension.

7. The method for improving polysaccharide yield according to claim 1, characterized in that, In step 2, before making up the volume, the pH of the enzymatic hydrolysis system is adjusted to 4.5-5.

0.

8. The method for improving polysaccharide yield according to claim 1, characterized in that, The pigment adsorption conditions in step 5 include: using LX-8 resin, with the amount of LX-8 resin being 10%~15% of the volume of the solution after enzyme inactivation, a temperature of 25~30℃, a stirring speed of 40~60 r / min, and an adsorption time of 2~3 hours.

9. The method according to claim 8, characterized in that, Before use, the LX-8 resin described in step 5 is soaked in 95% ethanol, washed with deionized water until there is no alcohol odor, then soaked in 5% hydrochloric acid solution, washed with deionized water until neutral, and finally soaked in 5% sodium hydroxide solution, washed with deionized water until neutral.

10. The method according to claim 1, characterized in that, The gradient elution conditions include: controlling the elution rate at 2~3 BV / h; and performing elution in the following order: (1) Use a 10% low-concentration ethanol aqueous solution with an elution volume of 1.0~1.5 BV; (2) Replace with a 30% medium-concentration ethanol aqueous solution, with an elution volume of 1.3~2.0 BV; (3) Replace with a 50% high-concentration ethanol aqueous solution, with an elution volume of 1.7~2.5 BV; Collect the effluent generated during the elution process of 50% ethanol aqueous solution in step (3).

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