A Highly Efficient Extraction Method for Ganoderma lucidum Polysaccharides

Abstract

This invention discloses a highly efficient extraction method for Ganoderma lucidum polysaccharides, relating to the field of polysaccharide extraction. The method includes: mixing Ganoderma lucidum powder with sodium bicarbonate, adding an aqueous citric acid solution, and using the generated carbon dioxide gas to pressurize and then instantly depressurize for physical cell wall disruption, followed by solid-liquid separation to obtain a clear, disrupted solution; subjecting the clear, disrupted solution to foam flotation to enrich it into a polysaccharide-rich solution; adding a thermosensitive affinity polymer (N-isopropylacrylamide) grafted onto a hydroxypropyl cellulose backbone to the polysaccharide-rich solution, followed by low-temperature adsorption and then heating to co-precipitate the polymer and polysaccharides; desorbing the precipitate in a low-temperature acidic buffer solution, followed by heating to regenerate the polymer and precipitate; and dialyzing and freeze-drying the supernatant to obtain the Ganoderma lucidum polysaccharide product. This invention can efficiently disrupt the dense cell wall structure of Ganoderma lucidum fruiting bodies, allowing for the full release of intracellular polysaccharides, significantly improving the extraction rate. Furthermore, the entire process is carried out at room temperature, effectively protecting the bioactivity of the polysaccharides.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction, specifically to a highly efficient method for extracting Ganoderma lucidum polysaccharides. Background Technology

[0002] Ganoderma lucidum polysaccharides are one of the main active ingredients of Ganoderma lucidum. They have a variety of pharmacological activities such as immunomodulation, anti-tumor, and antioxidant. They have broad application prospects in the fields of functional food and medicine. At present, the conventional extraction methods of Ganoderma lucidum polysaccharides mainly adopt hot water extraction or alkaline extraction, combined with ethanol precipitation for purification.

[0003] However, the above-mentioned traditional methods have many shortcomings: hot water extraction is time-consuming and requires repeated extraction 2-3 times, which not only consumes a lot of energy, but also easily leads to the degradation of polysaccharide structure and loss of biological activity. Although ethanol precipitation is widely used, it consumes a lot of organic solvents and has high recovery costs. In addition, a large amount of impurities such as proteins and pigments are co-precipitated during the precipitation process, resulting in a crude polysaccharide purity of only 40%-60%. Subsequent deproteinization and decolorization treatments are required, which is complicated and has high production costs. The cell wall structure of Ganoderma lucidum fruiting body is dense and contains components such as chitin and glucan. Conventional extraction methods have limited effect on cell wall disruption, which seriously affects the polysaccharide dissolution rate. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a highly efficient extraction method for Ganoderma lucidum polysaccharides, so as to solve the technical problems of high cost and low dissolution rate of general Ganoderma lucidum polysaccharide extraction methods.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a highly efficient extraction method for Ganoderma lucidum polysaccharides, comprising the following steps:

[0006] (1) Pulverize the dried Ganoderma lucidum fruiting body to 80-100 mesh to obtain Ganoderma lucidum powder; mix the Ganoderma lucidum powder and food-grade sodium bicarbonate powder at a mass ratio of 1:0.2-0.4, put them into a pressure-resistant container with a quick-opening release valve, and then inject citric acid aqueous solution equivalent to 2.5-3.5 times the mass of Ganoderma lucidum powder. The molar ratio of citric acid to sodium bicarbonate added in the step is 1:1.5-1:1.8. Control the filling coefficient of the liquid in the container to be 60%-70%. Seal the container and make the carbon dioxide gas generated by the reaction of acid and sodium bicarbonate form a pressure of 0.8-1.5 MPa in the container. Then quickly open the release valve and let the material spray out into the atmospheric pressure collection container within 1 second to obtain the cell wall broken mixture; filter the cell wall broken mixture through a 100-200 mesh filter cloth or centrifuge at low speed to remove solid residues to obtain a clear cell wall broken liquid.

[0007] (2) The clarified and broken liquid obtained in step (1) is introduced into a foam flotation column, and soybean lecithin equivalent to 0.02% to 0.05% of the volume of the clarified and broken liquid is added. The mixture is stirred evenly, and then clean air is introduced from the bottom of the flotation column at a flow rate of 0.3 to 0.5 L / min. Bubbles with a diameter of 0.1 to 1 mm are formed through the porous distributor at the bottom of the column. The bubbles rise and carry the polysaccharides to the top of the column to form foam. The foam is collected and defoamed to obtain the polysaccharide enriched liquid.

[0008] (3) Add 0.8% to 1.5% of the mass of the polysaccharide enrichment solution from step (2) to the thermosensitive affinity polymer. Stir slowly at 4 to 8°C for 40 to 60 minutes, then raise the temperature to 32 to 36°C and maintain it for 10 to 15 minutes to allow the polymer to change from a dissolved state to a precipitated state and adsorb the polysaccharide and precipitate together. Filter with a 100 to 200 mesh filter cloth or collect the precipitate by low-speed centrifugation. The thermosensitive affinity polymer is a reversible thermosensitive material prepared by introducing poly(N-isopropylacrylamide) side chains through chemical grafting with hydroxypropyl cellulose as the backbone. The grafting rate of poly(N-isopropylacrylamide) is 25% to 35%.

[0009] (4) The precipitate collected in step (3) is redispersed in a cold acetic acid-sodium acetate buffer solution at pH 4.6-5.2 and temperature 4-8℃. The mixture is stirred for 30-40 minutes to allow the polymer to redissolve and release the polysaccharide. The temperature is then raised to 32-36℃ to allow the polymer to precipitate again. The polymer precipitate is separated by centrifugation to obtain a supernatant containing polysaccharide. The supernatant is dialyzed in deionized water for 12-24 hours using a dialysis bag with a molecular weight cutoff of 3000-5000 Da. Finally, it is freeze-dried to obtain the Ganoderma lucidum polysaccharide product.

[0010] The citric acid aqueous solution in step (1) is prepared from edible citric acid. After the citric acid and sodium bicarbonate react in the molar ratio described above, the pH of the resulting clear cell wall breaking solution is controlled within the range of 4.8 to 5.2.

[0011] The design pressure of the pressure vessel in step (1) is not less than 2MPa. The quick-opening release valve is a ball valve, and its action time from fully closed to fully open does not exceed 0.5 seconds. The ratio of the valve port diameter to the container diameter is 1:3 to 1:4.

[0012] The height-to-diameter ratio of the foam flotation column in step (2) is 6:1 to 8:1. The porous distributor is a sintered glass plate or a microporous ceramic plate with a pore size of 10 to 50 micrometers. The foam is collected by overflowing from the top of the column into the collection tank. Defoaming is achieved by stirring with mechanical paddles at 50 to 80 rpm or by spraying a small amount of food-grade anhydrous ethanol.

[0013] In step (2), while the flotation operation is being carried out, the temperature of the feed liquid inside the column is kept between 25 and 35°C, and the flotation time is 20 to 30 minutes.

[0014] The method for preparing the thermosensitive affinity polymer in step (3) is as follows: Hydroxypropyl cellulose is dissolved in deionized water, nitrogen is passed through to remove oxygen, and cerium ammonium nitrate initiator is added. N-isopropylacrylamide monomer is added dropwise at 30-35°C and reacted for 4-6 hours. The product is purified by dialysis using a dialysis bag with a molecular weight cutoff of 10000 Da and freeze-dried to obtain the graft copolymer. The mass ratio of hydroxypropyl cellulose to N-isopropylacrylamide monomer is 1:3-4.

[0015] The stirring speed in step (3) is 40-60 rpm. The precipitate after heating is white flocculent or fine granular. Phase separation can be judged by visual inspection.

[0016] The pH of the cold acetic acid-sodium acetate buffer solution in step (4) is 4.8 and the temperature is 5°C. The polymer precipitate separated by centrifugation is washed twice with cold deionized water at 5°C and can be directly used for the next batch of step (3) for recycling. The number of times it can be reused is no less than 5 times.

[0017] The Ganoderma lucidum powder used in step (1) is first dried with hot air at 40-50℃ until the moisture content is less than 8%, and then pulverized.

[0018] In summary, the present invention has the following main beneficial effects:

[0019] 1. This invention utilizes the gas generated by the acid-base reaction to be released in a very short time, producing strong shear force, shock wave and cavitation effect, which can efficiently break the dense cell wall structure of Ganoderma lucidum fruiting body, so as to fully release the intracellular polysaccharides, significantly improve the extraction rate, and the whole process is carried out at room temperature, which effectively protects the biological activity of polysaccharides.

[0020] 2. The foam flotation step of this invention utilizes the amphiphilicity of polysaccharides to initially enrich them and remove a large number of water-soluble impurities. The temperature-sensitive affinity polymer captures polysaccharides through specific intermolecular affinity. Adsorption and desorption can be reversibly controlled simply by changing the temperature, avoiding the large consumption of organic solvents and high-temperature concentration operations required by traditional alcohol precipitation methods. The product purity can be stably maintained at over 90%, and the polymer can be recycled. The process is green, environmentally friendly, and low-cost. Attached Figure Description

[0021] Figure 1 This is a process flow diagram of a highly efficient extraction method for Ganoderma lucidum polysaccharides according to the present invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0023] The embodiments of the present invention will now be described.

[0024] This invention provides a highly efficient extraction method for Ganoderma lucidum polysaccharides, comprising the following core steps: pretreatment and mixing of materials, in-situ gas pressurization followed by instantaneous depressurization and cell wall disruption, foam flotation enrichment of polysaccharides, reversible adsorption and precipitation purification using temperature-sensitive affinity polymers, and product acquisition and reagent recycling.

[0025] Step 1: Material Pretreatment and Mixing

[0026] First, dried Ganoderma lucidum fruiting bodies are selected as raw materials. To ensure pulverization efficiency and subsequent reaction efficiency, it is preferable to pre-dry the Ganoderma lucidum fruiting bodies in a hot air circulating oven at 40-50℃ until the moisture content is below 8% (by mass). The dried Ganoderma lucidum fruiting bodies are then pulverized using a pulverizer and passed through an 80-100 mesh standard sieve to obtain Ganoderma lucidum powder with a particle size of 80-100 mesh. This particle size range ensures that the cell walls are effectively broken down while avoiding difficulties in subsequent filtration caused by excessively fine powder.

[0027] Next, the obtained Ganoderma lucidum powder and food-grade sodium bicarbonate powder are dry-mixed at a mass ratio of 1:0.2-0.4. For example, 100 kg of Ganoderma lucidum powder and 30 kg of sodium bicarbonate powder are thoroughly mixed in a V-type mixer for 15 minutes to obtain a mixed powder. The mixed powder is then placed in a specially designed pressure-resistant container with a design pressure of not less than 2 MPa to ensure operational safety. A key feature of this container is its quick-opening release valve, preferably a ball valve structure. The ball valve's action time from fully closed to fully open does not exceed 0.5 seconds to achieve instantaneous pressure relief. The ratio of the valve port diameter to the container diameter is designed to be 1:3 to 1:4, which helps ensure effective shear force and cavitation effect when the material is ejected.

[0028] Then, a citric acid aqueous solution equivalent to 2.5 to 3.5 times the mass of Ganoderma lucidum powder is injected into the mixed powder in the pressure-resistant container. The citric acid aqueous solution is prepared by mixing food-grade monohydrate citric acid or anhydrous citric acid with deionized water. The key preparation basis is to ensure that the molar ratio of citric acid in the solution to sodium bicarbonate added in the step is 1:1.5 to 1:1.8. This ratio makes the citric acid excess, which on the one hand promotes the rapid and complete reaction, and on the other hand, after the reaction is completed, a buffer system of sodium citrate and residual citric acid is generated, which can automatically adjust and stabilize the pH value of the final cell wall-breaking mixture in the range of 4.8 to 5.2. This weakly acidic environment is extremely beneficial to the stability and activity of soybean lecithin as a foam stabilizer in the subsequent foam flotation step.

[0029] After all materials are added, the filling coefficient of the liquid in the container is controlled at 60%–70%, leaving sufficient space for the gas phase. The container is then sealed, and stirring is started to allow the acid and sodium bicarbonate to react rapidly, producing carbon dioxide gas. As the gas accumulates, the pressure inside the container gradually rises to 0.8–1.5 MPa. At this point, the quick-release valve is quickly and fully opened. Driven by the huge pressure difference, the high-pressure material inside the container is ejected through the valve into an atmospheric pressure collection container within 1 second. At the moment of ejection, the material undergoes intense shearing, collision, and gas expansion cavitation, which efficiently breaks down the cell walls of Ganoderma lucidum and releases a large amount of cell contents into the liquid phase, resulting in a cell-wall-broken mixture.

[0030] Then, the cell wall-broken mixture is filtered through a plate and frame filter press with a 100-200 mesh filter cloth, or centrifuged at low speed using a horizontal screw centrifuge to remove a large amount of solid residue such as cell wall fragments, and a clear cell wall-broken liquid is collected.

[0031] Step 2: Enrichment of polysaccharides by foam flotation

[0032] The clarified and broken-cell liquid obtained in step one is introduced into a froth flotation column with a height-to-diameter ratio of 6:1 to 8:1. The bottom of the flotation column is equipped with a porous distributor with a pore size of 10 to 50 micrometers, preferably a sintered glass plate or a microporous ceramic plate. Soybean lecithin, at a volume of 0.02% to 0.05%, is added to the liquid in the column as a foaming agent and collector. The agitator is turned on and stirred at a speed of 40 to 60 rpm until homogeneous.

[0033] Subsequently, clean compressed air is introduced through the gas distributor at the bottom of the column at a flow rate of 0.3 to 0.5 L / min, forming a large number of microbubbles with a diameter between 0.1 and 1 mm. Because the Ganoderma lucidum polysaccharide molecule contains hydrophilic groups such as polyhydroxy and amino groups, and hydrophobic amino acid residues on the peptide chain, it is amphiphilic and can preferentially adsorb onto the gas-liquid interface of the bubbles. During the rising process, the bubbles carry the soybean lecithin-polysaccharide complex with adsorbed polysaccharides to the top of the flotation column, forming a foam layer rich in polysaccharides.

[0034] Throughout the flotation operation, the temperature of the feed solution inside the column is maintained between 25 and 35°C through a jacketed or constant-temperature chamber. This temperature range helps reduce the viscosity of the feed solution, improves bubble rise, and enhances polysaccharide adsorption efficiency. The flotation process lasts for 20 to 30 minutes, during which foam continuously flows out through the overflow port at the top of the column and enters a collection tank. In the collection tank, physical defoaming is performed by slow stirring with mechanical impellers at a speed of 50 to 80 rpm, or chemical defoaming is performed by spraying a very small amount (e.g., 0.1% of the foam volume) of food-grade anhydrous ethanol. The liquid obtained after defoaming is the polysaccharide-enriched solution. This step not only enriches the target product but also removes most of the unadsorbed impurities such as salts, monosaccharides, and pigments.

[0035] Step 3: Reversible adsorption and precipitation of thermosensitive affinity polymers

[0036] The key material used in this step is a thermosensitive affinity polymer, and its preparation method is illustrated in the following specific example:

[0037] In a reactor equipped with nitrogen protection, a condenser, and mechanical stirring, 1 kg of hydroxypropyl cellulose was dissolved in 100 L of deionized water. The solution was heated to 30–35 °C and high-purity nitrogen was introduced for 30 minutes to remove dissolved oxygen. Then, under nitrogen protection, 0.5 L of an initiator solution containing 0.1 mol of cerium ammonium nitrate was slowly added dropwise. After initiation for 10 minutes, 3.5 kg of N-isopropylacrylamide monomer dissolved in an appropriate amount of deionized water was added dropwise. The dropping rate was controlled to complete the reaction within 4–6 hours. The temperature was maintained at 30–35 °C throughout the reaction. After the reaction, the product was dialyzed in deionized water for 48 hours using a dialysis bag with a molecular weight cutoff of 10,000 Da to remove unreacted monomers and homopolymers. Finally, it was freeze-dried to obtain a white fibrous thermosensitive affinity polymer. The grafting rate of poly(N-isopropylacrylamide) on the hydroxypropyl cellulose backbone under these conditions was approximately 25%–35%. This polymer has unique temperature response characteristics, with its low critical dissolution temperature around 32°C.

[0038] The grafting rate of the above-mentioned thermosensitive affinity polymer was determined by gravimetric method. The specific procedure was as follows: the graft copolymer solution purified by dialysis was freeze-dried to constant weight (denoted as W1) and accurately weighed; another batch of ungrafted hydroxypropyl cellulose raw material of the same quality was subjected to the same drying treatment and weighed (denoted as W0). The grafting rate was calculated using the following formula: Grafting rate (%) = [(W1-W0) / W0] × 100%. It was determined that within the above feed ratio range, the grafting rate of the obtained polymer was consistently between 25% and 35%.

[0039] Take the polysaccharide enrichment solution obtained in step two, cool it to 4–8°C, and then add 0.8%–1.5% of the above-mentioned thermosensitive affinity polymer by mass of the enrichment solution. Stir slowly at 40–60 rpm for 40–60 minutes at a low temperature of 4–8°C to allow the polymer to completely dissolve and expand its molecular chains. At this time, the hydroxypropyl cellulose backbone and poly(N-isopropylacrylamide) side chains on the polymer chains work synergistically, fully contacting and binding with the Ganoderma lucidum polysaccharide molecules through weak interactions such as hydrogen bonding and hydrophobic interactions.

[0040] Subsequently, the system was heated to 32–36°C with continuous slow stirring and maintained at this temperature for 10–15 minutes. When the temperature rose above the lower critical solution temperature of the polymer, the poly(N-isopropylacrylamide) segments underwent a rapid conformational change, changing from an extended hydrophilic state to a contracted hydrophobic state, causing the entire polymer-polysaccharide complex to precipitate from the solution, forming a white flocculent or fine granular precipitate visible to the naked eye. The phase separation process was clearly discernible. The precipitate was collected by filtration through a 100–200 mesh filter cloth or by low-speed centrifugation (e.g., centrifugation at 3000 rpm for 15 minutes).

[0041] Step 4: Desorption of polysaccharides and purification of the product

[0042] The precipitate collected in step three was redispersed in 5-10 times its weight of a cold acetic acid-sodium acetate buffer solution at pH 4.8 and maintained at 4-8°C. Under this low-temperature acidic environment, the thermosensitive affinity polymer was transformed back into a soluble state, the molecular chains unfurled, and the bound polysaccharides were released into the liquid phase. The mixture was stirred for 30-40 minutes to allow for complete desorption of the polysaccharides. Then, the system was heated again to 32-36°C to allow the polymer to redefine. The polymer was separated from the polysaccharide-containing supernatant by centrifugation or filtration. The separated polymer precipitate was washed twice with 3-5 times its weight of cold deionized water at 5°C. The recovered polymer could be directly recycled in the next batch of step three. Experiments showed that after more than 5 reuses, its adsorption capacity for polysaccharides did not decrease significantly.

[0043] The recyclability of the thermosensitive affinity polymer involved in this invention was verified by the following method: The recovered polymer precipitate was redispersed in a fresh polysaccharide enrichment solution according to step (3), and an adsorption-precipitation-desorption cycle was performed under the same conditions. After each cycle, the adsorption capacity (Qe) of the batch of polymer for polysaccharides was measured and compared with the adsorption capacity (Q0) of the first use. The adsorption capacity retention rate (%) = (Qe / Q0) × 100%. The experiment showed that after 5 reuses, the adsorption capacity retention rate could still be maintained at more than 92%, proving that it could be reused at least 5 times.

[0044] The supernatant rich in polysaccharides is dialyzed in flowing deionized water for 12 to 24 hours using a dialysis bag with a molecular weight cutoff of 3000 to 5000 Da to completely remove small molecule salts such as sodium citrate and sodium acetate. Finally, the dialyzed polysaccharide solution is freeze-dried at -50°C and a pressure of less than 10 Pa to obtain a white flocculent or powdered product of Ganoderma lucidum polysaccharide with a purity of more than 90%.

[0045] Example 1

[0046] (1) Take Ganoderma lucidum fruiting bodies with a moisture content of 6%, crush them through a 100-mesh sieve to obtain 10 kg of Ganoderma lucidum powder, mix them evenly with 3 kg of food-grade sodium bicarbonate, and put them into a pressure-resistant container with a design pressure of 2.5 MPa and equipped with a quick-opening ball valve (the ratio of valve diameter to container diameter is 1:3.5, and the opening time is 0.3 seconds). Pour in 30 kg of citric acid aqueous solution (of which citric acid content is 2.8 kg, so that the molar ratio of citric acid to sodium bicarbonate is about 1:1.6), and control the material filling coefficient to 65%. The reaction is closed, and when the pressure rises to 1.2 MPa, the valve is opened instantly, and the material is sprayed into an atmospheric pressure tank. Filter with a 200-mesh filter cloth to obtain a clear cell wall-breaking liquid, and the pH is measured to be 5.0.

[0047] (2) The cell wall-breaking liquid was introduced into a flotation column with a height-to-diameter ratio of 7:1, and soybean lecithin equivalent to 0.03% of its volume was added. Air was introduced at 30°C at a rate of 0.4 L / min, and bubbles were generated by passing through a 50-micron microporous ceramic plate. The flotation was continued for 25 minutes. The foam was collected and defoamed to obtain the enriched liquid.

[0048] (3) Cool the enrichment solution to 5°C, add 1.0% by mass of thermosensitive affinity polymer (hydroxypropyl cellulose to N-isopropylacrylamide monomer in a mass ratio of 1:3.5, with a grafting rate of 30%), stir at 50 rpm for 50 minutes, then heat to 34°C and hold for 12 minutes, and centrifuge to collect the precipitate.

[0049] (4) The precipitate was dispersed in sodium acetate buffer at pH 4.8 and 5℃ at a ratio of 1:8. After stirring for 35 minutes, the temperature was raised to 34℃ and then centrifuged. The supernatant was dialyzed with a 5000Da dialysis bag for 18 hours and then freeze-dried to obtain 157g of Ganoderma lucidum polysaccharide product with a purity of 93.5% and an extraction rate of 1.57%.

[0050] Example 2

[0051] This embodiment is basically the same as embodiment 1, except that in step (1), the mass ratio of Ganoderma lucidum powder to sodium bicarbonate is 1:0.2, the molar ratio of citric acid to sodium bicarbonate is 1:1.8, and the pH of the final cell wall breaking solution is 5.2.

[0052] The final product yielded 148 grams of Ganoderma lucidum polysaccharide with a purity of 92.8% and an extraction rate of 1.48%.

[0053] Example 3

[0054] This embodiment is basically the same as that of embodiment 1, except that in step (3), the amount of thermosensitive affinity polymer is 0.8% of the mass of the enrichment solution, the stirring adsorption temperature is 8°C, and the time is 60 minutes.

[0055] The final product yielded 139 grams of Ganoderma lucidum polysaccharide with a purity of 91.9% and an extraction rate of 1.39%.

[0056] Example 4

[0057] This embodiment is basically the same as embodiment 1, except that in step (2), the flotation temperature is controlled at 25°C, the air flow rate is 0.3L / min, and the flotation time is 30 minutes.

[0058] The final yield was 152 grams of Ganoderma lucidum polysaccharide product with a purity of 93.1% and an extraction rate of 1.52%.

[0059] Comparative Example 1

[0060] 10 kg of the same Ganoderma lucidum powder as in Example 1 was added to 300 kg of deionized water at a material-to-liquid ratio of 1:30. The mixture was extracted at 95°C with stirring for 3 hours, and the extraction was repeated twice. The extracts were combined, concentrated, and then precipitated with 3 times the volume of anhydrous ethanol. The precipitate was collected by centrifugation, dialyzed, and then freeze-dried. A total of 385 g of crude polysaccharide was obtained, but the purity was only 45.6%. Based on the pure product, the yield of polysaccharide for the same amount was significantly lower than in Example 1.

[0061] Comparative Example 2

[0062] This comparative example is basically the same as Example 1, except that in step (1), sodium bicarbonate and citric acid are not added. Instead, Ganoderma lucidum powder is directly mixed with water and homogenized three times at 20 MPa using a high-pressure homogenizer to break the cell wall. The subsequent steps are the same.

[0063] The final yield was 102 grams of Ganoderma lucidum polysaccharide product with a purity of 88.7% and an extraction rate of 1.02%. This demonstrates that the in-situ instantaneous depressurization bubble cell disruption technology has a more efficient effect on breaking down specific cell wall structures and dissolving polysaccharides.

[0064] Comparative Example 3

[0065] This comparative example is basically the same as Example 1. The difference is that in step (3), no temperature-sensitive affinity polymer is added. Instead, the enriched liquid obtained by foam flotation is directly concentrated and precipitated with 4 times the volume of 80% ethanol at 4°C for 12 hours. The precipitate is obtained by centrifugation and then lyophilized by dialysis.

[0066] The final yield was 175 grams of Ganoderma lucidum polysaccharide product, but the purity was only 81.2%. Although the extraction rate was 1.75%, the high yield came at the cost of purity, and the product contained a large amount of co-precipitated impurities and pigments.

[0067] Comparative Example 4

[0068] This comparative example is basically the same as Example 3, except that an equal amount of chitosan quaternary ammonium salt is used as a polysaccharide precipitant, the complex is precipitated by adjusting the pH to 9.0, and then desorbed with acid solution at pH 3.0.

[0069] The final yield was 128 grams of Ganoderma lucidum polysaccharide product with a purity of 90.5% and an extraction rate of 1.28%. Compared with Example 3, it can be seen that the temperature-sensitive polymer of the present invention not only has milder operating conditions (only temperature variation is required), but also achieves higher yield while ensuring high purity through specific affinity.

[0070] Although embodiments of the present invention have been shown and described, these specific embodiments are merely explanations of the invention and are not intended to limit it. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. After reading this specification, those skilled in the art may make modifications, substitutions, and variations to the embodiments as needed without departing from the principles and spirit of the invention, but such modifications, substitutions, and variations are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A highly efficient extraction method for Ganoderma lucidum polysaccharides, characterized in that, Includes the following steps: (1) Pulverize the dried Ganoderma lucidum fruiting body to 80-100 mesh to obtain Ganoderma lucidum powder; mix the Ganoderma lucidum powder and food-grade sodium bicarbonate powder at a mass ratio of 1:0.2-0.4, put them into a pressure-resistant container with a quick-opening release valve, and then inject citric acid aqueous solution equivalent to 2.5-3.5 times the mass of Ganoderma lucidum powder. The molar ratio of citric acid to sodium bicarbonate added in the step is 1:1.5-1:1.

8. Control the filling coefficient of the liquid in the container to be 60%-70%. Seal the container and make the carbon dioxide gas generated by the reaction of acid and sodium bicarbonate form a pressure of 0.8-1.5 MPa in the container. Then quickly open the release valve and let the material spray out into the atmospheric pressure collection container within 1 second to obtain the cell wall broken mixture; filter the cell wall broken mixture through a 100-200 mesh filter cloth or centrifuge at low speed to remove solid residues to obtain a clear cell wall broken liquid. (2) The clarified and broken liquid obtained in step (1) is introduced into a foam flotation column, and soybean lecithin equivalent to 0.02% to 0.05% of the volume of the clarified and broken liquid is added. The mixture is stirred evenly, and then clean air is introduced from the bottom of the flotation column at a flow rate of 0.3 to 0.5 L / min. Bubbles with a diameter of 0.1 to 1 mm are formed through the porous distributor at the bottom of the column. The bubbles rise and carry the polysaccharides to the top of the column to form foam. The foam is collected and defoamed to obtain the polysaccharide enriched liquid. (3) Add 0.8% to 1.5% of the mass of the polysaccharide enrichment solution from step (2) to the thermosensitive affinity polymer. Stir slowly at 4 to 8°C for 40 to 60 minutes, then raise the temperature to 32 to 36°C and maintain it for 10 to 15 minutes to allow the polymer to change from a dissolved state to a precipitated state and adsorb the polysaccharide and precipitate together. Filter with a 100 to 200 mesh filter cloth or collect the precipitate by low-speed centrifugation. The thermosensitive affinity polymer is a reversible thermosensitive material prepared by introducing poly(N-isopropylacrylamide) side chains through chemical grafting with hydroxypropyl cellulose as the backbone. The grafting rate of poly(N-isopropylacrylamide) is 25% to 35%. (4) The precipitate collected in step (3) is redispersed in a cold acetic acid-sodium acetate buffer solution at pH 4.6-5.2 and temperature 4-8℃. The mixture is stirred for 30-40 minutes to allow the polymer to redissolve and release the polysaccharide. The temperature is then raised to 32-36℃ to allow the polymer to precipitate again. The polymer precipitate is separated by centrifugation to obtain a supernatant containing polysaccharide. The supernatant is dialyzed in deionized water for 12-24 hours using a dialysis bag with a molecular weight cutoff of 3000-5000 Da. Finally, it is freeze-dried to obtain the Ganoderma lucidum polysaccharide product.

2. The efficient extraction method for Ganoderma lucidum polysaccharides according to claim 1, characterized in that: The citric acid aqueous solution in step (1) is prepared from edible citric acid. After the citric acid and sodium bicarbonate react in the molar ratio described above, the pH of the resulting clear cell wall breaking solution is controlled within the range of 4.8 to 5.

2.

3. The efficient extraction method for Ganoderma lucidum polysaccharides according to claim 1, characterized in that: The design pressure of the pressure vessel in step (1) is not less than 2MPa. The quick-opening release valve is a ball valve, and its action time from fully closed to fully open does not exceed 0.5 seconds. The ratio of the valve port diameter to the container diameter is 1:3 to 1:

4.

4. The efficient extraction method for Ganoderma lucidum polysaccharides according to claim 1, characterized in that: The height-to-diameter ratio of the foam flotation column in step (2) is 6:1 to 8:

1. The porous distributor is a sintered glass plate or a microporous ceramic plate with a pore size of 10 to 50 micrometers. The foam is collected by overflowing from the top of the column into the collection tank. Defoaming is achieved by stirring with mechanical paddles at 50 to 80 rpm or by spraying a small amount of food-grade anhydrous ethanol.

5. The efficient extraction method for Ganoderma lucidum polysaccharides according to claim 1, characterized in that: In step (2), while the flotation operation is being carried out, the temperature of the feed liquid inside the column is kept between 25 and 35°C, and the flotation time is 20 to 30 minutes.

6. The efficient extraction method for Ganoderma lucidum polysaccharides according to claim 1, characterized in that: The method for preparing the thermosensitive affinity polymer in step (3) is as follows: Hydroxypropyl cellulose is dissolved in deionized water, nitrogen is passed through to remove oxygen, and cerium ammonium nitrate initiator is added. N-isopropylacrylamide monomer is added dropwise at 30-35°C and reacted for 4-6 hours. The product is purified by dialysis using a dialysis bag with a molecular weight cutoff of 10000 Da and freeze-dried to obtain the graft copolymer. The mass ratio of hydroxypropyl cellulose to N-isopropylacrylamide monomer is 1:3-4.

7. The efficient extraction method for Ganoderma lucidum polysaccharides according to claim 1, characterized in that: The stirring speed in step (3) is 40-60 rpm. The precipitate after heating is white flocculent or fine granular. Phase separation can be judged by visual inspection.

8. The efficient extraction method for Ganoderma lucidum polysaccharides according to claim 1, characterized in that: The pH of the cold acetic acid-sodium acetate buffer solution in step (4) is 4.8 and the temperature is 5°C. The polymer precipitate separated by centrifugation is washed twice with cold deionized water at 5°C and can be directly used for the next batch of step (3) for recycling. The number of times it can be reused is no less than 5 times.

9. The efficient extraction method for Ganoderma lucidum polysaccharides according to claim 1, characterized in that: The Ganoderma lucidum powder used in step (1) is first dried with hot air at 40-50℃ until the moisture content is less than 8%, and then pulverized.

Images