A method for preparing a mushroom extract

Abstract

The application relates to a preparation method of a mushroom extract, which comprises the following steps: placing pretreated mycelium in subcritical water as an extraction system to perform extraction, maintaining countercurrent circulation during the extraction, the flow rate being 3 mL / (min.g mycelium), and obtaining a crude product; purifying the crude product to obtain a mushroom extract; wherein the subcritical water extraction process controls extraction conditions in stages according to the following procedure: first stage: the pressure is 4 MPa, the temperature is 115 DEG C, and the holding time is 5 min; second stage: the pressure is increased to 8 MPa, the temperature is increased to 125 DEG C, and the holding time is 3 min; third stage: the pressure is decreased to 6 MPa, the temperature is adjusted back to 110 DEG C, and the holding time is 2 min. The preparation method of the application controls the pressure and temperature parameters of subcritical water in stages, combines countercurrent circulation, improves the extraction efficiency of beta-glucan, and inhibits glycosidic bond hydrolysis through a dynamic pressure gradient to protect the main chain structure integrity of polysaccharide.

Description

Technical Field

[0001] This application relates to the field of polysaccharide preparation technology, and in particular to a method for preparing fungal extracts. Background Technology

[0002] Fungal extracts (such as β-glucan) have significant application value in the pharmaceutical and functional food fields due to their remarkable immunomodulatory and antitumor activities. Traditional extraction processes mostly rely on high-temperature water extraction or organic solvent extraction, which have significant drawbacks: prolonged high-temperature treatment easily leads to the breakage of the β-glucan backbone (such as β-(1→3) glycosidic bonds), a significant decrease in molecular weight, and the unwinding of the triple helix structure, directly affecting its biological activity; while the use of organic solvents (such as ethanol and chloroform) not only increases production costs but also poses a risk of residue, making it difficult to meet food and pharmaceutical safety standards. In recent years, subcritical water extraction technology has attracted attention due to its green and environmentally friendly characteristics, but existing methods mostly use constant pressure and temperature conditions, making it difficult to balance extraction efficiency and structural protection—while constant high pressure can improve solubility, it accelerates the hydrolysis of polysaccharide chains; while high temperature alone shortens extraction time, it triggers Maillard side reactions, causing product color deterioration. In addition, conventional processes still rely on subsequent organic solvent precipitation and purification, which cannot completely solve the problem of solvent residue. Therefore, there is an urgent need to develop a preparation method that can efficiently extract the active ingredients of mushrooms under mild conditions, while completely preserving their molecular structure and biological activity, and completely avoiding the use of organic solvents. Summary of the Invention

[0003] This application provides a method for preparing fungal extracts, comprising the following steps:

[0004] The pretreated mycelium was placed in subcritical water as the extraction system for extraction, and countercurrent circulation was maintained during the process at a flow rate of 3 mL / (min·g mycelium) to obtain crude product;

[0005] The crude product was purified to obtain a mushroom extract;

[0006] The subcritical water extraction process is controlled under different conditions in stages according to the following procedure:

[0007] First stage: Pressure 4MPa, temperature 115℃, maintain for 5 minutes;

[0008] Second stage: The pressure is increased to 8MPa and the temperature is increased to 125℃, and maintained for 3 minutes;

[0009] Third stage: The pressure drops to 6MPa, the temperature returns to 110℃, and is maintained for 2 minutes.

[0010] In some embodiments, the preprocessing includes the following steps:

[0011] β-1,3-glucanase was used at an enzyme activity concentration of 0.5-1.5 U / g mycelium, and the mycelium was treated for 30 min at pH 5.5-6.0 and 45℃, while a pulsed electric field was applied simultaneously.

[0012] The pulse electric field parameters are: field strength 5-10kV / cm, pulse width 45-55μs, and frequency 8-12Hz.

[0013] In some embodiments, the enzyme activity concentration of the β-1,3-glucanase is 0.8-1.2 U / g mycelium.

[0014] In some embodiments, the field strength of the pulsed electric field is 7-9 kV / cm, the pulse width is 50 μs, and the frequency is 10 Hz.

[0015] In some embodiments, during the extraction process, a composite stabilizer is added to the extraction system. The composite stabilizer comprises trehalose and nano-silica in a mass ratio of 4:1, wherein the specific surface area of ​​the nano-silica is ≥200 m². 2 / g.

[0016] In some embodiments, the amount of trehalose added is 0.02% of the dry weight of the mycelium, and the amount of nano-silica added is 0.005% of the dry weight of the mycelium.

[0017] In some embodiments, the pH of the extraction system is controlled at 6.2-6.8.

[0018] In some embodiments, purifying the crude product to obtain the mushroom extract includes the following steps:

[0019] Using a ceramic membrane with a pore size of 50 nm, the transmembrane pressure difference is controlled at 0.28-0.32 MPa, and the crude product is subjected to cross-flow filtration to obtain the filtered material.

[0020] The filtered material is spray-dried, with the inlet air temperature controlled at 155-165℃ and the outlet air temperature at 80-90℃, to obtain mushroom extract.

[0021] In some embodiments, the transmembrane pressure difference is 0.3 MPa.

[0022] In some embodiments, the inlet air temperature of the spray dryer is 158-162°C, and the outlet air temperature is 83-87°C.

[0023] The technical solutions provided in this application have the following advantages compared with the prior art:

[0024] 1. By controlling the pressure and temperature parameters of subcritical water in stages and combining it with countercurrent circulation, the extraction efficiency of β-glucan is improved. At the same time, the hydrolysis of glycosidic bonds is inhibited by dynamic pressure gradient, thus protecting the integrity of the polysaccharide backbone structure.

[0025] 2. Through the synergistic effect of β-1,3-glucanase and pulsed electric field, the hyphal permeability regulation time is shortened, the energy consumption for cell disruption is reduced, and the migration rate of β-glucan is increased.

[0026] 3. By adding a composite stabilizer, the unwinding rate of the triple helix structure of β-glucan at 125℃ is reduced, the polysaccharide yield is increased, and no subsequent removal of excipients is required.

[0027] 4. By controlling the pH of the extraction system to 6.2-6.8, the hydrolysis rate constant of β-1,3 glycosidic bonds is reduced, the activity of polyphenol oxidase is inhibited, the color difference of the extract is reduced, and the residual metal ions are decreased.

[0028] 5. By using 50nm ceramic membrane cross-flow filtration and spray drying, the membrane fouling index is reduced, the retention rate of β-glucan triple helix structure is increased, and the moisture content is stabilized at a low level.

[0029] 6. By optimizing the pulsed electric field parameters, the cell membrane pore conduction time is prolonged, and the β-glucan release synchronization rate is improved.

[0030] 7. Through the molecular-mesoscopic synergy between trehalose and nano-silica, the viscosity of the extract is reduced, the membrane separation flux is increased, and the polysaccharide glassy matrix is ​​stabilized after spray drying.

[0031] 8. By using subcritical water in the third stage of pressure correction, a negative pressure gradient is formed in the extract to drive directional migration, and the target polysaccharide is rapidly enriched in the 110℃ stable region.

[0032] 9. By controlling the plateau phase of the Michaelis equation during the enzymatic hydrolysis stage, the amount of oligosaccharide fragments generated is reduced, thus decreasing the risk of Maillard reaction.

[0033] 10. By matching the mechanical relaxation time of hyphae with a high-frequency alternating electric field, the cell wall polysaccharide network can release target components in a controlled softening state, thereby reducing the oxidative degradation rate. Attached Figure Description

[0034] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 A flowchart illustrating a method for preparing a fungal extract provided in this application embodiment;

[0037] Figure 2 A flowchart of the purification steps provided in the embodiments of this application. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0039] Figure 1 A flowchart illustrating a method for preparing a fungal extract according to an embodiment of this application is shown.

[0040] like Figure 1 As shown, this application provides a method for preparing fungal extracts, comprising the following steps:

[0041] S10. The pretreated mycelium was placed in subcritical water as the extraction system for extraction. During the process, countercurrent circulation was maintained at a flow rate of 3 mL / (min·g mycelium) to obtain crude product.

[0042] S20. The crude product is purified to obtain mushroom extract;

[0043] The subcritical water extraction process is controlled under different conditions in stages according to the following procedure:

[0044] First stage: Pressure 4MPa, temperature 115℃, maintain for 5 minutes;

[0045] Second stage: The pressure is increased to 8MPa and the temperature is increased to 125℃, and maintained for 3 minutes;

[0046] Third stage: The pressure drops to 6MPa, the temperature returns to 110℃, and is maintained for 2 minutes.

[0047] This preparation method achieves efficient and mild extraction of β-glucan by controlling the pressure and temperature parameters of subcritical water in stages, combined with a countercurrent circulation fluid dynamics design. In the first stage (4 MPa / 115℃), the lower pressure promotes the penetration of subcritical water into the intercellular spaces of mycelium cells, softening the cell wall structure, while avoiding the breakage of glycosidic bonds caused by high temperature; the shear force generated by the countercurrent circulation (3 mL / (min·g)) accelerates the diffusion of intracellular substances. In the second stage (8 MPa / 125℃), the increased pressure reduces the dielectric constant of subcritical water to a level close to methanol, enhancing its solubility for β-glucan, while the brief high temperature (3 minutes) disrupts cell membrane integrity while inhibiting the hydrolysis of the polysaccharide backbone through pressure constraint. In the third stage (6 MPa / 110℃), the reverse regulation of the pressure gradient brings the extraction system into dynamic equilibrium, maintaining the polysaccharide in a soluble state while slowing down molecular thermal motion through cooling, thus protecting the stability of β-(1→3) glycosidic bonds.

[0048] This phased control strategy achieves a dynamic balance between extraction efficiency and structural protection at the molecular scale through the coordinated oscillation of pressure and temperature: the first phase, gentle permeation, pre-relaxes the cell wall, reducing mechanical damage caused by subsequent high-pressure shocks; the second phase, short-duration high-pressure and high-temperature, breaks through the mass transfer bottleneck, increasing the yield of β-glucan to 28.7%, while the 8MPa pressure increases the ion product of water, inhibiting glycosidic bond hydrolysis under acidic conditions; the third phase, the negative pressure gradient (ΔP = 2MPa) generated by pressure correction drives the directional migration of the extract, combined with the continuous renewal effect of countercurrent circulation, enabling the target polysaccharide to rapidly accumulate in the 110℃ stable region.

[0049] In some embodiments, the preprocessing includes the following steps:

[0050] β-1,3-glucanase was used at an enzyme activity concentration of 0.5-1.5 U / g mycelium, and the mycelium was treated for 30 min at pH 5.5-6.0 and 45℃, while a pulsed electric field was applied simultaneously.

[0051] The pulse electric field parameters are: field strength 5-10kV / cm, pulse width 45-55μs, and frequency 8-12Hz.

[0052] The pretreatment stage utilizes the synergistic effect of β-1,3-glucanase and pulsed electric field to construct a dual biophysical cell disruption mechanism: In a slightly acidic environment of pH 5.5-6.0, β-1,3-glucanase (0.5-1.5 U / g) specifically hydrolyzes β-1,3-glycosidic bonds in the hyphal cell wall, creating localized cracks in the dense polysaccharide network. At this point, a pulsed electric field of 5-10 kV / cm is applied, forming a transient electroporation effect with a pulse width of 45-55 μs—the electric field force establishes micron-sized pores in the phospholipid bilayer of the cell membrane, while the dielectrophoresis effect induced by the high-frequency alternating electric field (8-12 Hz) drives the intracellular β-glucan to migrate out of the cell. The oligosaccharide fragments produced by enzymatic hydrolysis form an ionization gradient under the action of the electric field, which accelerates diffusion into the bulk solution through electroosmosis. The temperature control condition of 45℃ maintains enzyme activity while avoiding premature denaturation of cell contents due to high temperature.

[0053] This synergistic pretreatment significantly improves the efficiency of subsequent subcritical water extraction: enzymatic hydrolysis directionally weakens the cell wall structure strength, reducing the energy consumption of cell wall disruption by the pulsed electric field, while the electroporation effect induced by the electric field increases the diffusion rate of the enzymatic hydrolysis products. With the synergy of both, the mycelial permeability regulation time is significantly shortened compared to conventional enzymatic hydrolysis; the optimized range of electric field strength of 5-10 kV / cm ensures membrane permeability while avoiding polysaccharide oxidative degradation caused by excessively high electric field strength.

[0054] In some embodiments, the enzyme activity concentration of the β-1,3-glucanase is 0.8-1.2 U / g mycelium.

[0055] When the enzyme concentration is below 0.8 U / g, the number of β-1,3-glycosidic bonds hydrolyzed per unit time is insufficient, resulting in a minimal increase in cell wall porosity and an inability to effectively synergize with the electroporation effect of the subsequent pulsed electric field. Conversely, when the concentration exceeds 1.2 U / g, competitive adsorption of enzyme molecules on the hyphal surface intensifies, not only wasting enzyme resources but also generating a large number of oligosaccharide fragments (DP<3) due to localized over-hydrolysis. These fragments are prone to Maillard reactions during the subsequent subcritical water extraction stage. An activity concentration of 0.8–1.2 U / g places the enzymatic hydrolysis reaction at the plateau phase of the Michaelis-Menten equation, where the binding of enzyme molecules to the substrate reaches dynamic saturation. This ensures a stable rate of 0.22–0.25 μmol of glycosidic bonds hydrolyzed per minute while avoiding non-specific cleavage caused by enzyme overload.

[0056] In some embodiments, the field strength of the pulsed electric field is 7-9 kV / cm, the pulse width is 50 μs, and the frequency is 10 Hz.

[0057] Under an electric field strength of 7-9 kV / cm, the transmembrane potential of the cell membrane reaches 1.05-1.35 V. At this point, the 50 μs pulse width ensures that the electric field energy remains effective for a certain duration after the cell membrane capacitance charging cycle, triggering bidirectional electroporation—the positive pulse period forms nanopores, while the negative pulse period stretches the pore structure through dielectroporation to prevent closure. The 10 Hz frequency matches the mechanical relaxation time of the hyphal cell wall, ensuring that the interval between each pulse is just enough to complete the elastic recovery of the pores, maintaining pore conductivity while avoiding membrane overload collapse. Under this parameter window, the electric field energy density and the glass transition temperature of β-glucan work synergistically, enabling the cell wall polysaccharide network to release target components in a controlled softening state.

[0058] The 7-9 kV / cm field strength increased the electroporation density, while the 50 μs pulse width allowed the single pulse duration to cover 3-4 cell membrane charge-discharge cycles, extending the pore conduction time to 1.8 times that of conventional parameters; the 10 Hz frequency modulation caused the peak migration rate of β-glucan to appear in the 5th-7th pulse cycle, matching the formation time of the enzymatic reaction process and improving the synchronization rate of intracellular polysaccharide release.

[0059] In some embodiments, during the extraction process, a composite stabilizer is added to the extraction system. The composite stabilizer comprises trehalose and nano-silica in a mass ratio of 4:1, wherein the specific surface area of ​​the nano-silica is ≥200 m². 2 / g.

[0060] The composite stabilizer synergistically protects the β-glucan structure through molecular interface engineering and nano-confinement effects: trehalose, in a subcritical aqueous environment (115-125℃), forms a dynamic hydrogen bond network with the β-(1→3) glycosidic bonds of the polysaccharide chain through hydroxyl groups. Its glass transition temperature forms a "molecular buffer layer" within the extraction temperature window, inhibiting excessive vibration of the sugar chain at high temperatures; nano-silica (specific surface area...)

[0061] ≥200m 2 The silica (g) adsorbs polysaccharide molecules through abundant silanol groups on its surface, and its nanoscale pores disperse thermal stress through steric hindrance. At the same time, its high specific surface area allows each gram of silica to load a huge amount of polysaccharide molecules, forming a "nano-armor" protective layer. The 4:1 mass ratio optimizes the synergistic balance between hydrogen bond donors (trehalose) and physical barriers (silica). When the temperature rises to 125°C, the hydroxyl donor density of trehalose just covers the potential breakage sites of β-glucan chains, while the silica loading (0.2 g / g polysaccharide) effectively limits the range of chain segment movement.

[0062] By incorporating a composite stabilizing system, the structural integrity of β-glucan is significantly improved under harsh extraction conditions. The trehalose-silica composite system increases the upper limit of the extraction temperature tolerance, allowing for a high β-(1→3) bond retention rate even at 125℃. Compared with traditional single stabilizer solutions (such as using 0.1% EDTA alone), the polysaccharide yield is increased by 41% without the need for subsequent additive removal steps and without excipient residues.

[0063] In some embodiments, the amount of trehalose added is 0.02% of the dry weight of the mycelium, and the amount of nano-silica added is 0.005% of the dry weight of the mycelium.

[0064] The combined addition of trehalose and nano-silica achieves polysaccharide structural stability through synergistic effects at the molecular and mesoscale: 0.02% trehalose forms a dynamic hydrogen bond network in a subcritical water environment, with its hydroxyl groups specifically binding to the C2 / C4 hydroxyl groups of β-glucan. At high temperatures (125°C), a local low dielectric environment is formed through a water molecule replacement mechanism (each trehalose molecule can bind about 8 water molecules), inhibiting the vibrational unwinding of the polysaccharide backbone; 0.005% nano-silica constructs a three-dimensional physical support network in the flow system through van der Waals forces between surface silanol groups and the hydrophobic regions of the polysaccharide.

[0065] This composite stabilizing system provides dual protection for the structural integrity of β-glucan under harsh extraction conditions: the hydrogen bond protection of trehalose reduces the unwinding rate of the triple helix structure at 125°C, while the physical support of nano-silica reduces the viscosity of the extract, significantly improving membrane separation flux; the optimized concentration ratio (4:1) allows trehalose to completely coat the silica surface, forming a "sugar coating-nanocore" composite. This structure locks in the polysaccharide molecule conformation through the glass transition of trehalose during the spray drying stage, keeping the moisture content of the final product at a low level.

[0066] In some embodiments, the pH of the extraction system is controlled at 6.2-6.8.

[0067] By controlling the pH of the extraction system between 6.2 and 6.8, the quality of the product can be significantly improved.

[0068] 1. The buffer system counteracts the acidic environment caused by the self-ionization of subcritical water, thereby reducing the hydrolysis rate constant of β-1,3 glycosidic bonds and increasing the molecular weight retention rate.

[0069] 2. Weakly acidic conditions inhibit polyphenol oxidase activity, reduce the color difference of the extract, and avoid interference of browning products with the immunomodulatory activity of polysaccharides.

[0070] 3. Metal chelation eliminates the catalytic effect of divalent ions, keeping the conductivity of the polysaccharide solution at a low level, ensuring the efficiency of subsequent membrane separation, and avoiding polymerization degradation during storage caused by residual metal ions.

[0071] Figure 2 A flowchart of the purification steps provided in an embodiment of this application is shown.

[0072] like Figure 2 As shown, in some embodiments, the purification of the crude product to obtain the mushroom extract includes the following steps:

[0073] S21. Using a ceramic membrane with a 50nm pore size, the transmembrane pressure difference is controlled at 0.28-0.32MPa to perform cross-flow filtration on the crude product to obtain the filter material;

[0074] S22. Spray dry the filtered material, controlling the inlet air temperature to 155-165℃ and the outlet air temperature to 80-90℃, to obtain mushroom extract.

[0075] The ceramic membrane design with a 50nm pore size can trap denatured protein aggregates and cell debris of >300kDa, while allowing the target β-glucan (hydrodynamic radius of about 12nm) to pass through efficiently. The tangential flow velocity generated by the cross-flow mode continuously washes the membrane surface, keeping the membrane fouling index at a low level. During the spray drying stage, the inlet air temperature of 155-165℃ causes the surface of the atomized droplets to instantly form a dense skin layer, locking the helical structure of the internal polysaccharides. The outlet air temperature of 80-90℃ induces the trehalose protectant to form a glassy matrix through a gradient cooling mechanism, inhibiting water diffusion and avoiding local overheating that could lead to glycosidic bond breakage.

[0076] In some embodiments, the transmembrane pressure difference is 0.3 MPa, the inlet air temperature of the spray dryer is 158-162°C, and the outlet air temperature is 83-87°C.

[0077] During the ceramic membrane separation process, a transmembrane pressure difference of 0.3 MPa allows the extract to pass through the membrane channel with a pore size of 50 nm in turbulent flow. This pressure difference threshold can effectively retain β-glucan with a molecular weight >10 kDa (Stokes radius of about 12 nm) while avoiding the concentration polarization phenomenon on the membrane surface caused by excessive pressure (>0.5 MPa).

[0078] When spray drying uses an inlet air temperature of 158-162℃, a temperature gradient above the glass transition temperature is instantly formed on the surface of the atomized droplets (particle size 20-50μm), causing the water to evaporate at a rate of >104℃ / s. Meanwhile, the core temperature remains below 70℃ due to the hydrogen bond protection of trehalose. The outlet air temperature setting of 83-87℃ ensures that the material temperature at the bottom of the drying tower is ≤45℃, preventing the unwinding of the triple helix structure of β-glucan.

[0079] The method of the present invention will now be described in detail with reference to embodiments, comparative examples and experimental data.

[0080] Example 1

[0081] This embodiment provides a method for preparing fungal extracts, the process including the following steps:

[0082] Step 1: Take 20L of Grifola frondosa liquid fermentation broth and separate it into wet mycelium (72% water content) using a plate and frame filter press (5μm filter cloth pore size). Weigh 1kg of wet mycelium (280g dry weight) and place it in an enzymatic reaction vessel. Add citrate-disodium hydrogen phosphate buffer (0.1M concentration) at pH 5.8 to the vessel to adjust the liquid-to-solid ratio to 1:3 (w / w). Add β-1,3-glucanase (enzyme activity 1.0U / g mycelium dry weight) and maintain the reaction system temperature at 45℃. Simultaneously turn on the pulsed electric field generator, setting the field strength to 8kV / cm, pulse width to 50μs, and frequency to 10Hz, and treat for 30 minutes with magnetic stirring (200rpm). After treatment, centrifuge (4000g, 10min) to collect the mycelium, wash twice with deionized water, and set for later use.

[0083] Step 2: Load the pretreated mycelium into a countercurrent circulation extraction tank, and add 0.02% trehalose (0.056g) and 0.005% nano silica (specific surface area 220m²) based on the dry weight of the mycelium. 2 (g, 0.014g) was added, and the pH of the system was adjusted to 6.5. Deionized water was added to a solid-liquid ratio of 1:25 (w / v). The high-pressure pump was started to increase the system pressure to 4 MPa, and the temperature was heated to 115°C and maintained for 5 minutes, during which the countercurrent circulation flow rate was controlled at 3 mL / (min·g mycelium). Subsequently, the pressure was increased to 8 MPa at a rate of 2 MPa / min, and the temperature was simultaneously increased to 125°C and maintained for 3 minutes. Finally, the pressure was decreased to 6 MPa at a rate of 1 MPa / min, and the temperature was adjusted back to 110°C and maintained for 2 minutes. The outlet temperature was monitored in real time and kept ≤35°C. The extract was collected.

[0084] Step 3: After the extract is cooled to 40°C using a plate heat exchanger, it is pumped into a 50nm ceramic membrane system (membrane area 0.5m²). 2 The transmembrane pressure differential was set to 0.3 MPa, and the crossflow velocity to 4 m / s. The system was run continuously until the filtrate volume reached 85% of the feed volume. The retentate was returned to the extraction tank for circulation, while the permeate entered the temporary storage tank.

[0085] Step 4: The membrane permeate is concentrated to a solid content of 15% using a vacuum concentrator (45℃, -0.08MPa), and then dried using a centrifugal spray dryer (atomizing disc speed 18000rpm). The inlet air temperature is set to 160℃, the outlet air temperature to 85℃, and the internal negative pressure to -200Pa. The powder at the bottom of the drying tower is collected and passed through a 100-mesh sieve to obtain the β-glucan extract.

[0086] Example 2

[0087] This embodiment provides a method for preparing fungal extracts, the process including the following steps:

[0088] Step 1: Take 20L of Grifola frondosa liquid fermentation broth and separate it into wet mycelium (72% water content) using a plate and frame filter press (5μm filter cloth pore size). Weigh 1kg of wet mycelium (280g dry weight) and place it in an enzymatic reaction vessel. Add citrate-disodium hydrogen phosphate buffer (0.1M concentration) at pH 5.8 to the vessel to adjust the liquid-to-solid ratio to 1:3 (w / w). Add β-1,3-glucanase (enzyme activity 0.8U / g mycelium dry weight) and maintain the reaction system temperature at 45℃. Simultaneously turn on the pulsed electric field generator, setting the field strength to 7kV / cm, pulse width to 50μs, and frequency to 10Hz, and treat for 30 minutes with magnetic stirring (200rpm). After treatment, centrifuge (4000g, 10min) to collect the mycelium, wash twice with deionized water, and set for later use.

[0089] Step 2: Load the pretreated mycelium into a countercurrent circulation extraction tank, and add 0.02% trehalose (0.056g) and 0.005% nano silica (specific surface area 220m²) based on the dry weight of the mycelium. 2 (g, 0.014g) was added, and the pH of the system was adjusted to 6.3. Deionized water was added to a solid-liquid ratio of 1:25 (w / v). The high-pressure pump was started to increase the system pressure to 4 MPa, and the temperature was heated to 115°C and maintained for 5 minutes, during which the countercurrent circulation flow rate was controlled at 3 mL / (min·g mycelium). Subsequently, the pressure was increased to 8 MPa at a rate of 2 MPa / min, and the temperature was simultaneously increased to 125°C and maintained for 3 minutes. Finally, the pressure was decreased to 6 MPa at a rate of 1 MPa / min, and the temperature was adjusted back to 110°C and maintained for 2 minutes. The outlet temperature was monitored in real time and kept ≤35°C. The extract was collected.

[0090] Step 3: After the extract is cooled to 40°C using a plate heat exchanger, it is pumped into a 50nm ceramic membrane system (membrane area 0.5m²). 2 The transmembrane pressure differential was set to 0.28 MPa, and the crossflow velocity was 4 m / s. The system was run continuously until the filtrate volume reached 85% of the feed volume. The retentate was returned to the extraction tank for circulation, while the permeate entered the temporary storage tank.

[0091] Step 4: The membrane permeate is concentrated to a solid content of 15% using a vacuum concentrator (45℃, -0.08MPa), and then dried using a centrifugal spray dryer (atomizing disc speed 18000rpm). The inlet air temperature is set to 158℃, the outlet air temperature to 83℃, and the negative pressure inside the tower to -200Pa. The powder at the bottom of the drying tower is collected and passed through a 100-mesh sieve to obtain the β-glucan extract.

[0092] Example 3

[0093] This embodiment provides a method for preparing fungal extracts, the process including the following steps:

[0094] Step 1: Take 20L of Grifola frondosa liquid fermentation broth and separate it into wet mycelium (72% water content) using a plate and frame filter press (5μm filter cloth pore size). Weigh 1kg of wet mycelium (280g dry weight) and place it in an enzymatic reaction vessel. Add citrate-disodium hydrogen phosphate buffer (0.1M concentration) at pH 5.8 to the vessel to adjust the liquid-to-solid ratio to 1:3 (w / w). Add β-1,3-glucanase (enzyme activity 1.2U / g mycelium dry weight) and maintain the reaction system temperature at 45℃. Simultaneously turn on the pulsed electric field generator, setting the field strength to 9kV / cm, pulse width to 50μs, and frequency to 10Hz, and treat for 30 minutes under magnetic stirring (200rpm). After treatment, centrifuge (4000g, 10min) to collect the mycelium, wash twice with deionized water, and set for later use.

[0095] Step 2: Load the pretreated mycelium into a countercurrent circulation extraction tank, and add 0.02% trehalose (0.056g) and 0.005% nano silica (specific surface area 220m²) based on the dry weight of the mycelium. 2 (g, 0.014g), adjust the pH of the system to 6.7. Inject deionized water to a solid-liquid ratio of 1:25 (w / v), start the high-pressure pump to increase the system pressure to 4MPa, heat to 115℃ and maintain for 5 minutes, during which the countercurrent circulation flow rate is controlled at 3mL / (min·g mycelium). Then increase the pressure to 8MPa at a rate of 2MPa / min, and simultaneously increase the temperature to 125℃ and maintain for 3 minutes. Finally, decrease the pressure to 6MPa at a rate of 1MPa / min, and reduce the temperature back to 110℃ and maintain for 2 minutes. Monitor the outlet temperature in real time to ≤35℃, and collect the extract.

[0096] Step 3: After the extract is cooled to 40°C using a plate heat exchanger, it is pumped into a 50nm ceramic membrane system (membrane area 0.5m²). 2 The transmembrane pressure differential was set to 0.32 MPa, and the crossflow velocity was 4 m / s. The system was run continuously until the filtrate volume reached 85% of the feed volume. The retentate was returned to the extraction tank for circulation, while the permeate entered the temporary storage tank.

[0097] Step 4: The membrane permeate is concentrated to a solid content of 15% using a vacuum concentrator (45℃, -0.08MPa), and then dried using a centrifugal spray dryer (atomizing disc speed 18000rpm). The inlet air temperature is set to 162℃, the outlet air temperature to 87℃, and the internal negative pressure to -200Pa. The powder at the bottom of the drying tower is collected and passed through a 100-mesh sieve to obtain the β-glucan extract.

[0098] Comparative Example 1

[0099] This comparative example provides a method for preparing fungal extracts, the process comprising the following steps:

[0100] Step 1: Take 20L of Grifola frondosa liquid fermentation broth and separate it into wet mycelium (72% water content) using a plate and frame filter press (5μm filter cloth pore size). Weigh 1kg of wet mycelium (280g dry weight) and place it in an enzymatic reaction vessel. Add citrate-disodium hydrogen phosphate buffer (0.1M concentration) at pH 5.8 to the vessel to adjust the liquid-to-solid ratio to 1:3 (w / w). Add β-1,3-glucanase (enzyme activity 0.4U / g mycelium dry weight) and maintain the reaction system temperature at 45℃. Simultaneously turn on the pulsed electric field generator, setting the field strength to 4kV / cm, pulse width to 50μs, and frequency to 10Hz, and treat for 30 minutes with magnetic stirring (200rpm). After treatment, centrifuge (4000g, 10min) to collect the mycelium, wash twice with deionized water, and set for later use.

[0101] Step 2: Load the pretreated mycelium into a countercurrent circulation extraction tank, and add 0.02% trehalose (0.056g) and 0.005% nano silica (specific surface area 220m²) based on the dry weight of the mycelium. 2 (g, 0.014g), adjust the pH of the system to 6.0. Inject deionized water to a solid-liquid ratio of 1:25 (w / v), start the high-pressure pump to increase the system pressure to 4 MPa, heat to 115℃ and maintain for 5 minutes, during which the countercurrent circulation flow rate is controlled at 3 mL / (min·g mycelium). Then increase the pressure to 8 MPa at a rate of 2 MPa / min, and simultaneously increase the temperature to 125℃ and maintain for 3 minutes. Finally, decrease the pressure to 6 MPa at a rate of 1 MPa / min, and restore the temperature to 110℃ and maintain for 2 minutes. Monitor the outlet temperature in real time to ≤35℃, and collect the extract.

[0102] Step 3: After the extract is cooled to 40°C using a plate heat exchanger, it is pumped into a 50nm ceramic membrane system (membrane area 0.5m²). 2 The transmembrane pressure differential was set to 0.3 MPa, and the crossflow velocity to 4 m / s. The system was run continuously until the filtrate volume reached 85% of the feed volume. The retentate was returned to the extraction tank for circulation, while the permeate entered the temporary storage tank.

[0103] Step 4: The membrane permeate is concentrated to a solid content of 15% using a vacuum concentrator (45℃, -0.08MPa), and then dried using a centrifugal spray dryer (atomizing disc speed 18000rpm). The inlet air temperature is set to 160℃, the outlet air temperature to 85℃, and the internal negative pressure to -200Pa. The powder at the bottom of the drying tower is collected and passed through a 100-mesh sieve to obtain the β-glucan extract.

[0104] Comparative Example 2

[0105] This comparative example provides a method for preparing fungal extracts, the process comprising the following steps:

[0106] Step 1: Take 20L of Grifola frondosa liquid fermentation broth and separate it into wet mycelium (72% water content) using a plate and frame filter press (5μm filter cloth pore size). Weigh 1kg of wet mycelium (280g dry weight) and place it in an enzymatic reaction vessel. Add citrate-disodium hydrogen phosphate buffer (0.1M concentration) at pH 5.8 to the vessel to adjust the liquid-to-solid ratio to 1:3 (w / w). Add β-1,3-glucanase (enzyme activity 1.0U / g mycelium dry weight) and maintain the reaction system temperature at 45℃. Simultaneously turn on the pulsed electric field generator, setting the field strength to 8kV / cm, pulse width to 50μs, and frequency to 10Hz, and treat for 30 minutes with magnetic stirring (200rpm). After treatment, centrifuge (4000g, 10min) to collect the mycelium, wash twice with deionized water, and set for later use.

[0107] Step 2: Load the pretreated mycelium into a countercurrent circulation extraction tank, and add 0.015% trehalose (0.042g) and 0.005% nano-silica (specific surface area 180m²) based on the dry weight of the mycelium. 2 (g, 0.014g) was added, and the pH of the system was adjusted to 6.5. Deionized water was added to a solid-liquid ratio of 1:25 (w / v). The high-pressure pump was started to increase the system pressure to 4 MPa, and the temperature was heated to 115°C and maintained for 5 minutes, during which the countercurrent circulation flow rate was controlled at 3 mL / (min·g mycelium). Subsequently, the pressure was increased to 8 MPa at a rate of 2 MPa / min, and the temperature was simultaneously increased to 125°C and maintained for 3 minutes. Finally, the pressure was decreased to 6 MPa at a rate of 1 MPa / min, and the temperature was adjusted back to 110°C and maintained for 2 minutes. The outlet temperature was monitored in real time and kept ≤35°C. The extract was collected.

[0108] Step 3: After the extract is cooled to 40°C using a plate heat exchanger, it is pumped into a 50nm ceramic membrane system (membrane area 0.5m²). 2 The transmembrane pressure differential was set to 0.3 MPa, and the crossflow velocity to 4 m / s. The system was run continuously until the filtrate volume reached 85% of the feed volume. The retentate was returned to the extraction tank for circulation, while the permeate entered the temporary storage tank.

[0109] Step 4: The membrane permeate is concentrated to a solid content of 15% using a vacuum concentrator (45℃, -0.08MPa), and then dried using a centrifugal spray dryer (atomizing disc speed 18000rpm). The inlet air temperature is set to 170℃, the outlet air temperature to 75℃, and the negative pressure inside the tower to -200Pa. The powder at the bottom of the drying tower is collected and passed through a 100-mesh sieve to obtain the β-glucan extract.

[0110] Experimental methods

[0111] 1. The total polysaccharide content was determined by the phenol-sulfuric acid method, and the yield was calculated by detecting the specific peak area of ​​β-1,3-glucan using high performance liquid chromatography (HPLC).

[0112] 2. The molecular weight distribution of the extract was determined by gel permeation chromatography (GPC), with the untreated mycelial extract as the baseline (100%).

[0113] 3. Use a colorimeter to measure the Lab* value of the extract and calculate the color difference with the standard white plate;

[0114] 4. The intensity of the characteristic peak of the triple helix of β-glucan was detected in the wavelength range of 195-250 nm using circular dichroism spectroscopy (CD).

[0115] 5. Record the percentage decrease in flux after the ceramic membrane has been running for 1 hour;

[0116] 6. The moisture content of the powder after spray drying and standing for 1 day was determined using the Karl Fischer method.

[0117] The experimental results are shown in Table 1.

[0118] Table 1 Comparison of Key Performance Indicators between Examples and Comparative Examples

[0119]

[0120] Analysis of Experimental Results

[0121] The experimental data show that Examples 1-3 exhibited stable performance in key indicators such as β-glucan yield (27.9-29.1%), molecular weight retention (90.8-92.5%), and helical structure retention (87.6-89.4%), significantly outperforming the comparative examples. Example 2, by reducing enzyme activity to 0.8 U / g and electric field strength to 7 kV / cm, resulted in a slight decrease in yield of 0.8%, but the molecular weight retention remained above 90%, demonstrating that parameter adjustments within the scope of the claims can maintain the stability of enzyme-electric field synergistic cell disruption. In Example 3, with enzyme activity of 1.2 U / g and electric field strength of 9 kV / cm, the yield increased to 29.1%, but the color difference slightly increased to 3.8, possibly due to the slight participation of oligosaccharide fragments generated by enzymatic hydrolysis in the Maillard reaction, but overall remained within a controllable range.

[0122] Comparative Example 1 suffered from insufficient cell wall porosity due to enzyme activity (0.4 U / g) and field strength (4 kV / cm) below the lower limit of the claims, hindering β-glucan release and causing the yield to plummet to 18.4%. Simultaneously, the pH of 6.0 deviated from the claim range (6.2-6.8), accelerating acidic hydrolysis of glycosidic bonds, resulting in a molecular weight retention of only 76.3%. In Comparative Example 2, the imbalanced ratio of trehalose to nano-silica (3:1) and insufficient specific surface area of ​​the nanomaterials (180 m²) further exacerbated the problem. 2 / g), which leads to a decrease in thermal protection ability during the high-temperature stage (125℃), with the helical structure retention rate being only 54.7%; the excessive inlet air temperature of spray drying (170℃) further damages the polysaccharide conformation, and the moisture content rises to 5.2%, indicating that the glassy matrix is ​​destroyed and the powder's resistance to moisture absorption decreases.

[0123] The differences in membrane separation performance further validated the synergistic effect of the technical solutions: the membrane flux decline rate of the example group (12.3-14.7%) was significantly lower than that of the comparative group (28.5-34.2%). This was attributed to the chelating effect of the composite stabilizer on the viscosity of the extract and the pH control on metal ions, which reduced the deposition of membrane contaminants. In contrast, in comparative example 2, the stabilizer failed, resulting in an increase in macromolecular impurities in the extract and a more severe membrane flux decline. At the same time, the excessively high drying temperature led to increased hygroscopicity of the powder and excessive moisture content.

[0124] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0125] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a mushroom extract, characterized by, Includes the following steps: Pretreatment of mycelium: β-1,3-glucanase was used at an enzyme activity concentration of 0.5-1.5 U / g mycelium. The mycelium was treated for 30 minutes at pH 5.5-6.0 and a temperature of 45℃, while a pulsed electric field was applied simultaneously. The pulsed electric field parameters were: field strength 5-10 kV / cm, pulse width 45-55 μs, and frequency 8-12 Hz. The pretreated mycelium is placed in a subcritical water as an extraction system, a composite stabilizer is added to the extraction system for extraction, during which countercurrent circulation is maintained, the flow rate is 3 mL / (min.g mycelium), a crude product is obtained, the pH of the extraction system is controlled at 6.2-6.8, and the composite stabilizer is trehalose and nanosilica with a mass ratio of 4:1, wherein the specific surface area of the nanosilica is ≥200 m 2 / g. The crude product is purified to obtain mushroom extract, wherein the purification includes cross-flow filtration and spray drying, and the inlet air temperature of the spray drying is 158-162℃. The subcritical water extraction process is controlled under different conditions in stages according to the following procedure: First stage: Pressure 4MPa, temperature 115℃, maintain for 5 minutes; Second stage: The pressure is increased to 8MPa and the temperature is increased to 125℃, and maintained for 3 minutes; Third stage: The pressure drops to 6MPa, the temperature returns to 110℃, and is maintained for 2 minutes.

2. The production method according to claim 1, characterized by, The enzyme activity concentration of the β-1,3-glucanase is 0.8-1.2 U / g mycelium.

3. The preparation method according to claim 1, characterized in that, The pulsed electric field has a field strength of 7-9 kV / cm, a pulse width of 45-55 μs, and a frequency of 8-12 Hz.

4. The preparation method according to claim 1, characterized in that, The amount of trehalose added is 0.02% of the dry weight of the mycelium, and the amount of nano-silica added is 0.005% of the dry weight of the mycelium.

5. The preparation method according to claim 1, characterized in that, The purification of the crude product to obtain the mushroom extract includes the following steps: Using a ceramic membrane with a pore size of 50 nm, the transmembrane pressure difference is controlled at 0.28-0.32 MPa, and the crude product is subjected to cross-flow filtration to obtain the filtered material. The filtered material is spray-dried, and the outlet air temperature is controlled at 80-90℃ to obtain mushroom extract.

6. The preparation method according to claim 5, characterized in that, The transmembrane pressure difference is 0.3 MPa.

7. The preparation method according to claim 6, characterized in that, The outlet air temperature of the spray dryer is 83-87℃.

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