Preparation method of edible mushroom active extract and food application thereof

Abstract

This invention discloses a method for preparing an active extract of edible fungi and its food application. A mixture of various edible fungi fruiting bodies is placed in the same continuous process flow. After low-temperature preservation pretreatment, it is sequentially hydrolyzed in three enzymatic hydrolysis units, with continuous transfer between units and simultaneous pulsed ultrasound. The hydrolysate is filtered and then extracted with an organic solvent to separate an organic phase and an aqueous phase. The organic phase is concentrated, subjected to column chromatography, and low-temperature crystallization to obtain fat-soluble active crystals. The aqueous phase is fractionated with ethanol and separated by multi-stage membrane filtration to obtain high-molecular-weight polysaccharide fractions and medium-molecular-weight polysaccharide fractions. The former is modified by sulfation to obtain sulfated polysaccharides, and the latter is dried to obtain a polysaccharide extract. The active components are compounded according to product requirements to obtain the active extract of edible fungi. When this active extract of edible fungi is used as a food ingredient, the polysaccharide extract and the sulfated polysaccharides synergistically form an aqueous thickening system, in which the fat-soluble active crystals are dispersed.

Description

Technical Field

[0001] This invention relates to the fields of food science and engineering and natural product chemical engineering technology, specifically to a method for preparing an active extract of edible fungi and its food application. Background Technology

[0002] Edible fungi are a group of large fungi with important nutritional value. They are rich in active ingredients such as polysaccharides, terpenoids, sterols, and polypeptides, and have significant value in the development of food ingredients and functional factors. Among them, Hericium erinaceus, Pleurotus ostreatus, and Tremella fuciformis are representative edible fungi widely cultivated in my country, and the extraction and utilization of their active ingredients has always been a research hotspot in the fields of food science and natural product chemistry.

[0003] Hericium erinaceus polysaccharide is mainly β-glucan, possessing high molecular weight and good water solubility and viscosity. Lovastatin from Pleurotus ostreatus is a fat-soluble lactone small molecule compound, which is relatively sensitive to heat and acid / alkali environments. Tremella fuciformis polysaccharide is an acidic heteropolysaccharide with a wide molecular weight distribution and often tightly bound to impurities such as proteins and pigments. These three active ingredients exhibit significant differences in chemical properties: Hericium erinaceus polysaccharide and Tremella fuciformis polysaccharide are water-soluble macromolecules, while lovastatin is a fat-soluble small molecule. This presents a significant challenge to achieving the co-extraction and targeted separation of multiple active ingredients within the same processing system.

[0004] Currently, the extraction of active ingredients from edible fungi mainly employs methods such as hot water extraction, enzymatic hydrolysis, ultrasound-assisted extraction, and microwave-assisted extraction. Hot water extraction is simple and inexpensive, but suffers from drawbacks such as high extraction temperatures, long extraction times, and easy degradation of heat-sensitive components. Enzymatic hydrolysis utilizes enzymes such as cellulase, pectinase, and protease to degrade the cell walls of edible fungi, effectively increasing the dissolution rate of active ingredients; however, single enzymatic hydrolysis conditions cannot adequately address the differences in cell wall composition among different fungal species. Ultrasound-assisted extraction disrupts cell wall structure through cavitation, offering advantages such as short extraction time and high efficiency; however, using ultrasound alone makes it difficult to selectively release multiple active ingredients from complex matrices. In recent years, combined enzymatic hydrolysis and ultrasound extraction technologies have been extensively studied, but these still primarily target single-species, single-product extraction, and differentiated extraction schemes for multiple fungal species and multiple active ingredients in mixed raw materials are still lacking.

[0005] In terms of separation and purification, the purification of edible fungal polysaccharides typically employs methods such as membrane filtration, macroporous adsorption resins, column chromatography, and ethanol fractionation. Membrane filtration technology achieves fractional separation of components based on molecular weight differences and has been widely used in the purification of single polysaccharides. However, when dealing with complex polysaccharide systems from mixed sources, the molecular weight distribution ranges of polysaccharides from different fungal strains significantly overlap, making precise separation difficult to achieve using membrane filtration alone. While ethanol fractionation can perform preliminary separation based on differences in polysaccharide solubility, its purity is often insufficient when used alone, and the precipitation process can easily lead to the loss of active ingredients. Macroporous adsorption resins and column chromatography are mostly used for the purification of single target products; in mixed polysaccharide systems, competitive adsorption can easily lead to a decrease in separation efficiency.

[0006] In the preparation of edible fungi complex polysaccharides, there are existing technical reports on the extraction of complex polysaccharides by mixing multiple edible fungi raw materials. For example, shiitake mushroom, tremella, and hericium erinaceus powders are mixed in a certain proportion, and then subjected to defatting, ultrasonic water extraction, filtration, concentration, washing, and drying to obtain complex polysaccharides. However, these methods all use the same extraction conditions to process the mixed raw materials, without considering the differences in the chemical composition of the cell walls of different edible fungi, nor designing differentiated release conditions for the active ingredients from different strains. Since the cell walls of hericium erinaceus are mainly composed of chitin-cellulose complex matrix, the cell walls of oyster mushroom are mainly composed of cellulose-hemicellulose, and the fruiting bodies of tremella are characterized by polysaccharide colloidal structures, the responses of the three cell wall structures to enzymatic hydrolysis conditions are quite different. Using the same extraction conditions will inevitably lead to insufficient release or excessive degradation of the active ingredients of some strains. In addition, there are no reports in the existing technology of designing sequential enzymatic hydrolysis conditions for the differences in the cell wall matrix of different strains in the mixed raw materials in the same process flow, and simultaneously achieving the directional separation of fat-soluble components and water-soluble polysaccharides.

[0007] Regarding the chemical modification of polysaccharides, the sulfation modification of Tremella fuciformis polysaccharides has been reported in the literature. Previous studies have used the chlorosulfonic acid-pyridine method to sulfate purified single Tremella fuciformis polysaccharides to improve their water solubility and rheological properties. However, these studies all used purified single Tremella fuciformis polysaccharides as raw materials for chemical modification and did not involve the selective sulfation of Tremella fuciformis polysaccharides in mixed polysaccharide systems, nor did they integrate this process with the extraction processes of Hericium erinaceus polysaccharides and lovastatin from Pleurotus ostreatus.

[0008] In summary, the existing technology lacks an integrated preparation method that can achieve partitioned sequential enzymatic hydrolysis, directional separation, and functional modification of mixed raw materials containing two or more different cell wall matrix types in the same continuous process flow, taking advantage of the differences in cell wall matrix and the differences in the physicochemical properties of active ingredients. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing active extracts of edible fungi. The method involves placing a mixture of various edible fungi fruiting bodies in the same continuous process flow, and obtaining the corresponding active components through partitioned sequential enzymatic hydrolysis and continuous separation. This solves the problems of insufficient release of active ingredients, excessive degradation, and insufficient separation accuracy of multiple components caused by the uniform processing of mixed raw materials in the prior art.

[0010] The "multiple edible fungi" mentioned in this invention refers to a combination of edible fungi fruiting bodies containing two or more different cell wall matrix types, such as fungi with chitin-cellulose complex walls (e.g., Hericium erinaceus), fungi with cellulose-hemicellulose walls (e.g., Pleurotus ostreatus), and fungi with polysaccharide gelatinous walls (e.g., Tremella fuciformis). Since the core of this invention lies in utilizing the differences in cell wall matrices among different fungal species to achieve directed sequential degradation through pH gradient and enzyme system matching, this process can be extended to other combinations of edible fungi with similar differences in cell wall structure.

[0011] Another objective of this invention is to provide an application of the edible fungus active extract prepared by the above-mentioned method as a food ingredient, utilizing the differences in the natural physicochemical properties of each active component to synergistically form a stable multiphase dispersion system in the food system.

[0012] The method for preparing active extracts of edible fungi according to the present invention involves placing a mixture of various edible fungi fruiting bodies as raw materials in the same continuous process flow, and obtaining the corresponding active components through partitioned sequential enzymatic hydrolysis and continuous separation and directional extraction, including the following steps: S1. Mix various edible fungi fruiting bodies, and then wash, freeze-dry, pulverize and defatted them at low temperature to obtain a pretreated mixed raw material; S2. The pretreated mixed raw materials are sequentially enzymatically hydrolyzed in the first, second and third enzymatic hydrolysis units. The materials are continuously transported between units and pulsed ultrasound is applied synchronously during the enzymatic hydrolysis process. The pH gradient and enzyme system differences between the units are used to perform directional sequential degradation of the cell wall matrix of different bacterial species in the mixed raw materials. S3. After filtering the enzymatic hydrolysate obtained from step S2 through sequential enzymatic hydrolysis, the organic phase and aqueous phase are separated by extraction with an organic solvent. The organic phase is concentrated, subjected to column chromatography and low-temperature crystallization to obtain lipid-soluble active crystals. The aqueous phase is separated according to molecular weight differences by ethanol fractionation and multi-stage membrane filtration to obtain high molecular weight polysaccharide fraction and medium molecular weight polysaccharide fraction. The high molecular weight polysaccharide fraction is modified by directional sulfation to obtain sulfated polysaccharide, and the medium molecular weight polysaccharide fraction is concentrated and dried to obtain polysaccharide extract. S4. Combine the active components obtained in step S3 according to product requirements to obtain edible fungi active extract.

[0013] Preferably, the various edible fungi fruiting bodies include Hericium erinaceus, Pleurotus ostreatus, and Tremella fuciformis.

[0014] Further, the specific operation of the partitioned sequential enzymatic hydrolysis in step S2 is as follows: the first enzymatic hydrolysis unit controls alkaline conditions to carry out alkaline extraction and hydrolysis, selectively loosening the protein-encapsulating structure on the surface of Tremella polysaccharide; the second enzymatic hydrolysis unit adjusts to weakly acidic conditions and adds a complex enzyme system composed of cellulase, pectinase and neutral protease to directionally degrade the gel layer in the cell wall of Hericium erinaceus and the residual protein connection structure of Tremella erinaceus; the third enzymatic hydrolysis unit adjusts to near-neutral conditions and adds a complex enzyme system composed of cellulase, hemicellulase and alkaline protease to gently release the heat-sensitive lovastatin and water-soluble polysaccharides in Pleurotus ostreatus.

[0015] Furthermore, the first enzymatic hydrolysis unit has a pH of 8.5–10.5, a temperature of 60–80°C, and a time of 0.5–1.5 h; the second enzymatic hydrolysis unit has a pH of 4.5–6.0, a temperature of 45–55°C, and a time of 1.5–3 h; and the third enzymatic hydrolysis unit has a pH of 6.0–7.5, a temperature of 40–50°C, and a time of 2–4 h.

[0016] Preferably, the frequency of the pulsed ultrasound in step S2 is 20–40 kHz, the power density is 100–350 W / L, and the pulse mode is on for 2–5 s and off for 1–2 s.

[0017] Preferably, the organic solvent in step S3 is ethyl acetate or ethanol with a volume fraction of 75–95%, the extraction temperature is 30–50°C, and the number of extractions is 2–3; the low-temperature crystallization temperature is 4–10°C, and the crystallization time is 12–48 h.

[0018] Preferably, the ethanol fractionation in step S3 is assisted by controlling the ethanol volume fraction to 35–55% to selectively precipitate high molecular weight polysaccharides. The supernatant is then separated into medium molecular weight polysaccharide fractions by multi-stage membrane filtration. After redissolving the precipitate, it is then desalted and purified by multi-stage membrane filtration to obtain high molecular weight polysaccharide fractions. The multi-stage membrane filtration is a three-stage tandem membrane filtration system, consisting of a microfiltration membrane with a pore size of 0.1–0.45 μm, an ultrafiltration membrane with a molecular weight cutoff of 10–50 kDa, and a nanofiltration membrane with a molecular weight cutoff of 200–500 Da. The directional sulfation modification is performed using the chlorosulfonic acid-pyridine method at a reaction temperature of 60–80 °C for 2–4 h.

[0019] Preferably, the mass ratio of the mixed hericium erinaceus, oyster mushroom and tremella in step S1 is (3–5):(2–4):(1–3); the mass ratio of the compound in step S4 is hericium erinaceus polysaccharide extract: lovastatin crystal: sulfated tremella polysaccharide = (5–15):(0.5–2):(3–8).

[0020] Preferably, the freeze-drying conditions in step S1 are -45°C to -55°C and vacuum degree ≤5Pa; the defatting is supercritical CO2 defatting, with an extraction pressure of 25–35MPa and an extraction temperature of 35–45°C.

[0021] Preferably, each enzymatic hydrolysis unit in step S2 is an independent reactor, and the units are connected by a closed pipeline. An online cleaning nozzle is installed in the pipeline to rinse the pipeline with purified water or pH transition buffer. After the reaction of each unit is completed, solid-liquid separation is performed first, and the solid material is washed before entering the next enzymatic hydrolysis unit to avoid the inhibition of enzyme activity in the next unit by the liquid enzyme system and acid / alkaline environment of the previous unit.

[0022] This invention also provides an application of the edible fungus active extract prepared according to the above-described method as a food ingredient. The polysaccharide extract and sulfated polysaccharide synergistically constitute an aqueous thickening system. The fat-soluble active crystals are dispersed in this aqueous thickening system. The amount of the edible fungus active extract added to the food is 0.5–10 wt%, and the median particle size D of the fat-soluble active crystals in the aqueous thickening system is... 50 The value is 1–20 μm, and the D 50 The result was obtained by dispersing the active extract of the edible fungi in an aqueous food matrix at an addition rate of 5 wt%.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to incorporate a mixture of various edible fungi fruiting bodies into the same continuous process flow. Through a partitioned sequential enzymatic hydrolysis strategy, appropriate pH gradients and enzyme systems are set for the differences in cell wall matrix of different fungi (chitin-cellulose complex wall of Hericium erinaceus, cellulose-hemicellulose wall of Pleurotus ostreatus, and polysaccharide colloidal wall of Tremella fuciformis). This overcomes the problem of insufficient release or excessive degradation of some fungi active ingredients caused by the uniform treatment of mixed raw materials in the prior art, and achieves efficient and simultaneous dissolution of the three active components. 2. In the separation stage, this invention employs a continuous separation sequence of multi-stage membrane filtration assisted by ethanol fractional precipitation. By controlling the volume fraction of ethanol, selective pre-precipitation of high molecular weight polysaccharides is achieved, which effectively solves the problem of unclear membrane separation boundaries caused by overlapping molecular weight distributions of polysaccharides from different bacterial strains, and significantly improves the separation purity of each polysaccharide fraction. 3. This invention achieves simultaneous acquisition of fat-soluble active crystals and water-soluble polysaccharide extracts in the same process flow through continuous operation of liquid-liquid extraction, column chromatography and low-temperature crystallization. This avoids the problems of lengthy process and high energy consumption caused by separate extraction and purification in traditional processes, and improves process integration and material utilization. 4. The present invention sets up independent reactors between enzymatic hydrolysis units, online cleaning and solid-liquid separation and refeeding mechanism, which effectively avoids cross-contamination and inactivation of different pH environments and enzyme systems in pipelines, ensures the process stability of partitioned sequential enzymatic hydrolysis, and is suitable for industrial continuous production. 5. When the edible fungi active extract prepared by this invention is used as a food ingredient, it utilizes the interfacial activity of the polysaccharide extract, the polyanionic thickening properties of the sulfated polysaccharide, and the lipophilicity of the lipophilic active crystals to synergistically form a stable aqueous thickening system and a lipophilic dispersion system in the food system. It can achieve uniform dispersion of lipophilic components in the aqueous food matrix without the need for additional synthetic emulsifiers and synthetic thickeners, which is in line with the development trend of clean label food ingredients. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the preparation process of the active extract of edible fungi of the present invention. Detailed Implementation

[0025] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but as long as they are within the scope of the present invention, they are protected by patent law.

[0026] The following example uses a combination of Hericium erinaceus, Pleurotus ostreatus, and Tremella fuciformis to illustrate the process of the present invention. Those skilled in the art can adjust the process parameters of each enzymatic hydrolysis unit according to the raw material composition to achieve targeted extraction of active components from other edible fungi combinations.

[0027] I. Experimental Section To verify the directional degradation mechanism of partitioned sequential enzymatic hydrolysis and the synergistic stabilizing effect of the compound products, this invention systematically carried out process analysis experiments (including tracking the composition of the dissolutions of each enzymatic hydrolysis unit, characterizing the cell wall microstructure, and monitoring enzyme activity residues) and application performance verification experiments (including rheological synergistic effect analysis, microscopic dispersion morphology observation, and accelerated storage stability test). The relevant experimental methods and results are detailed below.

[0028] To determine the reasonable range of each process parameter in claim 1, a pre-treated raw material consisting of dried fruiting bodies of Hericium erinaceus, Pleurotus ostreatus, and Tremella fuciformis mixed in a mass ratio of 4:3:2 was used as a benchmark. A single-factor rotation method was employed to screen the boundaries of key process parameters. Except for the factor under investigation, all other conditions followed the conventional parameters of Example 1.

[0029] (I) Screening of process parameters for the first enzymatic hydrolysis unit The first enzymatic hydrolysis unit's function is to selectively loosen the protein-encapsulating structure on the surface of Tremella fuciformis polysaccharide, while also ensuring the preservation of the molecular weight of Hericium erinaceus polysaccharide and the chemical stability of Lovastatin from Pleurotus ostreatus.

[0030] pH screening: At a fixed temperature of 70℃ and a duration of 1.0 h, the dissolution rate of *Tremella fuciformis* polysaccharides, the molecular weight retention rate of *Hericium erinaceus* polysaccharides, and the retention rate of lovastatin were investigated within the pH range of 7.0–11.0. The results showed that below pH 8.5, the dissolution rate of *Tremella fuciformis* polysaccharides was less than 70%, and the protein encapsulation structure was insufficiently loosened; above pH 10.5, the molecular weight retention rate of *Hericium erinaceus* polysaccharides was less than 80%, and the retention rate of lovastatin was less than 85%. Based on these findings, the effective pH range was determined to be 8.5–10.5.

[0031] Temperature screening: With a fixed pH of 9.0 and a treatment time of 1.0 h, various indicators were examined within a temperature range of 50–85℃. Results showed that below 60℃, the dissolution rate of Tremella fuciformis polysaccharides was less than 72%; above 80℃, the retention rate of lovastatin was less than 85%. The effective temperature range was determined to be 60–80℃.

[0032] Time-based screening: With a fixed pH of 9.0 and temperature of 70℃, various indicators were examined within a time range of 0.2–2.0 h. Results showed that when the time was less than 0.5 h, the dissolution rate of *Tremella fuciformis* polysaccharides was less than 68%; when the time was greater than 1.5 h, the molecular weight retention rate of *Hericium erinaceus* polysaccharides showed a decreasing trend. The effective time range was determined to be 0.5–1.5 h.

[0033] (II) Screening of process parameters for the second enzymatic hydrolysis unit The second enzymatic hydrolysis unit is responsible for the targeted degradation of the gelatinous layer in the cell wall of Hericium erinaceus and the residual protein linkage structure of Tremella fuciformis.

[0034] Screening of compound enzyme ratios: Under fixed pH 5.0, temperature 50℃, and time 2.0 h, the mass ratios of cellulase, pectinase, and neutral protease were investigated. Results showed that when the cellulase ratio was less than 2 parts, the yield of Hericium erinaceus polysaccharides was less than 14%; when the pectinase ratio was less than 1 part, insufficient degradation of the middle lamella led to difficulties in subsequent filtration; and when the neutral protease ratio was less than 1 part, the removal rate of protein impurities was less than 75%. The effective range for the three enzyme mass ratios was determined to be (2–4):(1–2):(1–2).

[0035] Temperature and time screening: An enzyme ratio of 3:1.5:1.5 was used to investigate the yield and molecular weight retention of Hericium erinaceus polysaccharides within a temperature range of 40–60℃ and a time range of 1.0–4.0 h. Results showed that when the temperature was below 45℃ or the time was below 1.5 h, the polysaccharide yield was less than 15%; when the temperature was above 55℃ or the time was above 3 h, the molecular weight retention was less than 80%. Therefore, the optimal temperature range was determined to be 45–55℃, and the optimal time range was 1.5–3 h.

[0036] (III) Screening of process parameters for the third enzymatic hydrolysis unit The third enzymatic hydrolysis unit functions to gently release heat-sensitive lovastatin and water-soluble polysaccharides from oyster mushrooms.

[0037] Screening of compound enzyme ratios: Under fixed pH 7.0, temperature 45℃, and time 2.5 h, the mass ratios of cellulase, hemicellulase, and alkaline protease were investigated. Results showed that when the cellulase ratio was less than 3 parts, the yield of oyster mushroom polysaccharides was less than 12%; when the hemicellulase ratio was less than 1 part, the degradation of the cell wall hemicellulose layer was insufficient; and when the alkaline protease ratio was less than 1 part, the removal rate of protein impurities was less than 70%. The effective range for the three enzyme mass ratios was determined to be (3–5):(1–2):(1–3).

[0038] Temperature and time screening: An enzyme-to-enzyme ratio of 4:1.5:2 was used to investigate the retention and yield of lovastatin within a temperature range of 35–55℃ and a time range of 1.5–5.0 h. Results showed that when the temperature was below 40℃ or the time was below 2 h, the lovastatin yield was less than 0.30%; when the temperature was above 50℃ or the time was above 4 h, the lovastatin retention was less than 80%. Therefore, the optimal temperature range was determined to be 40–50℃ and the optimal time range was 2–4 h.

[0039] (iv) Screening of ethanol fractionation precipitation concentration With other separation conditions kept constant, the effects of ethanol volume fractions of 25–65% on the precipitation selectivity of Tremella fuciformis polysaccharides and the co-precipitation rate of Hericium erinaceus polysaccharides were investigated. The results showed that when the ethanol volume fraction was below 35%, the precipitation rate of Tremella fuciformis polysaccharides was below 60%, with a large amount of polysaccharides remaining in the supernatant, leading to cross-contamination during subsequent membrane filtration. When the ethanol volume fraction was above 55%, the co-precipitation rate of Hericium erinaceus polysaccharides was above 15%, affecting the separation purity. The effective concentration range for ethanol fractionation precipitation was determined to be 35–55%.

[0040] (v) Screening of compound ratios The effects of different mass ratios of Hericium erinaceus polysaccharide extract, lovastatin crystals, and sulfated Tremella fuciformis polysaccharide on the dispersion stability (centrifugal stability and static stability) of the complex in an aqueous system were investigated. The results showed that when the Hericium erinaceus polysaccharide extract was less than 5 parts, the viscosity of the aqueous system was insufficient, and the dispersion stability of lovastatin was poor; when it was more than 15 parts, the viscosity of the system was too high, which was not conducive to subsequent food processing. When the lovastatin crystals were less than 0.5 parts, the lipid dispersed phase was insufficient; when they were more than 2 parts, oil rings were easily formed. When the sulfated Tremella fuciformis polysaccharide was less than 3 parts, the thickening and stabilizing effects were insufficient; when it was more than 8 parts, the system had an overly viscous texture. The effective range of the combined mass ratio was determined to be (5–15):(0.5–2):(3–8).

[0041] II. Implementation Section (a) Examples and comparative examples.

[0042] Example 1

[0043] S1. Pretreatment: Take dried fruiting bodies of Hericium erinaceus, Pleurotus ostreatus, and Tremella fuciformis, mix them in a mass ratio of 4:3:2, gently wash them in purified water at 10℃ for 15 minutes, drain them, and freeze-dry them at -50℃ and a vacuum of 3Pa for 24 hours. Then, pulverize them at low temperature to a particle size of 50μm. Place the pulverized mixed raw materials in a supercritical CO2 extraction device, control the extraction pressure at 30MPa, the extraction temperature at 40℃, and the CO2 flow rate at 20kg / h, and defatting them for 2 hours to obtain the pretreated mixed raw materials.

[0044] S2. Sequential enzymatic hydrolysis in zones: The pretreated mixed raw materials are sequentially hydrolyzed in three independent jacketed stirred reactors. The reactors are connected by a closed pipeline, which is equipped with a high-pressure rotary cleaning nozzle.

[0045] First enzymatic hydrolysis unit: The pretreated mixed raw materials were placed in a buffer solution with pH 9.0 and hydrolyzed at 70°C for 1 hour with stirring. Second enzymatic hydrolysis unit: The output from the first unit is filtered through a bottom screen, and the solid is washed with purified water and transferred to the second reactor. The pH is adjusted to 5.0, and a complex enzyme system consisting of cellulase, pectinase and neutral protease in a mass ratio of 3:1.5:1.5 is added. Enzymatic hydrolysis is carried out at 50°C for 2 hours, while pulsed ultrasound (frequency 25kHz, power density 200W / L, pulse mode on 3s / off 1s) is applied simultaneously. Third enzymatic hydrolysis unit: The output of the second unit is filtered through the bottom screen, and the solid is washed with purified water and transferred to the third reactor. The pH is adjusted to 7.0, and a complex enzyme system consisting of cellulase, hemicellulase and alkaline protease in a mass ratio of 4:1.5:2 is added. Enzymatic hydrolysis is carried out at 45℃ for 2.5 hours, and pulsed ultrasound is applied simultaneously (parameters are the same as in the second unit).

[0046] S3. Continuous Separation: The effluent from the third enzymatic hydrolysis unit was filtered through a plate and frame filter to remove solids. The filtrate was extracted three times at 40°C using ethyl acetate as the extractant, with an extractant-to-filtrate volume ratio of 1:5 each time. The organic phases were combined. The organic phase was concentrated under reduced pressure to 1 / 15 of its original volume at 45°C and loaded onto a silica gel column. Elution was performed using a petroleum ether-ethyl acetate gradient. The fraction containing lovastatin was collected, allowed to stand at 4°C for 24 hours to crystallize, filtered, and washed with cold ethanol to obtain lovastatin crystals.

[0047] Aqueous phase: The ethanol volume fraction was controlled at 45%, stirred at room temperature for 30 min, allowed to stand for 2 h to precipitate, and then centrifuged. After redissolving the precipitate, it was successively filtered through a 0.22 μm microfiltration membrane, concentrated through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and desalted and purified through a nanofiltration membrane with a molecular weight cutoff of 300 Da to obtain a high molecular weight Tremella fuciformis polysaccharide fraction. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the permeate was then concentrated through a nanofiltration membrane with a molecular weight cutoff of 300 Da to obtain a medium molecular weight Hericium erinaceus polysaccharide fraction.

[0048] The high molecular weight Tremella polysaccharide fraction was dissolved in anhydrous formamide, and chlorosulfonic acid-pyridine reagent (chlorosulfonic acid to pyridine volume ratio 1:4, polysaccharide to chlorosulfonic acid mass-volume ratio 1g:10mL) was slowly added dropwise under ice bath conditions. The reaction was carried out at 70℃ for 3h. After the reaction was completed, the solution was neutralized to pH 7.0 with saturated sodium bicarbonate solution, dialyzed, precipitated with alcohol, and freeze-dried to obtain sulfated Tremella polysaccharide.

[0049] Medium molecular weight Hericium erinaceus polysaccharide fraction was concentrated under vacuum and freeze-dried to obtain Hericium erinaceus polysaccharide extract.

[0050] S4. Compounding: Hericium erinaceus polysaccharide extract, lovastatin crystals and sulfated Tremella fuciformis polysaccharide are compounded in a mass ratio of 10:1:5 to obtain an active extract of edible fungi.

[0051] Example 2

[0052] This embodiment verifies the feasibility of process parameters under the lower limit boundary and raw material ratio conditions, and is applicable to production in low-temperature environments during winter and scenarios where high-fineness food ingredients need to be prepared subsequently.

[0053] S1. Pretreatment: Hericium erinaceus, oyster mushroom, and Tremella fuciformis were mixed in a mass ratio of 3:2:1. Due to the low proportion of Tremella fuciformis, the total amount of its protein-encapsulated structure was reduced. To verify the minimum effective strength of alkali extraction hydrolysis, the washing water temperature was controlled at 4℃, and the freeze-drying temperature was set to the equipment's minimum operating temperature of -45℃ (vacuum degree 5Pa). To meet the particle size requirements of the raw materials for subsequent gel candy applications, the material was ultra-finely pulverized to a particle size of 10μm. The supercritical CO2 defatting parameters were set to a pressure of 25MPa and a temperature of 35℃.

[0054] S2. Sequential enzymatic digestion by partition: First enzymatic hydrolysis unit: pH 8.5, 60℃, 0.5h; Second enzymatic hydrolysis unit: pH 4.5, 45℃, cellulase: pectinase: neutral protease = 2:1:1, enzymatic hydrolysis for 1.5h, sonication at 20kHz, 100W / L, on for 2s / off for 1s; Third enzymatic hydrolysis unit: pH 6.0, 40℃, cellulase: hemicellulase: alkaline protease = 3:1:1, enzymatic hydrolysis for 2 hours, sonication at 20kHz, 100W / L, on for 2 seconds and off for 1 second.

[0055] S3. Separation and purification: Extracted twice at 30℃ using 75% ethanol (v / v) as the extractant; crystallization temperature 10℃, time 12h; ethanol fractionation precipitation concentration 35%. Membrane filtration: successively using 0.45μm microfiltration membrane, 10kDa ultrafiltration membrane, and 500Da nanofiltration membrane. Sulfation reaction: 60℃, 2h.

[0056] S4. Compound: Hericium erinaceus polysaccharide extract: Lovastatin crystals: Sulfated Tremella fuciformis polysaccharide = 5:0.5:3.

[0057] Example 3

[0058] This embodiment verifies the feasibility of process parameters under the upper limit boundary and the upper limit of raw material ratio, and is applicable to production in high-temperature environments in summer and scenarios where the proportion of tremella in raw materials is high and extraction efficiency needs to be enhanced.

[0059] S1. Pretreatment: Lion's mane mushroom, oyster mushroom, and white fungus are mixed in a mass ratio of 5:4:3. Due to the high proportion of white fungus and its large total amount of gelatinous structure, enhanced pretreatment efficiency is required. Washing water temperature is 15℃, followed by freeze-drying at -55℃ (vacuum degree 2Pa). Because the subsequent enzymatic hydrolysis time is extended, ultrafine pulverization to a particle size of 100μm is sufficient to meet the cell wall breaking requirements. Supercritical CO2 defatting is performed at a pressure of 35MPa and a temperature of 45℃.

[0060] S2. Sequential enzymatic digestion by partition: First enzymatic hydrolysis unit: pH 10.5, 80℃, 1.5h; Second enzymatic hydrolysis unit: pH 6.0, 55℃, cellulase: pectinase: neutral protease = 4:2:2, enzymatic hydrolysis for 3 hours, sonication at 40kHz, 350W / L, on for 5 seconds and off for 2 seconds; Third enzymatic hydrolysis unit: pH 7.5, 50℃, cellulase: hemicellulase: alkaline protease = 5:2:3, enzymatic hydrolysis for 4 hours, sonication at 40kHz, 350W / L, 5s on / 2s off.

[0061] S3. Separation and purification: Extracted three times at 50°C using 95% ethanol (v / v) as the extractant; crystallization temperature 4°C, time 48 h; ethanol fractionation precipitation concentration 55%. Membrane filtration was performed sequentially using a 0.1 μm microfiltration membrane, a 50 kDa ultrafiltration membrane, and a 200 Da nanofiltration membrane. Sulfation esterification reaction was performed at 80°C for 4 h.

[0062] S4. Compound: Hericium erinaceus polysaccharide extract: Lovastatin crystals: Sulfated Tremella fuciformis polysaccharide = 15:2:8.

[0063] Example 3

[0064] The same process parameters as in Example 1 were used, with the difference that the three enzymatic hydrolysis units were independent 50L stainless steel stirred reactors connected by DN50 stainless steel sealed pipes, with a set of high-pressure rotary cleaning nozzles installed every 500mm inside the pipes. After the reaction in each unit was completed, the material underwent solid-liquid separation through a 200-mesh screen at the bottom of the reactor. The solid material was washed twice with purified water equivalent to three times its weight in solids and then transferred to the next unit via a screw conveyor. Before transferring material from the second unit to the third unit, the pipes were rinsed with phosphate buffer solution at pH 6.0. Testing showed that the residual pectinase activity in the effluent from the third enzymatic hydrolysis unit was 3.2% of the initial activity, and the residual neutral protease activity was 1.8% of the initial activity.

[0065] Example 4

[0066] Based on Example 4, the volume of the three enzymatic hydrolysis unit reactors was increased to 2000L, while the remaining structural design and operating parameters remained unchanged. Dried fruiting bodies of Hericium erinaceus, Pleurotus ostreatus, and Tremella fuciformis mixed in a mass ratio of 4:3:2 were used as raw materials, with 200kg of material fed per batch, and the process was repeated for 5 consecutive batches.

[0067] The retention rate of lovastatin and the yield of Hericium erinaceus polysaccharide in the third enzymatic hydrolysis unit were measured in each batch, and the relative standard deviation (RSD) between batches was calculated. The results showed that the RSD between batches of lovastatin retention was 2.8%, and the RSD between batches of Hericium erinaceus polysaccharide yield was 3.1%, demonstrating that this process has good reproducibility and process stability at the industrial pilot scale.

[0068] During continuous material transfer, after each batch of material was transferred between units, a high-pressure rotary cleaning nozzle inside the pipeline was activated to flush the pipeline with purified water at a volume three times that of the pipeline. The flushing solution was collected, and the protein residue in the flushing solution was detected using the BCA method. The results showed that no visible residue was found after wiping the inner wall of the pipeline after flushing, and the protein concentration in the flushing solution was reduced to less than 1.5% of the residue before flushing, verifying that the online cleaning system can effectively avoid cross-contamination between units.

[0069] Comparative Example 1 Extracts were extracted separately from Hericium erinaceus, Pleurotus ostreatus, and Tremella fuciformis using existing technologies. Hericium erinaceus: Hericium erinaceus polysaccharide was obtained by enzymatic hydrolysis at pH 5.0 weak acid (cellulase: pectinase: neutral protease = 3:1.5:1.5) at 50℃ for 2 hours with ultrasound assistance, followed by resin purification and vacuum drying. Oyster mushroom: enzymatic hydrolysis at near-neutral pH 7.0 (cellulase: hemicellulase: alkaline protease = 4:1.5:2) at 45℃ for 2.5 h, followed by ethyl acetate extraction, column chromatography, and crystallization to obtain lovastatin; Tremella: alkali extraction and hydrolysis at pH 9.0, 70℃, 1h, followed by membrane filtration and sulfation modification to obtain sulfated tremella polysaccharide.

[0070] The three individual extracts were mixed in the ratio of 10:1:5 as described in Example 1 to obtain the comparative complex.

[0071] Comparative Example 2 The order of the three enzymatic hydrolysis units in Example 1 was adjusted to: Unit 2 (weakly acidic) → Unit 1 (alkaline) → Unit 3 (near neutral), and the remaining process parameters were exactly the same as in Example 1.

[0072] Comparative Example 3 The first enzymatic hydrolysis unit in Example 1 is omitted. The pretreated mixed raw materials are directly fed into the second enzymatic hydrolysis unit for weak acid hydrolysis. The remaining process parameters are exactly the same as in Example 1.

[0073] Comparative Example 4 In step S3 of Example 1, the aqueous phase is not subjected to ethanol fractionation and precipitation, but is directly filtered and separated sequentially through a 0.22μm microfiltration membrane, a 30kDa ultrafiltration membrane, and a 300Da nanofiltration membrane. The remaining process parameters are exactly the same as in Example 1.

[0074] Comparative Example 5 The sequential enzymatic hydrolysis in Example 1 was replaced with enzymatic hydrolysis under uniform conditions: the pretreated mixed raw materials were placed in a buffer solution of pH 6.0, and a uniform complex enzyme system consisting of cellulase, pectinase, neutral protease, hemicellulase and alkaline protease was added. The mixture was enzymatically hydrolyzed at 50°C for 4 hours. The ultrasonic parameters were the same as in Example 1. No partitioning treatment was performed. The remaining steps were the same as in Example 1.

[0075] (II) Effect Detection and Data Analysis The products prepared in the above examples and comparative examples were tested as follows: yield and purity of Hericium erinaceus polysaccharide (phenol-sulfuric acid method), yield and retention of lovastatin (HPLC method, C18 column, detection wavelength 238 nm), dissolution rate and protein content of Tremella fuciformis polysaccharide (phenol-sulfuric acid method and Coomassie brilliant blue method), degree of sulfate substitution (ion chromatography), molecular weight distribution (GPC method), and infrared spectroscopy (KBr pellet method, 4000–4000 cm⁻¹). -1 Particle size distribution (laser particle size analyzer).

[0076] The results are shown in Table 1.

[0077] Table 1 Comparison of process effects between the examples and comparative examples

[0078] Note: Comparative Example 1 uses separate extraction processes. Hericium erinaceus polysaccharide is purified by resin and Tremella fuciformis polysaccharide is purified by single membrane filtration. The mixed raw material ethanol fractionation-assisted multi-stage membrane filtration separation system described in this invention was not used. Therefore, the two indicators "the proportion of Tremella fuciformis polysaccharide in the ultrafiltration residue" and "the proportion of Hericium erinaceus polysaccharide in the ultrafiltration permeate" could not be determined.

[0079] (III) Mechanism Verification Experiment (1) Verification of changes in cell wall microstructure The pretreated mixed raw materials, solid residues after treatment by the first enzymatic hydrolysis unit, and solid residues after treatment by the second enzymatic hydrolysis unit in Example 1 were respectively taken, fixed with 2.5% glutaraldehyde, dehydrated with gradient ethanol, critical point dried and sputter-coated with gold, and their surface morphology was observed under a scanning electron microscope (SEM, accelerating voltage 5kV, magnification 5000×).

[0080] The results showed that the pretreated *Tremella fuciformis* cell wall surface was smooth, with a continuous protein coating layer completely covering the polysaccharide gel surface. After treatment with the first enzymatic hydrolysis unit (alkaline conditions), the protein coating layer on the cell wall surface showed obvious fragmentation and peeling, exposing the underlying polysaccharide network structure, and the surface porosity increased significantly, confirming the effective loosening effect of alkaline conditions on the protein coating structure on the *Tremella fuciformis* polysaccharide surface. After treatment with the second enzymatic hydrolysis unit (weakly acidic complex enzyme system), the gel layer structure of the *Hericium erinaceus* cell wall was significantly degraded, and the cell wall surface showed a loose fibrous network morphology, confirming the directional degradation effect of pectinase and cellulase on the gel layer in the *Hericium erinaceus* cell wall.

[0081] (2) Tracking the composition of the extracts from each enzymatic hydrolysis unit The supernatants from the first, second, and third enzymatic hydrolysis units in Example 1 were collected respectively. The total sugar content was determined by the phenol-sulfuric acid method, the protein content was determined by the Coomassie brilliant blue method, and the monosaccharide composition was determined by HPLC (column: Agilent Zorbax Carbohydrate column, mobile phase: acetonitrile-water (75:25, v / v), detector: ELSD).

[0082] The results showed that in the supernatant of the first enzymatic hydrolysis unit, the molar proportion of mannose and glucuronic acid, characteristic monosaccharides of Tremella fuciformis, reached 78.3%, and the protein dissolution accounted for 62.5% of the total protein dissolution, indicating that this stage mainly released Tremella fuciformis polysaccharides and their bound proteins. In the supernatant of the second enzymatic hydrolysis unit, glucose accounted for 72.6% of the total sugars, which is consistent with the characteristic monosaccharide composition of Hericium erinaceus β-glucan. In the supernatant of the third enzymatic hydrolysis unit, HPLC detected that the release of lovastatin accounted for 92.4% of the total release, proving that the mild conditions effectively avoided the degradation of heat-sensitive lovastatin and achieved its main release.

[0083] (3) Verification of residual enzyme activity inhibition between enzymatic digestion units In the continuous processing apparatus of Example 4, after the reaction of the second enzymatic hydrolysis unit is completed and before the solid material is washed and transferred to the third enzymatic hydrolysis unit, the residual activity of pectinase in the washed solid material is detected (using the DNS colorimetric method, with polygalacturonic acid as the substrate, to determine the amount of reducing sugar generated). At the same time, the residual activity of pectinase in the effluent after the reaction of the third enzymatic hydrolysis unit is detected.

[0084] The results showed that the residual pectinase activity in the solid material after washing was only 2.1% of the initial addition amount; no detectable level of pectinase activity was detected in the effluent of the third enzymatic hydrolysis unit (detection limit 0.01 U / mL), proving that the solid-liquid separation and washing operation effectively eliminated the inhibitory interference of the acidic enzyme system in the previous unit on the enzymatic hydrolysis under the near-neutral conditions of the third unit.

[0085] III. Application Section The following application examples all use the edible fungus active extract or Hericium erinaceus polysaccharide extract prepared in Example 1 as raw materials to investigate their processing adaptability and performance in different food forms.

[0086] Application Example 1 (Liquid Food) The edible fungus active extract prepared in Example 1 was dissolved in a citric acid-sodium citrate buffer solution at pH 4.0 at a dosage of 5 wt%, supplemented with 0.1 wt% xanthan gum, stirred and dissolved, and then sterilized at 121°C for 15 min. The resulting product was then bottled to obtain a clear liquid food.

[0087] Centrifugation stability: No stratification or precipitation was observed at 3000 rpm for 30 min; Static stability: After standing at 25℃ for 30 days, the sample remained clear and transparent, with a lovastatin retention rate (HPLC detection) of 96.2%. Particle size distribution: The mass median particle size (D50) of lovastatin crystals, as determined by laser particle size analyzer, was 5 μm.

[0088] The method for determining the median particle size D50 of the lovastatin crystals is as follows: 5 wt% of the active extract of edible fungi is dispersed in a citrate-sodium citrate buffer solution at pH 4.0, supplemented with 0.1 wt% xanthan gum. After stirring and dissolving, the mixture is sterilized at 121℃ for 15 min and measured using a laser particle size analyzer. The median particle size D50 is calculated based on the mass distribution.

[0089] A control liquid sample was prepared using the control complex prepared in Comparative Example 1 under the same conditions (5 wt% addition, same buffer system, same sterilization conditions). After centrifugation at 3000 rpm for 30 min, a small amount of flocculent precipitate appeared in the control sample. After standing at 25℃ for 15 days, a clear precipitate layer appeared at the bottom, the lovastatin retention rate decreased to 82.5%, and the lovastatin D50 was 28 μm with uneven distribution.

[0090] (1) Thickening synergy and micro-dispersion verification The following three groups of samples were prepared into aqueous solutions of the same concentration (2wt%): Group A was the Hericium erinaceus polysaccharide extract prepared in Example 1; Group B was the sulfated Tremella fuciformis polysaccharide prepared in Example 1; and Group C was a mixture of the two in a mass ratio of 10:5 (consistent with the Hericium erinaceus polysaccharide:sulfated Tremella fuciformis polysaccharide ratio of 10:5 in Example 1). A rotational rheometer (TA Instruments DHR-2, conical rotor, 40 mm diameter, 2° cone angle, 25°C, shear rate scan range 0.1–100 s) was used. -1 The apparent viscosity of each group of samples was measured.

[0091] The results showed that at a shear rate of 10 s⁻¹ -1 At that time, the apparent viscosity of group A was 125 mPa·s, the apparent viscosity of group B was 98 mPa·s, while the apparent viscosity of the compound of group C reached 285 mPa·s, which were 2.28 times and 2.91 times that of groups A and B when they existed alone, respectively. This is far greater than the arithmetic sum of their viscosities (223 mPa·s), proving that Hericium erinaceus polysaccharide and sulfated Tremella fuciformis polysaccharide have a significant thickening synergistic effect in the aqueous phase.

[0092] Another liquid food sample from Application Example 1, sterilized at 121℃, was stained with phosphotungstic acid and its microstructure was observed under a transmission electron microscope (TEM, accelerating voltage 80kV, magnification 10000×). The results showed that Hericium erinaceus polysaccharide and sulfated Tremella fuciformis polysaccharide intertwined in the aqueous phase to form a continuous three-dimensional network structure with nanopores (approximately 50–200 nm). Lovastatin crystals (exhibiting electronically dense, irregular particles) were uniformly dispersed within the pores of this network structure. An adsorbed coating layer of polysaccharide chains was visible on the crystal surface, and no aggregation or fusion of free crystals was observed. Particle size analysis of 100 randomly selected crystal particles from the TEM images showed that approximately 96% of the crystal particles were distributed within the range of 1–20 μm, consistent with the D50 of 5 μm measured by a laser particle size analyzer.

[0093] (2) Comparison of long-term storage stability The following three control samples were prepared and dispersed in citrate-sodium citrate buffer at pH 4.0, with the final lovastatin concentration controlled at 0.1 mg / mL: Group I: Application of the active edible fungus extract of the present invention in Example 1 (containing Hericium erinaceus polysaccharide + sulfated Tremella fuciformis polysaccharide + lovastatin crystals); Group II: Blank buffer containing no polysaccharides, with an equal volume of lovastatin crystals (unembedded) added directly; Group III: Buffer solution containing only Hericium erinaceus polysaccharide extract (concentration same as Group I), with an equal amount of lovastatin crystals added.

[0094] The three groups of samples were sealed and placed in a 40℃ constant temperature incubator for accelerated preservation for 6 months. Samples were taken at 0, 1, 3 and 6 months, and the retention rate of lovastatin was determined by HPLC (C18 column, detection wavelength 238nm).

[0095] The results showed that after 6 months, the retention rate of lovastatin in group I was 94.2%; the retention rate in group II (blank buffer control) was only 52.1%; and the retention rate in group III (containing only Hericium erinaceus polysaccharide) was 73.5%. These results indicate that the three-dimensional network structure formed by Hericium erinaceus polysaccharide and sulfated Tremella fuciformis polysaccharide provides effective physical encapsulation and spatial confinement protection for lipid-soluble lovastatin crystals, significantly inhibiting crystal aggregation, sedimentation, and chemical degradation. Its protective effect is far superior to that of a single polysaccharide system or a system without adjuvants.

[0096] Application Example 2 (Gel-like Food) Take the edible fungus active extract prepared in Example 1, add it at 8 wt% and mix it with gelatin (4 wt%), pectin (1 wt%) and water, heat it to 60°C to dissolve it evenly, pour it into a mold, and cool it to form a gel-like food.

[0097] Meanwhile, a blank gel control was prepared: gelatin (4wt%), pectin (1wt%) and water were mixed in the same proportion and molded without adding any edible fungi active extracts.

[0098] Texture testing (texture analyzer, P / 0.5-inch cylindrical probe, compression speed 1mm / s, compression distance 10mm): Gel containing compound extracts: hardness 285g, elasticity 0.92, chewiness 245g·mm; Blank gel: hardness 305g, elasticity 0.83, chewiness 258g·mm.

[0099] Water retention test: After being placed at 4℃ for 7 days, the dehydration shrinkage rate of the gel containing the compound extract was 2.1%, while the dehydration shrinkage rate of the blank gel was 5.8%.

[0100] Application Example 3 (Probiotic Preparation) The Hericium erinaceus polysaccharide extract prepared in Example 1 was dissolved in purified water at a concentration of 15% w / v, and Bifidobacterium lyophilized powder (live count 1×10⁻⁶) was added. 11 (CFU / g), stirred to uniformly coat the bacterial cell surface with polysaccharides, and then freeze-dried under vacuum to produce a polysaccharide-probiotic complex powder. Scanning electron microscopy revealed a continuous polysaccharide coating layer on the bacterial cell surface.

[0101] Meanwhile, a blank control was prepared: a maltodextrin solution of the same concentration was used to replace the Hericium erinaceus polysaccharide extract, and the mixture was mixed with an equal amount of Bifidobacterium lyophilized powder and then lyophilized under the same conditions.

[0102] Simulated gastric acid environment test (hydrochloric acid-pepsin solution at pH 2.5, 37℃, 2h): Polysaccharide-probiotic compound powder: probiotic survival rate is 68.5%; Maltodextrin-Probiotics Control: Probiotic survival rate was 28.4%.

[0103] Simulated intestinal resuscitation test (samples treated with gastric acid were washed with PBS buffer and then placed in phosphate buffer at pH 6.8 at 37°C for 4 hours): Polysaccharide-probiotic compound powder: Live bacteria count recovered to 89.2% of the initial value; Maltodextrin-probiotic control: The number of live bacteria recovered to 65.3% of the initial value.

[0104] The above application examples show that the edible fungi active extract prepared by the present invention exhibits good processing adaptability in liquid, gel and powder food systems, and has technical advantages in terms of dispersion stability, texture retention and probiotic protection compared with the sample prepared in Comparative Example 1 and the blank control.

Claims

1. A method for preparing an active extract of edible fungi, characterized in that, A mixture of various edible fungi fruiting bodies is placed in the same continuous process flow, and the corresponding active components are obtained through zoned sequential enzymatic hydrolysis and continuous separation and directional extraction. The process includes the following steps: S1. Mix various edible fungi fruiting bodies, and then wash, freeze-dry, pulverize and defatted them at low temperature to obtain a pretreated mixed raw material; S2. The pretreated mixed raw materials are sequentially enzymatically hydrolyzed in the first, second and third enzymatic hydrolysis units. The materials are continuously transported between units and pulsed ultrasound is applied synchronously during the enzymatic hydrolysis process. The pH gradient and enzyme system differences between the units are used to perform directional sequential degradation of the cell wall matrix of different bacterial species in the mixed raw materials. S3. After filtering the enzymatic hydrolysate obtained from step S2 through sequential enzymatic hydrolysis, the organic phase and aqueous phase are separated by extraction with an organic solvent. The organic phase is concentrated, subjected to column chromatography and low-temperature crystallization to obtain lipid-soluble active crystals. The aqueous phase is separated according to molecular weight differences by ethanol fractionation and multi-stage membrane filtration to obtain high molecular weight polysaccharide fraction and medium molecular weight polysaccharide fraction. The high molecular weight polysaccharide fraction is modified by directional sulfation to obtain sulfated polysaccharide, and the medium molecular weight polysaccharide fraction is concentrated and dried to obtain polysaccharide extract. S4. Combine the active components obtained in step S3 according to product requirements to obtain edible fungi active extract.

2. The method for preparing an active extract of edible fungi according to claim 1, characterized in that, The various edible fungi fruiting bodies include Hericium erinaceus, Pleurotus ostreatus, and Tremella fuciformis.

3. The method for preparing an active extract of edible fungi according to claim 2, characterized in that, The specific operation of the partitioned sequential enzymatic hydrolysis in step S2 is as follows: the first enzymatic hydrolysis unit controls alkaline conditions for alkaline extraction and hydrolysis, selectively loosening the protein-encapsulated structure on the surface of Tremella polysaccharides; the second enzymatic hydrolysis unit adjusts to weakly acidic conditions and adds a complex enzyme system composed of cellulase, pectinase and neutral protease to directionally degrade the gel layer in the cell wall of Hericium erinaceus and the residual protein connection structure of Tremella erinaceus; the third enzymatic hydrolysis unit adjusts to near-neutral conditions and adds a complex enzyme system composed of cellulase, hemicellulase and alkaline protease to gently release the heat-sensitive lovastatin and water-soluble polysaccharides in Pleurotus ostreatus; the frequency of the pulsed ultrasound in step S2 is 20–40 kHz, the power density is 100–350 W / L, and the pulse mode is on for 2–5 s / off for 1–2 s.

4. The method for preparing an active extract of edible fungi according to claim 3, characterized in that, The first enzymatic hydrolysis unit has a pH of 8.5–10.5, a temperature of 60–80℃, and a time of 0.5–1.5 h; the second enzymatic hydrolysis unit has a pH of 4.5–6.0, a temperature of 45–55℃, and a time of 1.5–3 h; and the third enzymatic hydrolysis unit has a pH of 6.0–7.5, a temperature of 40–50℃, and a time of 2–4 h.

5. The method for preparing an active extract of edible fungi according to claim 1, characterized in that, The organic solvent in step S3 is ethyl acetate or ethanol with a volume fraction of 75–95%, the extraction temperature is 30–50°C, and the number of extractions is 2–3; the low-temperature crystallization temperature is 4–10°C, and the crystallization time is 12–48 h.

6. The method for preparing an active extract of edible fungi according to claim 1, characterized in that, The ethanol fractionation aid in step S3 is as follows: the volume fraction of ethanol is controlled at 35–55% to selectively precipitate high molecular weight polysaccharides. The supernatant is separated into medium molecular weight polysaccharide fractions by multi-stage membrane filtration. After redissolving the precipitate, it is purified by membrane filtration to obtain high molecular weight polysaccharide fractions. The multi-stage membrane filtration is a three-stage series membrane filtration system, consisting of a microfiltration membrane with a pore size of 0.1–0.45 μm, an ultrafiltration membrane with a molecular weight cutoff of 10–50 kDa, and a nanofiltration membrane with a molecular weight cutoff of 200–500 Da. The directional sulfation modification is carried out using the chlorosulfonic acid-pyridine method, with a reaction temperature of 60–80 °C and a reaction time of 2–4 h.

7. The method for preparing an active extract of edible fungi according to claim 2, characterized in that, The mass ratio of the mixed hericium erinaceus, oyster mushroom and tremella in step S1 is (3–5):(2–4):(1–3); the mass ratio of the compound in step S4 is hericium erinaceus polysaccharide extract: lovastatin crystal: sulfated tremella polysaccharide = (5–15):(0.5–2):(3–8).

8. The method for preparing an active extract of edible fungi according to claim 1, characterized in that, The freeze-drying conditions in step S1 are -45℃ to -55℃ and vacuum degree ≤5Pa; the defatting is supercritical CO2 defatting, with an extraction pressure of 25–35MPa and an extraction temperature of 35–45℃.

9. The method for preparing an active extract of edible fungi according to claim 1, characterized in that, In step S2, each enzymatic hydrolysis unit is an independent reactor, and the units are connected by a closed pipeline. The pipeline is equipped with an online cleaning nozzle to rinse the pipeline with purified water or pH transition buffer. After the reaction of each unit is completed, solid-liquid separation is performed first, and the solid material is washed before entering the next enzymatic hydrolysis unit to avoid the inhibition of enzyme activity in the next unit by the liquid enzyme system and acid / alkaline environment of the previous unit.

10. The application of an edible fungus active extract prepared according to any one of claims 1 to 9 as a food ingredient, characterized in that, The polysaccharide extract and sulfated polysaccharide synergistically constitute an aqueous thickening system. The fat-soluble active crystals are dispersed in this aqueous thickening system. The amount of the edible fungus active extract added to the food is 0.5–10 wt%, and the median particle size D50 of the fat-soluble active crystals in the aqueous thickening system is 1–20 μm. 50 The result was obtained by dispersing the active extract of the edible fungi in an aqueous food matrix at an addition rate of 5 wt%.

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