Enzymatic extraction process of lentinus edodes and application thereof

Abstract

This invention relates to the field of edible fungi deep processing technology, and provides a process for enzymatic extraction of shiitake mushrooms and its application. The process includes the following steps: 1) Cell wall disruption enzymatic hydrolysis: using cellulase to break down the cell wall and release the contents; 2) Proteolytic hydrolysis: using alkaline and neutral proteases to hydrolyze proteins into polypeptides, simultaneously releasing flavonoids bound to or embedded in the proteins; 3) Directed degradation of polysaccharides: after adjusting the pH, using β-1,3-glucan endonuclease to controllably degrade the molecular weight of large shiitake polysaccharides to a weight-average molecular weight of 5 × 10⁻⁶. 3 -5×10 4 Da's alcohol-soluble window; 4) Alcohol-soluble full component collection: After enzyme inactivation, ethanol is added to dissolve the degraded polysaccharide fragments, peptides and flavonoids in the alcohol-water system; This invention realizes the simultaneous enrichment of soluble polysaccharide degradation products, peptides and flavonoids in a single product for full component extraction. The resulting extract has excellent alcohol solubility and high bioavailability, and has broad application prospects in the field of health food.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi deep processing technology, and particularly relates to an enzymatic extraction process for shiitake mushrooms and its application. Background Technology

[0002] Shiitake mushrooms (Lentinula edodes) belong to the order Agaricales, family Ganodermacetes, and genus Lentinus. They are a large fungus used in both food and medicine. They are rich in polysaccharides (mainly β-glucan), eritadenine, dietary fiber, nucleotides, and various essential amino acids. Modern pharmacological studies have shown that shiitake mushroom extracts possess biological activities such as regulating immunity, lowering blood lipids, anti-tumor activity, and antioxidant activity. Therefore, the deep processing of shiitake mushrooms and the extraction technology of their active ingredients have always been a research hotspot in the fields of food science, medicine, and biotechnology.

[0003] Currently, the extraction technologies for active ingredients in shiitake mushrooms mainly include the following: Water extraction and alcohol precipitation: This is the most traditional and widely used method for extracting polysaccharides from shiitake mushrooms. Its basic principle is to utilize the characteristic that polysaccharides are easily soluble in hot water but poorly soluble in organic solvents. After hot water extraction, ethanol is added for precipitation and separation. This technology is relatively simple to operate and has a low cost, but it usually suffers from problems such as high extraction temperature, long time consumption, and high energy consumption. Prolonged high-temperature treatment can easily lead to the degradation of polysaccharide molecular chains, resulting in low polysaccharide yield and a significant decrease in biological activity. At the same time, this process mainly targets polysaccharide components, and the extraction rate of other components in shiitake mushrooms, including proteins and small molecules with lipid-lowering activity (such as lentinan), is extremely low, resulting in a waste of raw material resources.

[0004] Enzyme-assisted extraction method: In the prior art, such as the patent CN101709320B which discloses a method for preparing shiitake mushroom polypeptide powder, cellulase is used to break the cell wall, followed by hydrolysis with compound protease and flavor protease. This type of method solves the problems of cell wall breaking and protein hydrolysis, but its process endpoint is still to collect all water-soluble components (total hydrolysate). This process does not solve the problem of product application limitations caused by the coexistence of macromolecular polysaccharides with other small molecule active ingredients. For example, macromolecular polysaccharides cause high viscosity of the product aqueous solution, making filtration difficult, and poor stability in some applications (such as high-alcohol system health products).

[0005] Physically Assisted Extraction: In recent years, physical enhancement technologies such as ultrasonic-assisted extraction, microwave-assisted extraction, and high-pressure homogenization have been applied to the extraction process of shiitake mushrooms. These technologies accelerate the mass transfer process through mechanical, cavitation, or thermal effects, significantly shortening the extraction time. However, existing physically assisted technologies still have limitations in practical applications. For example, although ultrasonic treatment can break cells, localized instantaneous high temperatures and strong shear forces may damage the activity of heat-sensitive components. Microwave extraction is prone to localized overheating, resulting in uneven temperature distribution in the extraction system and affecting the stability of product quality.

[0006] Therefore, existing technologies lack a process that can systematically solve the following key technical contradiction: how to simultaneously and efficiently retain lentinan (or its active form), polypeptides, and flavonoids in a single process flow, and obtain a complete product with uniform composition and excellent application performance (such as good alcohol solubility). This is precisely the technical problem that this invention aims to solve. Summary of the Invention

[0007] This invention provides a process and application for enzymatic extraction of shiitake mushrooms, aiming to solve the above-mentioned problems.

[0008] This invention transforms the traditional approach of separating based on differences in physical solubility into an approach of enzymatically tailoring molecular weight to alter solubility. By precisely controlling the enzymatic hydrolysis process, large polysaccharides that are originally poorly soluble in alcohol are directionally degraded into specific molecular weight fragments with excellent alcohol solubility, allowing them to combine with peptides and flavonoids in the same alcohol-water system, thereby achieving true synergistic retention of all components.

[0009] This invention is achieved through an enzymatic extraction process for shiitake mushrooms, comprising the following steps: Step 1: Cell wall breaking Crush the shiitake mushroom raw material to 60-80 mesh or make it into a paste, and add water at a material-to-liquid ratio of 1:6-12 (w / v, i.e., weight in grams: volume in milliliters). Under constant temperature and stirring at 30-65℃, add cellulase at 0.5-2 wt% based on the dry weight of the raw material and react for 0.5-3 hours. The purpose of this step is to use cellulase to break down the tough cell wall skeleton of shiitake mushrooms, so as to promote the efficient dissolution of large molecules such as polysaccharides and proteins from the cells.

[0010] Step 2: Proteolysis and flavonoid release Add an alkaline solution (such as food-grade sodium hydroxide solution) to the system in step 1) to adjust the pH to 7.0-9.0; add 0.5-3 wt% alkaline protease and 0.5-2 wt% neutral protease based on the dry weight of the raw materials, and stir at a constant temperature of 40-60℃ for 1-3 hours; the alkaline protease provides efficient endonuclease activity, rapidly hydrolyzing large protein molecules into polypeptide fragments; the neutral protease has strong exonuclease activity, which can further trim the polypeptides into smaller peptides and free amino acids; more importantly, this enzymatic hydrolysis can destroy flavonoid complexes that are bound to or embedded in proteins, efficiently releasing flavonoid compounds.

[0011] After the reaction is complete, the system is heated to 85-90℃ and held for 10-20 minutes to irreversibly inactivate alkaline and neutral proteases, and then cooled to room temperature. This enzyme inactivation process ensures that the directional degradation of polysaccharides will not be interfered with by residual protease activity in subsequent steps.

[0012] Step 3: Targeted Degradation of Polysaccharides Step 2) After cooling, add acid (such as food-grade hydrochloric acid solution) to adjust the pH to 4.5-6.0. Add 0.5-2 wt% β-1,3-glucanase based on the dry weight of the raw materials, and react at 45-55℃ for 1-3 hours. By precisely controlling the amount of β-1,3-glucanase, reaction time, pH, and temperature, the molecular weight of lentinan is "tailored" to a specific alcohol-soluble window, i.e., a weight-average molecular weight of 5 × 10⁻⁶. 3 -5×10 4 Da; if the molecular weight is higher than this range, the polysaccharide will precipitate in the subsequent alcohol solution; if it is lower than this range, it will be over-degraded into oligosaccharides or monosaccharides, losing the original advanced biological activity of the polysaccharide; only within this range can the polysaccharide degradation products simultaneously possess excellent alcohol solubility and high immune activity.

[0013] Step 4: Collection of alcohol-soluble components After step 3), inactivate the enzyme in the enzymatic hydrolysate (e.g., heat to 85-90℃ and hold for 10-30 minutes), then add ethanol to completely dissolve or highly disperse the degraded polysaccharides, peptides, and flavonoids in the alcohol-water system. Finally, perform solid-liquid separation and collect the clear liquid phase.

[0014] In a preferred embodiment, after enzyme inactivation, ethanol can be directly added to the enzyme hydrolysate until the system volume fraction is 60%-85%, stirred thoroughly, and then the solid and liquid phases are separated and the liquid phase is collected.

[0015] In a preferred embodiment, in order to reduce the total amount of ethanol used and reduce the interference of enzymatic hydrolysis residue on subsequent operations, the enzyme-inactivated hydrolysate can be separated into solid and liquid phases before adding ethanol to remove undigested residues and collect the supernatant; then ethanol is added to the supernatant to a final concentration of 60%-85%, and the liquid phase is collected after solid-liquid separation.

[0016] In another, more preferred embodiment, to further improve the purity of the target active ingredient, the present invention also provides a stepwise gradient alcohol dissolution process, specifically: First-stage alcohol dissolution (low-alcohol dissociation): Add ethanol to the clarified liquid to be treated, adjust the ethanol volume fraction to 30%-40%, stir and then separate the solid and liquid. At this concentration, some precipitate will preferentially precipitate in the solution, which can be removed by filtration or centrifugation, and the first-stage clarified liquid can be collected. This step is beneficial to improve the purity and clarity of subsequent products.

[0017] Second-stage alcohol dissolution (high-ethanol collection): Add ethanol to the first-stage supernatant until the final concentration is 60%-85%, stir thoroughly to completely dissolve the target polysaccharide degradation products, peptides and flavonoids in the system, and then perform solid-liquid separation again to collect the second-stage supernatant, which is the final liquid phase.

[0018] Step 5: Drying The alcohol-water solution collected in step 4) is subjected to vacuum distillation to recover ethanol; the remaining concentrate is made into powder by spray drying or freeze drying to obtain the enzymatic hydrolysis extract of all components of shiitake mushroom.

[0019] The product obtained by the above method of the present invention is a complete enzymatic hydrolysis extract of shiitake mushrooms; the product is also rich in: a weight-average molecular weight of 5 × 10⁻⁶. 3 -5×10 4 The extract contains Da's lentinan degradation products, active polypeptides with a molecular weight of less than 3000 Da (preferably accounting for more than 60% of the total polypeptides), and flavonoids; by total weight, the extract contains ≥30% lentinan degradation products, ≥15% polypeptides, and ≥1.5% flavonoids.

[0020] Compared with the prior art, the embodiments of this application have the following main advantages: Abandoning the traditional process of alcohol precipitation of polysaccharides and discarding the supernatant, this invention achieves for the first time the efficient coexistence of polysaccharides (active fragments), peptides, and flavonoids in the same product, significantly improving the comprehensive utilization rate of raw materials.

[0021] By selectively tailoring the extract to molecular weight, the industry pain points of poor solubility and high solution viscosity of macromolecular polysaccharides have been addressed. The resulting extract can be completely dissolved in ethanol of 60% or higher concentrations, producing a clear and transparent solution, which greatly expands its application scope in the development of dosage forms for health products (such as oral liquids and functional beverages).

[0022] The entire process is carried out at a relatively mild temperature. In particular, the use of a stepwise gradient alcohol dissolution process avoids the excessive damage to heat-sensitive components such as flavonoids caused by traditional high-temperature enzyme inactivation, thus preserving the natural bioactivity of each component to the greatest extent. Attached Figure Description

[0023] Figure 1 This is a flowchart of an enzymatic extraction process for shiitake mushrooms provided by the present invention. Detailed Implementation

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0025] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0026] The enzyme preparations used in the following examples are all commercially available enzymes: cellulase (endoglucanase, 10,000 U / g), β-1,3-glucanase (5,000 U / g), alkaline protease (200,000 U / g), and neutral protease (100,000 U / g); food-grade anhydrous ethanol, commercially available.

[0027] In the following examples, the weight-average molecular weight of polysaccharides was determined by high-performance gel permeation chromatography (HPGPC); the molecular weight distribution of peptides was determined by high-performance liquid chromatography (HPLC); the polysaccharide content was determined by the phenol-sulfuric acid method; the peptide content was determined by the Kjeldahl method; and the flavonoid content was determined by spectrophotometry.

[0028] Example 1: Preparation method of full-component enzymatic hydrolysis extract from dried shiitake mushrooms 10 kg of dried shiitake mushroom fruiting bodies (moisture content approximately 10%) were pulverized to 60-80 mesh, added to 60 liters of water, stirred, and heated to 50°C. Under constant temperature and stirring, 100 g of cellulase was added, and the reaction was allowed to proceed for 2 hours. Then, the pH was adjusted to 8.0 with food-grade NaOH solution, and 150 g of alkaline protease and 50 g of neutral protease were added. The reaction was allowed to proceed at 55°C for 2 hours. After the reaction, the system was heated to 90°C and held for 10 minutes to inactivate the enzymes, then cooled to room temperature. The pH was adjusted to 5.5 with food-grade HCl solution, and then 100 g of β-1,3-glucan endonuclease was added. The reaction was allowed to proceed precisely at 50°C for 2 hours to control the polysaccharide molecular weight within the target range.

[0029] After enzymatic hydrolysis, the system was heated to 90°C and kept at that temperature for 30 minutes to inactivate the enzyme. After cooling to room temperature, the system was filtered using a plate and frame filter, and the clear filtrate was collected. Food-grade anhydrous ethanol was slowly added to the filtrate while stirring continuously until the ethanol volume fraction in the system reached 75%. The solution was allowed to stand for 1 hour. The solution was then centrifuged or filtered again to collect the clear alcohol-water solution. The ethanol was first recovered under reduced pressure at 60°C and a vacuum of -0.09 MPa. The concentrated solution was then freeze-dried to obtain 5.3 kg of light brown powder, with a yield of 53.0%.

[0030] Upon testing, the product obtained in this embodiment contained: polysaccharide degradation products (weight average molecular weight 5 × 10⁻⁶). 3 -5×10 4 The content of Da is 40.5%; the content of polypeptides (<3000 Da portion accounts for 90% of the total peptides) is 25.2%; the content of flavonoids is 2.8%; the product's solubility in 70% ethanol (1% concentration) is: completely dissolved, forming a clear and transparent liquid.

[0031] Example 2: Preparation method using stepwise gradient alcohol dissolution 10 kg of dried shiitake mushroom fruiting bodies were pulverized to 60-80 mesh, added to 60 liters of water, stirred, and heated to 50°C. Under constant temperature and stirring, 100 g of cellulase was added, and the reaction was allowed to proceed for 2 hours. Then, the pH was adjusted to 8.0 with food-grade NaOH solution, and 150 g of alkaline protease and 50 g of neutral protease were added, reacting at 55°C for 2 hours. After the reaction, the system was heated to 90°C and held for 10 minutes to inactivate the enzymes, then cooled to room temperature. The pH was adjusted to 5.5 with food-grade HCl solution, and 100 g of β-1,3-glucanase was added, reacting precisely at 50°C for 2 hours. After enzymatic hydrolysis, the system was heated to 90°C and held for 30 minutes to inactivate the enzymes. After cooling to room temperature, the system was filtered through a plate and frame filter to remove the enzymatic hydrolysis residue, and the clear filtrate was collected, totaling approximately 65 liters.

[0032] (Stepwise gradient alcohol dissolution step – first stage alcohol dissolution) While stirring, slowly add food-grade anhydrous ethanol to the above filtrate to adjust the ethanol volume fraction in the system to 35%; a small amount of flocculent precipitate appears in the system. Continue stirring for 30 minutes, let stand for 1 hour, filter by plate and frame filter, collect the first stage supernatant, and discard the filter cake.

[0033] (Stepwise gradient alcohol dissolution step - second stage alcohol dissolution) While stirring continuously, continue to slowly add food-grade anhydrous ethanol to the first stage clear liquid until the ethanol volume fraction in the system reaches 75%; stir thoroughly for 1 hour and let stand for 30 minutes; at this time, the solution is a completely dissolved system of the target polysaccharide degradation products, peptides and flavonoids; filter by plate and frame filter and collect the clear second stage alcohol-water solution.

[0034] The solution was first subjected to reduced pressure at 60℃ and a vacuum of -0.09 MPa to recover ethanol. The concentrated solution was then freeze-dried to obtain 4.8 kg of light brown powder, with a yield of 48.0%.

[0035] Upon testing, the product obtained in this embodiment contained: polysaccharide degradation products (weight average molecular weight 5 × 10⁻⁶). 3 -5×10 4 The content of Da is 45.3%; the proportion of target molecular weight polysaccharide fragments in the total polysaccharide reaches 92.5%; the content of polypeptides (<3000 Da portion accounts for 88% of the total peptides) is 24.8%; the content of flavonoids is 3.0%; the product's solubility in 70% ethanol (1% concentration) is: completely dissolved, forming a clear and transparent liquid.

[0036] Comparative analysis: Compared with the process of adding alcohol to 75% in one step in Example 1, this example removes some impurities with 35% ethanol first, and then adds alcohol to 75% to collect the target component. The target molecular weight polysaccharide fragment accounts for 92.5% of the total polysaccharide in the product, and the clarity of the solution is visibly improved. This shows that the stepwise gradient alcohol dissolution process can effectively separate non-target components and significantly improve the purity and quality of the product.

[0037] Example 3: Preparation method of full-component enzymatic hydrolysis extract from shiitake mushroom stems 10 kg of dried shiitake mushroom stems (moisture content approximately 8%) were pulverized to 60-80 mesh, following the same steps as in Example 1. The powder was then spray-dried (inlet air temperature 190°C, outlet air temperature 90°C) to obtain 4.2 kg of yellowish-brown powder, with a yield of 42.0%.

[0038] Testing revealed that the product obtained in this embodiment contained 52.1% polysaccharide degradation products, 15.5% polypeptides, and 1.9% flavonoids. The product exhibited good solubility in 70% ethanol.

[0039] Example 4: Preparation method of full-component enzymatic hydrolysis extract from fresh shiitake mushrooms 30 kg of fresh shiitake mushrooms (moisture content approximately 85%, dry matter approximately 4.5 kg) were washed, drained, and directly pulped. After determining the solid content, water was added until the total water volume in the system was equivalent to 10 times the dry matter volume (approximately 45 liters). All subsequent enzymatic hydrolysis and post-processing steps were the same as in Example 1. Finally, after freeze-drying, 2.8 kg of light brown powder was obtained, with a yield of 51.8% on a dry weight basis.

[0040] Testing revealed that the product obtained in this embodiment contained 31.0% polysaccharide degradation products, 38.5% polypeptides, and 2.5% flavonoids. The product exhibited excellent solubility in 70% ethanol.

[0041] Comparative Example 1 Ten kilograms of dried shiitake mushroom fruiting bodies (moisture content approximately 10%) were pulverized to 60-80 mesh. Following the same steps as in Example 1, steps 1 (cell wall disruption), 2 (enzymatic hydrolysis), and 3 (polysaccharide-directed degradation) were performed. After enzymatic hydrolysis, the system was heated to 90°C and held at that temperature for 30 minutes to inactivate the enzymes. After cooling to room temperature, without plate and frame filtration, food-grade anhydrous ethanol was slowly added directly to the system containing the enzymatic hydrolysis residue, while continuously stirring until the ethanol volume fraction reached 75%. The mixture was then allowed to stand for 1 hour. Solid-liquid separation was then performed, and the clear alcohol-water solution was collected. This solution was subjected to reduced pressure at 60°C and a vacuum of -0.09 MPa to recover the ethanol. The concentrated solution was then freeze-dried to obtain 5.8 kilograms of brown powder, with a yield of 58.0%.

[0042] Upon testing, the products obtained in this comparative example contained: polysaccharide degradation products (weight average molecular weight 5 × 10⁻⁶). 3 -5×10 4 The content of Da is 32.1%; the proportion of target molecular weight polysaccharide fragments in the total polysaccharide is 78.3%; the content of polypeptide is 21.5%; the content of flavonoid is 2.1%; the product's solubility in 70% ethanol (1% concentration) is basically dissolved, but the solution is slightly turbid with a small amount of suspended particles.

[0043] Comparative analysis: Compared with Example 1, although the yield of this comparative example was slightly higher (58.0% vs 53.0%), the proportion of the target polysaccharide fragment in the total polysaccharide (78.3% vs over 85%), the clarity of the solution, and the content of active ingredients were all significantly inferior. This indicates that pre-separation to remove enzymatic hydrolysis residues before adding alcohol can effectively reduce the adsorption and entrainment of the residues on the target components, and significantly improve the purity and quality of the product.

[0044] Comparative Example 2 Take 10 kg of dried shiitake mushroom fruiting bodies (similar to those in Example 1), and perform step 1 (cell wall disruption) and step 2 (enzymatic hydrolysis) according to the same steps as in Example 1. After enzyme inactivation and cooling in step 2, do not perform step 3 (directional polysaccharide degradation). After enzymatic hydrolysis, heat the system to 90°C and incubate for 30 minutes to inactivate the enzyme. After cooling to room temperature, filter using a plate and frame filter and collect the clear filtrate. Concentrate the filtrate to 1 / 3 of its original volume, and then slowly add food-grade anhydrous ethanol until the ethanol volume fraction in the system reaches 75%. Let it stand for 12 hours to precipitate. Discard the supernatant, collect the precipitate, and freeze-dry it to obtain 3.2 kg of off-white powder, with a yield of 32.0%.

[0045] Testing revealed that the product obtained in this comparative example (i.e., the target product of the traditional process—polysaccharide precipitate) contained 78.5% polysaccharide (weight-average molecular weight > 1 × 10⁻⁶). 5 The product is mainly composed of undegraded macromolecular components; the polypeptide content is 4.2%; the flavonoid content is 0.3%; the product's solubility in 70% ethanol (1% concentration) is almost insoluble, resulting in a large amount of turbid precipitate.

[0046] Comparative Analysis: This comparative example simulates the traditional water extraction and alcohol precipitation process, collecting only the alcohol precipitate as the product while discarding the supernatant rich in peptides and flavonoids. Compared with the product collected by alcohol solubility of all components in Example 1, although the product of the traditional method has a high polysaccharide content, almost all peptides and flavonoids are lost, and the obtained polysaccharides have extremely poor alcohol solubility due to their large molecular weight. This fully demonstrates the significant advantages of the present invention's enzyme-based targeted molecular weight trimming + full-component alcohol solubility collection technology in achieving synergistic retention of multiple active ingredients and improving product application performance.

[0047] Experimental Example 1: Verification of the effect of polysaccharide degradation degree on product alcohol solubility and activity Under the same conditions as in Example 1, experimental groups with different molecular weight degradation endpoints were set up, and the alcohol solubility and immunomodulatory activity of the products were measured (based on the ability to stimulate RAW264.7 macrophages to produce NO). Group A was prepared by reducing the amount of β-1,3-glucanase in step 3 to 20 g and shortening the reaction time to 0.5 hours; Group C was prepared by increasing the enzyme amount to 200 g and extending the reaction time to 4 hours; and Group D was prepared by increasing the enzyme amount to 500 g and extending the reaction time to 8 hours. The results are shown in Table 1 below. As shown in the table above, the molecular weight of polysaccharides can be precisely controlled at a weight-average molecular weight of 5 × 10⁻⁶. 3 -5×10 4 Only when the molecular weight of Da is within the alcohol solubility threshold range can the product achieve both complete alcohol solubility and high bioactivity; if the molecular weight is too large, the solubility will be poor, and if it is too small, the activity will be significantly reduced.

[0048] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0049] It should be understood that the disclosed technical solutions in the embodiments provided in this application can be implemented in other ways. For example, the embodiments described above are merely illustrative, and the division of the units described above is only a logical functional division. In actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or communication connections shown or discussed may be indirect coupling or communication connections between devices or units through some interfaces, and may be in telecommunications or other forms.

[0050] The units described above as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.

Claims

1. A process for enzymatic extraction of shiitake mushrooms, characterized in that, Includes the following steps: 1) Cell wall breaking: Crush or grind the shiitake mushroom raw material, add water, add cellulase at 30-65℃, and react for 0.5-3 hours; 2) Proteolysis and flavonoid release: Adjust the pH of the reaction system obtained in step 1) to 7.0-9.0, add alkaline protease and neutral protease, and react at 40-60℃ for 1-3 hours; after the reaction is completed, heat the system to 85-90℃ and hold for 10-20 minutes to inactivate the enzymes, and then cool. 3) Polysaccharide directional degradation: adjust the pH of the reaction system obtained in step 2) to 4.5-6.0, add β-1, 3-glucan endo-enzyme, and react at 45-55°C for 1-3 hours to degrade the macromolecular lentinan into active fragments with a weight average molecular weight of 5×10 3 Da. 4 Da. 4) Collection of alcohol-soluble components: After inactivating the enzyme in the enzymatic hydrolysate obtained in step 3), add ethanol to the system until the ethanol volume fraction is 60%-85%, stir thoroughly to dissolve, separate the solid and liquid phases, and collect the liquid phase; 5) Drying: After recovering the ethanol, the resulting liquid phase is concentrated and dried to obtain the enzymatic hydrolysis extract of all components of shiitake mushroom.

2. The enzymatic extraction process for shiitake mushrooms as described in claim 1, characterized in that, The shiitake mushroom raw materials are dried shiitake fruiting bodies, dried shiitake stems, or fresh shiitake mushrooms.

3. The enzymatic extraction process for shiitake mushrooms as described in claim 1, characterized in that, In step 2), the amount of alkaline protease used is 0.5-3 wt% of the dry weight of the shiitake mushroom raw material, and the amount of neutral protease used is 0.5-2 wt% of the dry weight of the shiitake mushroom raw material.

4. The enzymatic extraction process for shiitake mushrooms as described in claim 1, characterized in that, In step 3), the amount of β-1,3-glucan endonuclease used is 0.5-2 wt% of the dry weight of the shiitake mushroom raw material.

5. The enzymatic extraction process for shiitake mushrooms as described in claim 1, characterized in that, In step 4), before adding ethanol, the enzyme hydrolysate after enzyme inactivation is first separated into solid and liquid components to remove the enzyme hydrolysis residue and collect the clear liquid; then ethanol is added to the clear liquid.

6. The enzymatic extraction process for shiitake mushrooms as described in claim 5, characterized in that, Step 4) involves adding ethanol to the clarified liquid and performing solid-liquid separation again using a stepwise gradient alcohol dissolution process: First, add ethanol to the clarified liquid until the volume fraction reaches 30%-40%, stir, and then separate the solid and liquid phases to collect the first-stage clarified liquid; then, continue adding ethanol to the first-stage clarified liquid until the volume fraction reaches 60%-85%, stir, and then separate the solid and liquid phases to collect the liquid phase.

7. The enzymatic extraction process for shiitake mushrooms as described in claim 1, characterized in that, In step 5), the drying method is spray drying or freeze drying.

8. The enzymatic hydrolysis extract of all components of shiitake mushroom prepared by any one of the processes described in claims 1-7, characterized in that, The extract contains a weight-average molecular weight of 5 × 10⁻⁶. 3 -5×10 4 Da contains lentinan degradation products, peptides with a molecular weight of less than 3000 Da, and flavonoids.

9. The enzymatic hydrolysis extract of all components of shiitake mushroom as described in claim 8, characterized in that, By total weight, the extract contains ≥30% lentinan degradation products, ≥15% polypeptides, and ≥1.5% flavonoids.

10. The application of the enzymatic hydrolysis extract of shiitake mushroom as described in claim 8 or 9 in the preparation of health food with functions of enhancing immunity and anti-fatigue.

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