A hericium erinaceus glycosylated polypeptide extract, and a preparation method and application thereof

Abstract

This invention discloses a glycosylated polypeptide extract from Hericium erinaceus, its preparation method, and its applications, belonging to the technical field of Hericium erinaceus extracts. The technical solution includes: alkaline extraction, where Hericium erinaceus waste powder is subjected to alkaline extraction; the pH of the resulting extract is adjusted to the isoelectric point of Hericium erinaceus protein; the precipitate is repeatedly washed until neutral to obtain Hericium erinaceus protein and polysaccharide precipitates; enzymatic hydrolysis, where the Hericium erinaceus protein and polysaccharide precipitates are added to water, and an enzyme preparation is added for enzymatic hydrolysis to obtain an enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides; polypeptide glycosylation modification, where the enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides undergoes a gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction; after the reaction, freeze-drying yields the Hericium erinaceus glycosylated polypeptide extract. This invention enables the resource utilization of Hericium erinaceus waste, while simultaneously preparing a highly active, highly stable glycosylated polypeptide extract that improves the water solubility of lipid-soluble extracts, reducing production costs and expanding its application range.

Description

Technical Field

[0001] This invention belongs to the technical field of Hericium erinaceus extract, and particularly relates to a Hericium erinaceus glycosylated polypeptide extract, its preparation method, and its application. Background Technology

[0002] Hericium erinaceus, belonging to the order Hericium, family Hericiumceae, and genus Hericium, is a precious medicinal and edible fungus in my country, often described as "mountain delicacy, bird's nest." Hericium erinaceus is rich in nutrients, including proteins, polysaccharides, oligosaccharides, sterols, terpenes, phenols, and various essential minerals.

[0003] Currently, the deep processing of Hericium erinaceus polysaccharide extract generates a large amount of waste residue, which is usually discarded as waste, resulting in resource waste and environmental pressure. Research has found that Hericium erinaceus waste residue still contains a large amount of unextracted soluble proteins, peptides, and acidic heteropolysaccharides. These residual components still possess good biological activity and have extremely high resource utilization value.

[0004] In existing technologies, the utilization of Hericium erinaceus (monkey head mushroom) waste residue is limited, and conventional extraction methods suffer from low extraction efficiency, difficulty in separating proteins and polysaccharides, and easy degradation of active ingredients, resulting in the inability to fully recover and utilize the effective components in the waste residue. Meanwhile, polysaccharides (especially acidic heteropolysaccharides) and peptides in Hericium erinaceus are important bioactive substances, but single polysaccharides or peptides suffer from poor stability, insufficient water solubility, and incomplete expression of bioactivity. Therefore, developing a method for efficiently extracting proteins and polysaccharides from Hericium erinaceus waste residue can not only solve the problem of resource waste from Hericium erinaceus waste residue but also prepare highly active and stable glycosylated products, which has significant economic and environmental value. Summary of the Invention

[0005] To address the problems of resource waste, low extraction efficiency of effective components, and insufficient bioactivity of polysaccharides and peptides in existing technologies related to Hericium erinaceus waste residue, this invention provides a Hericium erinaceus glycosylated peptide extract, its preparation method, and its applications. This invention enables the resource utilization of Hericium erinaceus waste residue, while simultaneously preparing a highly active, highly stable glycosylated peptide extract that can improve the water solubility of fat-soluble extracts, thereby reducing production costs and expanding its application scope.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: This invention provides a method for preparing a glycosylated polypeptide extract of Hericium erinaceus, comprising the following steps: S1. Alkaline extraction: The powdered residue of Hericium erinaceus is subjected to alkaline extraction. The pH of the resulting extract is adjusted to the isoelectric point of Hericium erinaceus protein. The precipitate is repeatedly washed until neutral to obtain Hericium erinaceus protein and polysaccharide precipitate. S2. Enzymatic hydrolysis: The protein and polysaccharide precipitate of Hericium erinaceus are added to water, and an enzyme preparation is added for enzymatic hydrolysis to obtain the enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides. S3. Polypeptide glycosylation modification: The enzymatic hydrolysate of Hericium erinaceus peptides and polysaccharides was subjected to a gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction. After the reaction was completed, freeze-drying was performed to obtain Hericium erinaceus glycosylated peptide extract.

[0007] Preferably, in step S1, the Hericium erinaceus waste powder is prepared by the following method: The remaining waste residue from water extraction of Hericium erinaceus polysaccharide was obtained by drying, crushing and sieving in sequence.

[0008] Preferably, in step S2, the solids of the enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide have an acidic polysaccharide content of 40%-50% and a polypeptide content of 30%-40%.

[0009] Preferably, in step S3, the enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is adjusted to a solid content of 10-30 mg / mL before a gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction is carried out.

[0010] As a preferred option, the specific process of gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction is as follows: The enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is heated to 50-60℃ and kept at that temperature for 1-5 hours. Then, food-grade CO2 is slowly introduced into the system until the temperature drops to 20-30℃, at which point the introduction of food-grade CO2 is stopped. The system is then heated to 50-60℃ again and kept at that temperature for 1-5 hours. Then, food-grade CO2 is slowly introduced into the system until the temperature drops to 20-30℃, at which point the introduction of food-grade CO2 is stopped. This process is repeated 2-5 times. After the cycle is completed, the system is heated to 50-60℃ again to remove the CO2.

[0011] Preferably, in step S1, the extraction conditions for alkaline extraction of the Hericium erinaceus waste powder are as follows: The material-to-liquid mass ratio is 1:10-1:20, the extraction temperature is 60℃-100℃, the pH is 9-11, and the extraction time is 1h-4h. The isoelectric point (pH) of Hericium erinaceus protein is 3.5-4.5.

[0012] Preferably, in step S2, the enzyme preparation is one or both of medium-sized protease and alkaline protease.

[0013] Preferably, in step S2, the mass ratio of Hericium erinaceus protein and polysaccharide precipitate to water is 1:50-1:200, the enzyme dosage is 0.01g / L-1g / L, the hydrolysis pH is 7-10, the hydrolysis temperature is 30℃-60℃, and the hydrolysis time is 3h-6h.

[0014] In another aspect, the present invention provides a hericium erinaceus glycosylated polypeptide extract prepared according to a method for preparing a hericium erinaceus glycosylated polypeptide extract.

[0015] In another aspect, the present invention provides the use of the above-described Hericium erinaceus glycosylated polypeptide extract in improving the water solubility of fat-soluble extracts.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a method for preparing Hericium erinaceus glycosylated polypeptide extract. Using the waste residue remaining after water extraction of Hericium erinaceus polysaccharides as raw material, an alkaline extraction process is employed to simultaneously extract acidic Hericium erinaceus polysaccharides and proteins. The acidic polysaccharides, due to their inherent structural characteristics, are readily adaptable to glycosylation reactions without requiring additional modification. This method enables simultaneous glycosylation modification of polysaccharides and polypeptides in the extracted product, not only solving the resource waste problem caused by Hericium erinaceus waste residue but also yielding highly active and stable glycosylated products, possessing significant economic and environmental value.

[0017] This invention provides a glycosylated polypeptide extract of Hericium erinaceus, which has high activity and high stability, and can improve the water solubility of fat-soluble extracts, thereby realizing the resource utilization of Hericium erinaceus waste residue. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the extraction process of Hericium erinaceus glycosylated polypeptide extract provided in an embodiment of the present invention; Figure 2 To illustrate the grafting degree diagrams of the Hericium erinaceus glycosylated polypeptide extracts prepared in Examples 1-3 and Comparative Examples 1-3; Figure 3 To illustrate the dissolution diagrams of the lipid-soluble extract before and after modification with the Hericium erinaceus glycosylated peptide extract; Figure 4 To illustrate the dissolution diagrams of curcumin in different treatment groups in the examples. Detailed Implementation

[0019] The technical solutions in specific embodiments of the present invention will now be described in detail and completely with reference to the accompanying drawings. Obviously, the described embodiments are merely some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0020] This invention provides a method for preparing a glycosylated polypeptide extract of Hericium erinaceus, comprising the following steps: S1. Alkaline extraction: The powdered residue of Hericium erinaceus is subjected to alkaline extraction. The pH of the resulting extract is adjusted to the isoelectric point of Hericium erinaceus protein. The precipitate is repeatedly washed until neutral to obtain Hericium erinaceus protein and polysaccharide precipitate. S2. Enzymatic hydrolysis: The protein and polysaccharide precipitate of Hericium erinaceus are added to water, and an enzyme preparation is added for enzymatic hydrolysis to obtain the enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides. S3. Polypeptide glycosylation modification: The enzymatic hydrolysate of Hericium erinaceus peptides and polysaccharides was subjected to a gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction. After the reaction was completed, freeze-drying was performed to obtain Hericium erinaceus glycosylated peptide extract.

[0021] It should be noted that existing technologies have limited utilization of Hericium erinaceus (monkey head mushroom) waste residue, and conventional extraction methods suffer from low extraction efficiency, difficulty in separating proteins and polysaccharides, and easy degradation of active ingredients, resulting in the inability to fully recover and utilize the effective components in the waste residue. Meanwhile, polysaccharides (especially acidic heteropolysaccharides) and peptides in Hericium erinaceus are important bioactive substances, but single polysaccharides or peptides suffer from poor stability, insufficient water solubility, and inadequate bioactivity. Glycosylation modification is an effective means of improving the properties of biomolecules. By covalently binding polysaccharides and peptides, the water solubility, thermal stability, mucosal adhesion, and bioactivity of the product can be significantly improved. In traditional glycosylation reactions, polysaccharides usually require modification or other auxiliary methods to effectively bind with peptides; furthermore, conventional water extraction processes for Hericium erinaceus struggle to fully extract proteins, causing these protein resources to be lost with the waste residue.

[0022] This invention unexpectedly discovered during the protein extraction process of Hericium erinaceus that the waste residue from water-extracted Hericium erinaceus is still rich in acidic polysaccharides. The structural characteristics of these acidic polysaccharides differ significantly from those of conventionally water-extracted polysaccharides, and their structure is inherently suitable for glycosylation reactions. Therefore, this application first obtains acidic polysaccharides and proteins simultaneously through alkaline extraction, eliminating the need for additional polysaccharide modification, simplifying the glycosylation process, and reducing energy and reagent consumption. The use of gradient temperature-changing cyclic CO2-assisted catalysis further improves the efficiency and uniformity of the glycosylation reaction, avoiding damage to bioactive components from high temperatures. This achieves efficient recovery and in-situ glycosylation modification of acidic polysaccharides and proteins from Hericium erinaceus waste residue, significantly improving the resource utilization rate of the waste residue. The prepared glycosylated peptide extract possesses the dual activity advantages of both acidic polysaccharides and peptides, with significantly enhanced water solubility, thermal stability, and bioactivity.

[0023] In a preferred embodiment, in step S1, the Hericium erinaceus waste powder is prepared by the following method: The remaining waste residue from water extraction of Hericium erinaceus polysaccharide was obtained by drying, crushing and sieving in sequence.

[0024] Furthermore, the degree of grinding is 40-100 mesh.

[0025] In a preferred embodiment, in step S2, the solids of the enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide have an acidic polysaccharide content of 40%-50% and a polypeptide content of 30%-40%.

[0026] The above technical solution limits the content of acidic polysaccharides and polypeptides in the solids of the enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides. This range represents the optimal component ratio naturally formed after the extraction of Hericium erinaceus water extraction waste residue through alkaline extraction-enzymatic hydrolysis screening and synergistic extraction under adjusted reaction conditions. This ensures that the carboxyl sites of acidic polysaccharides and the amino sites of polypeptides maintain the optimal ratio, guaranteeing sufficient grafting and uniform structure in the glycosylation reaction. Simultaneously, it enables the product to form an amphiphilic structure with a suitable ratio of hydrophilic to hydrophobic groups, significantly improving the emulsifying properties and the solubility of lipid-soluble components. If the acidic polysaccharide content is too low, there will be insufficient grafting sites; if it is too high, the hydrophobic segments will be relatively lacking, making it impossible to form a stable solubilizing system.

[0027] In a preferred embodiment, in step S3, the enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is adjusted to a solid content of 10-30 mg / mL and then subjected to a gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction.

[0028] In a preferred embodiment, the specific process of gradient temperature-varying cyclic CO2-assisted catalytic glycosylation reaction is as follows: The enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is heated to 50-60℃ and kept at that temperature for 1-5 hours. Then, food-grade CO2 is slowly introduced into the system until the temperature drops to 20-30℃, at which point the introduction of food-grade CO2 is stopped. The system is then heated to 50-60℃ again and kept at that temperature for 1-5 hours. Then, food-grade CO2 is slowly introduced into the system until the temperature drops to 20-30℃, at which point the introduction of food-grade CO2 is stopped. This cycle is repeated 2-5 times. After the cycle is completed, the system is heated to 50-60℃ again to remove the CO2.

[0029] By employing the above technical solution, the system is first heated to 50-60℃ and held at this temperature, allowing the polypeptide molecular chains to fully extend and the carboxyl groups of the acidic polysaccharide to be fully exposed. This provides suitable kinetic conditions for the binding of carboxyl and amino groups, promoting the formation of glycosylated covalent bonds. Subsequently, CO2 is slowly introduced and the temperature is lowered to 20-30℃. On the one hand, the weakly acidic environment created by CO2 dissolving in water stabilizes the pH within the optimal range for glycosylation, ensuring continuous and efficient ionization of carboxyl groups. On the other hand, the cooling process stabilizes the conformation of the already formed glycosylated structure, preventing product dissociation or structural disorder. Multiple cycles can achieve a stepwise grafting process of "reaction-stabilization-re-reaction-re-stabilization," resulting in more uniform and complete glycosylation with a higher grafting degree. Simultaneously, the continuous introduction of CO2 can remove dissolved oxygen from the system, inhibiting polypeptide oxidation and product browning, and preserving the structural integrity of the product to the greatest extent.

[0030] In a preferred embodiment, in step S3, the enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is adjusted to a solid content of 20 mg / mL. The enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is heated to 55°C and kept at that temperature for 2 hours. Then, food-grade CO2 is slowly introduced into the system until the temperature drops to 25°C and the introduction of food-grade CO2 is stopped. The system is then heated to 55°C again and kept at that temperature for 2 hours. Then, food-grade CO2 is slowly introduced into the system until the temperature drops to 25°C and the introduction of food-grade CO2 is stopped. This cycle is repeated 3 times. After the cycle is completed, the system is heated to 55°C again. After the heating is completed, the system is freeze-dried to obtain the Hericium erinaceus glycosylated polypeptide extract.

[0031] In a preferred embodiment, in step S1, the extraction conditions for alkaline extraction of the Hericium erinaceus waste powder are as follows: The material-to-liquid mass ratio is 1:10-1:20, the extraction temperature is 60℃-100℃, the pH is 9-11, and the extraction time is 1h-4h. The isoelectric point (pH) of Hericium erinaceus protein is 3.5-4.5.

[0032] The above technical solution limits the extraction conditions for alkaline extraction of Hericium erinaceus waste powder. This is because the acidic polysaccharides from Hericium erinaceus contain a large number of carboxyl groups, which have low ionization and poor water solubility under neutral conditions. In a weakly alkaline environment, the carboxyl groups can fully ionize into carboxylate groups, significantly improving water solubility and allowing for complete dissolution from the waste. Simultaneously, alkaline conditions can disrupt the hydrogen bonds and hydrophobic bonds between polysaccharides, proteins, and cellulose, promoting the release of bound proteins. If the pH is too low, the acidic polysaccharides and proteins will not dissolve sufficiently; if the pH is too high, it will lead to protein denaturation and polysaccharide degradation, destroying the structures required for subsequent glycosylation. Therefore, pH 9-11 is the key range for achieving high extraction rates and high activity retention.

[0033] In a preferred embodiment, in step S1, the extraction conditions for alkaline extraction of the Hericium erinaceus waste powder are as follows: The material-to-liquid mass ratio was 1:15; the extraction temperature was 80℃, the pH was 10, the extraction time was 2.5h, and the isoelectric point (pH) of Hericium erinaceus protein was 4.0.

[0034] In a preferred embodiment, in step S2, the enzyme preparation is one or both of a medium protease and an alkaline protease.

[0035] In a preferred embodiment, in step S2, the mass ratio of Hericium erinaceus protein and polysaccharide precipitate to water is 1:50-1:200, the enzyme dosage is 0.01g / L-1g / L, the enzymatic hydrolysis pH is 7-10, the enzymatic hydrolysis temperature is 30℃-60℃, and the enzymatic hydrolysis time is 3h-6h.

[0036] The above technical solution defines the specific parameters of the enzymatic hydrolysis reaction, among which the hydrolysis time is crucial for achieving the ideal hydrolysis product. Too short a time (<3 h) leads to incomplete hydrolysis, resulting in a large amount of residual large protein molecules, which is detrimental to the efficient execution of subsequent glycosylation reactions; while too long a time (>6 h) easily causes excessive degradation of polypeptide chains and loss of active groups. Controlling the time within a 3-6 hour window allows for the precise acquisition of hydrolysis products with suitable molecular weight distribution, sufficient exposure of amino groups, and a high degree of matching with the polysaccharide ratio, thus directly and efficiently adapting to subsequent glycosylation reaction steps.

[0037] Meanwhile, precise control of enzyme dosage is crucial. Insufficient enzyme dosage (<0.01 g / L) leads to inadequate hydrolysis and an excessively high proportion of large protein molecules, hindering effective glycosylation. Excessive dosage (>1 g / L), while accelerating the reaction, significantly increases enzyme costs and may cause over-hydrolysis, resulting in too many exposed amino sites. Too many amino sites can easily trigger excessive glycosylation in subsequent steps, leading to a disordered product structure and decreased uniformity. Strictly limiting the enzyme dosage to the range of 0.01-1 g / L allows for a mild, controllable, and moderate hydrolysis process. This results in peptide products with relatively concentrated molecular weights and an appropriate number of exposed amino groups, where the amino groups and carboxyl sites of the acidic polysaccharide are highly matched in both number and space, significantly improving the uniformity of subsequent glycosylation grafting.

[0038] In a preferred embodiment, in step S2, the mass ratio of Hericium erinaceus protein and polysaccharide precipitate to water is 1:100, the enzyme dosage is 0.1 g / L, the hydrolysis pH is 8, the hydrolysis temperature is 45°C, and the hydrolysis time is 4 h.

[0039] In another aspect, the present invention provides a hericium erinaceus glycosylated polypeptide extract prepared according to a method for preparing a hericium erinaceus glycosylated polypeptide extract.

[0040] In another aspect, the present invention provides the use of the above-described Hericium erinaceus glycosylated polypeptide extract in improving the water solubility of fat-soluble extracts.

[0041] The Hericium erinaceus glycosylated polypeptide extract of the present invention has high activity and high stability, and can improve the water solubility of fat-soluble extracts.

[0042] To more clearly and in detail introduce the Hericium erinaceus glycosylated polypeptide extract, its preparation method, and its application provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0043] In the following examples and comparative examples: The waste residue remaining after water extraction of Hericium erinaceus polysaccharide is the waste residue recycled after the production of Hericium erinaceus polysaccharide extract (batch 20240228) from Qingdao Born High-Tech Biotechnology Co., Ltd. The medium-sized protease used was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., with an enzyme activity of 60 w U / g; The alkaline protease used was purchased from Nanning Pangbo Biotechnology Co., Ltd., with an enzyme activity of 50 w U / g.

[0044] The pure Hericium erinaceus polysaccharide used was Hericium erinaceus polysaccharide extract produced by Qingdao Born High-Tech Biotechnology Co., Ltd., with a purity ≥30%. The pure Hericium erinaceus polypeptide used was purchased from Bloomage Biotechnology (Shanxi) Co., Ltd., with a purity of ≥98%.

[0045] Example 1: Preparation of Hericium erinaceus glycosylated polypeptide extract 1. Pretreatment of Hericium erinaceus waste residue: The waste residue remaining after extracting polysaccharides from Hericium erinaceus was dried, crushed, and passed through a 40-mesh sieve to obtain 200g of pretreated waste residue powder. 2. Synergistic extraction of proteins and polysaccharides: Hericium erinaceus waste powder was extracted with NaOH alkaline water. The pH of the resulting extract was adjusted to the isoelectric point of Hericium erinaceus protein. The precipitate was repeatedly washed until neutral to ensure that the acid and alkali were washed away. The supernatant was discarded to obtain Hericium erinaceus protein and polysaccharide precipitate. The extraction conditions were as follows: a material-to-liquid mass ratio of 1:10, an extraction temperature of 60℃, a pH of 9, an extraction time of 1 hour, and the precipitate was recovered at an isoelectric point of 4.5.

[0046] 3. Enzymatic hydrolysis of proteins and polysaccharides: The precipitate was dissolved in water, the pH was adjusted, and an enzyme preparation was added for enzymatic hydrolysis to obtain an enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides. The enzyme preparation is a medium-sized protease; the mass ratio of precipitated solids to water in the enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is 1:50, the enzyme dosage is 0.01 g / L, the hydrolysis pH is 7, the hydrolysis temperature is 30℃, and the hydrolysis time is 3 h. Analysis showed that the acidic polysaccharide content and polypeptide content in the precipitated solids of the Hericium erinaceus polypeptide and polysaccharide hydrolysate were 45.7%.

[0047] 4. Polysaccharide-peptide glycosylation modification: The enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides was adjusted to a solid content of 10 mg / mL, placed in a container, and heated to 50°C for 1 hour. Food-grade CO2 was slowly introduced into the system until the temperature dropped to 20°C. After stopping the introduction of food-grade CO2, the system was heated to 50°C again for 1 hour. Food-grade CO2 was slowly introduced into the system until the temperature dropped to 20°C. After stopping the introduction of food-grade CO2, the system was heated to 50°C to remove CO2. After the heating was completed, the extract was freeze-dried to obtain 11.2 g of Hericium erinaceus glycosylated extract.

[0048] Example 2: Preparation of Hericium erinaceus glycosylated polypeptide extract 1. Pretreatment of Hericium erinaceus waste residue: The waste residue remaining after extracting polysaccharides from Hericium erinaceus was dried, crushed, and passed through a 60-mesh sieve to obtain 100g of pretreated waste residue powder. 2. Synergistic extraction of proteins and polysaccharides: Hericium erinaceus waste powder was extracted with NaOH alkaline water. The pH of the resulting extract was adjusted to the isoelectric point of Hericium erinaceus protein. The precipitate was repeatedly washed until neutral to ensure that the acid and alkali were washed away. The supernatant was discarded to obtain Hericium erinaceus protein and polysaccharide precipitate. The extraction conditions were as follows: a material-to-liquid mass ratio of 1:15, an extraction temperature of 80℃, a pH of 10, an extraction time of 2.5 h, and the precipitate was recovered at an isoelectric point of 4.0.

[0049] 3. Enzymatic hydrolysis of proteins and polysaccharides: The precipitate was dissolved in water, the pH was adjusted, and an enzyme preparation was added for enzymatic hydrolysis to obtain an enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides. The enzyme preparation consisted of a medium-sized protease and an alkaline protease in a 1:1 mass ratio. The mass ratio of the precipitated solids to water in the enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides was 1:100. The enzyme dosage was 0.01 g / L, the hydrolysis pH was 8, the hydrolysis temperature was 45℃, and the hydrolysis time was 4 h. After hydrolysis, the pH was adjusted to 7. Analysis showed that the acidic polysaccharide content and the polypeptide content in the precipitated solids of the Hericium erinaceus polypeptides and polysaccharides hydrolysate were 49.2%.

[0050] 4. Polysaccharide-peptide glycosylation modification: The enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides was adjusted to a solid content of 20 mg / mL, placed in a container, and heated to 55°C for 2 hours. Food-grade CO2 was slowly introduced into the system until the temperature dropped to 25°C. After stopping the introduction of food-grade CO2, the system was heated to 55°C again for 2 hours. Food-grade CO2 was slowly introduced into the system until the temperature dropped to 25°C. This cycle was repeated 3 times. The system was then heated to 55°C to remove CO2. After heating, the extract was freeze-dried to obtain 16.2 g of Hericium erinaceus glycosylated extract.

[0051] Example 3: Preparation of Hericium erinaceus glycosylated polypeptide extract 1. Pretreatment of Hericium erinaceus waste residue: The waste residue remaining after extracting polysaccharides from Hericium erinaceus was dried, crushed, and passed through an 80-mesh sieve to obtain 100g of pretreated waste residue powder. 2. Synergistic extraction of proteins and polysaccharides: Hericium erinaceus waste powder was extracted with NaOH alkaline water. The pH of the resulting extract was adjusted to the isoelectric point of Hericium erinaceus protein. The precipitate was repeatedly washed until neutral to ensure that the acid and alkali were washed away. The supernatant was discarded to obtain Hericium erinaceus protein and polysaccharide precipitate. The extraction conditions were as follows: a material-to-liquid mass ratio of 1:20, an extraction temperature of 100℃, a pH of 11, an extraction time of 4 hours, and the precipitate was recovered at an isoelectric point of 3.5.

[0052] 3. Enzymatic hydrolysis of proteins and polysaccharides: The precipitate was dissolved in water, the pH was adjusted, and an enzyme preparation was added for enzymatic hydrolysis to obtain an enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides. The enzyme preparation is a medium-sized protease. The mass ratio of precipitated solids to water in the enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides is 1:200, the enzyme dosage is 0.01 g / L, the hydrolysis pH is 10, the hydrolysis temperature is 60℃, and the hydrolysis time is 6 h. After hydrolysis, the pH is adjusted to 7. Analysis showed that the acidic polysaccharide content and polypeptide content in the precipitated solids of the Hericium erinaceus polypeptides and polysaccharides hydrolysate were 50.0%.

[0053] 4. Polysaccharide-peptide glycosylation modification: The enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides was adjusted to a solid content of 30 mg / mL, placed in a container, and heated to 60°C for 5 hours. Food-grade CO2 was slowly introduced into the system until the temperature dropped to 30°C. After stopping the introduction of food-grade CO2, the system was heated to 60°C again for 5 hours. Food-grade CO2 was slowly introduced into the system until the temperature dropped to 30°C. After repeating this cycle 5 times, the system was heated to 60°C to remove CO2. After heating, the extract was freeze-dried to obtain 18.0 g of Hericium erinaceus glycosylated extract.

[0054] Comparative Example 1 The difference between this comparative example and Example 3 is as follows: Omit the alkali extraction step; The specific extraction steps are as follows: 1. Pretreatment of Hericium erinaceus waste residue: The waste residue remaining after extracting polysaccharides from Hericium erinaceus was dried, crushed, and passed through an 80-mesh sieve to obtain 100g of pretreated waste residue powder. 2. Synergistic extraction of proteins and polysaccharides: Hericium erinaceus waste residue powder was extracted with pure water. The pH of the resulting extract was adjusted to the isoelectric point of Hericium erinaceus protein. The precipitate was repeatedly washed until neutral to ensure that the acid and alkali were washed away. The supernatant was discarded to obtain Hericium erinaceus protein and polysaccharide precipitate. The extraction conditions were as follows: the material-to-liquid mass ratio was 1:20, the extraction temperature was 100℃, the extraction time was 4h, and the precipitate was recovered when the isoelectric point pH was 3.5.

[0055] 3. Enzymatic hydrolysis of proteins and polysaccharides: The precipitate was dissolved in water, the pH was adjusted, and an enzyme preparation was added for enzymatic hydrolysis to obtain an enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides. The enzyme preparation is a medium-sized protease. The mass ratio of precipitated solids to water in the enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is 1:200, the enzyme dosage is 0.01 g / L, the hydrolysis pH is 10, the hydrolysis temperature is 60℃, and the hydrolysis time is 6 h. After hydrolysis, the pH is adjusted to 7. Analysis showed that the acidic polysaccharide content and polypeptide content in the precipitated solids of the Hericium erinaceus polypeptide and polysaccharide hydrolysate were 15.1%.

[0056] 4. Polysaccharide-peptide glycosylation modification: The enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides was adjusted to a solid content of 30 mg / mL, placed in a container, and heated to 60°C for 5 hours. Food-grade CO2 was slowly introduced into the system until the temperature dropped to 30°C. After stopping the introduction of food-grade CO2, the system was heated to 60°C again for 5 hours. Food-grade CO2 was slowly introduced into the system until the temperature dropped to 30°C. After repeating this cycle 5 times, the system was heated to 60°C to remove CO2. After heating, the extract was freeze-dried to obtain 5.3 g of Hericium erinaceus glycosylated extract.

[0057] Comparative Example 2 Glycosylation was performed using pure polysaccharides and pure peptides, and the actual mass ratio of added polysaccharides and peptides was consistent with that in Example 3. The specific steps were as follows: Take 30g of pure Hericium erinaceus polysaccharide extract (commercially available, purity ≥30%, equivalent to 9g of actual polysaccharide) and 6.72g of pure Hericium erinaceus polypeptide (commercially available, purity ≥98%, equivalent to 6.59g of actual polypeptide). Dissolve Hericium erinaceus protein and polysaccharide extract in water until the solid content is 30mg / mL. Place in a container and heat to 60℃ for 5 hours. Slowly introduce food-grade CO2 into the system until the temperature drops to 30℃. Stop introducing food-grade CO2 and then heat to 60℃ again for 5 hours. Slowly introduce food-grade CO2 into the system until the temperature drops to 30℃ and then stop introducing food-grade CO2. Repeat this cycle 5 times. Then heat to 60℃. After heating, freeze-dry the extract to obtain 36.72g of Hericium erinaceus glycosylated extract.

[0058] Comparative Example 3 The difference between this comparative example and Example 3 is that the gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction is omitted. The specific steps are as follows: 1. Pretreatment of Hericium erinaceus waste residue: The waste residue remaining after extracting polysaccharides from Hericium erinaceus was dried, crushed, and passed through an 80-mesh sieve to obtain 100g of pretreated waste residue powder. 2. Synergistic extraction of proteins and polysaccharides: Hericium erinaceus waste powder was extracted with NaOH alkaline water. The pH of the resulting extract was adjusted to the isoelectric point of Hericium erinaceus protein. The precipitate was repeatedly washed until neutral to ensure that the acid and alkali were washed away. The supernatant was discarded to obtain Hericium erinaceus protein and polysaccharide precipitate. The extraction conditions were as follows: a material-to-liquid mass ratio of 1:20, an extraction temperature of 100℃, a pH of 11, an extraction time of 4 hours, and the precipitate was recovered at an isoelectric point of 3.5.

[0059] 3. Enzymatic hydrolysis of proteins and polysaccharides: The precipitate was dissolved in water, the pH was adjusted, and an enzyme preparation was added for enzymatic hydrolysis to obtain an enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides. The enzyme preparation is a medium-sized protease. The mass ratio of precipitated solids to water in the enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides is 1:200, the enzyme dosage is 0.01 g / L, the hydrolysis pH is 10, the hydrolysis temperature is 60℃, and the hydrolysis time is 6 h. After hydrolysis, the pH is adjusted to 7. Analysis showed that the acidic polysaccharide content and polypeptide content in the precipitated solids of the Hericium erinaceus polypeptides and polysaccharides hydrolysate were 50.1%.

[0060] 4. Polysaccharide-peptide glycosylation modification: The enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides was adjusted to a solid content of 30 mg / mL, placed in a container, and reacted at 60°C, protected from light, under reduced pressure (-0.01 MPa) and controlled temperature (60°C) for 30 min. After the reaction, the extract was immediately stored at 4°C. 17.8 g of the Hericium erinaceus glycosylated extract was obtained by freeze-drying.

[0061] Experimental Example 1: Grafting Degree Determination (using the o-phthalaldehyde colorimetric method) 1. Experimental Method: Add 4.0 mL of OPA reagent to 200 μL of the sample solution (15 g / mL), react in a 35 °C water bath for 2 min, and measure the absorbance A340 at 340 nm.

[0062] Using distilled water as a sample blank, calculate according to the formula: DG grafting degree = (A0-A1) / A0×100 In the formula: A0, absorbance of the solution before the grafting reaction; A1, absorbance of the solution after the grafting reaction.

[0063] 2. Experimental Results: like Figure 2As shown, the grafting degrees of Examples 1-3 were 49.30%, 50.10%, and 51.27%, respectively, all remaining stable at around 50%, with Example 3 being slightly higher. The grafting degree of Comparative Example 1 was only 10.50%, significantly lower than that of the Examples; the grafting degree of Comparative Example 2 was 13.56%, also far lower than the levels of the Examples; the grafting degree of Comparative Example 3 was 40.89%, higher than Comparative Examples 1 and 2, but still lower than that of Examples 1-3.

[0064] Examples 1-3 employed an alkaline extraction combined with a gradient temperature-changing cyclic CO2-assisted catalytic glycosylation process, achieving a grafting degree of approximately 50%. This indicates that the process can efficiently promote covalent bonding between polysaccharides and peptides, resulting in a more complete glycosylation reaction.

[0065] Example 3 showed a slight improvement in grafting degree, possibly related to the use of a higher polysaccharide concentration and more precise reaction conditions (such as pH 6.0 and a reaction time of 2 hours), further confirming that optimizing process parameters can effectively improve grafting efficiency. Considering production cost factors, the process parameters of Example 2 were determined to be the optimal conditions.

[0066] Examples 1-3 employed an alkaline extraction + gradient temperature-changing cyclic CO2-assisted catalytic glycosylation process, achieving a grafting degree of approximately 50%, indicating that this process can efficiently promote the covalent binding of polysaccharides and peptides, resulting in a complete glycosylation reaction.

[0067] The grafting degree in Example 3 was slightly higher, which may be related to the higher polysaccharide concentration and more precise reaction conditions (such as pH 6.0 and 2h reaction), further verifying that optimizing process parameters can improve grafting efficiency. Considering cost factors in production, the parameter values ​​in Example 2 were taken as the optimal values.

[0068] Comparative Example 1 (without alkali extraction) had a grafting degree of only 10.50%, indicating that when not fully extracted, there were insufficient active sites such as carboxyl groups of polysaccharides and amino groups of polypeptides in the system that could participate in the reaction, resulting in a significant reduction in the degree of glycosylation reaction.

[0069] Comparative Example 2 involved reacting pure Hericium erinaceus polysaccharide with pure polypeptide, achieving a grafting degree of 13.56%, which was higher than Comparative Example 1 but still significantly lower than Examples 1-3.

[0070] The above results indicate that the acidic heteropolysaccharide-peptide complex extracted from waste residue by this invention has a molecular structure that is more suitable for glycosylation reactions (such as more complete exposure of carboxyl groups and easier participation of polypeptide amino groups in the reaction), and its grafting efficiency is better than that of pure raw materials, fully demonstrating the unique advantages of waste residue resource utilization.

[0071] Comparative Example 3 (which uses alkali extraction but only mild wet glycosylation without gradient temperature change cyclic CO2 assisted catalysis) has a grafting degree of 40.89%, which is higher than that of Comparative Example 1, but still lower than that of the other examples. This directly proves that the alkali extraction-gradient temperature change cyclic CO2 assisted catalytic glycosylation process plays a key role in achieving efficient glycosylation reaction.

[0072] Experimental Example 2: In vitro dissolution experiment Drug absorption depends on the drug's release from the formulation, its dissolution or solubility under physiological conditions, and the permeability of the gastrointestinal tract biomembranes. The drug's release from the formulation and its dissolution under physiological conditions are decisive factors; therefore, the in vitro dissolution rate may predict the drug's behavior in vivo.

[0073] 1. Comparison between improved and unimproved versions: Experimental methods: The experiment consisted of 6 treatments: Treatment 1: Modified curcumin: Take 3g of curcumin, add 7g of the glycosylated polypeptide extract from Example 3, and grind thoroughly to obtain modified curcumin.

[0074] Treatment 2: Modified resveratrol: Take 3g of resveratrol, add 7g of the glycosylated polypeptide extract from Example 1, and grind thoroughly to obtain modified resveratrol.

[0075] Treatment 3: Improved lycopene: Take 3g of lycopene, add 7g of the glycosylated polypeptide extract from Example 2, and grind thoroughly to obtain lycopene.

[0076] Process 4: Pure curcumin; Process 5: Pure resveratrol; Process 6: Pure lycopene; Following the dissolution and release determination method (rotating basket method) in Chinese Pharmacopoeia 2020 Edition 0931, experiments were conducted using an in vitro dissolution apparatus. The dissolution medium was 900 mL of 0.5% sodium dodecyl sulfate (SDS) solution, and the rotation speed was 75 rpm. 200 mg of each of the six treatments was taken and placed in No. 0 capsules. 5 mL samples were taken at 5 min, 10 min, 15 min, 20 min, 30 min, 60 min, 120 min, 180 min, and 240 min, and immediately replenished with an equal volume of dissolution medium (0.5% SDS) at the same temperature. Two replicates were set up, and the average value of the experimental data was taken. The samples were centrifuged for 10 min at 10000 rpm, and the supernatant was analyzed by high-performance liquid chromatography (HPLC).

[0077] Experimental results: Depend on Figure 3It can be seen that the in vitro dissolution rate of the glycosylated peptide modified groups (modified curcumin, modified resveratrol, and modified lycopene) increased rapidly within 30 minutes and reached 70% after 60 minutes.

[0078] The dissolution rates of pure curcumin, resveratrol, and lycopene stabilized at only around 36%, 32%, and 30%, respectively, after 60 minutes.

[0079] This demonstrates that glycosylated peptide modification can significantly improve the in vitro solubility of fat-soluble substances, solving the core problem of these substances' poor water solubility and difficulty in being absorbed by the human body.

[0080] Faster dissolution rate: The glycosylated peptide modified group (modified curcumin, modified resveratrol, modified lycopene) achieved a dissolution rate of over 30% within 10 minutes, while the unmodified group had a dissolution rate of less than 10% within 10 minutes.

[0081] This indicates that glycosylated peptide modification not only improves the final dissolution rate but also accelerates the dissolution rate, allowing the active substances to be released more quickly, which is beneficial for rapid onset of action and improved bioavailability.

[0082] Better dissolution stability: The dissolution of the glycosylated peptide modified group (modified curcumin, modified resveratrol, and modified lycopene) tended to stabilize after 60 minutes without significant decrease, indicating that the formed system was stable and not easily re-precipitated.

[0083] The unmodified group showed a slight decrease after reaching its peak, indicating that it is unstable in aqueous solution and is prone to recombination or precipitation.

[0084] 2. Curcumin in vitro dissolution experiment Experimental methods: The experiment consisted of 5 treatments: Treatment 1: Modified curcumin: Take 3g of curcumin, add 7g of the glycosylated polypeptide extract from Example 3, and grind thoroughly to obtain modified curcumin.

[0085] Process 2: Pure curcumin; Process 3: Take 3g of curcumin, add 7g of glycosylated polypeptide extract from Comparative Example 1, and grind thoroughly to obtain modified curcumin.

[0086] Process 4: Take 3g of curcumin, add 7g of glycosylated polypeptide extract from Comparative Example 2, and grind thoroughly to obtain modified curcumin.

[0087] Process 5: Take 3g of curcumin, add 7g of glycosylated polypeptide extract from Comparative Example 3, and grind thoroughly to obtain modified curcumin.

[0088] Following the dissolution and release determination method (rotating basket method) in Chinese Pharmacopoeia 2020 Edition 0931, experiments were conducted using an in vitro dissolution apparatus. The dissolution medium was 900 mL of 0.5% sodium dodecyl sulfate (SDS) solution, and the rotation speed was 75 rpm. 200 mg of each of the five treatments was taken and placed in No. 0 capsules. 5 mL samples were taken at 5 min, 10 min, 15 min, 20 min, 30 min, 60 min, 120 min, 180 min, and 240 min, and immediately replenished with an equal volume of dissolution medium (0.5% SDS) at the same temperature. Two replicates were set up, and the average value of the experimental data was taken. The samples were centrifuged for 10 min at 10000 rpm, and the supernatant was analyzed by high-performance liquid chromatography (HPLC).

[0089] Experimental results: The core drawback of the treatment is the lack of alkali extraction. Water extraction alone cannot meet the dissolution requirements of acidic polysaccharides and proteins, resulting in extremely low extraction efficiency. The acidic heteropolysaccharides (mainly containing glucuronic acid and galactose) in the Hericium erinaceus waste residue contain a large number of carboxyl groups (-COOH). These polysaccharides have low ionization and insufficient hydrophilicity in a neutral aqueous system, requiring mild alkali extraction conditions (pH 9-12) to ionize the carboxyl groups (-COOH → -COO). - To enhance the water solubility of polysaccharides, they must be fully dissolved from the cellulose skeleton of the waste residue. Simultaneously, the proteins in the Hericium erinaceus waste residue often form bound complexes with polysaccharides and cellulose. Water extraction alone cannot disrupt the hydrogen bonds and hydrophobic interactions of these complexes, hindering protein dissolution. Therefore, the alkali-free water extraction process results in extremely low extraction rates of both polysaccharides and proteins, directly leading to insufficient polysaccharide and protein substrate content in the subsequent glycosylation reaction, with a grafting degree of only 10.50%, far lower than the 49.30% in Example 1. Insufficient glycosylation is detrimental to the solubilizing properties of polysaccharide-peptide glycosylation products, which rely heavily on the amphiphilic structure of the "polysaccharide hydrophilic shell - peptide hydrophobic core." The formation of this structure requires sufficient grafting of polysaccharides and peptides. In Comparative Example 4, due to insufficient polysaccharide and protein substrates, the glycosylation reaction was extremely incomplete, failing to form a complete "hydrophilic shell" structure, resulting in weak solubilizing ability for curcumin. Experimental data show that the curcumin solubility of treatment 3 after 240 minutes was only 36.99%, which is basically the same as the water solubility of unmodified curcumin and far lower than the 78.18% of the example, fully demonstrating the limitations of the single water extraction process.

[0090] The reasons for the poor glycosylation effect of conventional treatment 4 are due to differences in raw material characteristics and sources: pure raw materials vs. waste residue raw materials. The low purity and incompatible types of pure polysaccharides lead to a lower degree of glycosylation grafting. In existing technologies, commercially available pure Hericium erinaceus polysaccharides are mostly prepared through a single water extraction process, resulting in generally low polysaccharide purity, with a content of only 10%~30%. Even when high-grade pure polysaccharides are selected for glycosylation, the proportion of effective active components is still lower than that of the polysaccharides extracted from waste residue in the examples. More importantly, the pure polysaccharides obtained by traditional water extraction are mostly neutral polysaccharides with extremely low carboxyl group content in their molecular structure. The carboxyl group (-COOH) is the core active site for the glycosylation reaction between polysaccharides and polypeptide amino groups (-NH2) to form amide bonds. Insufficient carboxyl groups result in a significantly lower degree of glycosylation grafting under existing process conditions compared to the examples, thus affecting the solubilization effect of the product on lipid-soluble components. The raw material ratios were not naturally compatible, resulting in poor glycosylation reaction. In the examples, the polysaccharides and peptides used for the glycosylation reaction were both derived from Hericium erinaceus waste residue. The two coexisted naturally in the waste residue and could be efficiently combined without artificial adjustment, ensuring the glycosylation reaction proceeded fully. In contrast, the pure polysaccharides and pure peptides used in Comparative Example 2 were prepared separately and then artificially proportioned. Although their ratios were similar to those in Example 2, the properties of Hericium erinaceus polysaccharides were not compatible with the glycosylation reaction of the peptides, resulting in insufficient improvement in the solubility of fat-soluble components in water, which was significantly lower than in the examples.

[0091] The core defect in addressing this issue is the lack of gradient-temperature-controlled cyclic CO2-assisted glycosylation, which fails to leverage the synergistic catalytic effect of gradient temperature changes and cyclic CO2. The degree of carboxyl ionization in Hericium erinaceus acidic polysaccharides is extremely sensitive to pH. During the glycosylation reaction, as carboxyl groups (-COOH) combine with amino groups (-NH2) to form amide bonds, the system slowly releases trace amounts of hydrogen ions, causing a slow local pH decrease (approximately 0.3-0.5). Without timely adjustment, this will reduce the degree of carboxyl ionization in the polysaccharides, gradually slowing the reaction rate. By cyclically replenishing CO2 (periodically adding trace amounts of food-grade CO2 and removing some dissolved gases), the system pH is stabilized in real-time within the optimal range of 5.5-6.0, continuously ensuring a high ionization state of the polysaccharide carboxyl groups (-COOH). - This design ensures high efficiency throughout the grafting reaction, while continuously removing dissolved oxygen and stabilizing pH through circulating CO2, preventing degradation and browning of active ingredients, resulting in a more complete product structure and superior solubility. Comparative Example 3 lacks two key innovative designs in the glycosylation stage: "gradient temperature control" and "CO2 circulation supply." This makes it unsuitable for the staged reaction characteristics of Hericium erinaceus acidic polysaccharide-peptide glycosylation, and also fails to maintain the dynamic optimal environment of the reaction system. Ultimately, this leads to a significant decrease in grafting efficiency, product structural integrity, and solubility.

Claims

1. A method for preparing a glycosylated polypeptide extract of Hericium erinaceus, characterized in that, Includes the following steps: S1. Alkaline extraction: The powdered residue of Hericium erinaceus is subjected to alkaline extraction. The pH of the resulting extract is adjusted to the isoelectric point of Hericium erinaceus protein. The precipitate is repeatedly washed until neutral to obtain Hericium erinaceus protein and polysaccharide precipitate. S2. Enzymatic hydrolysis: The protein and polysaccharide precipitate of Hericium erinaceus are added to water, and an enzyme preparation is added for enzymatic hydrolysis to obtain the enzymatic hydrolysate of Hericium erinaceus polypeptides and polysaccharides. S3. Polypeptide glycosylation modification: The enzymatic hydrolysate of Hericium erinaceus peptides and polysaccharides was subjected to a gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction. After the reaction was completed, freeze-drying was performed to obtain Hericium erinaceus glycosylated peptide extract.

2. The preparation method according to claim 1, characterized in that, In step S1, the Hericium erinaceus waste powder is prepared by the following method: The remaining waste residue from water extraction of Hericium erinaceus polysaccharide was obtained by drying, crushing and sieving in sequence.

3. The preparation method according to claim 2, characterized in that, In step S2, the solids of the enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide have an acidic polysaccharide content of 40%-50% and a polypeptide content of 30%-40%.

4. The preparation method according to claim 3, characterized in that, In step S3, the enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is adjusted to a solid content of 10-30 mg / mL before a gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction is carried out.

5. The preparation method according to claim 4, characterized in that, The specific process of gradient temperature-changing cyclic CO2-assisted catalytic glycosylation reaction is as follows: The enzymatic hydrolysate of Hericium erinaceus polypeptide and polysaccharide is heated to 50-60℃ and kept at that temperature for 1-5 hours. Then, food-grade CO2 is slowly introduced into the system until the temperature drops to 20-30℃, at which point the introduction of food-grade CO2 is stopped. The system is then heated to 50-60℃ again and kept at that temperature for 1-5 hours. Then, food-grade CO2 is slowly introduced into the system until the temperature drops to 20-30℃, at which point the introduction of food-grade CO2 is stopped. This cycle is repeated 2-5 times. After the cycle is completed, the system is heated to 50-60℃ again to remove the CO2.

6. The preparation method according to claim 3, characterized in that, In step S1, the extraction conditions for alkaline extraction of the Hericium erinaceus waste powder are as follows: The material-to-liquid mass ratio is 1:10-1:20, the extraction temperature is 60℃-100℃, the pH is 9-11, and the extraction time is 1h-4h. The isoelectric point (pH) of Hericium erinaceus protein is 3.5-4.

5.

7. The preparation method according to claim 3, characterized in that, In step S2, the enzyme preparation is one or both of medium-sized protease and alkaline protease.

8. The preparation method according to claim 7, characterized in that, In step S2, the mass ratio of Hericium erinaceus protein and polysaccharide precipitate to water is 1:50-1:200, the enzyme dosage is 0.01g / L-1g / L, the hydrolysis pH is 7-10, the hydrolysis temperature is 30℃-60℃, and the hydrolysis time is 3h-6h.

9. A hericium erinaceus glycosylated polypeptide extract prepared using the preparation method of the hericium erinaceus glycosylated polypeptide extract according to any one of claims 1-8.

10. The use of the Hericium erinaceus glycosylated polypeptide extract according to claim 9 in improving the water solubility of fat-soluble extracts.

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