Extraction method and application of tricholoma matsutake polysaccharide and polypeptide based on deep eutectic solvent

Abstract

The invention relates to the technical field of biological extracts, in particular to an extraction method and application of tricholoma matsutake polysaccharide and polypeptide based on a deep-eutectic solvent. The extraction method comprises the following steps: preparing a first eutectic solvent and a second eutectic solvent; preparing tricholoma matsutake dry powder; mixing the tricholoma matsutake dry powder with a first eutectic solvent, extracting, and centrifuging; a first extracting solution and first residues are obtained; treating the first residue, mixing the treated first residue with a second eutectic solvent, extracting, and centrifuging; a second extracting solution and second residues are obtained; then obtaining a tricholoma matsutake polysaccharide component A1 from the first extracting solution; obtaining the tricholoma matsutake polypeptide component B2 from the second extracting solution.

Description

Technical Field

[0001] This disclosure relates to the field of biological extract technology, specifically to a method and application for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent. Background Technology

[0002] Matsutake mushrooms are a rare edible and medicinal fungus, rich in bioactive components such as polysaccharides and polypeptides. Matsutake polysaccharides possess pharmacological activities including immunomodulation, antitumor activity, and antioxidant activity; while matsutake polypeptides have antioxidant, blood pressure-lowering, and metabolic-regulating functions. Therefore, efficient extraction of matsutake polysaccharides and polypeptides is of great significance for the in-depth development and utilization of matsutake resources.

[0003] Traditional extraction methods often employ water extraction and alcohol precipitation, a process characterized by cumbersome steps, low extraction efficiency, poor selectivity, and interference between polysaccharides and peptides during extraction, making effective separation difficult. Furthermore, prolonged high-temperature water extraction can lead to polysaccharide hydrolysis, peptide denaturation, and significant loss of active ingredients. While enzyme-assisted extraction can improve yield, the enzymatic hydrolysis process can break glycosidic or peptide bonds, damaging the natural structure, and enzyme preparations are expensive. In recent years, eutectic solvents (DES), as a green solvent system, have received widespread attention in the field of natural product extraction. DES are formed by hydrogen bond acceptors and donors interacting through hydrogen bonds, offering advantages such as high designability, good biocompatibility, and recyclability. However, existing DES extraction technologies are mostly designed for single components, lacking selective control for the simultaneous extraction of polysaccharides and peptides, making efficient separation of the two difficult. Therefore, developing a DES-based method for simultaneous extraction of matsutake polysaccharides and peptides, and achieving efficient extraction and separation of polysaccharides and peptides through stepwise selective extraction, is of practical significance for enhancing the utilization value of matsutake resources and promoting the advancement of intensive processing technology for edible and medicinal fungi. Summary of the Invention

[0004] The purpose of this disclosure is to provide a method and application for extracting matsutake polysaccharides and polypeptides based on eutectic solvents, in order to overcome the shortcomings of related technologies.

[0005] According to a first aspect of the present disclosure, a method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent is provided, the extraction method comprising the following steps:

[0006] Step 1: Prepare the first eutectic solvent and the second eutectic solvent;

[0007] Step 2: Prepare matsutake mushroom powder; mix the matsutake mushroom powder with the first eutectic solvent at a material-to-liquid ratio of 1:(15-25), and place it in a water bath at 50℃-70℃ for stirring and extraction for 1-3 hours; after extraction, centrifuge the obtained extract to obtain the first extract and the first residue;

[0008] Step 3: Process the first residue to obtain the processed first residue;

[0009] Step 4: Mix the treated first residue with the second eutectic solvent at a material-to-liquid ratio of 1:(12-20), and place it in a water bath at 70℃-80℃ for stirring and extraction for 1-3 hours; after extraction, centrifuge the resulting extract to obtain the second extract and the second residue; discard the second residue.

[0010] Step 5: The first extract was concentrated under reduced pressure to 1 / 3 of its original volume; then 3 times the volume of 95% ethanol was added for alcohol precipitation, and the mixture was allowed to stand overnight; then the precipitate was collected by centrifugation to obtain matsutake polysaccharide component A1; the supernatant obtained by centrifugation was concentrated, ammonium sulfate was added, and after standing, salting was performed; after centrifugation, the precipitate was collected, and after desalting and dialysis, it was freeze-dried to obtain matsutake polypeptide component A2;

[0011] Step 6: The second extract was concentrated to 1 / 4 of its original volume under reduced pressure; then 4 times the volume of 95% ethanol was added for alcohol precipitation, and the mixture was allowed to stand overnight; then the precipitate was collected by centrifugation, washed and dried to obtain matsutake polysaccharide component B1; the supernatant obtained by centrifugation was concentrated by ultrafiltration through a 3000 Da ultrafiltration membrane, and then freeze-dried to obtain matsutake polypeptide component B2.

[0012] In one aspect of the embodiments of this disclosure, the hydrogen bond donor of the first eutectic solvent is selected from glycerol or urea, and the hydrogen bond acceptor is selected from betaine or choline chloride; the hydrogen bond donor of the second eutectic solvent is selected from lactic acid, levulinic acid or malic acid, and the hydrogen bond donor includes a first hydrogen bond donor and a second hydrogen bond donor, wherein the first hydrogen bond acceptor is selected from zinc chloride, and the second hydrogen bond acceptor is selected from betaine or choline chloride.

[0013] In one aspect of the embodiments of this disclosure, the hydrogen bond donor of the first eutectic solvent is selected from glycerol, and the hydrogen bond acceptor is selected from betaine; the hydrogen bond donor of the second eutectic solvent is selected from lactic acid, and the hydrogen bond donor includes a first hydrogen bond donor and a second hydrogen bond donor, the first hydrogen bond acceptor is selected from zinc chloride, and the second hydrogen bond acceptor is selected from choline chloride.

[0014] In one aspect of this disclosure, the first eutectic solvent is prepared by the following steps:

[0015] Step 1-a: Add betaine and glycerin in a molar ratio of 1:(2.5-3.5) to a three-necked flask, heat in an oil bath to 90°C, and stir magnetically for 1-2 hours;

[0016] Step 2-a: Cool down to 60℃, add deionized water dropwise, the volume of deionized water added should be 35%-40% of the volume of the liquid formed in step 1-a; continue stirring for 30-60 minutes;

[0017] Step 3-a: Add anhydrous sodium sulfate; the mass of anhydrous sodium sulfate added is 1.2%-2.0% of the mass of the liquid formed in step 2-a; heat to 80°C, stir to dissolve, then cool naturally to room temperature, seal and store in the dark; obtain the first eutectic solvent.

[0018] In one aspect of this disclosure, the second eutectic solvent is prepared by the following steps:

[0019] Step 1-b: Add anhydrous zinc chloride and lactic acid in a molar ratio of 1:(2-3) to a three-necked flask, heat to 105°C in an oil bath under nitrogen protection, and stir magnetically for 45-75 min; then cool to 85°C and stir magnetically for 30-60 min.

[0020] Step 2-b: Further cool to 70°C, then add choline chloride; the molar amount of choline chloride added is 40%-60% of the molar amount of anhydrous zinc chloride; stir for 1-2 hours;

[0021] Step 3-b: Further cool to 50°C, then add mercaptoethanol and trehalose; the mass of mercaptoethanol added is 0.2%-0.5% of the mass of the liquid formed in step 2-b; the mass of trehalose added is 1.5%-2.5% of the mass of the liquid formed in step 2-a; stir for 20-45 minutes; then cool naturally to room temperature, then remove the nitrogen protection, seal and store in the dark; to obtain the second eutectic solvent.

[0022] In one aspect of this disclosure, the dried matsutake mushroom powder is prepared by the following steps:

[0023] Step 1-c: Provide matsutake fruiting bodies; dry the matsutake fruiting bodies with hot air to constant weight, then slice, pulverize and pass through a 60-80 mesh sieve to obtain the dried matsutake powder.

[0024] In one aspect of this disclosure, step 3 includes:

[0025] Step 3-1: Mix the first residue with deionized water at a material-to-liquid ratio of 1:(1-3) and stir at room temperature for 10-30 min; then centrifuge at 2000-4000 rpm for 5-15 min, discard the supernatant, collect the solid, and obtain the treated first residue.

[0026] According to a second aspect of the present disclosure, a matsutake polysaccharide is provided, the matsutake polysaccharide comprising matsutake polysaccharide component A1 extracted by the aforementioned method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent.

[0027] According to a third aspect of the present disclosure, a matsutake polypeptide is provided, the matsutake polypeptide comprising matsutake polypeptide component B2 extracted by the aforementioned method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent.

[0028] The technical solutions provided by the embodiments of this disclosure may include the following beneficial effects:

[0029] (1) This disclosure achieves stepwise selective extraction of polysaccharides and peptides: the first eutectic solvent inhibits peptide dissolution through salting-out effect and preferentially extracts polysaccharides; the second eutectic solvent utilizes the synergistic effect of the eutectic solvent and reducing agent to promote efficient dissolution of peptides, with a mass ratio of main product to secondary product of (5-10):1, resulting in good selectivity.

[0030] (2) The raw material utilization rate of this disclosure is relatively high: The extraction process provided by this disclosure realizes the full recovery of matsutake polysaccharides and polypeptides. The main product can be used for high value-added functional products, and the secondary product can be used for nutritional fortifiers, etc., with a high resource utilization rate.

[0031] (3) Due to the significant difference in the mass ratio of the primary and secondary products in the extraction process provided in this disclosure, namely, the yield of matsutake polysaccharide component A1 is significantly higher than that of matsutake polysaccharide component B1, and the yield of matsutake polypeptide component B2 is significantly higher than that of matsutake polypeptide component A2; therefore, the supernatant obtained by centrifugation in step 5 (containing matsutake polypeptide component A2) and the precipitate obtained by centrifugation in step 6 (containing matsutake polysaccharide component B1) can be temporarily left untreated, and the primary product can be treated first; these secondary products can be processed later, or used as raw materials for other products.

[0032] (4) The process conditions disclosed herein are mild and controllable; the extraction temperature is controlled at 50°C-80°C and the extraction time is controlled at 1-3 hours, avoiding the degradation of components caused by long-term high-temperature water extraction in traditional water extraction methods. The operation is simple and easy to scale up industrially.

[0033] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this disclosure. Attached Figure Description

[0034] Figure 1 The image shows the ion chromatogram of matsutake polysaccharide component A1 prepared in Example 1.

[0035] Figure 2 The image shows the ion chromatogram of Matsutake polysaccharide component B1 prepared in Example 1.

[0036] Among them, Fuc is fucose, Rha is rhamnose, Gal is galactose, Glc is glucose, Man is mannose, and GlcA is glucuronic acid. Detailed Implementation

[0037] Exemplary embodiments will now be described in detail. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this disclosure as detailed in the appended claims.

[0038] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. The embodiments described herein are illustrative in nature and are used to provide a basic understanding of this application. The embodiments of this application should not be construed as limiting this application.

[0039] For the sake of brevity, this article only discloses a few specific numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.

[0040] In this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0041] In this description, unless otherwise stated, "above" and "below" include the stated number.

[0042] Unless otherwise stated, the terms used in this disclosure have their common meanings as commonly understood by those skilled in the art. Unless otherwise stated, the values ​​of the parameters mentioned in this disclosure can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in the embodiments of this disclosure).

[0043] The term "about" is used to describe and indicate small variations. When used in conjunction with an event or situation, the term may refer to examples in which the event or situation occurred precisely or in examples in which the event or situation occurred very approximately. For example, when used in conjunction with numerical values, the term may refer to a range of variation less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. Additionally, quantities, ratios, and other numerical values ​​are sometimes presented in range format herein. It should be understood that such range format is for convenience and brevity and should be interpreted flexibly to include not only numerical values ​​explicitly specified as range limits but also all individual numerical values ​​or subranges covered within the range, as if each numerical value and subrange were explicitly specified.

[0044] The list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms can mean any combination of the listed items. For example, if items A and B are listed, then the phrase "at least one of A and B" means only A; only B; or A and B. In another instance, if items A, B, and C are listed, then the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A may contain a single component or multiple components. Item B may contain a single component or multiple components. Item C may contain a single component or multiple components.

[0045] In this disclosure, in the first eutectic solvent, sodium sulfate, as a strong electrolyte, completely dissociates. The hydration capacity of sodium ions and sulfate ions with water molecules is much stronger than that of the hydrophilic groups on the protein surface. The surface water molecules are attracted by inorganic ions, thereby destroying the hydration layer on the surface of the protein molecules. At the same time, sodium ions and sulfate ions compress the electric double layer on the protein surface, weakening the electrostatic repulsion. This exposes the hydrophobic regions of the protein molecules, which aggregate and precipitate through hydrophobic interactions, thus keeping the polypeptide components as much as possible in the first residue. Meanwhile, the polysaccharide molecules are rich in hydroxyl groups, which have strong hydrogen bonding with water molecules. They can still form a stable hydration layer with water molecules through hydroxyl groups to maintain dissolution, thereby achieving the selectivity of salting out proteins and preferential extraction of polysaccharides. In this disclosure, in the second eutectic solvent, zinc chloride coordinates with the carbonyl oxygen of the peptide bond in the polypeptide molecule to form a stable complex, enhancing the solubility of the polypeptide in the solvent; lactic acid provides a weakly acidic environment, promoting protein acid solubility; mercaptoethanol acts as a reducing agent to break the disulfide bonds within and between protein molecules, releasing the active polypeptide embedded in the cell wall or protein aggregates; trehalose, as a compatible solute, preferentially hydrates with the protein surface in a dehydrating environment; the synergistic effect of these components enables the efficient extraction of polypeptide components.

[0046] In this disclosure, a very small amount of sodium sulfate precipitation may be observed in the first eutectic solvent. This precipitation is either undissolved or precipitated, which is normal. Furthermore, the salting-out effect of sodium sulfate can continue to play a role in subsequent extraction.

[0047] In this disclosure, in a first eutectic solvent, glycerol can insert between polysaccharide molecular chains, and betaine can open the cell wall through hydration; the combination of the two can increase the polysaccharide yield.

[0048] The present disclosure will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present disclosure are obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0049] Example

[0050] Example 1:

[0051] Example 1 includes the following steps:

[0052] Betaine and glycerol were added to a three-necked flask at a molar ratio of 1:3. The mixture was heated in an oil bath to 90°C and magnetically stirred for 1.5 hours at a speed of 400 rpm to form a colorless, transparent, viscous liquid. The mixture was then cooled to 60°C, and 35% (v / v) of deionized water was added dropwise. The mixture was stirred for another 45 minutes. Then, 1.5 wt% of anhydrous sodium sulfate was added, the temperature was raised to 80°C, and the mixture was stirred until dissolved. The mixture was then allowed to cool naturally to room temperature and stored in a sealed container protected from light to obtain the first eutectic solvent of this embodiment.

[0053] Anhydrous zinc chloride and lactic acid were added to a three-necked flask at a molar ratio of 1:(2-3). Under nitrogen protection, the mixture was heated to 105°C in an oil bath and magnetically stirred for 60 min. Then, the temperature was lowered to 85°C and magnetically stirred for 45 min. Then, the temperature was lowered to 70°C, and choline chloride was added. The molar amount of choline chloride added was half that of anhydrous zinc chloride. The mixture was stirred for 1.5 h. Then, the temperature was lowered to 50°C, and 0.45 wt% mercaptoethanol and 2.0 wt% trehalose were added. The mixture was stirred for 30 min. Then, the mixture was allowed to cool naturally to room temperature. The nitrogen protection was then removed, and the mixture was sealed and stored away from light to obtain the second eutectic solvent of this embodiment.

[0054] Matsutake fruiting bodies are provided; the matsutake fruiting bodies are dried with hot air to constant weight, then sliced, pulverized and passed through a 60-mesh sieve to obtain matsutake powder; the matsutake powder is added to a first eutectic solvent at a material-to-liquid ratio of 1:20, placed in a 60℃ water bath, and magnetically stirred at 350 rpm for 2 hours; after extraction, the mixture is centrifuged at 4000 rpm for 10 minutes to obtain the first extract and the first residue.

[0055] The first residue was mixed with deionized water at a material-to-liquid ratio of 1:2 and stirred at room temperature for 20 minutes with a magnetic stirring speed of 350 rpm. Then, it was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the solid was collected to obtain the treated first residue.

[0056] The treated first residue was mixed with the second eutectic solvent at a material-to-liquid ratio of 1:16 and placed in a 75°C water bath. The mixture was magnetically stirred at 400 rpm for 2 hours. After extraction, the mixture was centrifuged at 4000 rpm for 10 minutes to obtain the second extract and the second residue. The second residue was discarded.

[0057] The first extract was concentrated under reduced pressure at 60℃ to 1 / 3 of its original volume, and 3 times its volume of 95% ethanol was added. The mixture was allowed to stand overnight at 4℃ for alcohol precipitation. The next day, the extract was centrifuged at 6000 rpm for 15 min, the precipitate was collected, and the precipitate was freeze-dried under vacuum to obtain matsutake polysaccharide component A1. The supernatant after centrifugation was evaporated at 50℃ to remove ethanol, concentrated, and ammonium sulfate was added to saturation to 60%. The mixture was allowed to stand at 4℃ for 4 h for salt precipitation, and the precipitate was collected after centrifugation at 6000 rpm for 15 min. The precipitate was desalted using a dialysis bag and then freeze-dried to obtain matsutake polypeptide component A2.

[0058] The second extract was concentrated to 1 / 4 of its original volume under reduced pressure at 60℃, and 4 times its volume of 95% ethanol was added. The mixture was allowed to stand overnight at 4℃ for alcohol precipitation. The next day, the precipitate was collected by centrifugation at 6000 rpm for 15 min, washed three times with anhydrous ethanol, and dried under vacuum to obtain matsutake polysaccharide component B1. The supernatant after centrifugation was concentrated by ultrafiltration using a 3000 Da ultrafiltration membrane and freeze-dried to obtain matsutake polypeptide component B2. The ion chromatogram of matsutake polysaccharide component A1 is shown below. Figure 1 As shown; the ion chromatogram of matsutake polysaccharide component B1 is as follows. Figure 2 As shown.

[0059] Example 2:

[0060] Example 2 includes the following steps:

[0061] Betaine and glycerol were added to a three-necked flask at a molar ratio of 1:3. The mixture was heated in an oil bath to 90°C and magnetically stirred for 1.5 hours at a speed of 400 rpm to form a colorless, transparent, viscous liquid. The mixture was then cooled to 60°C, and 35% (v / v) of deionized water was added dropwise. The mixture was stirred for another 45 minutes. The mixture was then allowed to cool naturally to room temperature and stored in a sealed container away from light to obtain the first eutectic solvent of this embodiment.

[0062] Anhydrous zinc chloride and lactic acid were added to a three-necked flask at a molar ratio of 1:(2-3). Under nitrogen protection, the mixture was heated to 105°C in an oil bath and magnetically stirred for 60 min. Then, the temperature was lowered to 85°C and magnetically stirred for 45 min. Then, the temperature was lowered to 70°C, and choline chloride was added. The molar amount of choline chloride added was half that of anhydrous zinc chloride. The mixture was stirred for 1.5 h. Then, the temperature was lowered to 50°C, and 0.45 wt% mercaptoethanol and 2.0 wt% trehalose were added. The mixture was stirred for 30 min. Then, the mixture was allowed to cool naturally to room temperature. The nitrogen protection was then removed, and the mixture was sealed and stored away from light to obtain the second eutectic solvent of this embodiment.

[0063] Matsutake fruiting bodies are provided; the matsutake fruiting bodies are dried with hot air to constant weight, then sliced, pulverized and passed through a 60-mesh sieve to obtain matsutake powder; 50g of matsutake powder (50g of matsutake powder is used as raw material in the following examples and comparative examples) is added to a first eutectic solvent at a material-to-liquid ratio of 1:20, placed in a 60℃ water bath, and magnetically stirred at 350rpm for 2h; after extraction, the mixture is centrifuged at 4000rpm for 10min to obtain the first extract and the first residue.

[0064] The first residue was mixed with deionized water at a material-to-liquid ratio of 1:2 and stirred at room temperature for 20 minutes with a magnetic stirring speed of 350 rpm. Then, it was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the solid was collected to obtain the treated first residue.

[0065] The treated first residue was mixed with the second eutectic solvent at a material-to-liquid ratio of 1:16 and placed in a 75°C water bath. The mixture was magnetically stirred at 400 rpm for 2 hours. After extraction, the mixture was centrifuged at 4000 rpm for 10 minutes to obtain the second extract and the second residue. The second residue was discarded.

[0066] The first extract was concentrated under reduced pressure at 60℃ to 1 / 3 of its original volume, and 3 times its volume of 95% ethanol was added. The mixture was allowed to stand overnight at 4℃ for alcohol precipitation. The next day, the extract was centrifuged at 6000 rpm for 15 min, the precipitate was collected, and the precipitate was freeze-dried under vacuum to obtain matsutake polysaccharide component A1. The supernatant after centrifugation was evaporated at 50℃ to remove ethanol, concentrated, and ammonium sulfate was added to saturation to 60%. The mixture was allowed to stand at 4℃ for 4 h for salt precipitation, and the precipitate was collected after centrifugation at 6000 rpm for 15 min. The precipitate was desalted using a dialysis bag and then freeze-dried to obtain matsutake polypeptide component A2.

[0067] The second extract was concentrated under reduced pressure at 60℃ to 1 / 4 of its original volume, and 4 times its volume of 95% ethanol was added. The mixture was allowed to stand overnight at 4℃ for alcohol precipitation. The next day, the extract was centrifuged at 6000 rpm for 15 min, the precipitate was collected, washed three times with anhydrous ethanol, and dried under vacuum to obtain matsutake polysaccharide component B1. The supernatant after centrifugation was concentrated by ultrafiltration using a 3000 Da ultrafiltration membrane and then freeze-dried to obtain matsutake polypeptide component B2.

[0068] The difference between Example 2 and Example 1 is that the first eutectic solvent in Example 2 does not contain anhydrous sodium sulfate.

[0069] Example 3:

[0070] Example 3 includes the following steps:

[0071] Betaine and glycerol were added to a three-necked flask at a molar ratio of 1:3. The mixture was heated in an oil bath to 90°C and magnetically stirred for 1.5 hours at a speed of 400 rpm to form a colorless, transparent, viscous liquid. The mixture was then cooled to 60°C, and 35% (v / v) of deionized water was added dropwise. The mixture was stirred for another 45 minutes. Then, 1.5 wt% of anhydrous sodium sulfate was added, the temperature was raised to 80°C, and the mixture was stirred until dissolved. The mixture was then allowed to cool naturally to room temperature and stored in a sealed container protected from light to obtain the first eutectic solvent of this embodiment.

[0072] Anhydrous zinc chloride and lactic acid were added to a three-necked flask at a molar ratio of 1:(2-3). Under nitrogen protection, the mixture was heated to 105°C in an oil bath and magnetically stirred for 60 min. Then, the temperature was lowered to 85°C and magnetically stirred for 45 min. Then, the temperature was lowered to 70°C, and choline chloride was added. The molar amount of choline chloride added was half that of anhydrous zinc chloride. The mixture was stirred for 1.5 h. Then, it was allowed to cool naturally to room temperature. The nitrogen protection was then removed, and the mixture was sealed and stored away from light to obtain the second eutectic solvent of this embodiment.

[0073] Matsutake fruiting bodies are provided; the matsutake fruiting bodies are dried with hot air to constant weight, then sliced, pulverized and passed through a 60-mesh sieve to obtain matsutake powder; the matsutake powder is added to a first eutectic solvent at a material-to-liquid ratio of 1:20, placed in a 60℃ water bath, and magnetically stirred at 350 rpm for 2 hours; after extraction, the mixture is centrifuged at 4000 rpm for 10 minutes to obtain the first extract and the first residue.

[0074] The first residue was mixed with deionized water at a material-to-liquid ratio of 1:2 and stirred at room temperature for 20 minutes with a magnetic stirring speed of 350 rpm. Then, it was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the solid was collected to obtain the treated first residue.

[0075] The treated first residue was mixed with the second eutectic solvent at a material-to-liquid ratio of 1:16 and placed in a 75°C water bath. The mixture was magnetically stirred at 400 rpm for 2 hours. After extraction, the mixture was centrifuged at 4000 rpm for 10 minutes to obtain the second extract and the second residue. The second residue was discarded.

[0076] The first extract was concentrated under reduced pressure at 60℃ to 1 / 3 of its original volume, and 3 times its volume of 95% ethanol was added. The mixture was allowed to stand overnight at 4℃ for alcohol precipitation. The next day, the extract was centrifuged at 6000 rpm for 15 min, the precipitate was collected, and the precipitate was freeze-dried under vacuum to obtain matsutake polysaccharide component A1. The supernatant after centrifugation was evaporated at 50℃ to remove ethanol, concentrated, and ammonium sulfate was added to saturation to 60%. The mixture was allowed to stand at 4℃ for 4 h for salt precipitation, and the precipitate was collected after centrifugation at 6000 rpm for 15 min. The precipitate was desalted using a dialysis bag and then freeze-dried to obtain matsutake polypeptide component A2.

[0077] The second extract was concentrated under reduced pressure at 60℃ to 1 / 4 of its original volume, and 4 times its volume of 95% ethanol was added. The mixture was allowed to stand overnight at 4℃ for alcohol precipitation. The next day, the extract was centrifuged at 6000 rpm for 15 min, the precipitate was collected, washed three times with anhydrous ethanol, and dried under vacuum to obtain matsutake polysaccharide component B1. The supernatant after centrifugation was concentrated by ultrafiltration using a 3000 Da ultrafiltration membrane and then freeze-dried to obtain matsutake polypeptide component B2.

[0078] The difference between Example 3 and Example 1 is that the second eutectic solvent in Example 3 does not contain mercaptoethanol and trehalose.

[0079] Example 4:

[0080] Example 4 includes the following steps:

[0081] Betaine and glycerol were added to a three-necked flask at a molar ratio of 1:3. The mixture was heated in an oil bath to 90°C and magnetically stirred for 1.5 hours at a speed of 400 rpm to form a colorless, transparent, viscous liquid. The mixture was then cooled to 60°C, and 35% (v / v) of deionized water was added dropwise. The mixture was stirred for another 45 minutes. Then, 1.5 wt% of anhydrous sodium sulfate was added, the temperature was raised to 80°C, and the mixture was stirred until dissolved. The mixture was then allowed to cool naturally to room temperature and stored in a sealed container protected from light to obtain the first eutectic solvent of this embodiment.

[0082] Choline chloride and lactic acid were added to a three-necked flask in a molar ratio of 1:1. Under nitrogen protection, the mixture was heated to 80°C in an oil bath and magnetically stirred for 60 min to form a colorless and transparent liquid. The mixture was then cooled to 50°C, and 0.45 wt% mercaptoethanol and 2.0 wt% trehalose were added. The mixture was stirred for 30 min and then allowed to cool naturally to room temperature. The nitrogen protection was then removed, and the mixture was sealed and stored away from light to obtain the second eutectic solvent of this embodiment.

[0083] Matsutake fruiting bodies are provided; the matsutake fruiting bodies are dried with hot air to constant weight, then sliced, pulverized and passed through a 60-mesh sieve to obtain matsutake powder; the matsutake powder is added to a first eutectic solvent at a material-to-liquid ratio of 1:20, placed in a 60℃ water bath, and magnetically stirred at 350 rpm for 2 hours; after extraction, the mixture is centrifuged at 4000 rpm for 10 minutes to obtain the first extract and the first residue.

[0084] The first residue was mixed with deionized water at a material-to-liquid ratio of 1:2 and stirred at room temperature for 20 minutes with a magnetic stirring speed of 350 rpm. Then, it was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the solid was collected to obtain the treated first residue.

[0085] The treated first residue was mixed with the second eutectic solvent at a material-to-liquid ratio of 1:16 and placed in a 75°C water bath. The mixture was magnetically stirred at 400 rpm for 2 hours. After extraction, the mixture was centrifuged at 4000 rpm for 10 minutes to obtain the second extract and the second residue. The second residue was discarded.

[0086] The first extract was concentrated under reduced pressure at 60℃ to 1 / 3 of its original volume, and 3 times its volume of 95% ethanol was added. The mixture was allowed to stand overnight at 4℃ for alcohol precipitation. The next day, the extract was centrifuged at 6000 rpm for 15 min, the precipitate was collected, and the precipitate was freeze-dried under vacuum to obtain matsutake polysaccharide component A1. The supernatant after centrifugation was evaporated at 50℃ to remove ethanol, concentrated, and ammonium sulfate was added to saturation to 60%. The mixture was allowed to stand at 4℃ for 4 h for salt precipitation, and the precipitate was collected after centrifugation at 6000 rpm for 15 min. The precipitate was desalted using a dialysis bag and then freeze-dried to obtain matsutake polypeptide component A2.

[0087] The second extract was concentrated under reduced pressure at 60℃ to 1 / 4 of its original volume, and 4 times its volume of 95% ethanol was added. The mixture was allowed to stand overnight at 4℃ for alcohol precipitation. The next day, the extract was centrifuged at 6000 rpm for 15 min, the precipitate was collected, washed three times with anhydrous ethanol, and dried under vacuum to obtain matsutake polysaccharide component B1. The supernatant after centrifugation was concentrated by ultrafiltration using a 3000 Da ultrafiltration membrane and then freeze-dried to obtain matsutake polypeptide component B2.

[0088] The difference between Example 4 and Example 1 is that the second eutectic solvent in Example 4 does not contain zinc chloride.

[0089] Comparative Example 1:

[0090] Comparative Example 1 includes the following steps:

[0091] Matsutake fruiting bodies were provided; the fruiting bodies were dried with hot air to constant weight, then sliced, pulverized, and passed through a 60-mesh sieve to obtain dried matsutake powder; the powder was added to deionized water at a material-to-liquid ratio of 1:20, and extracted in a 90℃ water bath with magnetic stirring at 400 rpm for 4 hours; after extraction, the mixture was centrifuged at 6000 rpm for 15 minutes to obtain the first extract and the first residue. The first residue was added to deionized water at a material-to-liquid ratio of 1:16, and extracted in a 90℃ water bath with magnetic stirring at 400 rpm for 4 hours; after extraction, the mixture was centrifuged at 6000 rpm for 15 minutes to obtain the second extract and the second residue, which was discarded. The first and second extracts were combined and concentrated under reduced pressure at 60°C to 1 / 4 of their original volume. Four volumes of 95% ethanol were added to the concentrated extract, and the mixture was allowed to stand overnight at 4°C for alcohol precipitation. The next day, the mixture was centrifuged at 6000 rpm for 15 min, the precipitate was collected, washed three times with anhydrous ethanol, and dried under vacuum to obtain the crude matsutake polysaccharide of this comparative example. The supernatant after centrifugation was rotary evaporated at 50°C to remove ethanol, yielding a concentrated solution. Ammonium sulfate was added to the solution until saturation reached 60%, and the mixture was allowed to stand at 4°C for 4 h for salt precipitation. The solution was then centrifuged at 6000 rpm for 15 min, the precipitate was collected, desalted using a dialysis bag, and then freeze-dried to obtain the crude matsutake polypeptide of this comparative example.

[0092] Content testing: The polysaccharide content of the extracts in Examples 1-4 and Comparative Example 1 was determined using the sulfuric acid-phenol method; the polypeptide content of the extracts in Examples 1-4 and Comparative Example 1 was determined using the biuret method; the mass and content values ​​of each component are shown in Table 1; where the former represents the mass of each component, and the latter represents the polysaccharide / polypeptide content of each component.

[0093] Table 1

[0094] Example A1 A2 B1 B2 Example 1 8.23g，78% 0.50g，45% 1.11g，52% 3.85g，82% Example 2 7.52g，68% 1.63g，41% 1.32g，48% 3.17g，76% Example 3 8.09g，77% 0.57g，44% 1.81g，36% 2.04g，55% Example 4 8.15g，77% 0.55g，45% 1.96g，29% 1.20g，32% Comparative Example 1 9.58g，49% - - 1.8g，28%

[0095] Comparing Examples 1 and 2, it can be seen that the absence of sodium sulfate leads to a decrease in polysaccharide selectivity in the first step, an increase in polypeptide co-dissolution, and a significant reduction in the distinction between primary and secondary products. Comparing Examples 1 and 3, it can be seen that the absence of mercaptoethanol results in the failure to break disulfide bonds, preventing the release of embedded proteins; the absence of trehalose leads to the denaturation of a small amount of polypeptides, resulting in a significant decrease in polypeptide selectivity in the second step. Comparing Examples 1 and 4, it can be seen that the absence of zinc chloride's coordination effect significantly reduces polypeptide extraction efficiency. Comparing Examples 1 and Comparative Example 1, it can be seen that the traditional method involves simultaneous dissolution of polysaccharides and polypeptides, making separation difficult; prolonged high temperatures lead to polysaccharide hydrolysis and polypeptide denaturation; the overall efficiency is significantly inferior to the stepwise selective extraction using a dual eutectic solvent disclosed in this invention.

[0096] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein.

Claims

1. A method for extracting matsutake polysaccharides and polypeptides from matsutake mushrooms based on a eutectic solvent, characterized in that, The extraction method includes the following steps: Step 1: Prepare the first eutectic solvent and the second eutectic solvent; Step 2: Prepare matsutake mushroom powder; mix the matsutake mushroom powder with the first eutectic solvent at a material-to-liquid ratio of 1:(15-25), and place it in a water bath at 50℃-70℃ for stirring and extraction for 1-3 hours; after extraction, centrifuge the obtained extract to obtain the first extract and the first residue; Step 3: Process the first residue to obtain the processed first residue; Step 4: Mix the treated first residue with the second eutectic solvent at a material-to-liquid ratio of 1:(12-20), and place it in a water bath at 70℃-80℃ for stirring and extraction for 1-3 hours; after extraction, centrifuge the resulting extract to obtain the second extract and the second residue; discard the second residue. Step 5: The first extract was concentrated under reduced pressure to 1 / 3 of its original volume; then 3 times the volume of 95% ethanol was added for alcohol precipitation, and the mixture was allowed to stand overnight; then the precipitate was collected by centrifugation to obtain matsutake polysaccharide component A1; the supernatant obtained by centrifugation was concentrated, ammonium sulfate was added, and after standing, salting was performed; after centrifugation, the precipitate was collected, and after desalting and dialysis, it was freeze-dried to obtain matsutake polypeptide component A2; Step 6: The second extract was concentrated to 1 / 4 of its original volume under reduced pressure; then 4 times the volume of 95% ethanol was added for alcohol precipitation, and the mixture was allowed to stand overnight; then the precipitate was collected by centrifugation, washed and dried to obtain matsutake polysaccharide component B1; the supernatant obtained by centrifugation was concentrated by ultrafiltration through a 3000 Da ultrafiltration membrane, and then freeze-dried to obtain matsutake polypeptide component B2.

2. The method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent according to claim 1, characterized in that, The hydrogen bond donor of the first eutectic solvent is selected from glycerol or urea, and the hydrogen bond acceptor is selected from betaine or choline chloride; the hydrogen bond donor of the second eutectic solvent is selected from lactic acid, levulinic acid or malic acid, and the hydrogen bond donor includes a first hydrogen bond donor and a second hydrogen bond donor, the first hydrogen bond acceptor is selected from zinc chloride, and the second hydrogen bond acceptor is selected from betaine or choline chloride.

3. The method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent according to claim 2, characterized in that, The hydrogen bond donor of the first eutectic solvent is selected from glycerol, and the hydrogen bond acceptor is selected from betaine; the hydrogen bond donor of the second eutectic solvent is selected from lactic acid, and the hydrogen bond donor includes a first hydrogen bond donor and a second hydrogen bond donor. The first hydrogen bond acceptor is selected from zinc chloride, and the second hydrogen bond acceptor is selected from choline chloride.

4. The method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent according to claim 3, characterized in that, The first eutectic solvent is prepared by the following steps: Step 1-a: Add betaine and glycerin in a molar ratio of 1:(2.5-3.5) to a three-necked flask, heat in an oil bath to 90°C, and stir magnetically for 1-2 hours; Step 2-a: Cool down to 60℃, add deionized water dropwise, the volume of deionized water added should be 35%-40% of the volume of the liquid formed in step 1-a; continue stirring for 30-60 minutes; Step 3-a: Add anhydrous sodium sulfate; the mass of anhydrous sodium sulfate added is 1.2%-2.0% of the mass of the liquid formed in step 2-a; heat to 80°C, stir to dissolve, then cool naturally to room temperature, seal and store in the dark; obtain the first eutectic solvent.

5. The method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent according to claim 3, characterized in that, The second eutectic solvent is prepared by the following steps: Step 1-b: Add anhydrous zinc chloride and lactic acid in a molar ratio of 1:(2-3) to a three-necked flask, heat to 105°C in an oil bath under nitrogen protection, and stir magnetically for 45-75 min; then cool to 85°C and stir magnetically for 30-60 min. Step 2-b: Further cool to 70°C, then add choline chloride; the molar amount of choline chloride added is 40%-60% of the molar amount of anhydrous zinc chloride; stir for 1-2 hours; Step 3-b: Further cool to 50°C, then add mercaptoethanol and trehalose; the mass of mercaptoethanol added is 0.2%-0.5% of the mass of the liquid formed in step 2-b; the mass of trehalose added is 1.5%-2.5% of the mass of the liquid formed in step 2-a; stir for 20-45 minutes; then cool naturally to room temperature, then remove the nitrogen protection, seal and store in the dark; to obtain the second eutectic solvent.

6. The method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent according to claim 1, characterized in that, The dried matsutake mushroom powder is prepared through the following steps: Step 1-c: Provide matsutake fruiting bodies; dry the matsutake fruiting bodies with hot air to constant weight, then slice, pulverize and pass through a 60-80 mesh sieve to obtain the dried matsutake powder.

7. The method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent according to claim 1, characterized in that, Step 3 includes: Step 3-1: Mix the first residue with deionized water at a material-to-liquid ratio of 1:(1-3) and stir at room temperature for 10-30 min; then centrifuge at 2000-4000 rpm for 5-15 min, discard the supernatant, collect the solid, and obtain the treated first residue.

8. A matsutake mushroom polysaccharide, characterized in that, The matsutake polysaccharide comprises matsutake polysaccharide component A1 extracted by the method for extracting matsutake polysaccharides and polypeptides based on a eutectic solvent as described in any one of claims 1-7.

9. A matsutake mushroom polypeptide, characterized in that, The matsutake polypeptide comprises matsutake polypeptide component B2 extracted by the simultaneous extraction method of matsutake polysaccharide and polypeptide based on eutectic solvent as described in any one of claims 1-7.

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