A method for extracting mycelium polysaccharide

Abstract

The application provides a mycelium polysaccharide extraction method and belongs to the technical field of polysaccharide extraction. The method comprises the following steps: the mycelium is sequentially subjected to impurity removal, cleaning and grinding to obtain mycelium slurry; the mycelium slurry is mixed with a freezing liquid and then subjected to cyclic freezing, and after thawing, a first extraction liquid is obtained; the first extraction liquid is mixed with enzymolysis granules and then subjected to enzymolysis, and after the enzymolysis is completed, a second extraction liquid is obtained by filtration; the second extraction liquid is added with sodium sulfate and a polysaccharide with a medium degree of polymerization to adjust the pH value and then subjected to ultrasonic treatment to obtain a third extraction liquid; and then the third extraction liquid is sequentially subjected to filtration, decolorization, desalination, concentration and drying to obtain the mycelium polysaccharide. The extraction method can extract macromolecular polysaccharides and further improve the functionality of the fungal polysaccharide product; moreover, the extraction has high purity and low protein content; the step of removing protein can be omitted, thereby saving a large amount of cost, and the process scheme is simple and suitable for large-scale promotion.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction technology, and specifically relates to a method for extracting mycelial polysaccharides. Background Technology

[0002] Edible fungi, as an important fungal resource in nature, are not only rich in nutrients such as protein, vitamins, and minerals, but also contain a variety of bioactive substances. Among them, edible fungal polysaccharides have attracted widespread attention from researchers and industry. Due to their unique physiological activities, such as immune regulation, anti-tumor, antioxidant, and hypoglycemic effects, edible fungal polysaccharides have shown great application potential in the pharmaceutical, health care, and food industries.

[0003] Polysaccharides range in molecular weight from several thousand to several million. The larger the molecular weight, the lower the water solubility. Therefore, while commercially available fungal polysaccharides possess high purity and certain efficacy, the lack of large-molecule polysaccharides reduces their overall effectiveness. Large polysaccharide molecules have poor water solubility, making complete extraction with water as a solvent difficult. Extraction processes for edible fungal polysaccharides mainly include water extraction, alcohol extraction, enzymatic hydrolysis, ultrasound-assisted extraction, and microwave-assisted extraction. Hot water extraction is the most basic and commonly used method, offering advantages such as simplicity and ease of implementation, but it suffers from low extraction efficiency and the presence of impurities in the extracted crude polysaccharide. Enzymatic hydrolysis utilizes enzymes to disrupt cell walls, accelerating the dissolution of intracellular polysaccharides; however, strict control of reaction temperature and pH is crucial during extraction. Ultrasonic extraction technology leverages the strong cavitation effect of ultrasound to disrupt cell tissues, increasing the frequency of polysaccharide molecule movement and thus accelerating the dissolution of intracellular polysaccharides, thereby improving polysaccharide yield. While the above extraction methods promote the precipitation of polysaccharides and increase the yield, they also increase the precipitation of non-polysaccharide substances, affecting the purity of the final polysaccharide. Among them, protein removal usually uses molecular sieve filtration to screen large molecular proteins through pore size, but this will reduce the yield of large molecular polysaccharides at the same time.

[0004] Therefore, there is an urgent need for a method to simultaneously extract small-molecule and large-molecule polysaccharides from mycelia while reducing the content of impurities. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for extracting mycelial polysaccharides. The mycelial polysaccharide extraction method of this invention can extract macromolecular polysaccharides to further improve the functionality of fungal polysaccharide products.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for extracting mycelial polysaccharides, comprising the following steps:

[0008] S1. The mycelium is sequentially subjected to impurity removal, washing, and grinding to obtain mycelium slurry;

[0009] S2. The mycelial slurry is mixed with the freezing liquid and then circulated and frozen. After thawing, the first extract is obtained.

[0010] S3. Mix the first extract with the enzymatically hydrolyzed particles and then perform enzymatic hydrolysis. After the enzymatic hydrolysis is completed, filter the mixture to obtain the second extract.

[0011] S4. After adding medium-polymerization degree isomalt / malt polysaccharide and sodium sulfate to the second extract, adjust the pH value and sonicate to obtain the third extract;

[0012] S5. Then the third extract is sequentially filtered, decolorized, desalted, concentrated, and dried to obtain mycelial polysaccharide.

[0013] Preferably, the mycelium in S1 is derived from shiitake mushrooms, *Sanghuang* fungus, black fungus, *Ganoderma lucidum*, *Dictyophora indicum*, and *Hericium erinaceus*. Edible fungi polysaccharides have significant effects in immune regulation, antitumor activity, antioxidation, and hypoglycemia. Currently, commercially available extracts are generally small-molecule polysaccharides, neglecting the efficacy of large-molecule polysaccharides. For example, larger-molecule polysaccharides have stronger antioxidant activity, possibly due to their better ability to scavenge free radicals. The polysaccharide component PEP-2, extracted, isolated, and purified from *Pleurotus eryngii*, can exert antitumor activity by arresting the cell cycle of liver cancer cells and inducing apoptosis. Therefore, the extraction and utilization of large-molecule polysaccharides from common edible fungi shows good promise.

[0014] Preferably, the mass ratio of mycelial slurry to cryosol in S2 is 1:(1-10).

[0015] Preferably, the composition of the cryosol in S2 is 80 wt% water, 15 wt% ethanol, 3 wt% sodium chloride, and 2 wt% propylene glycol. By adopting the above technical solution, the cryosol can rapidly lower the temperature, causing swelling due to the formation of intracellular ice particles and the increased salt concentration of the remaining cell fluid, thereby breaking down the cell structure and effectively destroying the cell structure and promoting the release of intracellular substances.

[0016] Preferably, the specific method of cyclic freezing in S2 is as follows: the mixture is cooled to -25°C at a cooling rate of 3°C / min, then naturally warmed to room temperature, and the cycle is repeated 3 times.

[0017] Preferably, the mass ratio of the first extract to the enzymatically hydrolyzed particles in S3 is 100:(1-5).

[0018] In this invention, the enzymatically hydrolyzed particles are easily separated and reused by immobilizing relevant enzymes that can hydrolyze cell walls; they can be derived from commercially available sources or prepared in-house.

[0019] More preferably, the enzymatically hydrolyzed particles are the pectinase complex particles described in patent CN118726329A.

[0020] Preferably, the enzymatic hydrolysis temperature in S3 is 35°C, and the enzymatic hydrolysis time is 10–24 h.

[0021] Preferably, the mass ratio of the second extract, medium-polymerization degree isomalt / maltose, and sodium sulfate in S4 is 100:(2-4):(5-8). In this invention, the inventors discovered that medium-polymerization degree isomalt / maltose can improve the solubility of macromolecular polysaccharides; medium-polymerization degree isomalt / maltose is obtained by gelatinizing linear dextrin and then adding maltose and glycosyltransferase. It can be prepared by referring to "Xue Naixiang, Wang Yanli, Dong Jingjing, et al. Enzymatic synthesis and solubilizing effect of medium-polymerization degree isomalt / maltose [J]. Food and Fermentation Industries, 2022, 48(13):141-147."

[0022] However, while improving the solubility of macromolecular polysaccharides, the inventors also encountered an increase in protein solubility, resulting in a decrease in the purity of the obtained polysaccharides. The inventors improved the purity of polysaccharides by reducing protein solubility and found that in this system, changing the pH and salting out could significantly inhibit high protein solubility without affecting the solubility and properties of the polysaccharides. Only desalination was required in the purification step, without the need for molecular sieve filtration.

[0023] Preferably, the molecular weight of the medium-polymerization degree isomalt / maltose in S4 is 6-8 kDa. By adopting the above technical solution, using an appropriate amount of medium-polymerization degree isomalt / maltose with a molecular weight of 6-8 kDa provides good solubility for macromolecular polysaccharides; too little will not have a functional effect, while too much will easily dissolve impurities.

[0024] Preferably, the pH value is adjusted to 4.2 to 4.5 in step S4.

[0025] Preferably, the decolorization is performed using activated carbon adsorption.

[0026] Preferably, the desalination is carried out using ion exchange resin, specifically by using a strong acid cation exchange resin D001 and a weak base anion exchange resin D301P in series for desalination.

[0027] Beneficial technical effects:

[0028] The mycelial polysaccharide extraction method of this invention can extract macromolecular polysaccharides to further improve the functionality of fungal polysaccharide products; moreover, the extraction purity is high and the protein content is low; the protein removal step can be omitted, thereby saving a lot of costs, and the process is simple and suitable for large-scale promotion. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0030] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0035] The enzymatically hydrolyzed particles in this invention were prepared according to the pectinase composite particles described in patent CN118726329A; the medium-polymerization degree isomalt / malt polysaccharide was prepared according to "Xue Naixiang, Wang Yanli, Dong Jingjing, et al. Study on enzymatic synthesis and solubilizing effect of medium-polymerization degree isomalt / malt polysaccharide [J]. Food and Fermentation Industries, 2022, 48(13):141-147.", and medium-polymerization degree isomalt / malt polysaccharides of 6.1, 7.3 and 8.1 kDa were obtained.

[0036] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0037] Example 1

[0038] This embodiment provides a method for extracting mycelial polysaccharides, including the following steps:

[0039] S1. The mycelium of shiitake mushrooms is successively subjected to impurity removal, washing, and grinding to obtain mycelium slurry;

[0040] S2. The mycelial slurry and the cryosol were mixed at a mass ratio of 1:1 and then cooled to -25°C at a cooling rate of 3°C / min. The mixture was then allowed to rise naturally to room temperature and the process was repeated 3 times. After the mixture was thawed, the first extract was obtained. The cryosol consisted of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0041] S3. Mix the first extract with the enzymatically hydrolyzed particles at a mass ratio of 100:1 and enzymatically hydrolyze at 35°C for 18 hours. After the enzymatic hydrolysis is completed, filter to obtain the second extract.

[0042] S4. After adding medium-polymerization degree isomalt / malt polysaccharide and sodium sulfate to the second extract, adjust the pH value to 4.3, and then sonicate to obtain the third extract; wherein the mass ratio of the second extract, medium-polymerization degree isomalt / malt polysaccharide (molecular weight 7.3kDa), and sodium sulfate is 100:3:6;

[0043] S5. Then, the third extract is subjected to centrifugation, filtration, decolorization, desalting, concentration, and drying to obtain mycelial polysaccharides; wherein the amount of activated carbon added is 2%, and the decolorization time is 2h; the specific method of ion exchange is: desalting is performed by using strong acid cation resin D001 and weak base anion resin D301P in series; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, and the desalting temperature is 35℃; the drying is performed by freeze drying.

[0044] Example 2

[0045] This embodiment provides a method for extracting mycelial polysaccharides, including the following steps:

[0046] S1. The mycelium of shiitake mushrooms is successively subjected to impurity removal, washing, and grinding to obtain mycelium slurry;

[0047] S2. The mycelial slurry and the cryosol were mixed at a mass ratio of 1:10 and then cooled to -25°C at a cooling rate of 3°C / min. The mixture was then allowed to rise naturally to room temperature and the mixture was circulated 3 times. After melting, the first extract was obtained. The cryosol consisted of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0048] S3. Mix the first extract with the enzymatically hydrolyzed particles at a mass ratio of 100:5 and enzymatically hydrolyze at 35°C for 10 hours. After the enzymatic hydrolysis is completed, filter to obtain the second extract.

[0049] S4. After adding medium-polymerization degree isomalt / malt polysaccharide and sodium sulfate to the second extract, adjust the pH value to 4.2, and then perform sonication to obtain the third extract; wherein the mass ratio of the second extract, medium-polymerization degree isomalt / malt polysaccharide (molecular weight 6.1kDa), and sodium sulfate is 100:2:5;

[0050] S5. Then, the third extract is subjected to centrifugation, filtration, decolorization, desalting, concentration, and drying to obtain mycelial polysaccharides; wherein the amount of activated carbon added is 1%, and the decolorization time is 1 h; the specific method of ion exchange is: desalting is performed by using strong acid cation resin D001 and weak base anion resin D301P in series; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, and the desalting temperature is 30℃; the drying is performed by freeze drying.

[0051] Example 3

[0052] This embodiment provides a method for extracting mycelial polysaccharides, including the following steps:

[0053] S1. The mycelium of shiitake mushrooms is successively subjected to impurity removal, washing, and grinding to obtain mycelium slurry;

[0054] S2. The mycelial slurry and the cryosol were mixed at a mass ratio of 1:5 and then cooled to -25°C at a cooling rate of 3°C / min. The mixture was then allowed to rise naturally to room temperature and the mixture was circulated 3 times. After melting, the first extract was obtained. The cryosol consisted of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0055] S3. Mix the first extract with the enzymatically hydrolyzed particles at a mass ratio of 100:3 and enzymatically hydrolyze at 35°C for 12 hours. After the enzymatic hydrolysis is completed, filter to obtain the second extract.

[0056] S4. After adding medium-polymerization degree isomalt / malt polysaccharide and sodium sulfate to the second extract, adjust the pH value to 4.5, and then sonicate to obtain the third extract; wherein the mass ratio of the second extract, medium-polymerization degree isomalt / malt polysaccharide (molecular weight 8.1kDa), and sodium sulfate is 100:4:8;

[0057] S5. Then, the third extract is subjected to centrifugation, filtration, decolorization, desalting, concentration, and drying to obtain mycelial polysaccharides; wherein the activated carbon addition is 3%, the decolorization time is 3h; the specific ion exchange method is: desalting is performed by using strong acid cation resin D001 and weak base anion resin D301P in series; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, the desalting temperature is 40℃; the drying is performed by freeze drying.

[0058] Example 4

[0059] The extraction method in this embodiment is the same as that in Embodiment 1, except that the mycelium is black fungus mycelium.

[0060] Example 5

[0061] The extraction method in this embodiment is the same as that in Embodiment 1, except that the mycelium is Hericium erinaceus mycelium.

[0062] Comparative Example 1

[0063] This comparative example provides a method for extracting mycelial polysaccharides, including the following steps:

[0064] S1. The mycelium of shiitake mushrooms is successively subjected to impurity removal, washing, and grinding to obtain mycelium slurry;

[0065] S2. The mycelial slurry and the cryosol were mixed at a mass ratio of 1:1 and then cooled to -25°C at a cooling rate of 3°C / min. The mixture was then allowed to rise naturally to room temperature and the process was repeated 3 times. After the mixture was thawed, the first extract was obtained. The cryosol consisted of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0066] S3. Mix the first extract with the enzymatically hydrolyzed particles at a mass ratio of 100:1 and enzymatically hydrolyze at 35°C for 18 hours. After the enzymatic hydrolysis is completed, filter to obtain the second extract.

[0067] S4. Add the second extract to medium-polymerization degree isomalt / malt polysaccharide and then sonicate to obtain the third extract; wherein the mass ratio of the second extract to medium-polymerization degree isomalt / malt polysaccharide (molecular weight 7.3kDa) is 100:3;

[0068] S5. Then, the third extract is subjected to centrifugation, filtration, decolorization, desalting, concentration, and drying to obtain mycelial polysaccharides; wherein the amount of activated carbon added is 2%, and the decolorization time is 2h; the specific method of ion exchange is: desalting is performed by using strong acid cation resin D001 and weak base anion resin D301P in series; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, and the desalting temperature is 35℃; the drying is performed by freeze drying.

[0069] Comparative Example 2

[0070] This comparative example provides a method for extracting mycelial polysaccharides, including the following steps:

[0071] S1. The mycelium of shiitake mushrooms is successively subjected to impurity removal, washing, and grinding to obtain mycelium slurry;

[0072] S2. The mycelial slurry and the cryosol were mixed at a mass ratio of 1:1 and then cooled to -25°C at a cooling rate of 3°C / min. The mixture was then allowed to rise naturally to room temperature and the process was repeated 3 times. After the mixture was thawed, the first extract was obtained. The cryosol consisted of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0073] S3. Mix the first extract with the enzymatically hydrolyzed particles at a mass ratio of 100:1 and enzymatically hydrolyze at 35°C for 18 hours. After the enzymatic hydrolysis is completed, filter to obtain the second extract.

[0074] S4. After adding sodium sulfate to the second extract, adjust the pH to 4.3, and then sonicate to obtain the third extract; wherein the mass ratio of the second extract to sodium sulfate is 100:6.

[0075] S5. Then, the third extract is subjected to centrifugation, filtration, decolorization, desalting, concentration, and drying to obtain mycelial polysaccharides; wherein the amount of activated carbon added is 2%, and the decolorization time is 2h; the specific method of ion exchange is: desalting is performed by using strong acid cation resin D001 and weak base anion resin D301P in series; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, and the desalting temperature is 35℃; the drying is performed by freeze drying.

[0076] Comparative Example 3

[0077] The extraction method in this embodiment is the same as in Example 1, except that the molecular weight of the medium-polymerization degree isomalt / malt polysaccharide in S4 is 2kDa.

[0078] Comparative Example 4

[0079] The extraction method in this embodiment is the same as in Example 1, except that the molecular weight of the medium-polymerization degree isomalt / malt polysaccharide in S4 is 10 kDa.

[0080] Comparative Example 5

[0081] The extraction method in this embodiment is the same as that in embodiment 1, except that the cryosol is replaced with water in S2.

[0082] Experimental Example 1

[0083] High-performance gel permeation chromatography (HPLC) can be used to determine the molecular weight and distribution of polysaccharides.

[0084] Chromatographic conditions:

[0085] Shimadzu DGU-20A High Performance Liquid Chromatograph;

[0086] High performance liquid chromatography column SB-806MHQ (8.0 mm × 300 nm);

[0087] TSKPWXL protective post (6.0mm×40mm);

[0088] Mobile phase: water; flow rate: 0.6 mL / min;

[0089] Column temperature: 35℃;

[0090] Injection volume 5 μL

[0091] Preparation of injection solution

[0092] Weigh 2 mg of each of the nine dextran standards (relative molecular weight 5000-500000 Da), dissolve in 1 mL of deionized water, centrifuge, and take the supernatant. Prepare polysaccharide solutions of the above examples and comparative examples using the same method. Determine the number-average molecular weight of the polysaccharides by HPLC-GPC, as shown in Table 1.

[0093] Table 1. Statistics on the molecular weight of polysaccharides

[0094]

[0095] As shown in Table 1, the content of macromolecular polysaccharides >100kDa in Examples 1 to 5 of the present invention is significantly increased. The content varies depending on the type of fungus extracted, but all show an increasing trend. It also shows that reducing protein solubility while using solubilization can increase the content of macromolecular polysaccharides. Comparative Example 5 shows that using quick-freezing liquid is more conducive to the efflux of macromolecular polysaccharides.

[0096] Experimental Example 2

[0097] The protein content of the polysaccharide solutions (prepared according to the method of Experimental Example 1) obtained in the examples and comparative examples was determined by Coomassie brilliant blue staining. The results are shown in Table 2.

[0098] Table 2

[0099] project Protein content % Example 1 3.28 Example 2 3.76 Example 3 3.41 Example 4 4.12 Example 5 3.97 Comparative Example 1 13.7 Comparative Example 2 3.02 Comparative Example 3 2.29 Comparative Example 4 5.27 Comparative Example 5 3.37

[0100] As shown in Table 2, the protein content in Examples 1 to 5 of this invention is low even without fine screening, indicating that the solubility of protein is reduced while solubilizing, thus eliminating the need for protein removal process. Even if the polysaccharide purity is further improved, the cost of protein removal is significantly reduced.

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting mycelium polysaccharide, characterized in that, The method comprises the following steps: S1. The mycelium is sequentially subjected to impurity removal, cleaning, and grinding to obtain a mycelium slurry; S2. The mycelium slurry is mixed with a freezing liquid, and then subjected to cyclic freezing, and after thawing, a first extraction liquid is obtained; S3. The first extraction liquid is mixed with enzymolysis granules, and then subjected to enzymolysis, and after the enzymolysis is completed, a second extraction liquid is obtained by filtration; S4. The second extraction liquid is added to medium-molecular-weight iso-malt / malt polysaccharides and sodium sulfate, and then subjected to ultrasonic treatment after the pH value is adjusted to obtain a third extraction liquid; S5. Then, the third extraction liquid is sequentially subjected to filtration, decolorization, desalination, concentration, and drying to obtain a mycelium polysaccharide. The freezing liquid in S2 comprises water 80wt%, ethanol 15wt%, sodium chloride 3wt%, and propylene glycol 2wt%. The enzymolysis granules in S3 are pectinase composite granules. The medium-molecular-weight iso-malt / malt polysaccharides in S4 have a molecular weight of 6-8kDa. The pH value in S4 is adjusted to 4.2-4.

5.

2. The extraction method according to claim 1, characterized in that, The mycelium in S1 is derived from shiitake mushrooms, Sanghun, black fungus, ganoderma lucidum, red bamboo, and monkey head mushroom.

3. The extraction method of claim 1, wherein, The mass ratio of the mycelium slurry to the freezing liquid in S2 is 1:(1-10).

4. The extraction method of claim 1, wherein, The cyclic freezing method in S2 is as follows: the mixed liquid is cooled to-25℃ at a cooling rate of 3℃ / min, and then naturally raised to room temperature and cycled for 3 times.

5. The extraction method of claim 1, wherein, The mass ratio of the first extraction liquid to the enzymolysis granules in S3 is 100:(1-5).

6. The extraction method of claim 1, wherein, The enzymolysis temperature in S3 is 35℃, and the enzymolysis time is 10-24h.

7. The extraction method of claim 1, wherein, The mass ratio of the second extraction liquid to the medium-molecular-weight iso-malt / malt polysaccharides to sodium sulfate in S4 is 100:(2-4):(5-8).

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