Extraction method of mycelium polysaccharide

Abstract

The invention provides an extraction method of mycelium polysaccharide, and belongs to the technical field of polysaccharide extraction. The method disclosed by the invention comprises the following steps: sequentially performing impurity removal, cleaning and grinding on mycelium to obtain mycelium slurry; mixing the mycelium slurry with a refrigerating fluid, and performing circulating freezing to obtain a first extracting solution after melting; mixing the first extracting solution with enzymolysis particles, performing enzymolysis, and filtering after the enzymolysis is completed to obtain a second extracting solution; adding isomalt / malt polysaccharide with medium polymerization degree and sodium sulfate into the second extracting solution, adjusting the pH value, and performing ultrasonic treatment to obtain a third extracting solution; sequentially filtering, decoloring, desalting, concentrating and drying the third extracting solution to obtain mycelium polysaccharide; the extraction method can be used for extracting macromolecular polysaccharide to further improve the functionality of fungus polysaccharide products; the extraction purity is high; the protein content is low; the step of removing protein can be omitted, so that a large amount of cost is saved, and the process scheme is simple and suitable for large-scale popularization.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for extracting mycelium polysaccharide. Background Art

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

[0003] The molecular weight of polysaccharides ranges from thousands to millions. The larger the molecular weight, the worse its water solubility. Therefore, although the fungus polysaccharides currently on the market have high purity and certain efficacy, their efficacy will be partially reduced due to the lack of large molecular weight polysaccharides. Polysaccharide macromolecules have poor water solubility, and it is difficult to extract them completely using water as a solvent in separation and extraction. The extraction process of edible fungus polysaccharides mainly includes water extraction, alcohol extraction, enzymatic hydrolysis, ultrasonic assisted extraction, microwave assisted extraction, etc. Among them, hot water extraction is the most basic and most commonly used polysaccharide extraction method, which has the advantages of simple operation and easy implementation, but the extraction efficiency is low, and the extracted crude polysaccharides contain impurities; enzymatic hydrolysis extraction uses enzymes to destroy cell walls to accelerate the dissolution of polysaccharides in cells. The temperature and pH of the reaction must be strictly controlled during the extraction process. Ultrasonic extraction technology uses the strong cavitation effect generated by ultrasound to destroy tissue cells, increase the movement frequency of polysaccharide molecules, thereby accelerating the dissolution of intracellular polysaccharides and increasing the yield of polysaccharides. Although the above extraction methods promote the precipitation of polysaccharides and improve 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 at the same time it will reduce the yield of large molecular polysaccharides.

[0004] Therefore, there is an urgent need for a method for simultaneously extracting small molecular polysaccharides and large molecular polysaccharides from mycelium and reducing the content of impurities. Summary of the invention

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

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for extracting mycelium polysaccharides, comprising the following steps:

[0008] S1. The mycelium is sequentially removed, cleaned, and ground to obtain a mycelium slurry;

[0009] S2. The mycelium slurry is mixed with the freezing liquid and then cyclically frozen to obtain a first extract after thawing;

[0010] S3. The first extract is mixed with the enzymatically hydrolyzed particles and then enzymatically hydrolyzed, and after the enzymatic hydrolysis is completed, the second extract is filtered to obtain;

[0011] S4. The second extract was added with medium polymerization degree isomalt / maltopolysaccharide, sodium sulfate, and then the pH value was adjusted and ultrasonicated to obtain a third extract;

[0012] S5. The third extract is then filtered, decolorized, desalted, concentrated, and dried in sequence to obtain mycelium polysaccharide.

[0013] Preferably, the mycelium in S1 is derived from shiitake mushrooms, mulberry, black fungus, ganoderma, red bamboo fungus, and hericium. Edible fungus polysaccharides have obvious effects in immunomodulation, anti-tumor, anti-oxidation, and hypoglycemic. The extracts currently available on the market are generally small-molecule polysaccharides, while the efficacy of large-molecule polysaccharides is ignored. For example, polysaccharides with larger molecular weights have stronger antioxidant activity, which may be due to the fact that large-molecule polysaccharides have better free radical scavenging ability. The polysaccharide component PEP-2 extracted, separated and purified from Pleurotus eryngii can play anti-tumor activity by blocking the cell cycle of liver cancer cells and inducing cell apoptosis. Therefore, there is a good prospect for the extraction and utilization of large-molecule polysaccharides in common edible fungi.

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

[0015] Preferably, the composition of the freezing liquid in S2 is 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol. By adopting the above technical solution, the freezing liquid can rapidly reduce the temperature, and the formation of ice particles in the cells and the increase in the salt concentration of the remaining cell fluid cause swelling, which breaks the cell structure, effectively destroys the cell structure, and promotes the release of intracellular substances.

[0016] Preferably, the specific method of cyclic freezing in S2 is: cooling the mixed liquid to -25°C at a cooling rate of 3°C / min and then naturally heating it to room temperature and cycling it 3 times.

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

[0018] In the present invention, the enzymatic granules are easily separated and reused by immobilizing the relevant enzymes that can enzymatically decompose the cell wall; the enzymatic granules can be obtained from commercial sources or prepared by themselves.

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

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

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

[0022] However, while the inventors improved the solubility of macromolecular polysaccharides, the solubility of proteins also increased, and the purity of the obtained polysaccharides decreased. The inventors improved the purity of polysaccharides by reducing the solubility of proteins, and found that in this system, the high solubility of proteins can be significantly inhibited by changing the pH and salting out, without affecting the solubility and performance of the polysaccharides. Desalting is only required in the purification step, and molecular sieve filtration is not required.

[0023] Preferably, the molecular weight of the medium-polymerization degree isomalt / malt polysaccharide in S4 is 6-8 kDa. By adopting the above technical solution, the solubility of the medium-polymerization degree isomalt / malt polysaccharide with a molecular weight of 6-8 kDa is better for macromolecular polysaccharides. Too little will not work, and too much will easily dissolve impurities.

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

[0025] Preferably, the decolorization is carried out by using an activated carbon adsorption method;

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

[0027] Beneficial technical effects:

[0028] The mycelium polysaccharide extraction method of the present invention can extract macromolecular polysaccharides to further improve the functionality of fungus polysaccharide products; the extraction purity is high and the protein content is low; the protein removal step can be omitted to save a lot of costs, and the process scheme is simple and suitable for large-scale promotion. DETAILED DESCRIPTION

[0029] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0030] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

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

[0034] The "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C unless otherwise specified.

[0035] The enzymatic hydrolysis particles in the present invention are prepared with reference to the pectinase composite particles described in patent CN118726329A; the medium-polymerization degree isomalt / malt polysaccharide can be prepared with reference to "Xue Naixiang, Wang Yanli, Dong Jingjing, et al. Study on enzymatic synthesis and solubilization 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 prepared.

[0036] The raw materials and instruments used in the following examples of the present invention are commercially available unless otherwise specified.

[0037] Example 1

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

[0039] S1. The shiitake mushroom mycelium is sequentially removed, cleaned, and ground to obtain a mycelium slurry;

[0040] S2. The mycelium slurry and the freezing liquid are mixed in a mass ratio of 1:1, cooled to -25°C at a cooling rate of 3°C / min, and then naturally raised to room temperature and cycled for 3 times to obtain the first extract after melting; wherein the freezing liquid is composed of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0041] S3. The first extract and the enzymatically hydrolyzed particles were mixed in a mass ratio of 100:1 and then enzymatically hydrolyzed at 35°C for 18 hours. After the enzymatic hydrolysis was completed, the second extract was filtered to obtain a second extract;

[0042] S4. The second extract was added with isomalt / maltopolysaccharide with medium polymerization degree and sodium sulfate, and the pH value was adjusted to 4.3, and then ultrasonicated to obtain a third extract; wherein the mass ratio of the second extract, isomalt / maltopolysaccharide with medium polymerization degree (molecular weight 7.3 kDa), and sodium sulfate was 100:3:6;

[0043] S5. The third extract is then centrifuged, decolorized, desalted, concentrated, and dried to obtain mycelium polysaccharide; wherein the amount of activated carbon added is 2%, and the decolorization time is 2 hours; the specific method of ion exchange is: using strong acid cation resin D001 and weak base anion resin D301P in series for desalination; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, and the desalination temperature is 35°C; and freeze drying is used for drying.

[0044] Example 2

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

[0046] S1. The shiitake mushroom mycelium is sequentially removed, cleaned, and ground to obtain a mycelium slurry;

[0047] S2. The mycelium slurry and the freezing liquid are mixed in a mass ratio of 1:10, cooled to -25°C at a cooling rate of 3°C / min, and then naturally raised to room temperature and cycled for 3 times to obtain the first extract after melting; wherein the freezing liquid is composed of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0048] S3. The first extract and the enzymatically hydrolyzed particles were mixed in a mass ratio of 100:5 and then enzymatically hydrolyzed at 35°C for 10 hours. After the enzymatic hydrolysis was completed, the second extract was filtered to obtain a second extract;

[0049] S4. The second extract was added with isomalt / maltopolysaccharide with medium polymerization degree and sodium sulfate, and the pH value was adjusted to 4.2, and then ultrasonicated to obtain a third extract; wherein the mass ratio of the second extract, isomalt / maltopolysaccharide with medium polymerization degree (molecular weight 6.1 kDa), and sodium sulfate was 100:2:5;

[0050] S5. The third extract is then centrifuged, decolorized, desalted, concentrated, and dried to obtain mycelium polysaccharide; wherein the amount of activated carbon added is 1%, and the decolorization time is 1 hour; the specific method of ion exchange is: using strong acid cation resin D001 and weak base anion resin D301P in series for desalination; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, and the desalination temperature is 30°C; and freeze drying is used for drying.

[0051] Example 3

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

[0053] S1. The shiitake mushroom mycelium is sequentially removed, cleaned, and ground to obtain a mycelium slurry;

[0054] S2. The mycelium slurry and the freezing liquid are mixed in a mass ratio of 1:5, and then cooled to -25°C at a cooling rate of 3°C / min, and then naturally raised to room temperature and cycled for 3 times to obtain the first extract after melting; wherein the freezing liquid is composed of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0055] S3. The first extract and the enzymatically hydrolyzed particles were mixed in a mass ratio of 100:3 and then enzymatically hydrolyzed at 35°C for 12 hours. After the enzymatic hydrolysis was completed, the second extract was filtered to obtain a second extract;

[0056] S4. The second extract was added with isomalt / maltopolysaccharide with medium polymerization degree and sodium sulfate, and the pH value was adjusted to 4.5, and then ultrasonicated to obtain a third extract; wherein the mass ratio of the second extract, isomalt / maltopolysaccharide with medium polymerization degree (molecular weight 8.1 kDa), and sodium sulfate was 100:4:8;

[0057] S5. The third extract is then centrifuged, decolorized, desalted, concentrated, and dried to obtain mycelium polysaccharide; wherein the amount of activated carbon added is 3%, and the decolorization time is 3 hours; the specific method of ion exchange is: using strong acid cation resin D001 and weak base anion resin D301P in series for desalination; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, and the desalination temperature is 40°C; and freeze drying is used for drying.

[0058] Example 4

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

[0060] Example 5

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

[0062] Comparative Example 1

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

[0064] S1. The shiitake mushroom mycelium is sequentially removed, cleaned, and ground to obtain a mycelium slurry;

[0065] S2. The mycelium slurry and the freezing liquid are mixed in a mass ratio of 1:1, cooled to -25°C at a cooling rate of 3°C / min, and then naturally raised to room temperature and cycled for 3 times to obtain the first extract after melting; wherein the freezing liquid is composed of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0066] S3. The first extract and the enzymatically hydrolyzed particles were mixed in a mass ratio of 100:1 and then enzymatically hydrolyzed at 35°C for 18 hours. After the enzymatic hydrolysis was completed, the second extract was filtered to obtain a second extract;

[0067] S4. The second extract was added to the medium polymerization degree isomalt / maltopolysaccharide and then subjected to ultrasound to obtain a third extract; wherein the mass ratio of the second extract to the medium polymerization degree isomalt / maltopolysaccharide (molecular weight 7.3kDa) was 100:3;

[0068] S5. The third extract is then centrifuged, decolorized, desalted, concentrated, and dried to obtain mycelium polysaccharide; wherein the amount of activated carbon added is 2%, and the decolorization time is 2 hours; the specific method of ion exchange is: using strong acid cation resin D001 and weak base anion resin D301P in series for desalination; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, and the desalination temperature is 35°C; and freeze drying is used for drying.

[0069] Comparative Example 2

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

[0071] S1. The shiitake mushroom mycelium is sequentially removed, cleaned, and ground to obtain a mycelium slurry;

[0072] S2. The mycelium slurry and the freezing liquid are mixed in a mass ratio of 1:1, cooled to -25°C at a cooling rate of 3°C / min, and then naturally raised to room temperature and cycled for 3 times to obtain the first extract after melting; wherein the freezing liquid is composed of 80wt% water, 15wt% ethanol, 3wt% sodium chloride, and 2wt% propylene glycol.

[0073] S3. The first extract and the enzymatically hydrolyzed particles were mixed in a mass ratio of 100:1 and then enzymatically hydrolyzed at 35°C for 18 hours. After the enzymatic hydrolysis was completed, the second extract was filtered to obtain a second extract;

[0074] S4. The pH value of the second extract was adjusted to 4.3 after adding sodium sulfate, and then ultrasonically obtained to obtain a third extract; wherein the mass ratio of the second extract to sodium sulfate was 100:6;

[0075] S5. The third extract is then centrifuged, decolorized, desalted, concentrated, and dried to obtain mycelium polysaccharide; wherein the amount of activated carbon added is 2%, and the decolorization time is 2 hours; the specific method of ion exchange is: using strong acid cation resin D001 and weak base anion resin D301P in series for desalination; wherein the ratio of strong acid cation resin D001 to weak base anion resin D301P is 2:1, and the desalination temperature is 35°C; and freeze drying is used for drying.

[0076] Comparative Example 3

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

[0078] Comparative Example 4

[0079] The extraction method of this embodiment is the same as that of embodiment 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 of this embodiment is the same as that of Embodiment 1, except that the freezing liquid is replaced by water in S2.

[0082] Experimental Example 1

[0083] High performance gel chromatography can be used to determine the molecular weight and distribution of polysaccharides

[0084] Chromatographic conditions:

[0085] Shimadzu DGU-20A high performance liquid chromatograph;

[0086] HPLC column SB-806MHQ (8.0 mm × 300 nm);

[0087] TSKPWXL guard column (6.0 mm × 40 mm);

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

[0089] Column temperature: 35°C;

[0090] Injection volume 5μL

[0091] Injection solution preparation

[0092] 2 mg of each of 9 dextran standards (relative molecular weight 5000-500000 Da) were weighed and dissolved in 1 mL of deionized water. The supernatant was collected after centrifugation. The polysaccharide solutions obtained in the above examples and comparative examples were prepared in the same way. The number average molecular weight of the polysaccharide was determined by HPLC-GPC method, as shown in Table 1.

[0093] Table 1 Statistics of polysaccharide molecular weight

[0094]

[0095] According to Table 1, the content of macromolecular polysaccharides > 100 kDa in Examples 1 to 5 of the present invention is significantly increased. The content varies depending on the extracted fungi, but all show a trend of increasing content; and it is shown that the content of macromolecular polysaccharides can be increased by reducing the dissolution of protein while increasing the solubility; Comparative Example 5 shows that the use of quick freezing solution is more conducive to the outflow of macromolecular polysaccharides.

[0096] Experimental Example 2

[0097] The protein content of the polysaccharide solutions obtained in the examples and comparative examples (prepared according to the method of Experimental Example 1) was detected by Coomassie Brilliant Blue staining. The test 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] According to Table 2, the protein content in Examples 1 to 5 of the present invention is low even without fine screening, which means that the solubility of protein is reduced while solubilizing, and the protein removal process can be omitted. Even if the purity of the polysaccharide is further improved, the cost of removing protein is significantly reduced.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting mycelium polysaccharides, characterized in that: The following steps are involved: S1. The mycelium is sequentially removed, cleaned, and ground to obtain a mycelium slurry; S2. The mycelium slurry is mixed with the freezing liquid and then cyclically frozen to obtain a first extract after thawing; S3. The first extract is mixed with the enzymatically hydrolyzed particles and then enzymatically hydrolyzed, and after the enzymatic hydrolysis is completed, the second extract is filtered to obtain; S4. The second extract was added with medium polymerization degree isomalt / maltopolysaccharide, sodium sulfate, and then the pH value was adjusted and ultrasonicated to obtain a third extract; S5. The third extract is then filtered, decolorized, desalted, concentrated, and dried in sequence to obtain mycelium polysaccharide.

2. The extraction method according to claim 1, characterized in that The mycelium in S1 is derived from Lentinus edodes, Phellinus linteus, Auricularia auricula, Ganoderma lucidum, Dictyophora rubra, and Hericium erinaceus.

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

4. The extraction method according to claim 1, characterized in that The composition of the freezing liquid in S2 is 80 wt % of water, 15 wt % of ethanol, 3 wt % of sodium chloride and 2 wt % of propylene glycol.

5. The extraction method according to claim 1, characterized in that The specific method of the cyclic freezing in S2 is: cooling the mixed liquid to -25°C at a cooling rate of 3°C / min and then naturally heating it to room temperature and circulating it for 3 times.

6. The extraction method according to claim 1, characterized in that The mass ratio of the first extract to the enzymatically hydrolyzed particles in S3 is 100:(1-5).

7. The extraction method according to claim 1, characterized in that The enzymatic hydrolysis temperature in S3 is 35° C., and the enzymatic hydrolysis time is 10 to 24 hours.

8. The extraction method according to claim 1, characterized in that The mass ratio of the second extract, medium-polymerization degree isomalt / maltopolysaccharide, and sodium sulfate in S4 is 100:(2-4):(5-8).

9. The extraction method according to claim 1, characterized in that The molecular weight of the medium-polymerization degree isomalt / malt polysaccharide in S4 is 6 to 8 kDa.

10. The extraction method according to claim 1, characterized in that The pH value in S4 is adjusted to 4.2-4.5.

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