Preparation method of pholiota nameko polysaccharide

Abstract

The invention relates to the field of biological extraction, in particular to a pholiota nameko polysaccharide preparation method. The invention provides a pholiota nameko polysaccharide preparation method which comprises the following steps: drying and crushing pholiota nameko sporocarp, and sequentially performing extrusion puffing treatment, water extraction treatment, enzymolysis treatment, alcohol precipitation treatment, concentration treatment and drying treatment to obtain the pholiota nameko polysaccharide. According to the method, the pholiota nameko powder is extruded by adopting extrusion puffing treatment, so that cell walls of pholiota nameko cells are destroyed, and the extraction rate of pholiota nameko polysaccharides is increased; the two steps of water extraction treatment and enzymolysis treatment further ensure the dissolution of pholiota nameko polysaccharide; the alcohol precipitation treatment can ensure the purity of the pholiota nameko polysaccharide. The four steps of extrusion puffing treatment, water extraction treatment, enzymolysis treatment and alcohol precipitation treatment are combined, so that the extraction rate of the pholiota nameko polysaccharide reaches 75-81%, and the purity of the pholiota nameko polysaccharide reaches 90.8-91.4%.

Description

Technical Field

[0001] The invention relates to the field of biological extraction, and in particular to a method for preparing Pleurotus eryngii polysaccharide. Background Art

[0002] Nameko, also known as Pholiota nameko, is a plant of the genus Pholiota in the family Strophariaceae of the order Agaricales. The cap of nameko is flat and hemispherical at first, then flat, yellowish brown, convex in the middle, reddish brown, brown around, and extremely sticky on the surface; the gills are white to grayish white, yellow, and then cinnamon; the flesh is light yellow; the stipe is cylindrical, slightly swollen at the base, yellowish brown; the ring is yellow; it grows in groups or clumps on the trunks of broad-leaved trees or rotten wood in autumn. Nameko is widely cultivated in China and Japan, mainly used as food and traditional medicine. Nameko is rich in various nutrients, such as 33-35% protein, more than 38% carbohydrates, and a variety of minerals and vitamins B1 and B2. It has good health benefits, such as anti-oxidation, anti-inflammatory, immune regulation, and anti-cancer.

[0003] Most of the polysaccharide extraction methods are to carry out liquid fermentation culture of mushroom mycelium or directly use fruiting bodies, extract by hot water extraction, enzyme extraction or acid-base extraction, and further precipitate by organic solvents to finally obtain polysaccharides. However, the polysaccharides extracted by this method have low purity and low extraction rate. For example, the extraction rate of nameko polysaccharides in the Chinese patent "Preparation method of nameko polysaccharide extract" is 72%, and the polysaccharide content is 84%; for another example, the purity of nameko polysaccharides in the Chinese patent "Preparation method of a nameko polysaccharide crude product" is 70%-75%. It can be seen that the nameko polysaccharides extracted by the prior art have low purity and low extraction rate. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing Pleurotus eryngii polysaccharide to solve the problems existing in the above-mentioned prior art. The Pleurotus eryngii polysaccharide prepared by the method provided by the present invention has high purity and high extraction rate.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for preparing Pleurotus eryngii polysaccharide, comprising the following steps:

[0007] After the Pleurotus ostreatus fruiting bodies are dried and crushed, they are sequentially subjected to extrusion puffing treatment, water extraction treatment, enzymolysis treatment, alcohol precipitation treatment, concentration treatment and drying treatment to obtain the Pleurotus ostreatus polysaccharide.

[0008] Preferably, the feeding speed of the extrusion puffing treatment is 13kg / h-15kg / h, the rotation speed is 200r / min-250r / min, and the temperature is 145°C-150°C.

[0009] Preferably, the water extraction treatment includes a first water extraction and a second water extraction;

[0010] The first water extraction comprises the steps of mixing the pretreated Pleurotus eryngii powder obtained by the extrusion puffing treatment with water, performing a first water extraction, and obtaining a first filtrate and a first filter residue;

[0011] The second water extraction comprises the steps of mixing the first filter residue with water, performing a second water extraction, and obtaining a second filtrate and a second filter residue.

[0012] Preferably, the mass ratio of the pretreated Pleurotus ostreatus powder to water is 1:10; the temperature of the first water extraction is 78° C., and the time is 1.5 h;

[0013] The mass ratio of the first filter residue to water is 1:3; the temperature of the second water extraction is 78° C. and the time is 1.5 h.

[0014] Preferably, the enzymatic hydrolysis treatment comprises the steps of mixing the second filter residue and the composite enzyme preparation, performing enzymatic hydrolysis treatment, and obtaining an enzymatic hydrolyzate.

[0015] Preferably, the composite enzyme preparation comprises trypsin, cellulase and pectinase; the enzyme activity of trypsin in the composite enzyme preparation is 500U / mL, the enzyme activity of cellulase is 800U / mL, and the enzyme activity of pectinase is 500U / mL;

[0016] The added amount of the composite enzyme preparation is 0.2%-0.25% of the mass of the second filter residue.

[0017] Preferably, the enzymatic treatment is carried out for 2 hours at a temperature of 45°C.

[0018] Preferably, the solvent used in the alcohol precipitation treatment is an ethanol solution; the volume percentage content of ethanol in the ethanol solution is 95%.

[0019] Preferably, the alcohol precipitation treatment is carried out at a temperature of 4°C and for 24 hours.

[0020] Preferably, the drying process is carried out for 2 hours at a temperature of 100°C.

[0021] The present invention discloses the following technical effects:

[0022] The present invention provides a method for preparing Nameko polysaccharide, comprising the steps of drying and crushing Nameko fruiting bodies, and then sequentially performing extrusion puffing treatment, water extraction treatment, enzymolysis treatment, alcohol precipitation treatment, concentration treatment and drying treatment to obtain the Nameko polysaccharide. The present invention adopts extrusion puffing treatment to extrude Nameko powder, destroys the cell wall of Nameko cells, thereby improving the extraction rate of Nameko polysaccharide; the two steps of water extraction treatment and enzymolysis treatment further ensure the dissolution of Nameko polysaccharide; and the alcohol precipitation treatment can ensure the purity of Nameko polysaccharide. The present invention combines the four steps of extrusion puffing treatment, water extraction treatment, enzymolysis treatment and alcohol precipitation treatment, so that the extraction rate of Nameko polysaccharide reaches 75-81% and the purity reaches 90.8-91.4%. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described 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.

[0025] 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.

[0026] 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 those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Unless otherwise specified, the methods used in the present invention are methods well known to those skilled in the art.

[0029] Example 1

[0030] The fruiting bodies of Pleurotus eryngii are dried and crushed to obtain Pleurotus eryngii powder; the particle size of the Pleurotus eryngii powder is 10 meshes;

[0031] The powder of Pleurotus eryngii is extruded and expanded by a twin-screw extruder to obtain pretreated Pleurotus eryngii powder; wherein the feeding speed of the twin-screw extruder during extrusion and expansion is 13 kg / h, the screw speed is 200 r / min, and the extrusion temperature is 150°C;

[0032] The pretreated Nameko powder was mixed with water in a mass ratio of 1:10, extracted with water at 78° C. for 1.5 h, and filtered to obtain a first filter residue and a second filtrate;

[0033] The first filter residue was mixed with water in a mass ratio of 1:5, extracted with water at 78° C. for 1.5 h, and filtered to obtain a second filter residue and a second filtrate;

[0034] Combine the two filtrates to obtain a water extract;

[0035] The second filter residue was mixed with water in a mass ratio of 1:3, and then a composite enzyme preparation was added for enzymolysis. After that, the enzyme was inactivated at 90°C for 10 minutes and filtered through a 0.25 μm filter membrane to obtain an enzymolysis solution; wherein the composite enzyme preparation was obtained by mixing trypsin (company: Yunbang Bio, product number: YB64034), cellulase (company: merck, CAS number: 9012-54-8) and pectinase (company: merck, CAS number: 9032-75-1) in a ratio of 1:1:2; the enzyme activity of trypsin in the composite enzyme preparation was 500 U / mL, the enzyme activity of cellulase was 800 U / mL, and the enzyme activity of pectinase was 500 U / mL; the addition amount of the composite enzyme preparation was 0.2% of the mass of the second filter residue, the temperature of enzymolysis was 45°C, and the time was 2 hours;

[0036] The water extract and the enzymatic hydrolyzate are mixed to obtain a mixed solution;

[0037] The mixed solution was mixed with 90% (volume percentage) ethanol, and then alcohol precipitation was performed, followed by concentration and drying to obtain Pleurotus eryngii polysaccharide, wherein the alcohol precipitation temperature was 4°C, the time was 24h, and the drying time was 2h, and the temperature was 100°C.

[0038] Example 2

[0039] The steps are the same as those in Example 1, except that the feeding rate during extrusion and puffing by the twin-screw extruder is 15 kg / h, the screw speed is 250 r / min, and the extrusion temperature is 145°C.

[0040] Comparative Example 1

[0041] The steps are the same as those in Example 1, except that the step of extruding and expanding the Pleurotus ostreatus powder by using a twin-screw extruder is omitted.

[0042] Comparative Example 2

[0043] The steps are the same as those in Example 1, except that the feeding rate during extrusion and puffing of the twin-screw extruder is 10 kg / h, the screw speed is 500 r / min, and the extrusion temperature is 120°C.

[0044] Comparative Example 3

[0045] The steps are the same as those in Example 1, except that the feeding rate during extrusion and puffing by the twin-screw extruder is 18 kg / h, the screw speed is 250 r / min, and the extrusion temperature is 155°C.

[0046] Example 3

[0047] The steps are the same as those in Example 1, except that the added amount of the complex enzyme preparation is 0.25% of the mass of the second filter residue.

[0048] Comparative Example 4

[0049] The steps are the same as those in Example 1, except that the added amount of the complex enzyme preparation is 0.4% of the mass of the second filter residue.

[0050] Comparative Example 5

[0051] The steps are the same as those in Example 1, except that the added amount of the complex enzyme preparation is 0.15% of the mass of the second filter residue.

[0052] Comparative Example 6

[0053] The steps are the same as those in Example 1, except that the composite enzyme preparation is obtained by mixing trypsin and cellulase in a ratio of 1:1; the enzyme activity of trypsin in the composite enzyme preparation is 500 U / mL, and the enzyme activity of cellulase is 800 U / mL.

[0054] Comparative Example 7

[0055] The steps are the same as those in Example 1, except that the composite enzyme preparation is obtained by mixing trypsin, cellulase and neutral protease in a ratio of 1:1:2; the enzyme activity of trypsin in the composite enzyme preparation is 500 U / mL, the enzyme activity of cellulase is 800 U / mL, and the enzyme activity of neutral protease is 500 U / mL.

[0056] Comparative Example 8

[0057] The steps are the same as those in Example 1, except that the steps of enzymatic hydrolysis and enzyme inactivation are omitted.

[0058] Comparative Example 9

[0059] The steps are the same as those in Example 1, except that the water extraction step is omitted, and the pretreated Pleurotus ostreatus powder is directly mixed with the powder in a mass ratio of 1:3 to perform enzymatic hydrolysis, enzyme inactivation and filtration.

[0060] Experimental Example 1

[0061] The extraction rate and purity of the Pleurotus eryngii polysaccharides prepared in Examples 1-3 and Comparative Examples 1-9 were investigated, and the results are shown in Table 1.

[0062] Table 1 Survey results of extraction rate and purity of Pleurotus eryngii polysaccharides obtained by different treatments

[0063]

[0064]

[0065] As shown in Table 1, the extraction rate of Nameko polysaccharide using the method provided by the present invention reaches more than 70%, and the purity reaches more than 90%. The present invention adopts twin-screw extruder extrusion puffing technology in the experimental process. Therefore, in order to verify the effect of the technology on the extraction rate and purity of Nameko polysaccharide, comparative examples 1-3 are set. It is found that omitting the step or changing the condition parameters of the step will affect the extraction rate and purity of Nameko polysaccharide, and will reduce the extraction rate and purity of Nameko polysaccharide; in order to verify the effect of the composite enzyme preparation on the extraction rate and purity of Nameko polysaccharide, the present invention sets comparative examples 4-7. It is found that changing the type and amount of enzyme in the composite enzyme preparation will also reduce the extraction rate and purity of Nameko polysaccharide. At the same time, in order to verify the importance of each experimental step, the present invention sets two extraction methods: twin-screw extruder extrusion puffing technology + water extraction (Comparative Example 8) and twin-screw extruder extrusion puffing technology + enzymolysis (Comparative Example 9). It is found that omitting the steps of water extraction or enzymolysis will also reduce the extraction rate and purity of Nameko polysaccharide. It can be seen that the extraction rate and purity of Pleurotus eryngii polysaccharide prepared according to the steps provided by the present invention can reach the best.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing Pleurotus eryngii polysaccharide, characterized in that: The following steps are involved: The Pleurotus ostreatus fruiting bodies are dried and crushed, and then subjected to extrusion puffing treatment, water extraction treatment, enzymatic hydrolysis treatment, alcohol precipitation treatment, concentration treatment and drying treatment in sequence to obtain the Pleurotus ostreatus polysaccharide.

2. The method for preparing Nameko polysaccharide according to claim 1, characterized in that: The feeding speed of the extrusion puffing treatment is 13kg / h-15kg / h, the rotation speed is 200r / min-250r / min, and the temperature is 145°C-150°C.

3. The method for preparing Nameko polysaccharide according to claim 1, characterized in that: The water extraction treatment includes a first water extraction and a second water extraction; The first water extraction comprises the steps of mixing the pretreated Pleurotus eryngii powder obtained by the extrusion puffing treatment with water, performing a first water extraction, and obtaining a first filtrate and a first filter residue; The second water extraction comprises the steps of mixing the first filter residue with water, performing a second water extraction, and obtaining a second filtrate and a second filter residue.

4. The method for preparing Nameko polysaccharide according to claim 3, characterized in that: The mass ratio of the pretreated Pleurotus ostreatus powder to water is 1:10; the temperature of the first water extraction is 78° C. and the time is 1.5 h; The mass ratio of the first filter residue to water is 1:3; the temperature of the second water extraction is 78° C. and the time is 1.5 h.

5. The method for preparing Nameko polysaccharide according to claim 3, characterized in that: The enzymatic hydrolysis treatment comprises the steps of mixing the second filter residue and the composite enzyme preparation, performing enzymatic hydrolysis treatment, and obtaining an enzymatic hydrolysis solution.

6. The method for preparing Nameko polysaccharide according to claim 5, characterized in that: The composite enzyme preparation comprises trypsin, cellulase and pectinase; the enzyme activity of trypsin in the composite enzyme preparation is 500U / mL, the enzyme activity of cellulase is 800U / mL, and the enzyme activity of pectinase is 500U / mL; The added amount of the composite enzyme preparation is 0.2%-0.25% of the mass of the second filter residue.

7. The method for preparing Nameko polysaccharide according to claim 1 or 5, characterized in that: The enzymatic treatment was carried out for 2 hours at a temperature of 45°C.

8. The method for preparing Nameko polysaccharide according to claim 1, characterized in that: The solvent used in the alcohol precipitation treatment is an ethanol solution; the volume percentage content of ethanol in the ethanol solution is 95%.

9. The method for preparing Nameko polysaccharide according to claim 1 or 8, characterized in that: The temperature of the alcohol precipitation treatment is 4° C. and the time is 24 hours.

10. The method for preparing Nameko polysaccharide according to claim 1, characterized in that: The drying process was carried out for 2 hours at a temperature of 100°C.

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