CN110563856A - extraction method of flammulina velutipes polysaccharide - Google Patents
extraction method of flammulina velutipes polysaccharide Download PDFInfo
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Abstract
the invention relates to the technical field of plant polysaccharide extraction, in particular to a method for extracting flammulina velutipes polysaccharide, which comprises the steps of cleaning fruiting bodies of flammulina velutipes, drying in the sun, grinding into powder, adding dried flammulina velutipes powder into distilled water, extracting in water bath, centrifuging an extracting solution, and concentrating centrifuged supernatant in vacuum at 50 ℃ to 1/4 of the original volume; the extraction rate of the flammulina velutipes polysaccharide obtained by the method is up to 8.21%, the extraction rate of the flammulina velutipes polysaccharide is greatly improved, the Saveg solution and the sugar solution are mixed and shaken uniformly, denatured protein is filtered out, the steps are repeated for many times, the content of the protein in the flammulina velutipes polysaccharide is basically 0, high-purity flammulina velutipes polysaccharide without protein can be obtained, and the biological activity of the flammulina velutipes polysaccharide is improved.
Description
Technical Field
The invention relates to the technical field of plant polysaccharide extraction, in particular to a method for extracting flammulina velutipes polysaccharide.
background
the golden mushroom is named as hirsutella velutipes, pleomorph mushrooms, winter mushrooms, needle mushrooms, broussonetia papyrifera and the like (Yanrui, Liukang dry, edible fungus health care function and dietary therapy method [ M ]. Beijing: Kingdun Press, 2000), and belongs to Agaricales, Tricholomataceae and Microflame species (in Ronli, Qixusheng, Songheng, golden mushroom research overview [ J ]. edible fungus academic, 2004, 11(4): 63-68). Golden and tender stems such as golden needles are named and are one of precious edible mushrooms, at present, researchers can produce golden mushroom polysaccharides from golden mushrooms, and numerous researches show that the golden mushroom polysaccharides have good biological activities of oxidation resistance, blood fat reduction, immunity improvement, aging resistance and the like and have high economic benefits, so that the golden mushroom polysaccharides in the golden mushrooms need to be extracted.
The prior art also presents a technical scheme about an extraction method of flammulina velutipes polysaccharide, and for example, a Chinese patent with the application number of 2008102222716 discloses a flammulina velutipes extract, and an obtaining method and application thereof. The method comprises the following steps of mixing needle mushroom fruiting bodies with water according to a ratio of 1: 3-5, homogenizing, extracting at 80-95 deg.C and pH 7-9 for 1-5 hr, and removing residue to obtain needle mushroom extractive solution containing needle mushroom polysaccharide.
the method can extract the flammulina velutipes polysaccharide, but in the method, the extraction yield of the flammulina velutipes is only 1.1254%, the extraction rate is very low, and the extracted flammulina velutipes polysaccharide contains more protein impurities, so that the purity of the flammulina velutipes polysaccharide is reduced, and the biological activity of the extracted flammulina velutipes polysaccharide is further reduced.
disclosure of Invention
this section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above problems.
in order to solve the technical problems, the invention provides the following technical scheme:
the extraction method of the flammulina velutipes polysaccharide comprises the following steps:
Crude extraction: cleaning needle mushroom fruiting bodies, drying in the sun, grinding into powder, adding distilled water into needle mushroom dry powder according to the feed liquid ratio of 1:15-40g/ml, extracting in a water bath at 50-100 ℃ for 5h, centrifuging the extracting solution at 3000r/min for 10min, and vacuum concentrating the centrifuged supernatant at 50 ℃ to 1/4 of the original volume;
fine extraction: dialyzing the concentrated solution after vacuum concentration in distilled water at 4 deg.C for 24-48h, centrifuging at 3000r/min for 10min, collecting supernatant, and freeze drying to obtain Flammulina velutipes polysaccharide;
And (3) storage: storing at-20 deg.C.
Preferably, in the crude extract, the feed-liquid ratio of the needle mushroom dry powder to the water is 1:20 g/ml.
Preferably, in the crude extract, the temperature of the water bath is 85 ℃.
Preferably, in the fine extraction, the dialysis time in the distilled water is 48 h.
preferably, the refining and the preserving also comprise re-purification;
And (3) re-purification: redissolving preserved flammulina velutipes polysaccharide with hot water, carrying out high-voltage pulse electric field treatment, inducing more polar groups to be exposed by electric field molecular polarization, inducing charged molecules to generate repeated violent movement, destroying protein fibrous structures remained in the flammulina velutipes polysaccharide, decomposing a compound formed by soluble proteins remained in the flammulina velutipes polysaccharide, thinning thick proteins, further reducing viscosity, dispersing molecules, redissolving the flammulina velutipes polysaccharide and the hot water according to a feed-liquid ratio of 1:20g/ml to obtain sugar solution, mixing the Saveg solution and the sugar solution at a volume ratio of 2:1 at a temperature of between 55 and 70 ℃, shaking uniformly, filtering out denatured proteins, repeating for multiple times, retaining filtrate, detecting the protein content in the filtrate, mixing the Saveg solution and the sugar solution at a volume ratio of 2:1 again, shaking uniformly when the proteins can be detected, filtering out the denatured proteins, repeating the step again, and when the protein can not be detected, freezing and drying the filtrate to obtain the needle mushroom polysaccharide without the protein.
preferably, in the re-purification, the protein content is detected by placing the filtrate in an ultraviolet analyzer and detecting the absorption peaks of protein and nucleic acid in the filtrate at wavelengths of 260 and 280 nm.
Preferably, impurities are removed between the crude extraction and the fine extraction;
Impurity removal: adding the concentrated solution obtained by crude extraction into a 100L macroporous adsorption resin column at the flow rate of 50-100L/h, washing the macroporous adsorption resin column with water at the flow rate of 100-200L/h, and removing unadsorbed impurities; washing the macroporous adsorption resin column by 15-20% ethanol solution at the flow rate of 30-50L/h to remove impurities with higher polarity; then, ethanol with the volume fraction of 65-70% is used as absorption liquid to wash the macroporous absorption resin column at the flow rate of 40-60L/h, and the washed absorption liquid is collected, and is dried by vacuum centrifugation for later use.
Compared with the prior art: according to the extraction method of the flammulina velutipes polysaccharide, the optimal extraction conditions are obtained through screening by a three-factor three-level orthogonal experiment, the extraction rate of the flammulina velutipes polysaccharide obtained by the method is up to 8.21%, the extraction rate of the flammulina velutipes polysaccharide is greatly improved, a sugar solution is obtained by re-dissolving the stored flammulina velutipes polysaccharide and hot water, the Saveg solution and the sugar solution are uniformly mixed, denatured protein is filtered out, the repetition is repeated for many times, the content of the protein in the flammulina velutipes polysaccharide is basically 0, the high-purity flammulina velutipes polysaccharide without the protein can be obtained, and the biological activity of the flammulina velutipes polysaccharide is improved.
Drawings
FIG. 1 shows the effect of water bath extraction temperature on the extraction yield of Flammulina velutipes polysaccharide in the method of the present invention;
FIG. 2 is a graph showing the effect of water bath extraction time on the extraction yield of Flammulina velutipes polysaccharide in the method of the present invention;
FIG. 3 shows the effect of feed liquid ratio between needle mushroom dry powder and water on needle mushroom polysaccharide extraction yield in the method of the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
the methods of the following examples are conventional unless otherwise specified.
The percentages given in the following examples are by weight unless otherwise specified.
Materials used in the following examples: the needle mushroom is purchased from cheap and fine supermarkets in Beijing; all reagents used were analytical grade.
Example 1
1) Cleaning fruiting body of needle mushroom, sun drying, grinding, adding needle mushroom dry powder into distilled water, extracting in water bath for 5 hr, centrifuging the extractive solution at 3000r/min for 10min, and vacuum concentrating the centrifuged supernatant at 50 deg.C to 1/4 of the original volume;
2) fine extraction: dialyzing the concentrated solution after vacuum concentration in distilled water at 4 deg.C for 24-48h, centrifuging at 3000r/min for 10min, collecting supernatant, and freeze drying to obtain Flammulina velutipes polysaccharide;
3) And (3) storage: storing at-20 deg.C;
Under the condition that the material-liquid ratio and the extraction time are kept unchanged, the polysaccharide yield at the extraction temperatures of 50 ℃, 60 ℃, 70, 80 ℃, 90 ℃ and 100 ℃ is respectively researched and tested, the obtained result is shown in figure 1, under the conditions that the extraction time is 5 hours and the material-liquid ratio is 1:15g/ml, when the extraction temperature is 50-80 ℃, the yield of the flammulina velutipes polysaccharide is gradually improved along with the increase of the extraction temperature, but after the extraction temperature exceeds 80 ℃, the yield of the flammulina velutipes polysaccharide begins to slowly decrease; the high temperature can decompose the flammulina velutipes polysaccharide so as to reduce the content of crude polysaccharide, so the optimal extraction temperature is 80 ℃;
Under the condition that the material-liquid ratio and the extraction temperature are kept unchanged, the polysaccharide yield is respectively researched and tested when the extraction time is 1, 2, 3, 4, 5h and 6h, the obtained result is shown in figure 2, under the conditions that the extraction temperature is 80 ℃ and the material-liquid ratio is 1:15g/ml, when the extraction time is 1-6 h, the yield of the flammulina velutipes polysaccharide is gradually improved along with the increase of the extraction time, but after the extraction time exceeds 5h, the increase amplitude of the flammulina velutipes polysaccharide yield is very small; considering the time cost of the extraction of the flammulina velutipes polysaccharide, the optimal extraction time is 5 h;
Respectively researching polysaccharide yields when the material-liquid ratio is 1:15, 1:20, 1:25, 1:30, 1:35g/ml and 1:40g/ml under the condition that the material-liquid ratio and the extraction time are kept unchanged, obtaining results as shown in figure 3, wherein under the conditions that the extraction time is 5 hours and the extraction temperature is 80 ℃, when the extraction temperature is 1:15-20g/ml, the yield of the flammulina velutipes polysaccharide is gradually improved along with the increase of the material-liquid ratio, but when the extraction temperature exceeds 1:20g/ml, the yield of the flammulina velutipes polysaccharide begins to slowly decrease; therefore, the optimal extraction ratio of the materials to the liquid is 801:20 g/ml;
the polysaccharide yield (%) -, polysaccharide content in the extract/total weight of raw materials x 100%;
designing orthogonal experiment for key factors (water bath extraction temperature, feed-liquid ratio and extraction time), and carrying out L according to 3-factor 3 level9(33) Performing an experiment;
Table 1 orthogonal experimental design and results of the method of the invention
The results of the orthogonal experiments obtained in table 1 were analyzed, and it was found from the results of the range analysis that: the influence of various factors on the yield of the polysaccharide in the flammulina velutipes extract is as follows from big to small: b extraction time>Ratio of material to liquid>A, extracting temperature; the best matching of the extraction conditions of the flammulina velutipes polysaccharide is as follows: a. the2B3C2Namely: the extraction time is 5h, the material-liquid ratio is 1:20g/ml, the extraction temperature is 80 ℃, and experiments show that the yield of the flammulina velutipes polysaccharide can reach 8.21 percent by using the scheme to extract the flammulina velutipes polysaccharide.
Example 2
1) Cleaning fruiting body of needle mushroom, sun drying, grinding, adding needle mushroom dry powder into distilled water, extracting in water bath for 5 hr, centrifuging the extractive solution at 3000r/min for 10min, and vacuum concentrating the centrifuged supernatant at 50 deg.C to 1/4 of the original volume;
2) Fine extraction: dialyzing the concentrated solution after vacuum concentration in distilled water at 4 deg.C for 24-48h, centrifuging at 3000r/min for 10min, collecting supernatant, and freeze drying to obtain Flammulina velutipes polysaccharide;
3) Redissolving the preserved flammulina velutipes polysaccharide with hot water, carrying out high-voltage pulse electric field treatment, inducing more polar groups to be exposed by electric field molecular polarization, inducing charged molecules to generate repeated violent movement, destroying the fibrous structure of protein remained in the flammulina velutipes polysaccharide, decomposing a compound formed by the soluble protein remained in the flammulina velutipes polysaccharide, thinning the thick protein, further reducing the viscosity, dispersing the molecules, enabling the protein to be more easily deformed to form an incompatible precipitate, redissolving the protein and the hot water according to the feed-liquid ratio of 1:20g/ml to obtain sugar liquid, mixing and shaking the Saveg liquid and the sugar liquid at the volume ratio of 2:1 at the temperature of 55-70 ℃, filtering out the denatured protein, repeating the steps for multiple times, reserving the filtrate, detecting the protein content in the filtrate, mixing and shaking the Saveg liquid and the sugar liquid at the volume ratio of 2:1 again when the protein can be detected, filtering out denatured protein, repeating the step again, and freeze-drying the filtrate to obtain the needle mushroom polysaccharide without protein when the protein cannot be detected;
4) And (3) storage: storing at-20 deg.C;
And (3) placing the filtrate in an ultraviolet tester, detecting absorption peaks of protein and nucleic acid in the filtrate at wavelengths of 260 nm and 280nm, mixing and shaking up the Saveg solution and the sugar solution in a volume ratio of 2:1 for the fourth time through a plurality of experiments, filtering out denatured protein, wherein the content of the protein is basically 0, so that the high-purity flammulina velutipes polysaccharide without protein can be obtained, and the biological activity of the flammulina velutipes polysaccharide is improved.
example 2
1) cleaning fruiting body of needle mushroom, sun drying, grinding, adding needle mushroom dry powder into distilled water, extracting in water bath for 5 hr, centrifuging the extractive solution at 3000r/min for 10min, and vacuum concentrating the centrifuged supernatant at 50 deg.C to 1/4 of the original volume;
2) adding the concentrated solution into a 100L macroporous adsorption resin column at the flow rate of 50-100L/h, washing the macroporous adsorption resin column with water at the flow rate of 100-200L/h, and removing unadsorbed impurities; washing the macroporous adsorption resin column by 15-20% ethanol solution at the flow rate of 30-50L/h to remove impurities with higher polarity; then, ethanol with the volume fraction of 65-70% is used as absorption liquid to wash the macroporous adsorption resin column at the flow rate of 40-60L/h, the washed absorption liquid is collected, and the absorption liquid is centrifuged and pumped out in vacuum for later use, so that impurities in the flammulina velutipes solution are further removed, and the purity of the product is improved;
3) fine extraction: dialyzing the concentrated solution after vacuum concentration in distilled water at 4 deg.C for 24-48h, centrifuging at 3000r/min for 10min, collecting supernatant, and freeze drying to obtain Flammulina velutipes polysaccharide;
4) redissolving preserved flammulina velutipes polysaccharide with hot water, carrying out high-voltage pulse electric field treatment, inducing more polar groups to be exposed by electric field molecular polarization, inducing charged molecules to generate repeated violent movement, destroying protein fibrous structures remained in the flammulina velutipes polysaccharide, decomposing a compound formed by soluble proteins remained in the flammulina velutipes polysaccharide, thinning thick proteins, further reducing viscosity, dispersing molecules, redissolving the flammulina velutipes polysaccharide and the hot water according to a feed-liquid ratio of 1:20g/ml to obtain sugar solution, mixing the Saveg solution and the sugar solution at a volume ratio of 2:1 at a temperature of between 55 and 70 ℃, shaking uniformly, filtering out denatured proteins, repeating for multiple times, retaining filtrate, detecting the protein content in the filtrate, mixing the Saveg solution and the sugar solution at a volume ratio of 2:1 again, shaking uniformly when the proteins can be detected, filtering out the denatured proteins, repeating the steps again, and when the protein cannot be detected, freeze-drying the filtrate to obtain the protein-free flammulina velutipes polysaccharide;
5) And (3) storage: storing at-20 deg.C.
While the invention has been described above with reference to an embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the various features of the embodiments disclosed herein may be used in any combination, provided that there is no structural conflict, and the combinations are not exhaustively described in this specification merely for the sake of brevity and conservation of resources. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (7)
1. The extraction method of the flammulina velutipes polysaccharide is characterized by comprising the following steps:
Crude extraction: cleaning needle mushroom fruiting bodies, drying in the sun, grinding into powder, adding distilled water into needle mushroom dry powder according to the feed liquid ratio of 1:15-40g/ml, extracting in a water bath at 50-100 ℃ for 5h, centrifuging the extracting solution at 3000r/min for 10min, and vacuum concentrating the centrifuged supernatant at 50 ℃ to 1/4 of the original volume;
fine extraction: dialyzing the concentrated solution after vacuum concentration in distilled water at 4 deg.C for 24-48h, centrifuging at 3000r/min for 10min, collecting supernatant, and freeze drying to obtain Flammulina velutipes polysaccharide;
And (3) storage: storing at-20 deg.C.
2. The extraction method of flammulina velutipes polysaccharide according to claim 1, wherein the extraction method comprises the following steps: in the crude extraction, the feed-liquid ratio of the needle mushroom dry powder to water is 1:20 g/ml.
3. the extraction method of flammulina velutipes polysaccharide according to claim 1, wherein the extraction method comprises the following steps: in the crude extract, the temperature of the water bath was 85 ℃.
4. The extraction method of flammulina velutipes polysaccharide according to claim 1, wherein the extraction method comprises the following steps: in the fine extraction, the dialysis time in the distilled water is 48 h.
5. The extraction method of flammulina velutipes polysaccharide according to claim 1, wherein the extraction method comprises the following steps: re-purification is also included between the fine extraction and the preservation;
And (3) re-purification: redissolving preserved flammulina velutipes polysaccharide with hot water, carrying out high-voltage pulse electric field treatment, inducing more polar groups to be exposed by electric field molecular polarization, inducing charged molecules to generate repeated violent movement, destroying protein fibrous structures remained in the flammulina velutipes polysaccharide, decomposing a compound formed by soluble proteins remained in the flammulina velutipes polysaccharide, thinning thick proteins, further reducing viscosity, dispersing molecules, redissolving the flammulina velutipes polysaccharide and the hot water according to a feed-liquid ratio of 1:20g/ml to obtain sugar solution, mixing the Saveg solution and the sugar solution at a volume ratio of 2:1 at a temperature of between 55 and 70 ℃, shaking uniformly, filtering out denatured proteins, repeating for multiple times, retaining filtrate, detecting the protein content in the filtrate, mixing the Saveg solution and the sugar solution at a volume ratio of 2:1 again, shaking uniformly when the proteins can be detected, filtering out the denatured proteins, repeating the step again, and when the protein can not be detected, freezing and drying the filtrate to obtain the needle mushroom polysaccharide without the protein.
6. the extraction method of flammulina velutipes polysaccharide according to claim 5, wherein the extraction method comprises the following steps: in the re-purification, the protein content is detected by placing the filtrate in an ultraviolet analyzer and detecting the absorption peaks of protein and nucleic acid in the filtrate at wavelengths of 260 and 280 nm.
7. the extraction method of flammulina velutipes polysaccharide according to claim 1, wherein the extraction method comprises the following steps: impurities are removed between the crude extraction and the fine extraction;
impurity removal: adding the concentrated solution obtained by crude extraction into a 100L macroporous adsorption resin column at the flow rate of 50-100L/h, washing the macroporous adsorption resin column with water at the flow rate of 100-200L/h, and removing unadsorbed impurities; washing the macroporous adsorption resin column by 15-20% ethanol solution at the flow rate of 30-50L/h to remove impurities with higher polarity; then, ethanol with the volume fraction of 65-70% is used as absorption liquid to wash the macroporous absorption resin column at the flow rate of 40-60L/h, and the washed absorption liquid is collected, and is dried by vacuum centrifugation for later use.
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| CN114409924A (en) * | 2021-11-25 | 2022-04-29 | 南京财经大学 | Flammulina velutipes polysaccharide-soybean protein gel suitable for 3D printing and preparation method thereof |
| CN116497633A (en) * | 2023-06-09 | 2023-07-28 | 京准化工技术(上海)有限公司 | Fluorine-free oil-proof packaging paper and preparation method thereof |
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| CN116497633B (en) * | 2023-06-09 | 2024-03-01 | 京准化工技术(上海)有限公司 | Fluorine-free oil-proof packaging paper and preparation method thereof |
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