CN115028755B - Preparation method of high molecular weight sparassis crispa beta-glucan - Google Patents
Preparation method of high molecular weight sparassis crispa beta-glucan Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention discloses a preparation method of high molecular weight sparassis crispa beta-glucan, which comprises the following steps: 1) Pulverizing Sparassis crispa, and degreasing with organic solvent; 2) Degreasing fungus powder, adding acetic acid solution, stirring and soaking, and then performing high-temperature high-pressure pyrolysis through a high-pressure reaction kettle to obtain pyrolysis extract; 3) Adjusting the pH value of the pyrolysis extracting solution to be slightly alkaline, and then carrying out solid-liquid separation to obtain beta-glucan extracting solution; 4) And (3) carrying out alcohol precipitation on the beta-glucan extract, collecting the precipitate, washing and freeze-drying to obtain the Sparassis crispa high molecular beta-glucan. The invention adopts the low-concentration acid pyrolysis technology under the conditions of high temperature and high pressure, reduces the damage to the target component of the Sparassis crispa beta-glucan, better keeps the biological activity of the Sparassis crispa beta-glucan, effectively breaks down the cell wall structure by acidolysis, hydrolyzes the impurity components such as alpha-glucan, protein and the like, and improves the hydrophilicity of the Sparassis crispa high molecular beta-glucan, thereby realizing the purposes of improving the extraction rate of the Sparassis crispa high molecular beta-glucan and the purity of the product.
Description
Technical Field
The Pediococcus guangdong She Xiu (Sparassis latifolia) has high nutritional and health promoting value. The research result shows that the Sparassis crispa has rich beta-glucan content and the research result of the activity shows that the Sparassis crispa has obvious immunoregulatory activity and immune-mediated antitumor activity. The active beta-glucan of the sparassis crispa which is researched in the prior report is mainly extracted from strong alkali solution, the molecular weight is more than 200 kilodaltons, the main chain structure is beta-1, 3-glucan with a 1-6 branch structure, the beta-glucan is difficult to dissolve in water, and the low concentration solution is gel.
Sparassis crispa beta-glucan mainly exists in cell walls and has the characteristics of high molecular weight, ordered structure and difficult water solubility. The ideal yield is difficult to obtain by adopting conventional hot water extraction, ultrasonic-assisted extraction and enzymatic assisted extraction, and the obtained extract has lower beta-glucan, only accounting for about 30 percent, and contains a large amount of other alpha-glucan with medium and low molecular weight. Currently, the feasible extraction methods for the high molecular weight beta-glucan component of Sparassis crispa mainly adopt strong alkali solution extraction and sodium hydroxide-urea system extraction. However, the methods have the defects of large consumption of alkali and urea, low extraction yield, deep color of an extracted product, high cost of subsequent desalting, decoloring and further purification due to a large amount of alpha-glucan and protein impurities, and the like, and the difficulty of industrialized large-scale production is high.
In order to solve the problems in the extraction of the Sparassis crispa high molecular weight beta-glucan, development and improvement are necessary to the extraction technology and method of Sparassis crispa high molecular weight beta-glucan, so that the dissolution efficiency of the high molecular weight beta-glucan in the Sparassis crispa cell wall in the extraction process is improved, the content of impurities such as alpha-glucan, protein and the like in the crude extract product is reduced, and the purity of the product is improved.
Disclosure of Invention
The invention aims to provide a preparation method of high molecular weight sparassis crispa beta-glucan, which mainly solves the technical problems of 1, improving the dissolution efficiency of the high molecular weight beta-glucan in sparassis crispa fruiting bodies. 2. Improves the purity of the Sparassis crispa high molecular weight beta-glucan product. 3. The structure and the bioactivity of the beta-glucan with high molecular weight of the Sparassis crispa are protected in the high-temperature high-pressure acid cracking process.
In order to achieve the above object, the technical idea of the present invention is as follows:
acid hydrolysis is a common method for hydrolyzing polysaccharides and proteins, and because the polysaccharides are linked by glycosidic bonds, i.e. carbon-oxygen-carbon bonds formed by acetal ketones, which are relatively stable under alkaline conditions, and under acidic conditions, the polysaccharides can be hydrolyzed into oligosaccharides or monosaccharides under acidic conditions because hydrogen ions can be added to oxygen atoms to promote cleavage of a carbon-oxygen bond. Therefore, the extraction is generally not carried out by adopting an acid extraction method in polysaccharide extraction so as to avoid damaging the structure and the function of the polysaccharide. In fact, however, polysaccharides are also difficult to hydrolyze under acidic conditions, and generally, polysaccharides which are easily dissolved and have disordered structures are easier to hydrolyze, and alpha-1, 4 glycosidic bonds are easier to hydrolyze than other types of glycosidic bonds.
Sparassis crispa beta-glucan is a linear main chain linked by beta- (1- & gt 3) glycosidic bonds and has a branched structure linked by beta- (1- & gt 6) glycosidic bonds, has extremely large molecular weight which can reach more than 2000kDa, mainly exists in cell walls of bacteria, has stable morphology in the cell wall structure, is in a three-dimensional coiled cross-linked structure, has limited exposure of polar groups, and is weaker in solvation effect and harder to dissolve. These characteristics result in the acid solution having lower efficiency of invading the inside of the structure and cleaving the glycosidic bond, and better stability under the hydrolysis of low concentration acid.
The high-temperature high-pressure treatment condition has the effects of promoting the stretching of the sugar chain structure, exposing the polar groups, increasing the charge quantity, improving the hydrophilicity and promoting the solvation of the polysaccharide on the high-molecular polysaccharide. According to the invention, by controlling the acid hydrolysis under high temperature and high pressure conditions, the alpha-1, 4 glucan and protein components which are easy to hydrolyze in the sparassis crispa cells and cell walls are destroyed, so that the cell walls are broken down, the hydrophilicity of insoluble beta-glucan is improved, and the high extraction yield and the product purity of sparassis crispa high-molecular beta-glucan are further obtained. The technology has the advantages of simple process, easy production and application, less acid and alkali consumption, high extraction efficiency, and high purity and good molecular weight uniformity of the crude product, and most of alpha-glucan and protein impurities are removed by controlling hydrolysis.
Based on the principle, the technical scheme adopted by the invention is as follows:
a preparation method of high molecular weight Sparassis crispa beta-glucan comprises the following steps;
1) Sparassis crispa crushing degreasing
Pulverizing Sparassis crispa fruiting body dry product to obtain powder, defatting with organic solvent, reflux extracting, filtering to obtain residue, and drying the residue to remove solvent;
2) High temperature and high pressure acid cracking
Adding 2-4% of acid solution (v/v) into the residue, controlling the feed-liquid ratio to be 20-30 (v/w), uniformly stirring to obtain mixed solution, stirring the mixed solution at room temperature for 8-12 h, loading the mixed solution into a high-pressure reaction kettle for high-temperature high-pressure pyrolysis, controlling the reaction temperature to be 130-140 ℃ and the pressure to be 0.18-0.30MPa, reacting for 2-4 h, cooling to 80 ℃ after the reaction is finished, and discharging to obtain pyrolysis liquid;
3) Alkalization and solid-liquid separation of cracking liquid
Adding alkali liquor into the pyrolysis extracting solution to adjust the pH value to 8.0-8.5, stirring at 80-90 ℃ for reacting for 2-3 hours, carrying out solid-liquid separation after the viscosity of the solution is obviously reduced, respectively collecting solid residues and supernatant, repeatedly washing the solid residues with a small amount of dilute alkali solution with the pH value of 8.0-8.5 for 1-2 times, centrifuging, collecting supernatant, and combining the supernatant to obtain the Sparassis crispa beta-glucan extracting solution;
4) Alcohol precipitation separation
Cooling the Sparassis crispa beta-glucan extract, adding 0.9-1.2 times of 95% ethanol solution, uniformly stirring, standing at 0-4 ℃ for precipitation for 8-12 h, centrifuging or filtering, collecting precipitate, washing with 50-60% ethanol for 1-2 times, and entering a drying procedure;
5) Vacuum freeze drying
Lyophilizing the precipitate collected in the step 4) in a vacuum environment to obtain the Sparassis crispa high molecular beta-glucan.
Further, the organic solvent used for degreasing in the step 1) is one or more of ethanol, ethyl acetate, petroleum ether and chloroform.
Further, the acid solution in the step 2) is one or more of hydrochloric acid, acetic acid, sulfuric acid and oxalic acid.
Further, the alkali used in the alkali liquor in the step 3) is one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and calcium carbonate.
Further, the solid-liquid separation mode in the step 3) is one or more of centrifugation and filtration modes.
The invention adopts a low-concentration acid cracking technology under high temperature and high pressure, and utilizes acid hydrolysis to hydrolyze alpha-1, 4 glucan and protein components which are easier to hydrolyze to destroy the cell wall structure of Sparassis crispa and increase the hydrophilicity of insoluble polymer beta-glucan, thereby achieving the purpose of improving the extraction rate and the product purity of the Sparassis crispa polymer beta-glucan.
Chinese patent application CN 105384842A discloses a method for extracting water-soluble beta-glucan from sparassis crispa fruiting body, which utilizes the principle of acid hydrolysis, but the basic technical route is to extract sparassis crispa beta-glucan by alkali, remove alpha-glucan by enzymolysis of alpha amylase, and hydrolyze target beta-glucan by acid to obtain a beta-glucan product with smaller molecular weight (2 kDa-200 kDa). Unlike the patent application, the present invention utilizes the acid hydrolysis of high temperature and high pressure under controllable condition, and the hydrolysis targets are mainly alpha-glucan and protein components in Sparassis crispa thallus, and the target component of high molecular beta-glucan is well protected, and the molecular weight of the finally obtained product is mainly distributed above 300 kilodaltons.
Compared with the prior art, the invention has the beneficial effects that:
the invention is characterized in that after degreasing and high-temperature high-pressure acid cracking, alpha-glucan and protein in bacteria are basically hydrolyzed into small molecular oligosaccharides, peptides, monosaccharides and amino acids, the small molecular oligosaccharides, peptides, monosaccharides and amino acids are synchronously removed along with supernatant fluid in an alcohol precipitation process, meanwhile, the hydrophilicity of high molecular beta-glucan in cell walls is increased, and then alkali water dissolution promotion, alcohol precipitation and vacuum freeze-drying are carried out to obtain the high molecular weight beta-glucan product of the Sparassis crispa. The method greatly improves the extraction rate of the Sparassis crispa high molecular beta-glucan, and the product yield can reach more than 20%. The product has high purity, the polysaccharide content can reach more than 93 percent, the weight average molecular weight is more than 3000kDa, and the polydispersity is less than 1.3. And the method is simple to operate, small in acid and alkali consumption and easy for large-scale and industrialized production.
Drawings
FIG. 1 is a gel permeation chromatogram of the product of example 2.
FIG. 2 is a gel permeation chromatogram of the comparative example product.
FIG. 3 is a nuclear magnetic resonance of the product of example 2 1 H spectrum.
FIG. 4 is a nuclear magnetic resonance of the product of example 2 13 C spectrogram.
FIG. 5 is the effect of Sparassis crispa high molecular weight beta-glucan on RAW264.7 macrophage IL-6 factor release.
FIG. 6 is the effect of Sparassis crispa high molecular weight beta-glucan on RAW264.7 macrophage IL-1 factor release.
FIG. 7 is a graph showing the effect of Sparassis crispa high molecular weight beta-glucan on TNF-alpha release from RAW264.7 macrophages.
Detailed Description
The invention is further illustrated below with reference to examples, which are to be understood as being for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1
A method for preparing high molecular weight sparassis crispa beta-glucan, comprising the following steps:
1) Sparassis crispa crushing degreasing
Pulverizing Sparassis crispa fruiting body 1kg, sieving with 800 mesh sieve to obtain powder, adding 20L absolute ethanol, reflux extracting for 3 hr, filtering to obtain residue, drying the residue, and volatilizing solvent.
2) High temperature and high pressure acid cracking
2% hydrochloric acid solution (v/v) was added to the residue, and the feed-to-liquid ratio was 1:20 (w/v) and uniformly stirring to obtain a mixed solution, stirring and soaking the mixed solution at room temperature for 12h, loading the mixed solution into a high-pressure reaction kettle for high-temperature high-pressure pyrolysis, controlling the reaction temperature to 135 ℃, controlling the pressure to 0.22MPa, reacting for 2h, cooling to 80 ℃ after the reaction is finished, and discharging to obtain a pyrolysis extracting solution.
3) Alkalization and solid-liquid separation of cracking liquid
And under the stirring condition, adding 5M sodium hydroxide solution to the pyrolysis extracting solution to adjust the pH to 8.5, stirring at 80 ℃ for reaction for 3 hours, centrifuging after the solution viscosity is obviously reduced, centrifuging at a rotational speed of 5000r/min for 15min, and taking supernatant. And (3) washing the solid residue for 1-2 times by using a small amount of 0.2% sodium hydroxide solution, centrifuging and collecting supernatant, and combining the supernatant to obtain the Sparassis crispa beta-glucan extract.
4) Alcohol precipitation separation
Cooling the sparassis crispa beta-glucan extract obtained in the step 3), adding an equal volume of 95% ethanol solution, stirring uniformly, standing at the temperature of 4 ℃ for precipitation for 8 hours, centrifuging at the speed of 5000r/min for 15min, discarding supernatant, collecting precipitate, washing the precipitate with 50% ethanol for 1-2 times, and sending into a drying procedure.
5) Vacuum freeze drying
And (3) drying the precipitate collected in the step (4) through vacuum freeze-drying to obtain the Sparassis crispa high molecular beta-glucan product.
Example 2
A method for preparing high molecular weight sparassis crispa beta-glucan, comprising the following steps:
1) Sparassis crispa crushing degreasing
Weighing Sparassis crispa fruiting body 1kg, pulverizing, sieving with 80 mesh sieve to obtain powder, adding 20L of 95% ethanol, reflux extracting for degreasing, filtering to obtain residue, and volatilizing solvent from the residue.
2) High temperature and high pressure acid cracking
Adding 20L of 3% acetic acid solution into the residue, stirring and soaking for 8 hours, loading into a high-pressure reaction kettle for high-temperature high-pressure pyrolysis, controlling the reaction temperature to 131 ℃, controlling the reaction pressure to be 0.18MPa, cooling to below 80 ℃ for 3 hours, and discharging to obtain pyrolysis extract.
3) Alkalization and solid-liquid separation of cracking liquid
155mL of 5M sodium hydroxide solution was added to the lysate under stirring to adjust the pH to 8.5, the mixture was stirred at 90℃for 2 hours, and the residue was removed by plate basket filtration through a 0.5 μm filter cloth to obtain about 14 liters of a filtrate (Sparassis crispa. Beta. -glucan extract).
4) Alcohol precipitation separation
Adding 15L of absolute ethyl alcohol into the filtrate, pushing the filtrate into a cold storage at 4 ℃ for standing and precipitating for 8 hours, filtering the filtrate by a plate basket with 0.5 micron pore size cloth to obtain a precipitate, washing the precipitate with 60% of ethyl alcohol for 2 times, and pumping the precipitate.
5) Vacuum freeze drying
Adding 5L distilled water into the precipitate, heating to 60 ℃, stirring until the distilled water is completely dissolved, pouring the mixture into a charging tray after cooling, pre-freezing the mixture at the temperature of minus 30 ℃ for 8 hours, pushing the mixture into a vacuum freeze dryer for freeze drying, and obtaining the Sparassis crispa high molecular weight beta-glucan with the water content of less than 5 percent, wherein the dry weight is about 200g.
Comparative example
Extraction of Sparassis crispa beta-glucan by alkali solution
1) Weighing Sparassis crispa fruiting body 1kg, pulverizing, sieving with 80 mesh sieve, adding 20L of 95% ethanol, reflux extracting for degreasing, and volatilizing ethanol from residue.
20 adding 10% sodium hydroxide solution 20L, stirring, soaking for 12 hr, heating to 95deg.C, reflux-stirring, extracting for 3 hr, cooling to below 80deg.C, discharging, and filtering with 0.5 micrometer filter cloth to remove residue to obtain filtrate about 15L.
3) Adding 16L of absolute ethyl alcohol into the filtrate, pushing the filtrate into a refrigerator at 4 ℃ for standing for 8 hours, filtering the filtrate by using a plate basket with 0.5 micron aperture cloth to obtain a precipitate, washing the precipitate with 60% of ethyl alcohol for 2 times, and pumping the precipitate.
4) Adding 5L distilled water into the precipitate, heating to 60 ℃, stirring until the distilled water is completely dissolved, pouring the mixture into a charging tray after cooling, pre-freezing the mixture for 8 hours at the temperature of minus 30 ℃, pushing the mixture into a vacuum freeze dryer to perform freeze-drying operation, and obtaining the high molecular weight beta-glucan of the Sparassis crispa, wherein the dry weight of the beta-glucan is about 130g.
The polysaccharide content, the extraction yield and the product molecular weight distribution of the sparassis crispa high molecular weight beta-glucan products obtained in the example 2 and the comparative example are measured, the comparative results are shown in the table 1, the high molecular weight beta-glucan product yield of the example 2 is 20.92%, and the alkali extraction method of the comparative example 1 is improved by 57.65%; the molecular weight distribution characteristics of the high molecular part are not greatly different from those of the alkali extraction method, and the weight average molecular weight is slightly improved due to the dissolution promotion effect of high-temperature high-pressure acid cracking treatment on the high molecular weight beta-glucan. The hydrolysis of high temperature and high pressure acid cracking results in the hydrolysis of alpha-glucan and protein into small molecular oligosaccharides, monosaccharides, polypeptides and amino acids, and the separation of the small molecular oligosaccharides, monosaccharides, polypeptides and amino acids from the target polysaccharide through 50% alcohol precipitation, so that the purity of the product is improved, the polysaccharide content reaches 92.32%, the content of the product is improved by 8.1% compared with the alkali extraction method, the peak content of the high molecular polysaccharide reaches 97.70%, and the product is 75.41% compared with the alkali extraction method, and the product also contains about 25% of impurity components such as medium molecular weight alpha-glucan and the like (figure 2).
Analysis of nuclear magnetic properties of the high molecular weight beta-glucan product obtained in example 2 (figures 3-4) shows that the polysaccharide structure of the product obtained in example 2 of the present invention is beta-glucan with a linear backbone linked by beta- (1-3) glycosidic bonds and a branched structure linked by beta- (1-6) glycosidic bonds, and the structural properties are identical to those of the active Sparassis crispa beta-glucan obtained by the reported alkaline extraction. The treatment of example 2 of the present invention was shown to not cause significant disruption of the primary structure of the high molecular weight beta-glucan component of Sparassis crispa.
After further purifying the sparassis crispa high molecular weight beta-glucan product harvested in example 2 and comparative example to a single peak, the in vitro biological activities of the two were compared by analysis of the effect of RAW264.7 on the release of macrophage factor. As shown in FIGS. 5-7, the high molecular weight beta-glucan component of the Sparassis crispa prepared in example 2 and comparative example has substantially the same trend in the release of cytokines IL-1 and IL-6, and the effect of both on the release of TNF-alpha cytokine at low concentration is substantially the same, but the product of example 2 shows significantly higher stimulatory activity on the release of TNF-alpha at high concentration (100 ug/mL).
Table 1 comparison of the technical index of sparassis crispa high molecular weight beta-glucan of example 2 and comparative example product
Example 3
A method for preparing high molecular weight sparassis crispa beta-glucan, comprising the following steps:
1) Sparassis crispa crushing degreasing
Pulverizing Sparassis crispa fruiting body dry product to obtain powder, defatting with ethyl acetate, reflux extracting, filtering to obtain residue, and drying the residue to remove solvent;
2) High temperature and high pressure acid cracking
Adding 3% hydrochloric acid solution (v/v) into the residues, controlling the feed-liquid ratio to be 30 (v/w), uniformly stirring to obtain mixed solution, stirring the mixed solution at room temperature for 8 hours, loading the mixed solution into a high-pressure reaction kettle for high-temperature high-pressure pyrolysis, controlling the reaction temperature to be 130 ℃, controlling the pressure to be 0.18MPa, reacting for 4 hours, cooling to 80 ℃ after the reaction is finished, and discharging to obtain pyrolysis liquid;
3) Alkalization and solid-liquid separation of cracking liquid
Adding potassium hydroxide solution into the pyrolysis extracting solution to adjust the pH to 8.0, stirring at 90 ℃ for reaction for 2 hours, centrifuging after the solution viscosity is obviously reduced, respectively collecting solid residues and supernatant, repeatedly washing the solid residues with a small amount of potassium hydroxide solution with the pH of 8.5 for 2 times, centrifuging, collecting supernatant, and combining the supernatant to obtain the Sparassis crispa beta-glucan extracting solution.
4) Ethanol precipitation centrifugation
Cooling the Sparassis crispa beta-glucan extract, adding 0.9 times of 95% ethanol solution, stirring uniformly, standing at 4deg.C for precipitation for 8 hr, centrifuging or filtering, collecting precipitate, washing with 50% ethanol for 2 times, and drying;
5) Vacuum freeze drying
Lyophilizing the precipitate collected in the step 4) in a vacuum environment to obtain the Sparassis crispa high molecular beta-glucan.
Example 4
A method for preparing high molecular weight sparassis crispa beta-glucan, comprising the following steps:
1) Sparassis crispa crushing degreasing
Pulverizing Sparassis crispa fruiting body dry product to obtain powder, defatting with ethyl acetate, reflux extracting, filtering to obtain residue, and drying the residue to remove solvent;
2) High temperature and high pressure acid cracking
Adding 4% hydrochloric acid solution (v/v) into the residues, controlling the feed-liquid ratio to 25 (v/w), uniformly stirring to obtain mixed solution, stirring the mixed solution at room temperature for 10h, loading the mixed solution into a high-pressure reaction kettle for high-temperature high-pressure pyrolysis, controlling the reaction temperature to 140 ℃, controlling the pressure to 0.27MPa, reacting for 2h, cooling to 80 ℃ after the reaction is finished, and discharging to obtain pyrolysis liquid;
3) Alkalization and solid-liquid separation of cracking liquid
Adding potassium hydroxide solution into the pyrolysis extracting solution to adjust the pH to 8.2, stirring at 85 ℃ for reaction for 2.5 hours, centrifuging after the solution viscosity is obviously reduced, respectively collecting solid residues and supernatant, repeatedly washing the solid residues with a small amount of potassium hydroxide solution with the pH of 8.2 for 1 time, centrifuging, collecting supernatant, and combining the supernatant to obtain the Sparassis crispa beta-glucan extracting solution.
4) Ethanol precipitation centrifugation
Cooling the Sparassis crispa beta-glucan extract, adding 1.2 times of 95% ethanol solution, stirring uniformly, standing at 0deg.C for precipitation for 12h, centrifuging or filtering, collecting precipitate, washing with 60% ethanol for 1 time, and drying;
5) Vacuum freeze drying
Lyophilizing the precipitate collected in the step 4) in a vacuum environment to obtain the Sparassis crispa high molecular beta-glucan.
Claims (6)
1. A method for preparing high molecular weight sparassis crispa beta-glucan, which is characterized by comprising the following steps:
1) Sparassis crispa crushing degreasing
Pulverizing Sparassis crispa fruiting body dry product to obtain powder, defatting with organic solvent, reflux extracting, filtering to obtain residue, drying the residue, and volatilizing solvent;
2) High temperature and high pressure acid cracking
Adding 3% acetic acid solution into the residues, controlling the feed-liquid ratio to be 20-30, stirring uniformly to obtain mixed solution, stirring the mixed solution at room temperature for 8-12 h, then loading the mixed solution into a high-pressure reaction kettle for high-temperature high-pressure pyrolysis, controlling the reaction temperature to be 130-140 ℃, controlling the pressure to be 0.18-0.3 MPa, and cooling and discharging after the reaction is finished to obtain pyrolysis liquid;
3) Alkalization and solid-liquid separation of cracking liquid
Adding alkali liquor into the pyrolysis extracting solution to adjust the pH value to 8.0-8.5, stirring and reacting for 2-3 hours at 80-90 ℃ to reduce the viscosity of the extracting solution, then carrying out solid-liquid separation, and respectively collecting solid residues and supernatant; washing the solid residue with a small amount of dilute alkali solution with pH value of 8.0-8.5 for 1-2 times, centrifuging, collecting supernatant, and combining all the collected supernatant to obtain Sparassis crispa beta-glucan extract;
4) Alcohol precipitation separation
Cooling the Sparassis crispa beta-glucan extract, adding 0.9-1.2 times of 95% ethanol solution, uniformly stirring, standing at 0-4 ℃ for precipitation for 8-12 h, centrifuging or filtering, collecting precipitate and washing;
5) Vacuum freeze drying
Lyophilizing the precipitate collected in the step 4) in a vacuum environment to obtain the Sparassis crispa high molecular beta-glucan.
2. The method for preparing high molecular weight sparassis crispa beta-glucan as claimed in claim 1, wherein the organic solvent used for degreasing in the step 1) is one or more of ethanol, ethyl acetate, petroleum ether and chloroform.
3. The method for preparing the high molecular weight sparassis crispa beta-glucan as claimed in claim 1, wherein in the step 2), the sparassis crispa beta-glucan is discharged after the high-temperature high-pressure pyrolysis reaction is finished and the material is cooled to a temperature below 80 ℃.
4. The method for preparing high molecular weight sparassis crispa beta-glucan as claimed in claim 1, wherein the alkaline solution in the step 3) is one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide and sodium carbonate.
5. The method for preparing high molecular weight sparassis crispa beta-glucan as claimed in claim 1, wherein the solid-liquid separation mode in the step 3) is one or more of centrifugation and filtration.
6. The method for preparing high molecular weight sparassis crispa beta-glucan as claimed in claim 1, wherein in step 4), the precipitate is washed with 50-60% ethanol for 1-2 times.
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