CN112079939A - Preparation method of agaricus blazei polysaccharide extract - Google Patents
Preparation method of agaricus blazei polysaccharide extract 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention discloses a preparation method of agaricus blazei polysaccharide extract, which comprises the following steps: sending the dried agaricus blazei murill in a constant weight state into a grinder to be ground into agaricus blazei murill powder; adding a wall breaking enzyme agent, and putting into an ultrasonic generator for low-frequency enzymolysis to obtain liquid; adding distilled water for dissolving, and stirring thoroughly; placing the mixture into an ultrasonic generator for high-frequency extraction; extracting with complex enzyme; inactivating enzyme of the extractive solution in boiling water; the agaricus blazei murill polysaccharide is obtained by concentrating, alcohol precipitating and centrifuging the extracting solution. The invention has the beneficial effect that the ultrasonic extraction method and the enzyme extraction method are combined to improve the extraction efficiency.
Description
Technical Field
The invention relates to a preparation method of agaricus blazei polysaccharide extract, which is applied to the technical field of biotechnology.
Background
Agaricus blazei muricus blazei Murr (also known as Agaricus blazei Murr) which is native to Brazil and Peru. It is a saprophytic bacterium growing in summer and autumn, living in high-temperature, humid and ventilated environment, and has almond fragrance and crisp and tender mouthfeel. The agaricus blazei murill is tender in cover, crisp in stipe, excellent in taste and pure in 5-7 g; the protein composition comprises 18 amino acids, 8 essential amino acids of human body are complete, and also contains various vitamins and ergosterol. The mannan contained in the composition has effects in inhibiting tumor (especially ascites carcinoma), treating hemorrhoid complicated by anal fistula, improving energy, and preventing and treating cardiovascular diseases.
The agaricus blazei polysaccharide is an effective active ingredient extracted from high-quality agaricus blazei fruiting bodies, is aromatic in smell, remarkable in effect, capable of enhancing energy of a human body, inhibiting growth of tumor cells, resisting tumors, absorbing and excreting carcinogens, reducing blood sugar, improving diabetes, reducing cholesterol and improving arteriosclerosis, and has high medicinal value and health care value.
Currently, the extraction method of agaricus blazei murill polysaccharide includes an ultrasonic extraction method, an acid-base extraction method, an ethanol extraction method, an enzyme extraction method, and the like. The ultrasonic extraction method has the defects of large ineffective energy loss and relatively high cost of complete extraction. The acid-base extraction method has high requirements on equipment, and the molecular structure of the polysaccharide is easily damaged by acid-base conditions. The ethanol extraction method has the problems of low extraction efficiency and general purity of the obtained polysaccharide. The enzyme extraction method has the problems of long reaction time and large sugar liquid filtering difficulty.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of agaricus blazei murill polysaccharide extract, wherein an ultrasonic extraction method and an enzyme extraction method are combined to improve the extraction efficiency.
The invention is realized by the following technical scheme.
A method for preparing polysaccharide extract of Agaricus blazei Murill comprises the following steps:
step (1): feeding dried Agaricus blazei Murill into a pulverizer, pulverizing to obtain Agaricus blazei Murill powder with fineness of 80-150 meshes, and sieving;
step (2): adding cell wall breaking enzyme, and performing low-frequency enzymolysis in an ultrasonic generator to obtain liquid, wherein the frequency of the ultrasonic generator is controlled at 30-40 KHZ;
and (3): adding 10-15 times of distilled water by mass, dissolving and fully stirring;
and (4): placing the mixture into an ultrasonic generator for high-frequency extraction, wherein the extraction time is controlled to be 30-45 minutes, and the frequency of the ultrasonic generator is regulated to be 120-140 KHZ;
and (5): extracting with complex enzyme in 40-55 deg.C constant temperature water bath for 40-60 min;
and (6): inactivating enzyme of the extractive solution in boiling water for 8-10 min;
and (7): concentrating the extractive solution, precipitating with ethanol, and centrifuging to obtain Agaricus blazei Murill polysaccharide, wherein the concentration degree is 1/6-1/4, and the centrifugation is carried out at 5500r/min of 5000-.
The invention relates to an extraction method of agaricus blazei murill polysaccharide, which is characterized in that an ultrasonic extraction method and a compound enzyme extraction method are organically combined, firstly, ultrasonic extraction is used as primary lifting, and a vibration effect, a cavitation effect and a heat effect generated by ultrasonic high-frequency extraction and high-frequency oscillation are utilized to act inside an agaricus blazei murill water-soluble mixture to form cell rupture and promote the dissolution of effective components in cells, so that the loss of the active components such as the agaricus blazei murill polysaccharide is small.
The agaricus blazei murill comprises the following components: polysaccharides, protein, fat, crude fiber, ergosterol, various amino acids and the like are extracted by ultrasonic waves, and a micro shock wave is formed by a cavitation effect, so that the cell rupture and the rupture of the water-soluble mixture can be accelerated, the whole rupture process is completed in the moment, and the absorbed energy is completely or mostly converted into heat energy by a heat effect medium, so that the temperature of the medium and the tissue temperature of medicinal materials are increased, the dissolving speed of active ingredients of the medicines is increased, and the agaricus blazei murill polysaccharide is dissolved out from the cells of the water-soluble mixture, and the activity of the agaricus blazei murill polysaccharide can be maximally retained due to the fact that the primary extraction belongs to a.
And secondly, the compound enzyme extraction is used for secondary extraction, the compound enzyme is adopted to further destroy the cell wall structure, the method has the characteristics of mild reaction conditions and high selectivity, the specificity of the enzyme can avoid the damage to substances outside the substrate, in addition, the enzyme extraction method also has an important function of being capable of aiming at low-content components and having outstanding targeting capability, targeted extraction is carried out on the agaricus blazei murill after primary extraction, and intercellular connective filaments can be decomposed through the enzyme, so that the dissolution of intracellular effective components is promoted, and therefore, the enzyme extraction and the ultrasonic extraction are combined with each other, and the extraction purity and the extraction efficiency of the agaricus blazei murill polysaccharide can be effectively improved.
As a further improvement of the invention, the wall-breaking enzyme agent in the step (2) is a water-soluble mixture of cellulase.
The method uses cellulase as a wall breaking enzyme agent, can pre-treat agaricus blazei before ultrasonic extraction, and has the mechanism of dissolving cell walls under the action of the cellulase. On one hand, the effective components are easier to extract by degrading the plant cell walls, thereby achieving the purpose of improving the extraction yield or reducing the solvent consumption; on the other hand, the composite can be selectively degraded aiming at most impurities (starch, pectin, protein and the like) in the plant medicine, so as to be beneficial to the subsequent ultrasonic extraction.
As a further improvement of the invention, when the ultrasonic extraction is carried out in the step (4), 3 to 5 mass percent of alumina particles are added, and the alumina particles are filtered by using a microfiltration membrane after the extraction is finished.
During ultrasonic extraction, the alumina particles can be fully contacted with cells and generate friction, and the generated heat can strengthen the heat effect of the ultrasonic extraction, so that the dissolution of the agaricus blazei murill polysaccharide from the cells is improved; in addition, the generated heat may further exert an effect on the agaricus blazei murrill polysaccharide to be decomposed into small molecules by breaking bonds of the large molecules of the agaricus blazei murrill polysaccharide.
As a further improvement of the invention, after the extraction in the step (4) is completed and before the step (5), the extract is sent to a centrifuge for centrifugation at 3500-.
As a further improvement of the invention, the compound enzyme in the step (5) comprises protease and pectinase.
The protease can break peptide bonds of proteins and hydrolyze free proteins in cells, the space structure of the protease is broken, the protease becomes loose, meanwhile, the protease can also hydrolyze egg tropine in proteoglycan and glycoprotein, the binding rate of the protease to polysaccharide is reduced, and the polysaccharide is easier to dissolve out.
As a further improvement of the invention, the pH value of the step (5) is adjusted to 5.0-5.8 before the complex enzyme is used for extraction.
Because the activity of the complex enzyme is sensitive to pH, the combination degree of the complex enzyme and the substrate can be improved by properly adjusting the pH value, and the extraction efficiency and the reaction speed are favorably improved.
As a further improvement of the invention, step (5) is to adjust the pH to 5.2 before using the complex enzyme for extraction.
The pH value is the optimal enzymolysis pH value of the compound enzyme (protease and pectinase), and the combination degree of the compound enzyme and the substrate reaches the highest degree under the condition.
As a further improvement of the present invention, step (5) is pH-adjusted using a sodium dihydrogen phosphate buffer and a citric acid buffer.
As a further improvement of the invention, the temperature of the thermostatic water bath in the step (5) is 50.5 ℃.
The constant temperature water bath is at a suitable temperature for extraction at 40-55 deg.C, the polysaccharide extraction rate is increased when the temperature is gradually increased from 40 deg.C, the polysaccharide extraction rate is highest when the temperature is about 50.5 deg.C, and the polysaccharide extraction rate is decreased with the increase of the temperature.
The invention has the beneficial effects that:
through the ultrasonic wave extraction method with the physics mode of extracting to agaricus blazei murill polysaccharide carry out preliminary extraction, use the enzyme extraction method to extract it again, both organic combination has compared single extraction mode, has improved agaricus blazei murill polysaccharide's extraction efficiency and extraction rate, in addition through broken wall processing in advance before the extraction and use the supplementary ultrasonic wave of strengthening of aluminium oxide granule to extract etc. further strengthened the effect.
Detailed Description
The present invention will be described in further detail below based on embodiments.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Example 1:
a method for preparing polysaccharide extract of Agaricus blazei Murill comprises the following steps:
step (1): sending dried Agaricus blazei Murill to a pulverizer, pulverizing to obtain Agaricus blazei Murill powder with fineness of 100 mesh, and sieving;
step (2): adding a wall-breaking enzyme agent, placing into an ultrasonic generator, and performing low-frequency enzymolysis to obtain a liquid, wherein the frequency of the ultrasonic generator is regulated to 35KHZ, and the wall-breaking enzyme agent is a water-soluble mixture of cellulase;
and (3): adding 10 mass times of distilled water, dissolving and fully stirring;
and (4): putting the mixture into an ultrasonic generator for high-frequency extraction, adding 3-5% of alumina particles by mass ratio, controlling the extraction time to be 40 minutes, regulating the frequency of the ultrasonic generator to be 135KHZ, filtering the alumina particles by using a microfiltration membrane, and then conveying the filtered alumina particles into a centrifuge for centrifuging at 3800r/min for 12 min;
and (5): adjusting pH to 5.0 with sodium dihydrogen phosphate and citric acid buffer solution, extracting with complex enzyme at 40 deg.C in constant temperature water bath for 45 min, wherein the complex enzyme is selected from 1:1 compound protease and pectinase, and the amount is 2-3% by mass;
and (6): inactivating enzyme in boiling water for 10 min;
and (7): concentrating the extractive solution, precipitating with ethanol, and centrifuging to obtain Agaricus blazei Murill polysaccharide, wherein the concentration degree is 1/4, and the centrifugation is carried out at 5200r/min for 10 min.
Example 2:
a method for preparing polysaccharide extract of Agaricus blazei Murill comprises the following steps:
step (1): sending dried Agaricus blazei Murill to a pulverizer, pulverizing to obtain Agaricus blazei Murill powder with fineness of 80 mesh, and sieving;
step (2): adding a wall-breaking enzyme agent, placing into an ultrasonic generator, performing low-frequency enzymolysis to obtain a liquid, wherein the frequency of the ultrasonic generator is regulated to 30KHZ, and the wall-breaking enzyme agent is a water-soluble mixture of cellulase;
and (3): adding 15 mass times of distilled water, dissolving and fully stirring;
and (4): putting the mixture into an ultrasonic generator for high-frequency extraction, adding 3-5% of alumina particles by mass ratio, controlling the extraction time to be 40 minutes, regulating the frequency of the ultrasonic generator to be 140KHZ, filtering the alumina particles by using a microfiltration membrane, and then conveying the filtered alumina particles into a centrifuge for centrifuging for 10 minutes at 4000 r/min;
and (5): adjusting pH to 5.5 with sodium dihydrogen phosphate and citric acid buffer solution, extracting with complex enzyme at 50 deg.C in thermostatic water bath for 50 min, wherein the complex enzyme is selected from 1:1 compound protease and pectinase, and the amount is 2-3% by mass;
and (6): inactivating enzyme in boiling water for 8 min;
and (7): concentrating the extractive solution, precipitating with ethanol, and centrifuging to obtain Agaricus blazei Murill polysaccharide, wherein the concentration degree is 1/5, and the centrifugation is carried out at 5500r/min for 8 min.
Example 3:
a method for preparing polysaccharide extract of Agaricus blazei Murill comprises the following steps:
step (1): sending the dried agaricus blazei murill in a constant weight state into a grinder to be ground into agaricus blazei murill powder, wherein the ground fineness is 150 meshes, and screening for later use;
step (2): adding a wall-breaking enzyme agent, placing into an ultrasonic generator, performing low-frequency enzymolysis to obtain a liquid, wherein the frequency of the ultrasonic generator is controlled at 40KHZ, and the wall-breaking enzyme agent is a water-soluble mixture of cellulase;
and (3): adding 12 times of distilled water by mass, and fully stirring after dissolving;
and (4): putting the mixture into an ultrasonic generator for high-frequency extraction, adding 3-5% of alumina particles by mass ratio, controlling the extraction time to be 45 minutes, regulating the frequency of the ultrasonic generator to be 140KHZ, filtering the alumina particles by using a microfiltration membrane, and then conveying the filtered alumina particles into a centrifuge for centrifuging at 3800r/min for 15 min;
and (5): adjusting pH to 5.2 with sodium dihydrogen phosphate and citric acid buffer solution, extracting with complex enzyme at 50.5 deg.C in thermostatic water bath for 40-60 min, wherein the complex enzyme is selected from 1:1 compound protease and pectinase, and the amount is 2-3% by mass;
and (6): inactivating enzyme in boiling water for 10 min;
and (7): concentrating the extractive solution, precipitating with ethanol, and centrifuging to obtain Agaricus blazei Murill polysaccharide, wherein the concentration degree is 1/6, and the centrifugation is carried out at 5200r/min for 6 min.
Example 4:
a method for preparing polysaccharide extract of Agaricus blazei Murill comprises the following steps:
step (1): sending dried Agaricus blazei Murill to a pulverizer, pulverizing to obtain Agaricus blazei Murill powder with fineness of 120 mesh, and sieving;
step (2): adding a wall-breaking enzyme agent, placing into an ultrasonic generator, performing low-frequency enzymolysis to obtain a liquid, wherein the frequency of the ultrasonic generator is controlled at 33KHZ, and the wall-breaking enzyme agent is a water-soluble mixture of cellulase;
and (3): adding 10-15 times of distilled water by mass, dissolving and fully stirring;
and (4): putting the mixture into an ultrasonic generator for high-frequency extraction, adding 3-5% of alumina particles by mass ratio, controlling the extraction time to be 42 minutes, regulating the frequency of the ultrasonic generator to be 128KHZ, filtering the alumina particles by using a microfiltration membrane, and then conveying the filtered alumina particles into a centrifuge for centrifugation for 14 minutes at 4000 r/min;
and (5): adjusting pH to 5.6 with sodium dihydrogen phosphate and citric acid buffer solution, extracting with complex enzyme at 52 deg.C in thermostatic water bath for 55 min, wherein the complex enzyme is selected from 1:1 compound protease and pectinase, and the amount is 2-3% by mass;
and (6): inactivating enzyme in boiling water for 10 min;
and (7): concentrating the extractive solution, precipitating with ethanol, and centrifuging to obtain Agaricus blazei Murill polysaccharide, wherein the concentration degree is 1/5, and the centrifugation is carried out at 5300r/min for 8 min.
Example 5:
a method for preparing polysaccharide extract of Agaricus blazei Murill comprises the following steps:
step (1): sending dried Agaricus blazei Murill to a pulverizer, pulverizing to obtain Agaricus blazei Murill powder with fineness of 120 mesh, and sieving;
step (2): adding a wall-breaking enzyme agent, placing into an ultrasonic generator, performing low-frequency enzymolysis to obtain a liquid, wherein the frequency of the ultrasonic generator is controlled at 33KHZ, and the wall-breaking enzyme agent is a water-soluble mixture of cellulase;
and (3): adding 11 times of distilled water by mass, and fully stirring after dissolving;
and (4): putting the mixture into an ultrasonic generator for high-frequency extraction, adding 3-5% of alumina particles by mass ratio, controlling the extraction time to be 42 minutes, regulating the frequency of the ultrasonic generator to be 125KHZ, filtering the alumina particles by using a microfiltration membrane, and then conveying the filtered alumina particles into a centrifuge for centrifuging at 3500r/min for 10 min;
and (5): adjusting pH to 5.8 with sodium dihydrogen phosphate and citric acid buffer solution, extracting with complex enzyme at 55 deg.C in thermostatic water bath for 55 min, wherein the complex enzyme is selected from 1:1 compound protease and pectinase, and the amount is 2-3% by mass;
and (6): inactivating enzyme in boiling water for 10 min;
and (7): concentrating the extractive solution, precipitating with ethanol, and centrifuging to obtain Agaricus blazei Murill polysaccharide, wherein the concentration degree is 1/5, and the centrifugation is carried out at 5300r/min for 8 min.
Comparative example: agaricus blazei Murill polysaccharide obtained by extracting with hot water is prepared by the following experimental method:
the experiment groups 1 to 5 are respectively the experiment group 1 to the experiment group 5, and the comparative example is the control group, wherein, each group quantitatively controls 50g of hericium erinaceus to carry out the experiment.
The data are shown in table 1:
TABLE 1 Experimental groups 1-5, control group experimental data table
Determining the conditions, respectively dissolving in 95% ethanol 20 times by mass under laboratory conditions, stirring thoroughly until no particulate organic matter is present, and measuring polysaccharide content in the experimental group 1-5 and the control group by HPLC OR GC (high performance liquid chromatography).
Experimental test data are shown in table 2:
in conclusion, the experimental data show that the extraction purity of the hericium erinaceus polysaccharide is improved by the extraction method of the hericium erinaceus polysaccharide, the process is simple, and the method is suitable for industrial production.
Claims (9)
1. A method for preparing agaricus blazei murill polysaccharide extract is characterized by comprising the following steps:
step (1): feeding dried Agaricus blazei Murill into a pulverizer, pulverizing to obtain Agaricus blazei Murill powder with fineness of 80-150 meshes, and sieving;
step (2): adding cell wall breaking enzyme, and performing low-frequency enzymolysis in an ultrasonic generator to obtain liquid, wherein the frequency of the ultrasonic generator is controlled at 30-40 KHZ;
and (3): adding 10-15 times of distilled water by mass, dissolving and fully stirring;
and (4): placing the mixture into an ultrasonic generator for high-frequency extraction, wherein the extraction time is controlled to be 30-45 minutes, and the frequency of the ultrasonic generator is regulated to be 120-140 KHZ;
and (5): extracting with complex enzyme in 40-55 deg.C constant temperature water bath for 40-60 min;
and (6): inactivating enzyme of the extractive solution in boiling water for 8-10 min;
and (7): concentrating the extractive solution, precipitating with ethanol, and centrifuging to obtain Agaricus blazei Murill polysaccharide, wherein the concentration degree is 1/6-1/4, and the centrifugation is carried out at 5500r/min of 5000-.
2. The method for preparing agaricus blazei murill polysaccharide extract according to claim 1, wherein the wall-breaking enzyme agent in the step (2) is an aqueous mixture of cellulase.
3. The method of preparing the polysaccharide extract of Agaricus blazei according to claim 1, wherein the ultrasonic extraction in the step (4) is performed by adding 3 to 5 mass% of alumina particles and filtering the alumina particles with a microfiltration membrane after the completion of the extraction.
4. The method for preparing polysaccharide extract of Agaricus blazei Murill according to claim 3, wherein the polysaccharide extract is centrifuged in a centrifuge at 3500 and 4000r/min for 10-15min after the extraction in step (4) and before the extraction in step (5).
5. The method for preparing the polysaccharide extract of Agaricus blazei according to claim 1, wherein the complex enzyme of step (5) comprises protease and pectinase.
6. The method for preparing polysaccharide extract of Agaricus blazei according to claim 5, wherein the pH in step (5) is adjusted to 5.0 to 5.8 before the complex enzyme extraction.
7. The method for preparing polysaccharide extract of Agaricus blazei according to claim 6, wherein the pH in step (5) is adjusted to 5.2 before the complex enzyme extraction.
8. The method of preparing the polysaccharide extract of Agaricus blazei according to claim 6 or 7, wherein the pH adjustment in step (5) is performed using a sodium dihydrogen phosphate buffer and a citric acid buffer.
9. The method for preparing agaricus blazei murill polysaccharide extract according to claim 5, wherein the temperature of the thermostatic water bath of the step (5) is 50.5 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115226727A (en) * | 2021-12-10 | 2022-10-25 | 金华市农业科学研究院(浙江省农业机械研究院) | Anti-plant virus composition, preparation containing composition and application thereof |
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Application publication date: 20201215 |