CN110272932B - Preparation method of ganoderma lucidum spore powder polysaccharide peptide - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a preparation method of ganoderma lucidum spore powder polysaccharide peptide. The preparation method of the ganoderma lucidum spore powder polysaccharide peptide comprises the steps of performing wall breaking treatment on the ganoderma lucidum spore powder, extracting by adopting supercritical carbon dioxide, performing enzymolysis on the degreased wall-broken ganoderma lucidum spore powder, extracting and filtering enzymolysis liquid, concentrating, drying, crushing and sieving. The method has the advantages of simpler and more convenient process, greenness, safety and convenience for industrial production; the treatment time is shortened, and the degeneration and inactivation of ganoderma lucidum functional components due to overlong treatment time are prevented; the enzyme used for enzymolysis does not damage the glycopeptide structure and is easy to filter; and an organic solvent is not used, so that the problem of organic solvent residue can be avoided. In addition, by the method, the yield of the glycopeptide can reach 9.50%, and the content of the polysaccharide peptide in the ganoderma lucidum spore powder of the sample is 36.11%.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of ganoderma lucidum spore powder polysaccharide peptide.
Background
Ganoderma lucidum is a fungus belonging to the genus Ganoderma of the family Polyporaceae of the class Basidiomycetes, and has a long history as a traditional rare traditional Chinese medicine for medicine and food. The spore powder is spore continuously ejected from the fungus tube after the Ganoderma encarpium is mature, is biologically called basidiospore, and is collected to be powder, called Ganoderma spore powder. The spore powder collects the essence part of Ganoderma, and has components such as triterpene, polysaccharide, adenosine, protein, enzymes, selenium element, etc. more abundant than Ganoderma. A large number of researches show that the ganoderma lucidum spore powder has various pharmacological effects of enhancing the immunity of organisms, inhibiting tumors, reducing blood sugar and blood fat, protecting the liver, resisting viruses, resisting radiation and the like. The Ganoderma spore has double-wall structure, is surrounded by hard chitin cellulose, has hard texture, and is acid and alkali resistant, and can prevent human body from absorbing and utilizing its effective components. In order to fully utilize the effective components in the ganoderma lucidum spore powder, the spore powder needs to be subjected to wall breaking so as to be convenient for the utilization of the effective components.
Many pharmacological activities of ganoderma lucidum are of great importance to ganoderma lucidum polysaccharide peptides. Ganoderan peptide is a glycoprotein, a combination of polysaccharides and proteins, consisting of repeating units of disaccharides, and generally contains a high proportion of polysaccharides. The present researches show that the ganoderma lucidum polysaccharide peptide has wide pharmacological activity, can improve the immunity of human bodies, improve the anoxia resistance of the human bodies, eliminate in-vivo free radicals, inhibit the generation of tumor cells, resist radiation, improve the capability of synthesizing DNA, RNA and protein of livers, bone marrow and blood, and has the health-care effects of delaying senility and prolonging the service life. In addition, the ganoderan peptide also has the effects of activating nonspecific antibodies in human bodies, enhancing immune specific reaction, inhibiting physiological activity of transplanted tumors and the like. The ganoderma lucidum polysaccharide peptide has obvious effects on cardiovascular diseases, asthma, allergy, neurasthenia, stomach heat and the like, and also has the effects of reducing blood pressure, reducing blood fat, relieving blood stasis, improving blood circulation, whitening skin and the like.
At present, ganoderma lucidum polysaccharide peptide mainly takes ganoderma lucidum mycelia and ganoderma lucidum sporocarp as raw materials, ganoderma lucidum extract is obtained through extraction treatment such as water extraction, alkali extraction and alcohol extraction, and then is subjected to membrane filtration treatment such as fine filtration, ultrafiltration and reverse osmosis, and then is subjected to purification treatment by combining with one or more of column chromatography methods such as an anion-cation resin column, a gel column, a medium-pressure column and a reverse-phase column, or only by combining fillers such as resin, silica gel and gel in the column without column packing. The method can separate and purify Ganoderma polysaccharide peptide by repeated membrane filtration and column chromatography with different principles, and obtain product with high purity and stable property. However, these processes have large loss, large equipment investment, high equipment cleaning and maintenance cost, time consumption, long production time and high production cost, and a large amount of organic solvent is required in the extraction process, so that the product may have solvent residues, and hidden troubles are brought to the edible safety of the product. In addition, the existing extraction and purification of ganoderma lucidum polysaccharide peptide also has the problems of low extraction efficiency, high extraction cost, low product quality, low content of effective components, poor stability and the like. For example, the existing extraction method finally obtains high-purity ganoderma lucidum polysaccharide peptide through multiple separation and purification procedures, but the production procedures are complicated, a plurality of high-precision separation and purification equipment is required to be applied, the investment is large, the cost is high, the industrial production is difficult to realize, and the product is difficult to popularize and apply.
CN201610221431.X discloses a method for comprehensive extraction and utilization of Ganoderma fruiting body and Ganoderma spore. The method needs decompression separation after supercritical extraction of the ganoderma lucidum spores, and shows that the ganoderma lucidum spore oil in the spore powder can not be fully extracted due to unreasonable parameter setting during supercritical extraction; the ganoderma lucidum fruiting body and the extraction residues are mixed for water body, the ganoderma lucidum fruiting body has high lignification degree and complex structure, the polysaccharide is generally combined with cellulose and lignin through physical or chemical bonds, the polysaccharide is difficult to extract by adopting a water extraction method, and multiple times of reflux extraction are needed; ethanol precipitation is carried out, and because the small molecular polysaccharide is dissolved in ethanol, part of active polysaccharide is lost by the ethanol precipitation, and the ethanol precipitate is washed by acetone, so that the problem of organic solvent residue exists. In fact, the method has obvious defects, such as long production time, high production cost, complex production procedures, low extraction efficiency, residual solvent in the product and the like.
CN200610097229.7 discloses a refining process of ganoderma lucidum spore polysaccharide, which adopts warm immersion water extraction, the extraction process takes longer time, needs to be carried out for a plurality of times, and after the extracting solutions are combined, the extracting solution needs to be stood for a longer time and then is filtered and concentrated; an alcohol precipitation step is adopted, and small molecular polysaccharides are dissolved in ethanol, so that part of active polysaccharides are lost by alcohol precipitation; the refining process comprises decolorization, deproteinization by a sevag method, dialysis, alcohol precipitation and washing, and the operation is complicated, a large amount of organic solvent is required (for example, deproteinization by the sevag method is carried out, chloroform-n-butanol (4. In addition, although the yield and the content of the polysaccharide are improved after refining, the content of the polysaccharide in the refined product of the ganoderma lucidum spore polysaccharide is calculated by anhydrous glucose, and the polysaccharide cannot be proved to be the micromolecular polysaccharide peptide.
CN201610853239.2 discloses a method for preparing glycopeptide of ganoderma spore powder, which comprises sequentially extracting with concentrated alcohol, extracting with diluted alcohol, mixing the concentrated alcohol extractive solution with the diluted alcohol extractive solution, defatting with diethyl ether, removing impurities, decolorizing, and purifying with resin, and preferably further extracting the residue with concentrated alcohol and diluted alcohol, precipitating with alcohol, and refining. The method uses a large amount of alcohol in the extraction process, needs to carry out extraction reflux for many times, uses ether for degreasing, and uses chloroform-n-butanol (4).
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for extracting polysaccharide peptide from ganoderma lucidum spore powder. The invention takes the defatted spore powder as the raw material, obtains the ganoderma lucidum polysaccharide peptide with stable property by a feasible and industrialized method, changes the defatted spore powder into valuables, solves the problems of low extraction efficiency and high extraction cost of the ganoderma lucidum polysaccharide peptide in the prior art and the problems of inactivation and low content of functional components of the ganoderma lucidum polysaccharide peptide in the final product, improves the effective utilization rate of the ganoderma lucidum spore powder, and plays a great role in promoting the development of the ganoderma lucidum industry.
The invention is realized by the following technical scheme:
a method for preparing polysaccharide peptide of ganoderma lucidum spore powder comprises the following steps:
(1) Wall breaking: performing low-temperature physical wall breaking treatment on the ganoderma lucidum spore powder to enable the wall breaking rate to reach more than 95%;
(2) Degreasing: extracting the wall-broken Ganoderma spore powder in supercritical carbon dioxide extraction device, separating twice, and collecting oil and defatted wall-broken Ganoderma spore powder;
(3) Enzymolysis: mixing the defatted wall-broken Ganoderma spore powder with purified water, adding alkaline solution to adjust pH to 5-10, and adding enzyme for enzymolysis;
(4) Extracting and filtering: heating the enzymolysis liquid to 90-100 ℃ under stirring, preserving heat for 1-2h to obtain an extracting solution, then separating, and collecting clear filtrate;
(5) Concentration: concentrating the filtrate to obtain extract with solid content of 35-55%;
(6) And (3) drying: vacuum drying the extract;
(7) Crushing and sieving: pulverizing, and sieving with 60-80 mesh sieve to obtain Ganoderma spore powder polysaccharide peptide powder.
In a preferred embodiment of the present invention, the low-temperature physical wall breaking is performed by using a vibrating ultrafine grinder. Specifically, charging Ganoderma spore powder into a vibration type ultrafine pulverizer, opening a cooling water switch, and breaking cell wall when the temperature of cooling water is constant to 3-5 deg.C for 25-35min to make the cell wall breaking rate reach above 95%.
In the preferred embodiment of the invention, the pump frequency of the extraction device is 25-30Hz, the flow rate of the supercritical carbon dioxide is more than or equal to 120L/h, the extraction pressure is 18-25MPa, the extraction temperature is 40-50 ℃, and the extraction time is 4-5h. More preferably, the flow rate of the supercritical carbon dioxide is 120 to 160L/h.
In the preferred embodiment of the invention, two times of separation are carried out, wherein the first-stage separation pressure is 8-10MPa, and the separation temperature is 40-50 ℃; the secondary separation pressure is 5-6MPa, and the separation temperature is 28-35 ℃.
In a preferred embodiment of the present invention, the concentration of the alkali solution is 2 to 3mol/L, and the alkali solution is one of sodium hydroxide, potassium hydroxide, calcium hydroxide and ammonia water.
In a preferred embodiment of the invention, a basic solution is added to adjust the pH to 7.0-8.5.
In a preferred embodiment of the invention, the enzyme is one or more of cellulase, complex protease, alkaline protease and neutral protease; the addition amount of the enzyme is 0.1-2% of the weight of the defatted wall-broken ganoderma lucidum spore powder.
In the preferred embodiment of the invention, the enzymolysis temperature is 40-55 ℃, and the enzymolysis time is 2-12h. More preferably, the time for enzymatic hydrolysis is 4-10h.
In the preferred embodiment of the invention, the ultrasonic treatment is carried out during the enzymolysis, the frequency of the ultrasonic treatment is 20-40KHz, and the time is 4-6h.
In the preferred embodiment of the invention, the concentration is carried out by double-effect vacuum energy-saving concentration equipment, the first-effect vacuum degree of the double-effect vacuum energy-saving concentration equipment is-0.06 Mpa, and the temperature is 70-80 ℃; the double-effect vacuum degree is-0.08 MPa, and the temperature is 50-60 ℃.
In a preferred embodiment of the invention, the temperature of the vacuum drying is 60-70 ℃ and the pressure is-0.07 to-0.098 MPa.
The invention has the beneficial effects that:
the invention adopts the degreased wall-broken ganoderma lucidum spore powder as the raw material, and obtains the ganoderma lucidum spore powder polysaccharide peptide through wall breaking, degreasing, enzymolysis, extraction, concentration, drying, crushing and sieving processes.
Compared with the method in the prior art, the method does not need to use fine filtration methods such as reverse osmosis, nanofiltration, ultrafiltration, microfiltration and the like for purification treatment, so that the equipment investment and the cost of use and maintenance are saved; the process is simpler, green and safe, and is convenient for industrial production; the treatment time is shortened, and the degeneration and inactivation of the ganoderma lucidum functional components caused by overlong treatment time are prevented; the enzyme used in enzymolysis does not damage the glycopeptide structure and is easy to filter; and an organic solvent is not used, so that the problem of organic solvent residue can be avoided. In addition, the defatted spore powder is directly extracted by water, the yield of glycopeptide is only 0.21%, and the polysaccharide peptide content of the ganoderma spore powder of the sample is only 1.95%; by the method, the yield of the glycopeptide can reach 9.50%, and the content of the polysaccharide peptide in the ganoderma lucidum spore powder of the sample is 36.11%.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
A method for preparing polysaccharide peptide of ganoderma lucidum spore powder comprises the following steps:
(1) Wall breaking: loading 100kg Ganoderma spore powder into a vibration type ultramicro pulverizer, opening a cooling water switch, and breaking wall for 30min after the temperature of cooling water is constant to 3-5 deg.C to make the wall breaking rate reach above 95%;
(2) Degreasing: putting the wall-broken ganoderma lucidum spore powder into a supercritical carbon dioxide extraction kettle, introducing supercritical carbon dioxide fluid with the flow rate of 120L/h, extracting for 4h under the conditions of the pump frequency of 27.5Hz, the extraction pressure of 21MPa and the temperature of 45 ℃, then separating under the conditions of the primary separation pressure of 9MPa, the temperature of 45 ℃, the secondary separation pressure of 5.5MPa and the temperature of 31 ℃, and collecting grease and degreased wall-broken ganoderma lucidum spore powder;
(3) Enzymolysis: mixing 50kg of defatted wall-broken ganoderma lucidum spore powder and 500kg of purified water, adding 2mol/L of sodium hydroxide solution under the stirring condition, adjusting the pH to 7.2, then adding 0.5kg of cellulase and 0.25kg of compound protease for enzymolysis, keeping stirring during the enzymolysis, maintaining the pH of the solution to be 7.2 +/-0.1 by adding 2mol/L of sodium hydroxide, and carrying out enzymolysis for 4 hours at the temperature of 48 ℃;
(4) Extraction and separation: heating the enzymolysis liquid to 95 ℃ under the stirring condition, preserving the heat for 2 hours to obtain an extracting solution, then separating slag and liquid in the extracting solution at the rotating speed of 5000rpm by using a centrifugal machine, and collecting clear filtrate;
(5) And (3) concentrating: concentrating the clear filtrate by double-effect vacuum energy-saving concentration equipment to obtain extract with solid content of 45% at the first-effect vacuum degree of-0.06 MPa and the temperature of 70 ℃, and the second-effect vacuum degree of-0.08 MPa and the temperature of 50 ℃;
(6) And (3) drying: vacuum drying the extract at 65 deg.C under-0.084 MPa for 12 hr;
(7) Crushing and sieving: pulverizing, and sieving with 80 mesh sieve to obtain Ganoderma spore powder polysaccharide peptide powder.
Example 2
A method for preparing polysaccharide peptide of ganoderma lucidum spore powder comprises the following steps:
(1) Wall breaking: loading 100kg Ganoderma spore powder into a vibration type superfine pulverizer, opening a cooling water switch, and breaking wall for 30min after the cooling water temperature is constant to 3-5 deg.C to make the wall breaking rate reach above 95%;
(2) Degreasing: putting the wall-broken ganoderma lucidum spore powder into a supercritical carbon dioxide extraction kettle, introducing a supercritical carbon dioxide fluid with the flow rate of 140L/h, extracting for 5h under the conditions of the pump frequency of 25Hz, the extraction pressure of 18MPa and the temperature of 50 ℃, then separating under the conditions of the primary separation pressure of 8MPa, the temperature of 50 ℃, the secondary separation pressure of 5MPa and the temperature of 35 ℃, collecting oil and degreased wall-broken ganoderma lucidum spore powder;
(3) Enzymolysis: mixing 50kg of defatted wall-broken ganoderma lucidum spore powder and 300kg of purified water, adding 2.5mol/L potassium hydroxide solution under the stirring condition, adjusting the pH to 8.5, then adding 0.25kg of cellulase and 0.325kg of alkaline protease for enzymolysis, keeping stirring during the enzymolysis period, maintaining the pH of the solution to 8.5 +/-0.1 by adding 2.5mol/L potassium hydroxide, and carrying out enzymolysis for 4 hours at the temperature of 42 ℃ under the ultrasonic condition;
(4) Extraction and separation: heating the enzymolysis liquid to 95 ℃ under the stirring condition, preserving the heat for 2 hours to obtain an extracting solution, filtering the extracting solution by a plate and frame filter, and collecting the filtrate after the filtrate is refluxed to be clear;
(5) Concentration: concentrating the clear filtrate by double-effect vacuum energy-saving concentration equipment to obtain extract with solid content of 55% at the first-effect vacuum degree of-0.06 MPa and the temperature of 70 ℃, and the second-effect vacuum degree of-0.08 MPa and the temperature of 50 ℃;
(6) And (3) drying: vacuum drying the extract at 70 deg.C under-0.07 MPa for 10 hr;
(7) Crushing and sieving: pulverizing, and sieving with 60 mesh sieve to obtain Ganoderma spore powder polysaccharide peptide powder.
Example 3
A method for preparing polysaccharide peptide of ganoderma lucidum spore powder comprises the following steps:
(1) Wall breaking: loading 100kg Ganoderma spore powder into a vibration type ultramicro pulverizer, opening a cooling water switch, and breaking wall for 30min after the temperature of cooling water is constant to 3-5 deg.C to make the wall breaking rate reach above 95%;
(2) Degreasing: putting the ganoderma lucidum spore powder after wall breaking into a supercritical carbon dioxide extraction kettle, introducing supercritical carbon dioxide fluid with the flow rate of 160L/h, extracting for 4h under the conditions of the pump frequency of 30Hz, the extraction pressure of 25MPa and the temperature of 40 ℃, then separating under the conditions of the primary separation pressure of 10MPa, the temperature of 40 ℃, the secondary separation pressure of 6MPa and the temperature of 28 ℃, and collecting grease and degreased wall-broken ganoderma lucidum spore powder;
(3) Enzymolysis: mixing 50kg of defatted wall-broken ganoderma lucidum spore powder and 250kg of purified water, adding 3.0mol/L sodium hydroxide solution under stirring, adjusting the pH to 7.5, then adding 0.3kg of neutral protease for enzymolysis, keeping stirring during the enzymolysis, maintaining the pH of the solution to 7.5 +/-0.1 by adding 3.0mol/L sodium hydroxide, and performing enzymolysis for 4 hours at 53 ℃ under ultrasonic conditions;
(4) Extraction and separation: heating the enzymolysis liquid to 95 ℃ under the stirring condition, preserving the heat for 2 hours to obtain an extracting solution, then separating slag and liquid in the extracting solution at the rotating speed of 4000rpm by using a centrifugal machine, and collecting clear filtrate;
(5) Concentration: concentrating the clear filtrate by double-effect vacuum energy-saving concentration equipment to obtain extract with solid content of 40% at the first-effect vacuum degree of-0.06 MPa and the temperature of 70 ℃, and the second-effect vacuum degree of-0.08 MPa and the temperature of 50 ℃;
(6) And (3) drying: vacuum drying the extract at 60 deg.C under-0.098 MPa for 8 hr;
(7) Crushing and sieving: pulverizing, and sieving with 60 mesh sieve to obtain Ganoderma spore powder polysaccharide peptide powder.
Example 4
A method for preparing polysaccharide peptide of ganoderma lucidum spore powder comprises the following steps:
(1) Wall breaking: loading 100kg Ganoderma spore powder into a vibration type ultramicro pulverizer, opening a cooling water switch, and breaking wall for 30min after the temperature of cooling water is constant to 3-5 deg.C to make the wall breaking rate reach above 95%;
(2) Degreasing: putting the wall-broken ganoderma lucidum spore powder into a supercritical carbon dioxide extraction kettle, introducing supercritical carbon dioxide fluid with the flow rate of 160L/h, extracting for 4h under the conditions of the pump frequency of 30Hz, the extraction pressure of 25MPa and the temperature of 40 ℃, then separating under the conditions of the primary separation pressure of 10MPa, the temperature of 40 ℃, the secondary separation pressure of 6MPa and the temperature of 28 ℃, collecting grease and degreased wall-broken ganoderma lucidum spore powder;
(3) Enzymolysis: mixing 50kg of defatted wall-broken ganoderma lucidum spore powder and 330kg of purified water, adding 2.5mol/L sodium hydroxide solution under the stirring condition, adjusting the pH to 7.0, then adding 0.3kg of cellulase and 0.3kg of compound protease for enzymolysis, keeping stirring during the enzymolysis period, maintaining the pH of the solution to 7.0 +/-0.1 by adding 2.5mol/L sodium hydroxide, and carrying out enzymolysis for 6 hours at the temperature of 48 ℃ under the ultrasonic condition;
(4) Extraction and separation: heating the enzymolysis liquid to 95 ℃ under the stirring condition, preserving the heat for 2 hours to obtain an extracting solution, then separating slag and liquid in the extracting solution at the rotating speed of 5000rpm by using a centrifugal machine, and collecting clear filtrate;
(5) And (3) concentrating: concentrating the clear filtrate by double-effect vacuum energy-saving concentration equipment to obtain extract with solid content of 45% at the first-effect vacuum degree of-0.06 MPa and the temperature of 70 ℃, and the second-effect vacuum degree of-0.08 MPa and the temperature of 50 ℃;
(6) And (3) drying: vacuum drying the extract at 60 deg.C under-0.098 MPa for 10 hr;
(7) Crushing and sieving: pulverizing, and sieving with 80 mesh sieve to obtain Ganoderma spore powder polysaccharide peptide powder.
Example 5
A method for preparing polysaccharide peptide of ganoderma lucidum spore powder comprises the following steps:
(1) Wall breaking: loading 100kg Ganoderma spore powder into a vibration type ultramicro pulverizer, opening a cooling water switch, and breaking wall for 30min after the temperature of cooling water is constant to 3-5 deg.C to make the wall breaking rate reach above 95%;
(2) Degreasing: putting the wall-broken ganoderma lucidum spore powder into a supercritical carbon dioxide extraction kettle, introducing a supercritical carbon dioxide fluid with the flow rate of 140L/h, extracting for 5h under the conditions of the pump frequency of 25Hz, the extraction pressure of 18MPa and the temperature of 50 ℃, then separating under the conditions of the primary separation pressure of 8MPa, the temperature of 50 ℃, the secondary separation pressure of 5MPa and the temperature of 35 ℃, collecting oil and degreased wall-broken ganoderma lucidum spore powder;
(3) Enzymolysis: mixing 50kg of defatted wall-broken ganoderma lucidum spore powder and 290kg of purified water, adding 2.5mol/L potassium hydroxide solution under stirring, adjusting the pH to 7.8, then adding 0.5kg of compound protease for enzymolysis, keeping stirring during the enzymolysis, maintaining the pH of the solution to 7.8 +/-0.1 by adding 2.5mol/L potassium hydroxide, and performing enzymolysis for 4 hours at 53 ℃ under ultrasonic conditions;
(4) Extraction and separation: heating the enzymolysis liquid to 95 ℃ under the stirring condition, preserving the heat for 2 hours to obtain an extracting solution, then separating slag and liquid in the extracting solution at the rotating speed of 6000rpm by using a centrifugal machine, and collecting clear filtrate;
(5) And (3) concentrating: concentrating the clear filtrate with double-effect vacuum energy-saving concentrating equipment to obtain extract with a solid content of 35% at a first-effect vacuum degree of-0.06 MPa and a temperature of 70 ℃, a second-effect vacuum degree of-0.08 MPa and a temperature of 50 ℃;
(6) And (3) drying: vacuum drying the extract at 60 deg.C under-0.098 MPa for 10 hr;
(7) Crushing and sieving: pulverizing, and sieving with 80 mesh sieve to obtain Ganoderma spore powder polysaccharide peptide powder.
Example 6
A method for preparing polysaccharide peptide of ganoderma lucidum spore powder comprises the following steps:
(1) Wall breaking: loading 100kg Ganoderma spore powder into a vibration type superfine pulverizer, opening a cooling water switch, and breaking wall for 30min after the cooling water temperature is constant to 3-5 deg.C to make the wall breaking rate reach above 95%;
(2) Degreasing: putting the ganoderma lucidum spore powder after wall breaking into a supercritical carbon dioxide extraction kettle, introducing supercritical carbon dioxide fluid with the flow rate of 120L/h, extracting for 4h under the conditions of the pump frequency of 27.5Hz, the extraction pressure of 21MPa and the temperature of 45 ℃, then separating under the conditions of the primary separation pressure of 9MPa, the temperature of 45 ℃, the secondary separation pressure of 5.5MPa and the temperature of 31 ℃, and collecting grease and degreased wall-broken ganoderma lucidum spore powder;
(3) Extraction and separation: extracting defatted wall-broken Ganoderma spore powder with water at 95 deg.C for 2 hr to obtain extractive solution, separating residue and liquid in the extractive solution at 5000rpm with centrifuge, and collecting clear filtrate;
(4) Concentration: concentrating the clear filtrate with double-effect vacuum energy-saving concentrating equipment to obtain extract with solid content of 45% at first-effect vacuum degree of-0.06 MPa and temperature of 70 deg.C, second-effect vacuum degree of-0.08 MPa and temperature of 50 deg.C;
(5) And (3) drying: vacuum drying the extract at 65 deg.C under-0.084 MPa for 12 hr;
(6) Crushing and sieving: pulverizing, and sieving with 80 mesh sieve to obtain Ganoderma spore powder polysaccharide peptide powder.
Test examples measurement of content of Ganoderma lucidum polysaccharide peptides
The content detection of the ganoderan peptide is carried out by adopting a high performance gel chromatography, and a Waters1525 type high performance liquid chromatography is adopted; waters2487 ultraviolet detector; chromatographic column TSK-GEL G4000PWXL 7.5X 300mm; mobile phase: ultrapure water; flow rate: 1.0mL/min; column temperature: 35 ℃; the sample size is 10 mu L; the detection wavelength λ =280nm.
Drawing of standard curve
Accurately weighing 10.0mg of dried polysaccharide peptide reference substance (the purity is more than or equal to 98 percent, and the national center for engineering and technology of grass and fungi), dissolving in a 10mL volumetric flask with water, and fixing the volume to obtain polysaccharide peptide reference substance stock solution with the concentration of 1.0 mg/mL; respectively transferring appropriate amount of polysaccharide peptide reference substance stock solution, diluting with water to 2mL, obtaining standard working solution with concentration of 100, 200, 300, 400, 500 μ g/mL, respectively, and drawing standard curve with reference substance concentration (μ g/mL) as abscissa and peak area as ordinate.
Sample solution preparation
Respectively weighing 50mg of the ganoderma lucidum polysaccharide peptide powder prepared in the embodiments 1-6, adding water to dissolve the ganoderma lucidum polysaccharide peptide powder and fixing the volume to 5.0mL; filtering, taking 1mL of filtrate, adding 3.0mL of absolute ethyl alcohol and mixing uniformly. Centrifuging at 10000rpm for 10min, dissolving the precipitate with water to 5.0mL, and filtering with 0.22 μm filter membrane to obtain sample filtrate.
Chromatography analysis
Injecting 10 μ L sample solution into chromatograph, running for 10min, taking polysaccharide peptide control concentration (μ g/ml) as abscissa and peak area as ordinate, drawing standard curve, and calculating Ganoderma polysaccharide peptide concentration in sample by using the curve.
The ganoderan peptide content was calculated by the following formula and the results are shown in table 1.
TABLE 1 Ganoderma lucidum polysaccharide peptide content in Ganoderma lucidum polysaccharide peptide powders prepared in examples 1-6
| Numbering | Yield of polysaccharide peptide,% | Sample polysaccharide peptide content% |
| Example 1 | 9.24 | 35.75 |
| Example 2 | 8.76 | 31.61 |
| Example 3 | 9.01 | 32.33 |
| Example 4 | 9.50 | 36.11 |
| Example 5 | 8.97 | 34.23 |
| Example 6 | 0.21 | 1.95 |
As can be seen from Table 1, the yield of the polysaccharide peptide prepared by the method is 8.76-9.50%, wherein the content of the polysaccharide peptide is 31.61-36.11%, and the yield and content index of the polysaccharide peptide are obviously better than those of the polysaccharide peptide prepared by preparing the ganoderma spore powder by pure water extraction.
Claims (2)
1. A method for preparing ganoderma lucidum spore powder polysaccharide peptide is characterized by comprising the following steps:
(1) Wall breaking: performing low-temperature physical wall breaking treatment on the ganoderma lucidum spore powder to enable the wall breaking rate to reach more than 95%;
(2) Degreasing: extracting the wall-broken Ganoderma spore powder in supercritical carbon dioxide extraction device, separating twice, and collecting oil and defatted wall-broken Ganoderma spore powder;
(3) Enzymolysis: mixing the defatted wall-broken Ganoderma spore powder with purified water, adding alkaline solution to adjust pH to 7.0-8.5, and adding enzyme for enzymolysis;
(4) Extraction and filtration: heating the enzymolysis liquid to 90-100 ℃ under stirring, preserving heat for 1-2h to obtain an extracting solution, separating, and collecting clear filtrate;
(5) Concentration: concentrating the filtrate to obtain extract with solid content of 35-55%;
(6) And (3) drying: vacuum drying the extract;
(7) Crushing and sieving: pulverizing, and sieving with 60-80 mesh sieve to obtain Ganoderma spore powder polysaccharide peptide powder;
wherein the low-temperature physical wall breaking is carried out by using a vibration type ultrafine grinder, the wall breaking temperature is 3-5 ℃, and the wall breaking time is 25-35min;
the pump frequency of the extraction device is 25-30Hz, the flow rate of the supercritical carbon dioxide is more than or equal to 120L/h, the extraction pressure is 18-25MPa, the extraction temperature is 40-50 ℃, and the extraction time is 4-5 hours;
the primary separation pressure of the two times of separation is 8-10MPa, and the separation temperature is 40-50 ℃; the secondary separation pressure is 5-6MPa, and the separation temperature is 28-35 ℃;
the enzyme is one or more of cellulase, compound protease, alkaline protease and neutral protease; the addition amount of the enzyme is 0.1-2% of the weight of the defatted wall-broken ganoderma lucidum spore powder, the enzymolysis temperature is 40-55 ℃, and the enzymolysis time is 4-6h;
carrying out ultrasonic treatment during the enzymolysis, wherein the frequency of the ultrasonic treatment is 20-40KHz, and the time is 4-6h;
filtering the extracting solution obtained in the step (4) by a plate and frame filter, refluxing the filtrate until the filtrate is clear, and then collecting the filtrate or separating slag and liquid in the extracting solution by using a centrifugal machine at the rotating speed of 4000-6000 rpm, and collecting the clear filtrate;
the concentration is carried out by adopting double-effect vacuum energy-saving concentration equipment; the one-effect vacuum degree of the double-effect vacuum energy-saving concentration equipment is-0.06 Mpa, and the temperature is 70-80 ℃; the double-effect vacuum degree is-0.08 MPa, and the temperature is 50-60 ℃.
2. The method for preparing ganoderma lucidum spore powder polysaccharide peptide according to claim 1, wherein the alkali solution is one of sodium hydroxide, potassium hydroxide, calcium hydroxide and ammonia water.
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| CN111363006B (en) * | 2020-01-16 | 2021-09-03 | 中南林业科技大学 | Ganoderma lucidum mycelium antihypertensive peptide and preparation method thereof |
| CN111671764A (en) * | 2020-05-06 | 2020-09-18 | 广东华夏友美生物科技有限公司 | Application of ganoderic acid polysaccharide in preparation of skin histamine inhibitor |
| CN113842450A (en) * | 2021-10-12 | 2021-12-28 | 南京中科药业有限公司 | Application of ganoderma lucidum spore powder polysaccharide peptide in preparation of medicine for treating cancer-induced fatigue |
| CN113831388A (en) * | 2021-10-15 | 2021-12-24 | 南京中科药业有限公司 | Ganoderma spore glycopeptide with effect of improving liver injury and preparation method thereof |
| CN113912750B (en) * | 2021-11-11 | 2022-11-11 | 开平健之源保健食品有限公司 | Method for extracting ganoderma lucidum fruiting body polysaccharide through fermentation pretreatment |
| CN115804450A (en) * | 2022-12-14 | 2023-03-17 | 江苏郢天富硒产业发展有限公司 | Deep processing technology of selenium-rich ganoderma lucidum spore powder, composition thereof and application of composition |
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