CN110922447A - Method for extracting triterpenic acid and polysaccharide from ganoderma lucidum - Google Patents
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Abstract
The invention relates to a method for extracting tripalmic acid and polysaccharide from lucid ganoderma, which comprises the following steps: s1, preprocessing; s2, ultrasonic enzymolysis: adding 5-12% of magnetic complex enzyme (W/W) and 40-55 times of deionized water (v/W) into the degreased ganoderma lucidum powder obtained in the step S1, uniformly stirring to obtain a mixed solution, adding a pH regulator into the mixed solution, controlling the pH to be 4-6, and extracting the mixed solution at 40-60 ℃ under the condition that the ultrasonic power is 150-300W for 8-10 h; s3, centrifuging the extracting solution to separate solid from liquid, and filtering solid residues to obtain filtrate; repeatedly leaching for 2-3 times, and mixing the above filtrates to obtain Ganoderma extract; recovering the magnetic compound enzyme from the centrifuged solid residue by using a magnetic material; s4, decolorizing and centrifuging to obtain Ganoderma extract containing tripalmic acid and polysaccharide.
Description
Technical Field
The invention belongs to the technical field of health-care product processing, and particularly relates to a method for extracting triterpenic acid and polysaccharide from lucid ganoderma.
Background
The ganoderma lucidum is also called black ganoderma lucidum, red ganoderma lucidum, purple ganoderma lucidum, Wannian mushroom, ganoderma lucidum and the like, has more than two thousand years of medicinal history, according to the record of Shennong Bencao Jing, the ganoderma lucidum has multiple peculiar effects of prolonging life, relieving cough and asthma and the like, the ganoderma lucidum contains multiple active substances, including polysaccharides, polypeptides, triterpenes, alkaloids, inorganic ions and the like, the ganoderma lucidum polysaccharides and the triterpenes are main active substances of the ganoderma lucidum, and in recent years, many scholars propose that the ganoderma lucidum polysaccharides have the effects of resisting tumors, resisting oxidation, resisting aging, improving the immunity of organisms and the like.
The ganoderma lucidum is characterized by containing rich polysaccharide and polysaccharide, wild ganoderma lucidum is also rich in trace elements, but the cell wall of plant cells is composed of cellulose, hemicellulose and the like, the structure is compact, the mode of direct taking is time-consuming and labor-consuming, the taking is inconvenient, most active ingredients in the ganoderma lucidum are difficult to absorb by a human body, and the bioavailability is low.
The methods for extracting ganoderic acid and ganoderan from natural products are different. In the traditional method, the water bath extraction method is a common method for extracting polysaccharide, but the method has low yield and takes long time. More importantly, some thermally unstable components are at risk of decomposition when heated at high temperatures for extended periods of time. The operation of the ultrasonic extraction method is relatively simple and can provide mild extraction conditions, and the ultrasonic extraction method is considered to be possible to be a main method for extracting triterpenic acid and polysaccharide in ganoderma lucidum in the future, but the method is low in efficiency and cannot meet the production requirement. Therefore, there is also a method for extracting effective components from plant ganoderma lucidum by using ultrasonic wave in combination with enzyme method, which can improve the yield of effective components, but has high requirement for ultrasonic operation, and because the activity of enzyme is reduced by increasing ultrasonic wave, the enzyme needs to be frequently replaced, which undoubtedly increases the production cost.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for extracting triterpenic acid and polysaccharide from lucid ganoderma, and a novel magnetic compound enzyme is prepared specially for lucid ganoderma extraction process and ultrasonic parameters, the magnetic compound enzyme is used synchronously with ultrasonic, so that the extraction efficiency of triterpenic acid and polysaccharide is improved, meanwhile, the magnetic compound enzyme can be recycled, the production cost is reduced, and in addition, the requirement of ultrasonic operation is reduced due to the use of the compound enzyme.
The technical scheme of the invention is as follows:
a method for extracting tripalmic acid and polysaccharide from ganoderma lucidum specifically comprises the following steps:
s1, preprocessing: pulverizing Ganoderma, and sieving; adding deionized water into Ganoderma powder, and mixing at a material-to-liquid ratio of 1: 40-60; then adding a degreasing agent for degreasing pretreatment, filtering with a filter membrane to remove lipids, and finally drying the ganoderma lucidum powder at the temperature of 60-70 ℃;
s2, ultrasonic enzymolysis: adding 5-12% of magnetic complex enzyme (W/W) and 40-55 times of deionized water (v/W) into the degreased ganoderma lucidum powder obtained in the step S1, uniformly stirring to obtain a mixed solution, adding a pH regulator into the mixed solution, controlling the pH to be 4-6, and extracting the mixed solution at 40-60 ℃ under the condition that the ultrasonic power is 150-300W for 8-10 h;
s3, centrifuging the extracting solution to separate solid from liquid, and filtering solid residues to obtain filtrate; repeatedly leaching for 2-3 times, and mixing the above filtrates to obtain Ganoderma extract; recovering the magnetic compound enzyme from the centrifuged solid residue by using a magnetic material;
s4, adding a decoloring agent into the ganoderma lucidum extract for decoloring, centrifuging and drying the solution to obtain the ganoderma lucidum extract containing tripalmonic acid and polysaccharide.
Further, the magnetic compound enzyme simultaneously fixes cellulase and pectinase on CTF-Fe3O4On the composite material.
Further, the preparation method of the magnetic complex enzyme comprises the following steps:
(1) preparing CTF with carboxyl-rich surface;
(2) dispersing the carboxyl-rich CTF prepared in the step (1) in 100-150mL of water, adding 0.4-0.6g of ferric trichloride hexahydrate and 0.4-0.65g of ferrous sulfate heptahydrate, ultrasonically dispersing for 10-30min, increasing the temperature to 60 ℃, adding 20mL3.5mol/L of ammonia water solution for reacting for 60 min, separating the product by using a magnet, washing and drying to obtain CTF-Fe3O4A composite material;
(3) taking 200g of CTF-Fe3O4Adding 200g of water into the composite material, adding 20mL of 6-arm polymer with the concentration of 5mg/mL, performing ultrasonic treatment for 1 hour, adding 40mg of EDC, stirring for 2 hours at room temperature, then adding 52mg of EDC, stirring for 12 hours, magnetically separating a product, and washing with deionized water to be neutral; drying in a drying oven to constant weight to synthesize P-CTF-Fe3O4A material;
(4) taking 100mg of P-CTF-Fe prepared in the step (3)3O4Adding 100mL of glutaraldehyde solution into the material, uniformly mixing, standing for 6-10 hours at 40-50 ℃, washing and recovering the P-CTF-Fe3O4Preparing a carrier material into a suspension, adding a mixed enzyme solution of cellulase and pectinase, uniformly stirring, standing for 24-32h at the temperature of 30-40 ℃, washing a magnet, recovering and drying to obtain the magnetic complex enzyme.
Further, the enzyme is mixed with P-CTF-Fe in the step (4)3O4The mass ratio of the materials is 0.3-0.5: 1.
Further, the mass ratio of the cellulase to the pectinase is 1: 0.2-0.6.
Further, the specific steps of the step (1) are as follows: taking 0.5-1.2g of CTF, adding 0.3-0.6g of sodium hydroxide, adding 200-300mL of deionized water, and carrying out ultrasonic treatment for 2-4 h; and then 0.5-0.6g of chloroacetic acid is added for ultrasonic treatment for 2h, and the CTF with rich carboxyl on the surface is obtained by washing and drying.
Further, the degreasing agent is one or more of alkali metal hydroxide, alkali metal carbonate or alkali metal bicarbonate.
Further, the decolorizing agent is activated carbon, the decolorizing time is 90min, the decolorizing temperature is 70-80 ℃, and the ratio of the activated carbon dosage to the ganoderma lucidum extract is 0.05-0.1: 1 (M/V).
Drawings
Fig. 1 is an XRD pattern of CTF prepared in example 2 of the present invention;
FIG. 2 shows CTF-Fe prepared in example 2 of the present invention3O4XRD of the composite material;
FIG. 3 shows P-CTF-Fe prepared in examples 1 to 3 of the present invention3O4XRD pattern of (a);
FIG. 4 shows P-CTF-Fe prepared in example 2 of the present invention3O4XPS spectra of (A).
The invention has the following beneficial effects:
1. the invention replaces the traditional single methods such as leaching and ultrasound by an ultrasonic enzymolysis method, improves the extraction rate of the ganoderma triterpenic acid and the ganoderma polysaccharide, and simultaneously provides a novel magnetic complex enzyme which can be directly recycled by a magnet and the like, so that the cost is reduced, and the magnetic complex enzyme has higher tolerance temperature and stronger stability; on the premise of increasing the ultrasonic power and prolonging the ultrasonic time, the ultrasonic probe still has higher activity.
2. The enzyme adopted by the invention is a novel magnetic complex enzyme, and CTF has larger specific surface area and abundant functional groups and is an ideal material for immobilized enzyme, so a novel catalytic system is designed, and a 6-arm polymer (6-arm-PEG-NH) is grafted on the CTF2) An amide is formed. The enzyme immobilization is achieved by the combination of cellulase and pectinase through glutaraldehyde. The hydrophilic PEG and remaining hydrophilic groups can help the enzyme retain more of the necessary water molecules, prevent deactivation by polymeric precipitation, and maintain its active conformation. Meanwhile, ferroferric oxide particles are carried on the CTF, and the immobilized enzyme is helped to be recovered by applying a proper magnetic field. Meanwhile, the enzymolysis efficiency is improved, the enzyme activity temperature range is increased, and the inactivation caused by the temperature change of the solution due to the ultrasonic is avoided.
3. The invention modifies abundant carboxyl on the CTF surface. Ferroferric oxide particles are loaded on the surface of the carboxylated CTF by a coprecipitation method, so that the carboxylated CTF is rich in magnetism and is beneficial to recycling the material. And then grafting the 6-arm polymer to the surface of the CTF through the acyl reaction of carboxyl and amino, and then connecting the amino of the carrier material and the amino of cellulase and pectinase through glutaraldehyde to form immobilized cellulase and pectinase.
Detailed Description
The invention will be further described with reference to preferred embodiments.
EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), CTF (covalent triazine organic polymer).
Example 1
Preparing a magnetic complex enzyme:
(1) taking 0.5g of CTF, adding 0.3g of sodium hydroxide, adding 200mL of deionized water, and carrying out ultrasonic treatment for 2 h; then adding 0.5g of chloroacetic acid, performing ultrasonic treatment for 2h, washing and drying to obtain CTF with rich carboxyl on the surface;
(2) dispersing the carboxyl-rich CTF prepared in the step (1) in 100-mL water of 150mL, adding 0.4g ferric chloride hexahydrate and 0.4g ferrous sulfate heptahydrate, performing ultrasonic dispersion for 10min, increasing the temperature to 60 ℃, adding 20mL3.5mol/L ammonia water solution for reaction for 60 min, separating the product by using a magnet, washing and drying to obtain CTF-Fe3O4A composite material;
(3) taking 200g of CTF-Fe3O4Adding 200g of water into the composite material, adding 20mL of 6-arm polymer with the concentration of 5mg/mL, performing ultrasonic treatment for 1 hour, adding 40mg of EDC, stirring for 2 hours at room temperature, then adding 52mg of EDC, stirring for 12 hours, magnetically separating a product, and washing with deionized water to be neutral; drying in a drying oven to constant weight to synthesize P-CTF-Fe3O4A material;
(4) taking 100mg of P-CTF-Fe prepared in the step (3)3O4Adding 100mL of glutaraldehyde solution into the material, uniformly mixing, standing for 6-10 hours at 40-50 ℃, washing and recovering the P-CTF-Fe3O4Preparing a carrier material into a suspension, adding a mixed enzyme solution of 25mg of cellulase and 5mg of pectinase, uniformly stirring, standing for 24-32h at the temperature of 30-40 ℃, washing a magnet, recovering and drying to obtain the magnetic complex enzyme.
Extracting tripalmic acid and polysaccharide from ganoderma lucidum:
s1, preprocessing: pulverizing Ganoderma, and sieving; adding 40L of deionized water into 1kg of ganoderma lucidum powder, and uniformly mixing; then adding 0.1kg of alkali metal hydroxide for degreasing pretreatment, filtering with a filter membrane to remove lipids, and finally drying the ganoderma lucidum powder at the temperature of 60-70 ℃;
s2, ultrasonic enzymolysis: adding 0.05kg of magnetic complex enzyme and 40L of deionized water into the degreased ganoderma lucidum powder obtained in the step S1, uniformly stirring to obtain a mixed solution, simultaneously adding sodium hydroxide into the mixed solution, controlling the pH value to be 4-6, and extracting the mixed solution for 8 hours at the temperature of 40 ℃ and under the condition that the ultrasonic power is 200W;
s3, centrifuging the extracting solution to separate solid from liquid, and filtering solid residues to obtain filtrate; repeatedly leaching for 3 times, and mixing the above filtrates to obtain Ganoderma extract; recovering the magnetic complex enzyme from the centrifuged solid residue by using a magnet;
s4, adding activated carbon into the ganoderma lucidum extract for decoloring, centrifuging and drying the solution to obtain the ganoderma lucidum extract containing tripalmitic acid and polysaccharide; decolorizing for 90min at 70-80 deg.C, and mixing the active carbon with Ganoderma extract at a ratio of 0.05: 1 (M/V).
Example 2
Preparing a magnetic complex enzyme:
(1) taking 1g of CTF, adding 0.5g of sodium hydroxide, adding 300mL of deionized water, and carrying out ultrasonic treatment for 3 h; then adding 0.6g of chloroacetic acid, performing ultrasonic treatment for 2h, washing and drying to obtain CTF with rich carboxyl on the surface;
(2) dispersing the carboxyl-rich CTF prepared in the step (1) in 150mL of water, adding 0.5g of ferric trichloride hexahydrate and 0.5g of ferrous sulfate heptahydrate, ultrasonically dispersing for 20min, increasing the temperature to 60 ℃, adding 20mL3.5mol/L of ammonia water solution, reacting for 60 min, separating a product by using a magnet, washing and drying to obtain CTF-Fe3O4A composite material;
(3) taking 200g of CTF-Fe3O4Adding 200g of water into the composite material, adding 20mL of 6-arm polymer with the concentration of 5mg/mL, performing ultrasonic treatment for 1 hour, adding 40mg of EDC, stirring for 2 hours at room temperature, then adding 52mg of EDC, stirring for 12 hours, magnetically separating a product, and washing with deionized water to be neutral; drying in a drying oven to constant weight to synthesize P-CTF-Fe3O4A material;
(4) taking 100mg of P-CTF-Fe prepared in the step (3)3O4Adding 100mL of glutaraldehyde solution into the material, uniformly mixing, standing for 6-10 hours at 40-50 ℃, washing and recovering the P-CTF-Fe3O4Preparing a carrier material into a suspension, adding a mixed enzyme solution of 25mg of cellulase and 15mg of pectinase, uniformly stirring, standing for 24-32h at the temperature of 30-40 ℃, washing a magnet, recovering and drying to obtain the magnetic complex enzyme.
Extracting tripalmic acid and polysaccharide from ganoderma lucidum:
s1, preprocessing: pulverizing Ganoderma, and sieving; adding 50L of deionized water into 1kg of ganoderma lucidum powder and mixing uniformly; then adding alkali metal hydroxide for degreasing pretreatment, filtering with a filter membrane to remove lipids, and finally drying the ganoderma lucidum powder at the temperature of 60-70 ℃;
s2, ultrasonic enzymolysis: adding 0.1kg of magnetic complex enzyme and 40L of deionized water into the degreased ganoderma lucidum powder obtained in the step S1, uniformly stirring to obtain a mixed solution, simultaneously adding sodium hydroxide into the mixed solution, controlling the pH value to be 5, and extracting the mixed solution for 10 hours at the temperature of 50 ℃ and under the condition that the ultrasonic power is 300W;
s3, centrifuging the extracting solution to separate solid from liquid, and filtering solid residues to obtain filtrate; repeatedly leaching for 3 times, and mixing the above filtrates to obtain Ganoderma extract; recovering the magnetic compound enzyme from the centrifuged solid residue by using a magnetic material;
s4, adding activated carbon into the ganoderma lucidum extract for decoloring, centrifuging and drying the solution to obtain the ganoderma lucidum extract containing tripalmitic acid and polysaccharide; the decoloring time is 90min, the decoloring temperature is 80 ℃, and the ratio of the active carbon consumption to the lucid ganoderma extract is 0.08: 1 (M/V).
Example 3
Preparing a magnetic complex enzyme:
(1) taking 2g of CTF, adding 0.6g of sodium hydroxide, adding 300mL of deionized water, and carrying out ultrasonic treatment for 4 h; then adding 0.6g of chloroacetic acid, performing ultrasonic treatment for 2h, washing and drying to obtain CTF with rich carboxyl on the surface;
(2) dispersing the carboxyl-rich CTF prepared in the step (1) in 150mL of water, adding 0.6g of ferric trichloride hexahydrate and 0.65g of ferrous sulfate heptahydrate, ultrasonically dispersing for 30min, and increasing the temperatureAdding 20mL3.5mol/L ammonia water solution to react for 60 minutes at the temperature of 60 ℃, separating the product by using a magnet, washing and drying to obtain CTF-Fe3O4A composite material;
(3) taking 200g of CTF-Fe3O4Adding 200g of water into the composite material, adding 20mL of 6-arm polymer with the concentration of 5mg/mL, performing ultrasonic treatment for 1 hour, adding 40mg of EDC, stirring for 2 hours at room temperature, then adding 52mg of EDC, stirring for 12 hours, magnetically separating a product, and washing with deionized water to be neutral; drying in a drying oven to constant weight to synthesize P-CTF-Fe3O4A material;
(4) taking 100mg of P-CTF-Fe prepared in the step (3)3O4Adding 100mL of glutaraldehyde solution into the material, uniformly mixing, standing at 50 ℃ for 10 hours, washing and recovering the P-CTF-Fe3O4Preparing a carrier material into a suspension, adding 35mg of cellulase and 15mg of pectinase mixed enzyme solution, uniformly stirring, standing for 32 hours at 40 ℃, washing a magnet, recovering and drying to obtain the magnetic complex enzyme.
Extracting tripalmic acid and polysaccharide from ganoderma lucidum:
s1, preprocessing: pulverizing Ganoderma, and sieving; adding 60L of deionized water into 1kg of ganoderma lucidum powder and mixing uniformly; adding alkali metal hydroxide for degreasing pretreatment, filtering with a filter membrane to remove lipids, and finally drying the ganoderma lucidum powder at 70 ℃;
s2, ultrasonic enzymolysis: adding 0.12kg of magnetic complex enzyme and 55L of deionized water into the degreased ganoderma lucidum powder obtained in the step S1, uniformly stirring to obtain a mixed solution, simultaneously adding sodium hydroxide into the mixed solution, controlling the pH value to be 5, and extracting the mixed solution for 10 hours at the temperature of 50 ℃ and under the condition that the ultrasonic power is 300W;
s3, centrifuging the extracting solution to separate solid from liquid, and filtering solid residues to obtain filtrate; repeatedly leaching for 3 times, and mixing the above filtrates to obtain Ganoderma extract; recovering the magnetic compound enzyme from the centrifuged solid residue by using a magnetic material;
s4, adding activated carbon into the ganoderma lucidum extract for decoloring, centrifuging and drying the solution to obtain the ganoderma lucidum extract containing tripalmitic acid and polysaccharide; decolorizing for 90min at 70-80 deg.C, and mixing the active carbon with Ganoderma extract at a ratio of 0.05-0.1: 1 (M/V).
Comparative example 1:
extracting tripalmic acid and polysaccharide from ganoderma lucidum:
s1, preprocessing: pulverizing Ganoderma, and sieving; adding 60L of deionized water into 1kg of ganoderma lucidum powder and mixing uniformly; adding alkali metal hydroxide for degreasing pretreatment, filtering with a filter membrane to remove lipids, and finally drying the ganoderma lucidum powder at 70 ℃;
s2, ultrasonic enzymolysis: adding 0.12kg of complex enzyme (25 mg of cellulase and 15mg of pectinase) and 55L of deionized water into the degreased ganoderma lucidum powder obtained in the step S1, uniformly stirring to obtain a mixed solution, simultaneously adding sodium hydroxide into the mixed solution, controlling the pH value to be 5, and extracting the mixed solution at 50 ℃ for 10 hours under the condition that the ultrasonic power is 300W;
s3, centrifuging the extracting solution to separate solid from liquid, and filtering solid residues to obtain filtrate; repeatedly leaching for 3 times, and mixing the above filtrates to obtain Ganoderma extract;
s4, adding activated carbon into the ganoderma lucidum extract for decoloring, centrifuging and drying the solution to obtain the ganoderma lucidum extract containing tripalmitic acid and polysaccharide; decolorizing for 90min at 70-80 deg.C, and mixing the active carbon with Ganoderma extract at a ratio of 0.05-0.1: 1 (M/V).
Comparative example 2:
extracting tripalmic acid and polysaccharide from ganoderma lucidum:
s1, preprocessing: pulverizing Ganoderma, and sieving; adding 60L of deionized water into 1kg of ganoderma lucidum powder and mixing uniformly; adding alkali metal hydroxide for degreasing pretreatment, filtering with a filter membrane to remove lipids, and finally drying the ganoderma lucidum powder at 70 ℃;
s2, ultrasonic enzymolysis: adding 0.12kg of 40mg of cellulase and 55L of deionized water into the degreased ganoderma lucidum powder in the step S1, uniformly stirring to obtain a mixed solution, simultaneously adding sodium hydroxide into the mixed solution, controlling the pH value to be 5, and extracting the mixed solution for 10 hours at the temperature of 50 ℃ and under the condition that the ultrasonic power is 300W;
s3, centrifuging the extracting solution to separate solid from liquid, and filtering solid residues to obtain filtrate; repeatedly leaching for 3 times, and mixing the above filtrates to obtain Ganoderma extract;
s4, adding activated carbon into the ganoderma lucidum extract for decoloring, centrifuging and drying the solution to obtain the ganoderma lucidum extract containing tripalmitic acid and polysaccharide; decolorizing for 90min at 70-80 deg.C, and mixing the active carbon with Ganoderma extract at a ratio of 0.05-0.1: 1 (M/V).
1. Load measurement
The steps of the method for preparing the magnetic complex enzyme are repeated for 3 times and the load is calculated according to the steps of the method for preparing the magnetic complex enzyme in the examples 1 to 3 respectively, wherein the immobilized carrier material is separated by a magnet, and the supernatant solution is reserved for measuring the enzyme concentration. The carrier material was washed twice with buffer solution to remove non-immobilized cellulase and pectinase. And (3) detecting the concentrations of the residual cellulase and the pectinase in the adsorption solution by using an ultraviolet-visible spectrophotometer by adopting a Coomassie brilliant blue method, and calculating the load capacity. The cellulase and pectinase loadings (C) can be calculated using the following formula:
in the formula, M1Expressed as protein content (mg), M, in the enzyme solution2Representing the protein content (mg) in the remaining supernatant.
| Number of times | 1 | 2 | 3 | Mean value of |
| Example 1 | 92.6％ | 93.1％ | 92.8％ | 92.8％ |
| Example 2 | 95.3％ | 94.8％ | 95.2％ | 95.1％ |
| Example 3 | 93.1％ | 92.8％ | 93.5％ | 93.1％ |
2. Cycling experiments
According to the steps of the embodiment 2, the magnetic compound enzyme recovered in the embodiment 2 is repeated for 5 times according to the steps of the embodiment 2, and the extraction of the ganoderma lucidum active substances is carried out, and the results are as follows:
| number of cycles | 1 | 2 | 3 | 4 | 5 |
| Triterpene acid yield (%) | 69.41 | 68.32 | 67.30 | 64.32 | 61.20 |
| Polysaccharide yield (%) | 35.48 | 35.11 | 34.60 | 32.10 | 31.21 |
From the above, the magnetic complex enzyme prepared in example 2 has good repeatability, which indicates that the magnetic complex enzyme prepared in the application has good market prospect.
3. Characterization of
FIG. 1 is an XRD pattern of CTF, and FIG. 2 is CTF-Fe3O4XRD of composite Material, FIG. 3 shows P-CTF-Fe prepared in examples 1-33O4The XRD patterns of (a-c in the figure correspond to examples 1-3 respectively), 3 peaks between 15-30 degrees in figure 1 are the crystalline form values of CTF, and it can be seen from figure 2 that 7 peaks correspond to Fe respectively3O4Diffraction peaks of the nanoparticle crystal phase at (111), (220), (311), (400), (422), (333) and (440) planes, and no peak of CTF occurred because of Fe3O4The peak of (c) masks the CTF peak; while Fe of 3 examples in FIG. 33O4The position of the peak of (A) is not shifted compared to FIG. 2, indicating that 6-arm-PEG-NH is grafted2Fe after polymer layer3O4The absence of significant changes in the crystalline form maintains greater integrity. From FIG. 4, characteristic peaks of C1s (282.7eV), N1s (400.1eV), O1s (530.6eV) he Fe2p (711.1eV) can be seen, and the peaks of these elements are shownThe characterization of XRD is combined to prove that the material is successfully synthesized.
4. Triterpene acid and polysaccharide content detection
The content of triterpenic acid is detected by a method in DB44/T496-2008 'determination of triterpenes in lucid ganoderma', and the content of polysaccharide is detected by a phenol-sulfuric acid method.
TABLE 1 yield of triterpenic acids and polysaccharides in Ganoderma lucidum extracts
| Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | |
| Triterpene acid yield (%) | 68.71 | 69.41 | 61.14 | 22.15 | 15.10 |
| Polysaccharide yield (%) | 36.16 | 35.48 | 38.12 | 14.40 | 8.21 |
From the above, the complex enzyme of comparative example 1 is 25mg of cellulase and 15mg of pectinase, and the single cellulase of comparative example 2 is adopted, so that the extraction rates of the triterpenic acid and the polysaccharide are lower than those of examples 1-3, on one hand, the enzyme activity is not higher than that of the complex enzyme, on the other hand, the magnetic complex enzyme has higher tolerance temperature and stronger stability, and cannot be inactivated under the conditions of high ultrasonic power and longer ultrasonic time, and the enzyme activity of the pectinase or cellulase is reduced under the conditions, so that the extraction rates of the triterpenic acid and the polysaccharide are low.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A method for extracting tripalmic acid and polysaccharide from lucid ganoderma is characterized in that: the method specifically comprises the following steps:
s1, preprocessing: pulverizing Ganoderma, and sieving; adding deionized water into Ganoderma powder, and mixing at a material-to-liquid ratio of 1: 40-60; then adding a degreasing agent for degreasing pretreatment, filtering with a filter membrane to remove lipids, and finally drying the ganoderma lucidum powder at the temperature of 60-70 ℃;
s2, ultrasonic enzymolysis: adding 5-12% of magnetic complex enzyme (W/W) and 40-55 times of deionized water (v/W) into the degreased ganoderma lucidum powder obtained in the step S1, uniformly stirring to obtain a mixed solution, adding a pH regulator into the mixed solution, controlling the pH to be 4-6, and extracting the mixed solution at 40-60 ℃ under the condition that the ultrasonic power is 150-300W for 8-10 h;
s3, centrifuging the extracting solution to separate solid from liquid, and filtering solid residues to obtain filtrate; repeatedly leaching for 2-3 times, and mixing the above filtrates to obtain Ganoderma extract; recovering the magnetic compound enzyme from the centrifuged solid residue by using a magnetic material;
s4, adding a decoloring agent into the ganoderma lucidum extract for decoloring, centrifuging and drying the solution to obtain the ganoderma lucidum extract containing tripalmonic acid and polysaccharide.
2. The method for extracting triterpenic acid and polysaccharide from ganoderma lucidum as claimed in claim 1, wherein the method comprises the following steps: the magnetic compound enzyme is prepared by simultaneously fixing cellulase and pectinase on CTF-Fe3O4On the composite material.
3. The method for extracting the tripalmic acid and the polysaccharide from the ganoderma lucidum as claimed in claim 2, wherein the preparation method of the magnetic complex enzyme is as follows:
(1) preparing CTF with carboxyl-rich surface;
(2) dispersing the carboxyl-rich CTF prepared in the step (1) in 100-150mL of water, adding 0.4-0.6g of ferric trichloride hexahydrate and 0.4-0.65g of ferrous sulfate heptahydrate, ultrasonically dispersing for 10-30min, increasing the temperature to 60 ℃, adding 20mL3.5mol/L of ammonia water solution for reacting for 60 min, separating the product by using a magnet, washing and drying to obtain CTF-Fe3O4A composite material;
(3) taking 200g of CTF-Fe3O4Adding 200g of water into the composite material, adding 20mL of 6-arm polymer with the concentration of 5mg/mL, performing ultrasonic treatment for 1 hour, adding 40mg of EDC, stirring for 2 hours at room temperature, then adding 52mg of EDC, stirring for 12 hours, magnetically separating a product, and washing with deionized water to be neutral; drying in a drying oven to constant weight to synthesize P-CTF-Fe3O4A material;
(4) taking 100mg of P-CTF-Fe prepared in the step (3)3O4Adding 100mL of glutaraldehyde solution into the material, uniformly mixing, standing for 6-10 hours at 40-50 ℃, washing and recovering the P-CTF-Fe3O4Preparing a carrier material into a suspension, adding a mixed enzyme solution of cellulase and pectinase, uniformly stirring, standing for 24-32h at the temperature of 30-40 ℃, washing a magnet, recovering and drying to obtain the magnetic complex enzyme.
4. The method for extracting triterpenic acid and polysaccharide from ganoderma lucidum as claimed in claim 3, wherein the method comprises the following steps: mixing the enzyme and the P-CTF-Fe in the step (4)3O4The mass ratio of the materials is 0.3-0.5: 1.
5. The method of claim 4, wherein the extraction of the triterpenic acid and polysaccharide from the ganoderma lucidum comprises: the mass ratio of the cellulase to the pectinase is 1: 0.2-0.6.
6. The method of claim 5, wherein the extraction of the triterpenic acid and polysaccharide from the ganoderma lucidum comprises: the specific steps of the step (1) are as follows: taking 0.5-1.2g of CTF, adding 0.3-0.6g of sodium hydroxide, adding 200-300mL of deionized water, and carrying out ultrasonic treatment for 2-4 h; and then 0.5-0.6g of chloroacetic acid is added for ultrasonic treatment for 2h, and the CTF with rich carboxyl on the surface is obtained by washing and drying.
7. The method of claim 6, wherein the extraction of the triterpenic acid and polysaccharide from the ganoderma lucidum comprises: the degreasing agent is one or more of alkali metal hydroxide, alkali metal carbonate or alkali metal bicarbonate.
8. The method of claim 7, wherein the extraction of the triterpenic acid and polysaccharide from the ganoderma lucidum comprises: the decolorizing agent is activated carbon, the decolorizing time is 90min, the decolorizing temperature is 70-80 ℃, and the ratio of the activated carbon to the lucid ganoderma extract is (0.05-0.1): 1 (M/V).
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