CN111620961A - Method for extracting ganoderan from ganoderma lucidum spore powder - Google Patents
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Abstract
A method for extracting ganoderma lucidum polysaccharide from ganoderma lucidum spore powder relates to the extraction of natural products. Drying wall-broken ganoderma lucidum spore powder, adding petroleum ether, carrying out degreasing treatment, volatilizing the spore powder obtained by suction filtration until no ether smell exists, and drying to obtain degreased ganoderma lucidum spore powder; adding ultrapure water into the defatted ganoderma lucidum spore powder, and placing the mixture into a self-sealing bag for ultrasonic treatment; vacuumizing and sealing the valve bag, and then carrying out ultrahigh pressure treatment on the valve bag; carrying out water bath at constant temperature on the treated ganoderma lucidum spore powder, and carrying out suction filtration to obtain a crude extract of ganoderma lucidum polysaccharide; adding macroporous resin, treating by a shaking table, and performing suction filtration to obtain supernatant, namely the degreased and deproteinized polysaccharide solution; concentrating the defatted deproteinized polysaccharide solution by rotary evaporation to one fourth of the original volume, adding four times of anhydrous ethanol into the concentrated solution, precipitating overnight, centrifuging to obtain polysaccharide precipitate, and freeze drying to obtain refined ganoderan powder. The method has the advantages of solvent saving, high efficiency, less impurities, and good antioxidant activity of the extracted ganoderan.
Description
Technical Field
The invention relates to extraction of natural products, in particular to a method for extracting ganoderma lucidum polysaccharide from ganoderma lucidum spore powder.
Background
Ganoderma Lucidum (Ganoderma Lucidum) is a polyperaceae (Polyporaceae) of Basidiomycetes, belongs to Ganoderma (Ganoderma) fungi, is a well-known traditional Chinese medicine in ancient and modern times, has the effect of prolonging life, can help cure various diseases, and is called Lingdan Miao medicine (Zhou, et al.2012). Ganoderma lucidum has received attention from many researchers in the fields of phytochemistry, pharmacy, biology, and the like, as a fungus for both food and medicine (Gao, et al 2006). Ganoderma spores (Ganoderma lucidum spore) are seeds of mature Ganoderma lucidum. The ganoderma lucidum spores contain all the genetic materials of ganoderma lucidum, the nutritive value is higher than that of other parts of ganoderma lucidum, and the medicinal value of ganoderma lucidum spores is very high (tang willow, et al.2011). Ganoderma spore powder containing multiple active chemical components has multiple pharmacological effects of regulating immunity, inhibiting tumor, resisting cancer, reducing blood glucose, reducing blood lipid, scavenging free radicals, resisting radiation nerve, cardiovascular disease, regulating respiratory system, etc. (Yuan, et al.2006; Bao X, et al.2001; Liu X, et al.2002). Among the active ingredients of ganoderma spores, polysaccharides are one of the most important active ingredients. Because the ganoderma lucidum spore has double-layer cell walls, the spore walls are hard and incomparable under the action of chitin, calcium and silicon, the resistance to acid and alkali is high, the oxidative decomposition is very difficult, and the wall breaking treatment is difficult to be carried out by using a common physical and chemical method, so that the characteristic not only hinders the absorption and utilization of the effective components of the ganoderma lucidum spore by a human body, but also limits the research of people on the effective components. The detection and control of the quality of the related products of the ganoderma lucidum are important links in the development of the functional food industry and the pharmaceutical industry, scientific and effective methods become problems to be solved urgently, an efficient extraction method is a prerequisite, the extraction of the polysaccharide provides a basis for the subsequent separation and purification, structural characterization and activity research of the polysaccharide, and the extraction of the polysaccharide plays a crucial role in the whole research process of the polysaccharide. Therefore, the research of the efficient polysaccharide extraction technology has important practical significance. The most common method for extracting ganoderan is the traditional hot water method, and in recent years, in the domestic and foreign researches of polysaccharide, new extraction technologies are gradually discovered, including microwave-assisted extraction, ultrasonic-assisted extraction, ultrahigh-pressure extraction, supercritical extraction and the like (huling, et al.2011; Ma C W, et al.2013; Prasad, et al.2010; Gao, et al.2017).
Disclosure of Invention
The invention aims to provide a method for extracting ganoderma lucidum polysaccharide from ganoderma lucidum spore powder, which combines ultrahigh pressure treatment and hot water extraction, can save solvent and process cost, has high extraction efficiency, and ensures that the extracted ganoderma lucidum polysaccharide has good antioxidant activity.
The invention comprises the following steps:
1) pretreatment of raw materials: drying wall-broken ganoderma lucidum spore powder, adding petroleum ether, carrying out degreasing treatment at room temperature, putting the spore powder obtained by suction filtration in a fume hood, volatilizing until no ether smell exists, and then drying to obtain degreased ganoderma lucidum spore powder;
2) infiltrating: adding ultrapure water into the defatted ganoderma lucidum spore powder, and placing the mixture into a self-sealing bag for ultrasonic treatment;
3) ultrahigh pressure treatment: vacuumizing and sealing the valve bag, and then carrying out ultrahigh pressure treatment on the valve bag;
4) hot water extraction: carrying out constant-temperature water bath on the ganoderma lucidum spore powder subjected to ultrahigh pressure treatment, and carrying out suction filtration to obtain a crude extract of ganoderma lucidum polysaccharide;
5) deproteinizing and depigmenting ganoderma lucidum polysaccharide: adding macroporous resin into the crude extract of ganoderan, treating by shaking table, and vacuum filtering to obtain supernatant, i.e. defatted deproteinized polysaccharide solution;
6) alcohol precipitation: concentrating the defatted deproteinized polysaccharide solution by rotary evaporation to one fourth of the original volume, adding four times of anhydrous ethanol into the concentrated solution, precipitating overnight, centrifuging to obtain polysaccharide precipitate, and freeze drying to obtain refined ganoderan powder.
In the step 1), the drying temperature can be 50-60 ℃, and the drying time can be 10-12 h; the feed-liquid ratio of the wall-broken ganoderma lucidum spore powder to the petroleum ether can be 1: 20-30 g/mL; the time of degreasing treatment can be 10-12 h; uniformly stirring at 200-1000 r/min in the degreasing treatment process; the volatilization time can be 4-6 h; the temperature of redrying can be 50 ~ 60 ℃.
In the step 2), the feed-liquid ratio of the defatted ganoderma lucidum spore powder to the ultrapure water can be 1 mg: 10-55 mL; the time of the ultrasonic treatment can be 5-10 min.
In the step 3), the vacuumizing time can be 90-120 s; the pressure maintaining time of the ultrahigh pressure treatment is 3-15 min, and the treatment pressure is 100-600 MPa; the preferable pressure maintaining time is 6-10 min.
In the step 4), the time of the water bath can be 30-60 min, and the temperature of the water bath can be 60 ℃.
In step 5), the feed-liquid ratio of the polysaccharide solution to the macroporous resin can be 1: 20 (g/mL); the time of the shaking table treatment can be 4-9 h, the temperature of the shaking table treatment is 28-35 ℃, and the rotating speed of the shaking table can be 200 r/min.
In step 6), the precipitation overnight may be performed at 4 ℃ overnight; the centrifugation time can be 12-15 min; the freeze-drying time may be 12 hours.
The invention provides a process for extracting ganoderma lucidum polysaccharide from ganoderma lucidum spore powder, which comprises the steps of degreasing, infiltrating, ultrahigh pressure treatment, hot water extraction, ethanol precipitation of polysaccharide, macroporous resin impurity removal, dialysis, secondary alcohol precipitation, vacuum freeze drying and the like to obtain refined ganoderma lucidum polysaccharide. The process provided by the invention combines the ultrahigh pressure and hot water extraction methods, and carries out fine purification on the ganoderma lucidum polysaccharide, only one-time extraction can achieve the extraction effect of the common method, the solvent is saved, the efficiency is high, the impurities are few, and high temperature or acid-base environment is not generated in the extraction and impurity removal process, so that the structure of the polysaccharide can be better protected.
Compared with the prior art, the invention has the following outstanding advantages:
(1) the invention can effectively remove lipid through the processes of petroleum ether degreasing and ultrasonic infiltration, and the petroleum ether can recover and obtain lipid substances in the ganoderma lucidum, thereby improving the utilization rate of raw materials, and simultaneously, the ultrasonic process can fully and uniformly mix solid and liquid.
(2) The invention firstly carries out ultrahigh pressure treatment on the spore powder and then carries out hot water extraction, thereby improving the extraction rate of the ganoderma lucidum polysaccharide.
(3) The invention adopts macroporous resin to remove impurities, can achieve the effect of removing protein and pigment, and the resin can be reused.
Detailed Description
The following examples further illustrate the invention. The features and advantages of the present invention will become more apparent as the description proceeds, but the exemplary embodiments are merely illustrative of the present invention and do not set any limit to the scope of the present invention.
Example 1
(1) Pretreatment of raw materials: drying wall-broken Ganoderma spore powder at 60 deg.C for 12h, adding petroleum ether at a ratio of material to liquid of 1: 30(g/mL), defatting at room temperature for 12h, and stirring at 500 r/min. Volatilizing the obtained spore powder in a fume hood for 6h until no ether smell exists, and drying at 60 ℃ to obtain defatted Ganoderma spore powder;
(2) infiltrating: adding ultrapure water into spore powder at a ratio of 1: 30mg/mL, and placing in a self-sealing bag under ultrasound for 5 min.
(3) Ultrahigh pressure treatment: vacuumizing the valve bag for 120s, sealing, and then carrying out ultrahigh pressure treatment on the valve bag for 10min at the treatment pressure of 400 MPa.
(4) Hot water extraction: and carrying out constant-temperature water bath on the spore powder subjected to the ultrahigh pressure treatment at the temperature of 60 ℃ for 60min, and filtering to obtain a crude ganoderma lucidum polysaccharide extracting solution.
(5) Deproteinizing and depigmenting ganoderma lucidum polysaccharide: adding macroporous resin into the crude Ganoderma polysaccharide extractive solution at a ratio of 1: 20(g/mL), treating in a shaking table at 28 deg.C for 6 hr, and filtering to obtain supernatant, i.e. defatted deproteinized polysaccharide solution;
(6) alcohol precipitation: performing rotary evaporation and concentration on the degreased and deproteinized polysaccharide solution to one fourth of the original volume, adding anhydrous ethanol with four times of the volume into the concentrated solution, precipitating at 4 ℃ overnight, centrifuging for 15min to obtain polysaccharide precipitate, and finally freeze-drying to obtain refined polysaccharide powder;
example 2
(1) Pretreatment of raw materials: drying wall-broken Ganoderma spore powder at 60 deg.C for 12h, adding petroleum ether at a ratio of material to liquid of 1: 30(g/mL), defatting at room temperature for 12h, and stirring at 1000 r/min. Volatilizing the obtained Ganoderma spore powder in a fume hood for 6 hr until no ether smell exists, and drying at 60 deg.C to obtain defatted Ganoderma spore powder;
(2) infiltrating: adding ultrapure water into defatted Ganoderma spore powder at a ratio of 1: 40mg/mL, and placing in a self-sealing bag under ultrasound for 5 min.
(3) Ultrahigh pressure treatment: vacuumizing the valve bag for 120s, sealing, and then carrying out ultrahigh pressure treatment on the valve bag for 10min at the treatment pressure of 500 MPa.
(4) Hot water extraction: and carrying out constant-temperature water bath on the spore powder subjected to the ultrahigh pressure treatment at the temperature of 60 ℃ for 60min, and filtering to obtain a crude ganoderma lucidum polysaccharide extracting solution.
(5) Deproteinizing and depigmenting ganoderma lucidum polysaccharide: adding macroporous resin into the crude Ganoderma polysaccharide extractive solution at a ratio of 1: 20(g/mL), treating in a shaking table at 28 deg.C for 6 hr, and filtering to obtain supernatant, i.e. defatted deproteinized polysaccharide solution;
(6) alcohol precipitation: performing rotary evaporation and concentration on the degreased and deproteinized polysaccharide solution to one fourth of the original volume, adding anhydrous ethanol with four times of the volume into the concentrated solution, precipitating at 4 ℃ overnight, centrifuging for 15min to obtain polysaccharide precipitate, and finally freeze-drying to obtain refined polysaccharide powder;
example 3
(1) Pretreatment of raw materials: drying wall-broken Ganoderma spore powder at 60 deg.C for 12h, adding petroleum ether at a ratio of material to liquid of 1: 30(g/mL), defatting at room temperature for 12h, and stirring at 1000 r/min. Volatilizing the obtained spore powder in a fume hood for 6h until no ether smell exists, and drying at 60 ℃ to obtain defatted Ganoderma spore powder;
(2) infiltrating: adding ultrapure water into spore powder at a ratio of 1: 40mg/mL, and placing in a self-sealing bag under ultrasound for 10 min.
(3) Ultrahigh pressure treatment: and vacuumizing the valve bag for 120s, sealing, and then carrying out ultrahigh pressure treatment on the valve bag for 8min at the treatment pressure of 500 MPa.
(4) Hot water extraction: and carrying out constant-temperature water bath on the spore powder subjected to the ultrahigh pressure treatment at the temperature of 60 ℃ for 60min, and filtering to obtain a crude ganoderma lucidum polysaccharide extracting solution.
(5) Deproteinizing and depigmenting ganoderma lucidum polysaccharide: adding macroporous resin into the crude Ganoderma polysaccharide extractive solution at a ratio of 1: 20(g/mL), treating in a shaking table at 28 deg.C for 6 hr, and filtering to obtain supernatant, i.e. defatted deproteinized polysaccharide solution;
(6) alcohol precipitation: performing rotary evaporation and concentration on the degreased and deproteinized polysaccharide solution to one fourth of the original volume, adding anhydrous ethanol with four times of the volume into the concentrated solution, precipitating at 4 ℃ overnight, centrifuging for 15min to obtain polysaccharide precipitate, and finally freeze-drying to obtain refined polysaccharide powder;
example 4
(1) Pretreatment of raw materials: drying wall-broken Ganoderma spore powder at 55 deg.C for 11h, adding petroleum ether at a ratio of material to liquid of 1: 25(g/mL), defatting at room temperature for 11h, and stirring at 800 r/min. Volatilizing the obtained spore powder after suction filtration in a fume hood for 5h until no ether smell exists, and then drying at the temperature of 55 ℃ to obtain defatted ganoderma lucidum spore powder;
(2) infiltrating: adding ultrapure water into spore powder at a ratio of 1: 55mg/mL, and placing in a self-sealing bag under ultrasound for 8 min.
(3) Ultrahigh pressure treatment: vacuumizing the valve bag for 100s, sealing, and then carrying out ultrahigh pressure treatment on the valve bag for 10min at the treatment pressure of 600 MPa.
(4) Hot water extraction: and (3) carrying out constant-temperature water bath on the spore powder subjected to the ultrahigh pressure treatment at the temperature of 60 ℃ for 45min, and filtering to obtain a crude ganoderma lucidum polysaccharide extracting solution.
(5) Deproteinizing and depigmenting ganoderma lucidum polysaccharide: adding macroporous resin into the crude Ganoderma polysaccharide extractive solution at a material-to-liquid ratio of 1: 20(g/mL), treating in a shaking table at 35 deg.C for 9 hr, and filtering to obtain supernatant, i.e. defatted deproteinized polysaccharide solution;
(6) alcohol precipitation: performing rotary evaporation and concentration on the degreased and deproteinized polysaccharide solution to one fourth of the original volume, adding four times of volume of absolute ethyl alcohol into the concentrated solution, precipitating at 4 ℃ overnight, centrifuging for 13min to obtain polysaccharide precipitate, and finally freeze-drying to obtain refined polysaccharide powder;
example 5
(1) Pretreatment of raw materials: drying wall-broken Ganoderma spore powder at 50 deg.C for 10h, adding petroleum ether at a ratio of material to liquid of 1: 20(g/mL), defatting at room temperature for 10h, and stirring at 200 r/min. Volatilizing the obtained Ganoderma spore powder in a fume hood for 4 hr until no ether smell exists, and drying at 50 deg.C to obtain defatted Ganoderma spore powder;
(2) infiltrating: adding ultrapure water into defatted Ganoderma spore powder at a ratio of 1: 10mg/mL, and placing in a self-sealing bag under ultrasound for 5 min.
(3) Ultrahigh pressure treatment: vacuumizing the valve bag for 90s, sealing, and then carrying out ultrahigh pressure treatment on the valve bag for 6min at the treatment pressure of 100 MPa.
(4) Hot water extraction: carrying out constant-temperature water bath on the spore powder subjected to the ultrahigh pressure treatment at the temperature of 60 ℃ for 30min, and filtering to obtain a crude ganoderma lucidum polysaccharide extracting solution.
(5) Deproteinizing and depigmenting ganoderma lucidum polysaccharide: adding macroporous resin into the crude Ganoderma polysaccharide extractive solution at a ratio of 1: 20(g/mL), treating in a shaking table at 30 deg.C for 4 hr, and filtering to obtain supernatant, i.e. defatted deproteinized polysaccharide solution;
(6) alcohol precipitation: concentrating the defatted and deproteinized polysaccharide solution by rotary evaporation to one fourth of the original volume, adding four times of anhydrous ethanol into the concentrated solution, precipitating at 4 deg.C overnight, centrifuging for 12min to obtain polysaccharide precipitate, and freeze drying to obtain refined polysaccharide powder.
The invention adopts the method of combining ultrahigh pressure treatment and hot water extraction to extract the ganoderma lucidum polysaccharide from the ganoderma lucidum spore powder, the extraction process has no high temperature or acid-base environment, the higher polysaccharide extraction rate can be achieved by only one-time extraction, the solvent and the process cost are saved, and the extracted ganoderma lucidum polysaccharide has good antioxidant activity. The invention extracts high-quality ganoderma lucidum polysaccharide with higher efficiency, provides a new method for the extraction technology of ganoderma lucidum polysaccharide, and has important significance for the development of ganoderma lucidum industry.
The method has the advantages that the ultrahigh pressure and hot water extraction method is combined, the solvent is saved, the efficiency is high, the impurities are few, high-temperature or acid-base environments are not generated in the extraction and impurity removal process, and the structure of the polysaccharide can be better protected.
The above description is only a preferred embodiment of the present invention, and it should be noted that the process of the present invention is also applicable to the refining of other natural polysaccharides, and it will be apparent to those skilled in the art that several modifications and touch-up can be made without departing from the technical principle of the present invention, and these modifications and touch-up should be regarded as the protection scope of the present invention.
Claims (10)
1. A method for extracting ganoderma lucidum polysaccharide from ganoderma lucidum spore powder is characterized by comprising the following steps:
1) pretreatment of raw materials: drying wall-broken ganoderma lucidum spore powder, adding petroleum ether, carrying out degreasing treatment at room temperature, putting the spore powder obtained by suction filtration in a fume hood, volatilizing until no ether smell exists, and then drying to obtain degreased ganoderma lucidum spore powder;
2) infiltrating: adding ultrapure water into the defatted ganoderma lucidum spore powder, and placing the mixture into a self-sealing bag for ultrasonic treatment;
3) ultrahigh pressure treatment: vacuumizing and sealing the valve bag, and then carrying out ultrahigh pressure treatment on the valve bag;
4) hot water extraction: carrying out constant-temperature water bath on the ganoderma lucidum spore powder subjected to ultrahigh pressure treatment, and carrying out suction filtration to obtain a crude extract of ganoderma lucidum polysaccharide;
5) deproteinizing and depigmenting ganoderma lucidum polysaccharide: adding macroporous resin into the crude extract of ganoderan, treating by shaking table, and vacuum filtering to obtain supernatant, i.e. defatted deproteinized polysaccharide solution;
6) alcohol precipitation: concentrating the defatted deproteinized polysaccharide solution by rotary evaporation to one fourth of the original volume, adding four times of anhydrous ethanol into the concentrated solution, precipitating overnight, centrifuging to obtain polysaccharide precipitate, and freeze drying to obtain refined ganoderan powder.
2. The method for extracting ganoderan from ganoderma lucidum spore powder according to claim 1, wherein in the step 1), the drying temperature is 50-60 ℃ and the drying time is 10-12 h.
3. The method for extracting ganoderan from ganoderma lucidum spore powder according to claim 1, wherein in the step 1), the material-to-liquid ratio of the wall-broken ganoderma lucidum spore powder to petroleum ether is 1: 20-30 g/mL.
4. The method for extracting ganoderan from ganoderma lucidum spore powder according to claim 1, wherein in the step 1), the time of the degreasing treatment is 10-12 h; uniformly stirring at 200-1000 r/min in the degreasing treatment process.
5. The method for extracting ganoderan from ganoderma lucidum spore powder according to claim 1, wherein in the step 1), the volatilization time is 4-6 h; the temperature of redrying can be 50 ~ 60 ℃.
6. The method for extracting ganoderan from ganoderma lucidum spore powder according to claim 1, wherein in step 1) and in step 2), the feed-liquid ratio of the defatted ganoderma lucidum spore powder to ultrapure water is 1 mg: 10-55 mL; the time of the ultrasonic treatment can be 5-10 min.
7. The method for extracting ganoderan from ganoderma lucidum spore powder according to claim 1, wherein in the step 3), the time for vacuumizing is 90-120 s; the pressure maintaining time of the ultrahigh pressure treatment is 3-15 min, and the treatment pressure is 100-600 MPa; the preferable pressure maintaining time is 6-10 min.
8. The method for extracting ganoderan from ganoderma lucidum spore powder according to claim 1, wherein in the step 4), the water bath time is 30-60 min, and the water bath temperature is 60 ℃.
9. The method of claim 1, wherein in step 5), the ratio of polysaccharide solution to macroporous resin is 1: 20 (g/mL); the time of the shaking table treatment can be 4-9 h, the temperature of the shaking table treatment is 28-35 ℃, and the rotating speed of the shaking table can be 200 r/min.
10. The method for extracting ganoderan from ganoderma lucidum spore powder according to claim 1, wherein in step 6), the overnight precipitation is performed at 4 ℃; the centrifugation time can be 12-15 min; the freeze-drying time may be 12 hours.
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CN116268254A (en) * | 2022-08-26 | 2023-06-23 | 广西师范大学 | Wild-imitation total ganoderma lucidum effervescent tablet with health care function and preparation method thereof |
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CN109400741A (en) * | 2018-11-06 | 2019-03-01 | 中科健康产业集团江苏药业有限公司 | A kind of isolation and purification method of ganoderma spore polysaccharide |
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CN109400741A (en) * | 2018-11-06 | 2019-03-01 | 中科健康产业集团江苏药业有限公司 | A kind of isolation and purification method of ganoderma spore polysaccharide |
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CN116268254A (en) * | 2022-08-26 | 2023-06-23 | 广西师范大学 | Wild-imitation total ganoderma lucidum effervescent tablet with health care function and preparation method thereof |
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