CN113069478A - Process for extracting effective components from culture medium after picking lucid ganoderma - Google Patents
Process for extracting effective components from culture medium after picking lucid ganoderma Download PDFInfo
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Abstract
The invention discloses a process for extracting effective components from a culture medium after picking lucid ganoderma, which comprises the following steps: (1) selecting culture medium without contamination of mixed bacteria after picking Ganoderma, spraying water on surface, culturing for 2-4 days, and collecting surface bacterial colony; (2) washing the obtained culture medium, standing the washing solution to remove the bottom precipitate to obtain a suspension containing Ganoderma mycelia; (3) separating and purifying the ganoderma lucidum mycelia in the suspension to obtain purified ganoderma lucidum mycelia; (4) mixing the colonies and Ganoderma mycelia, extracting with water for 3-5 times, mixing extractive solutions, standing to remove sediment, and collecting supernatant to obtain the extractive solution of effective components in culture medium after picking Ganoderma. The invention collects the ganoderma lucidum mycelium in the culture medium after picking the ganoderma lucidum, then separates and purifies the ganoderma lucidum mycelium, and finally extracts the effective components, the process is simple, the design is reasonable, and the extracted effective components come from the ganoderma lucidum, and the impurity components in the culture medium are not introduced.
Description
Technical Field
The invention belongs to the technical field of extraction of effective components of lucid ganoderma, and particularly relates to a process for extracting the effective components from a culture medium after lucid ganoderma is picked.
Background
The shape of the ganoderma lucidum is umbrella-shaped, pileus kidney-shaped, semicircular or nearly circular, is the fruiting body of the ganoderma lucidum which is a fungus in the family of polyporaceae, and is one of famous edible and medicinal fungi in China. Modern medicine proves that ganoderma lucidum contains a plurality of physiological regulation factors with obvious effect on human bodies, wherein the most important effective components are ganoderma lucidum polysaccharide and ganoderic acid. Ganoderan is mainly a monosaccharide polymer with physiological activity, has the effects of resisting tumor, improving the immunity of the body, removing free radicals, resisting aging, resisting thrombus, resisting blood coagulation and the like, and is a main effective component of ganoderma with the effects of strengthening body resistance and consolidating constitution. Ganoderic acid has activities of protecting liver, removing toxic substances, resisting oxidation, resisting bacteria, diminishing inflammation, resisting virus, inhibiting liver tumor cells and the like, and is one of main material bases of medicinal efficacy of ganoderma.
The ganoderma lucidum is also called Linzhongling, which takes the growth in the forest as the best and the highest drug effect, and is the wood-rotting fungus which is grown by dead fallen trees and mainly grows in a wetter place. The growth temperature of the ganoderma lucidum is 25-30 ℃, the ganoderma lucidum belongs to aerobic fungi, is very sensitive to light in the growth and development process, has obvious inhibition effect on the growth of hyphae by the light, and has the fastest growth speed under dark conditions.
Because of the limited wild resources of ganoderma, the technology of artificial cultivation of ganoderma is developed, wherein the culture medium is the source of nutrients for the growth of ganoderma. Because the lucid ganoderma sporocarp grows from lucid ganoderma hypha, the lucid ganoderma hypha often remains on the culture medium after the lucid ganoderma is cultivated, but most of the culture medium after the lucid ganoderma is picked is abandoned and not utilized at present, and the effective components of the lucid ganoderma in the culture medium are not effectively extracted, thereby causing waste. Some manufacturers treat and extract the ganoderma lucidum together with the culture medium when picking the ganoderma lucidum, so that impurity components of the culture medium are introduced into the ganoderma lucidum extract, and particularly polysaccharide components in the raw materials of the culture medium can be used as ganoderma lucidum polysaccharide, so that the purity of the active ingredients of the ganoderma lucidum is insufficient, and the drug effect is influenced.
In summary, how to design a process for extracting effective components from a culture medium after picking lucid ganoderma is a problem which needs to be solved urgently at present, so that the effective components in the culture medium after picking lucid ganoderma are fully extracted and utilized, and the extracted effective components come from the lucid ganoderma, and impurity components in the culture medium are not introduced.
Disclosure of Invention
The invention aims to solve the technical problems and provides a process for extracting effective components from a culture medium after lucid ganoderma is picked.
The invention realizes the aim through the following technical scheme, and a process for extracting effective components from a culture medium after lucid ganoderma is picked comprises the following steps:
(1) selecting culture medium without contamination of infectious microbes after picking Ganoderma, spraying water on surface, placing at 25-30 deg.C under sterile dark condition for 2-4 days, collecting bacterial colony on the surface of the culture medium, repeatedly culturing for 3-5 times, and mixing bacterial colonies on the surface of the culture medium to obtain Ganoderma bacterial colony;
(2) washing the culture medium obtained in the step (1) to obtain a washing solution, standing the washing solution for 1-2h, and removing bottom sediment to obtain a suspension containing ganoderma lucidum mycelia;
(3) separating and purifying the ganoderma lucidum mycelia in the suspension obtained in the step (2) to obtain purified ganoderma lucidum mycelia;
(4) and (3) combining the ganoderma lucidum colonies and the ganoderma lucidum mycelia purified in the step (3), placing the ganoderma lucidum colonies and the ganoderma lucidum mycelia in a pulverizer with the speed of 300-400r/min for pulverizing, sieving by a 50-60-mesh sieve, performing reflux extraction for 3-5 times at 105 ℃ by using 8-12 times of water, combining the extracting solutions, standing for 6-8h, removing bottom sediment, and taking supernatant to obtain the extracting solution of the effective components in the culture medium after picking the ganoderma lucidum.
Further, the method for washing the culture medium in the step (2) comprises the following steps:
s1, washing the culture medium obtained in the step (1) with water for 2-3 times, spreading the culture medium in an open container, and keeping the thickness of the culture medium in the container to be 1-1.2 cm;
s2, adding fine sand into the culture medium in the container, uniformly stirring, spraying castor oil into the culture medium by using a nozzle, uniformly stirring, transferring the materials into an elution column, eluting for 2-3 times by using 8-10 times of 60-70% ethanol solution, and merging the eluates;
s3, distilling the eluent obtained in the step S2 under reduced pressure to remove ethanol, then placing the eluent in a separation funnel, and collecting the lower layer liquid to obtain the washing liquid.
Further, the mass ratio of the culture medium to the fine sand and the castor oil in the container is 1: (0.3-0.5): (0.7-0.8).
Furthermore, the elution column is an organic glass tube without a bottom, and a 30-40 mesh filter screen is arranged at the bottom of the organic glass tube.
Further, the separation and purification treatment method of the ganoderma lucidum mycelia in the step (3) comprises the following steps:
A. adding 1/3 mass Ganoderma colonies into the suspension obtained in step (2), mixing uniformly, and dispersing in a dispersion machine of 400-500r/min for 20-30min to obtain seed suspension;
B. mixing gelatin with 6-9 times of water to obtain gel, granulating the gel with a granulator to obtain 20-40 mesh gel particles, and coating gauze on the surface of the gel particles to obtain a fixed carrier;
C. inoculating the seed suspension into a liquid culture medium according to the inoculation amount of 10-12%, adding a fixed carrier, performing shake culture at 24-26 ℃ and 180-;
D. washing the collected mycelium pellets with sterile water, removing gauze on the fixed carrier, and collecting mycelium on the gauze to obtain the purified ganoderma lucidum mycelium.
Further, the colonies in step (4) are the mass of 2/3 remaining from the colonies collected in step (1).
Further, the liquid medium in step C is: 200mL of clear water, 10g of glucose, 0.1g of peptone, 0.8g of KH2PO40, 0.0 g of MgSO41, 10.1g of VB10, and 1.0g of yeast powder.
Further, the mass ratio of the gelatin to the seed suspension is (0.1-0.3): 1, the mesh number of the gauze is 200 meshes and 300 meshes.
The above operations are carried out under aseptic conditions, and all the instruments are used after sterilization.
The invention has the beneficial effects that:
(1) the invention adopts a special process, firstly collects the ganoderma lucidum mycelia in the culture medium after picking the ganoderma lucidum, then separates and purifies the ganoderma lucidum mycelia, and finally extracts the effective components, the process is simple, the design is reasonable, and the extracted effective components come from the ganoderma lucidum, and the impurity components in the culture medium are not introduced;
(2) in the invention, the culture medium after picking the lucid ganoderma is cultured at first from the beginning of the total extraction process, so that the lucid ganoderma hyphae in the culture medium are subjected to expanded culture, the quality of the lucid ganoderma hyphae is improved, and raw materials are provided for the subsequent separation and purification of the lucid ganoderma hyphae;
(3) when the ganoderma lucidum mycelia in the culture medium are collected, the fine sand is added to increase gaps of the culture medium, so that the action area is increased, and then the castor oil is sprayed, so that the culture medium is lubricated, the adsorption between the culture medium and the ganoderma lucidum mycelia is damaged, and the castor oil contains a large number of hydroxyl groups, can be adsorbed together with the ganoderma lucidum mycelia (the main components of the cell walls of fungi are polysaccharides and contain a large number of hydroxyl groups), and then is eluted by using ethanol, and the ganoderma lucidum mycelia in the culture medium can be collected;
(4) when the ganoderma lucidum mycelia are separated and purified, the ganoderma lucidum mycelia are cultured to be mycelium pellets, and then the mycelium pellets are separated independently to extract effective components, so that the purity of the ganoderma lucidum effective components is effectively improved;
(5) when the ganoderma lucidum mycelia are separated and purified, firstly, the obtained 1/3-quality bacterial colony is added when a seed suspension is prepared, so that the concentration of the mycelia in the seed suspension can be increased, and the ganoderma lucidum mycelia is suitable for culturing the mycelia to grow into mycelium pellets;
(6) when the ganoderma lucidum mycelia are separated and purified, the fixed carrier is added when the mycelium pellets are cultured, so that the mycelium is favorably adsorbed on the fixed carrier and continues to grow to form the mycelium pellets, and the gauze is arranged on the surface of the fixed carrier, so that the mycelium pellets can be separated and collected only by removing the gauze;
(7) when the ganoderma lucidum mycelia are separated and purified and the fixed carrier is prepared, gelatin is selected to be prepared into gel particles, the gelatin contains a large number of hydroxyl groups and can be tightly adsorbed with the ganoderma lucidum mycelia, and the gelatin contains amino acid and can provide nutrition for the growth of the mycelia, so that most of peptone in a liquid culture medium can be replaced.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a process for extracting effective components from a culture medium after lucid ganoderma is picked, which comprises the following steps:
(1) selecting culture medium without contamination of infectious microbes after picking Ganoderma, spraying water on the surface, standing at 25 deg.C in sterile dark for 2 days, collecting bacterial colony on the surface of the culture medium, repeating the culture for 3 times, and mixing bacterial colonies on the surface of the culture medium to obtain Ganoderma bacterial colony;
(2) washing the culture medium obtained in the step (1) to obtain a washing solution, standing the washing solution for 1h, and removing bottom sediment to obtain a suspension containing ganoderma lucidum mycelia;
(3) separating and purifying the ganoderma lucidum mycelia in the suspension obtained in the step (2) to obtain purified ganoderma lucidum mycelia;
(4) and (3) combining the ganoderma lucidum colonies and the ganoderma lucidum mycelia purified in the step (3), placing the ganoderma lucidum colonies and the ganoderma lucidum mycelia purified in the step (3) into a grinder with the speed of 300r/min for grinding, sieving by a 50-mesh sieve, performing reflux extraction for 3 times at 100 ℃ by using 8 times of water, combining extracting solutions, standing for 6h, removing bottom sediment, and taking supernatant to obtain the extracting solution of the effective components in the culture medium after picking the ganoderma lucidum.
Example 2
On the basis of embodiment 1, this embodiment also provides a process for extracting effective components from the culture medium after picking the ganoderma lucidum, and the washing treatment method of the culture medium in step (2) is as follows:
s1, washing the culture medium obtained in the step (1) with water for 2 times, spreading the culture medium in an open container, and keeping the thickness of the culture medium in the container to be 1 cm;
s2, adding fine sand into the culture medium in the container, uniformly stirring, spraying castor oil into the culture medium by using a nozzle, uniformly stirring, transferring the materials into an elution column, eluting for 2 times by using 8 times of 60% ethanol solution, and merging the eluates;
s3, distilling the eluent obtained in the step S2 under reduced pressure to remove ethanol, then placing the eluent in a separation funnel, and collecting the lower layer liquid to obtain the washing liquid.
The mass ratio of the culture medium in the container to the fine sand and the castor oil is 1: 0.3: 0.7.
the elution column is an organic glass tube without a bottom, and a 30-mesh filter screen is arranged at the bottom of the organic glass tube.
The rest is the same as in example 1.
Example 3
On the basis of embodiment 1, this embodiment also provides a process for extracting effective components from the culture medium after picking the ganoderma lucidum, and the separation and purification treatment method of ganoderma lucidum mycelia in step (3) is as follows:
A. adding 1/3 mass Ganoderma colonies into the suspension obtained in step (2), mixing uniformly, and dispersing in a 400r/min dispersion machine for 20min to obtain seed suspension;
B. mixing gelatin with 6 times of water to obtain gel, granulating the gel with a granulator to obtain 20-mesh gel particles, and coating gauze on the surfaces of the gel particles to obtain a fixed carrier;
C. inoculating the seed suspension into a liquid culture medium according to the inoculation amount of 10%, then adding a fixed carrier, placing the fixed carrier in a shaking table at 24 ℃ and 180r/min for culturing until ganoderma lucidum mycelia are gathered on the fixed carrier and grow to form stable mycelium pellets, and collecting the mycelium pellets;
D. washing the collected mycelium pellets with sterile water, removing gauze on the fixed carrier, and collecting mycelium on the gauze to obtain the purified ganoderma lucidum mycelium.
The Ganoderma lucidum colonies in step (4) are now the mass of the residual 2/3 of the Ganoderma lucidum colonies collected in step (1).
The liquid culture medium in the step C is as follows: 200mL of clear water, 10g of glucose, 0.1g of peptone, 0.8g of KH2PO40, 0.0 g of MgSO41, 10.1g of VB10, and 1.0g of yeast powder.
The mass ratio of the gelatin to the seed suspension is 0.1: 1, the mesh number of the gauze is 200 meshes.
The rest is the same as in example 1.
Example 4
The embodiment provides a process for extracting effective components from a culture medium after lucid ganoderma is picked, which comprises the following steps:
(1) selecting culture medium without contamination of infectious microbes after picking Ganoderma, spraying water on the surface, standing at 25 deg.C in sterile dark for 2 days, collecting bacterial colony on the surface of the culture medium, repeating the culture for 3 times, and mixing bacterial colonies on the surface of the culture medium to obtain Ganoderma bacterial colony;
(2) washing the culture medium obtained in the step (1) to obtain a washing solution, standing the washing solution for 1h, and removing bottom sediment to obtain a suspension containing ganoderma lucidum mycelia;
(3) separating and purifying the ganoderma lucidum mycelia in the suspension obtained in the step (2) to obtain purified ganoderma lucidum mycelia;
(4) and (3) combining the ganoderma lucidum colonies and the ganoderma lucidum mycelia purified in the step (3), placing the ganoderma lucidum colonies and the ganoderma lucidum mycelia purified in the step (3) into a grinder with the speed of 300r/min for grinding, sieving by a 50-mesh sieve, performing reflux extraction for 3 times at 100 ℃ by using 8 times of water, combining extracting solutions, standing for 6h, removing bottom sediment, and taking supernatant to obtain the extracting solution of the effective components in the culture medium after picking the ganoderma lucidum.
The method for washing and treating the culture medium in the step (2) comprises the following steps:
s1, washing the culture medium obtained in the step (1) with water for 2 times, spreading the culture medium in an open container, and keeping the thickness of the culture medium in the container to be 1 cm;
s2, adding fine sand into the culture medium in the container, uniformly stirring, spraying castor oil into the culture medium by using a nozzle, uniformly stirring, transferring the materials into an elution column, eluting for 2 times by using 8 times of 60% ethanol solution, and merging the eluates;
s3, distilling the eluent obtained in the step S2 under reduced pressure to remove ethanol, then placing the eluent in a separation funnel, and collecting the lower layer liquid to obtain the washing liquid.
The mass ratio of the culture medium in the container to the fine sand and the castor oil is 1: 0.3: 0.7.
the elution column is an organic glass tube without a bottom, and a 30-mesh filter screen is arranged at the bottom of the organic glass tube.
The separation and purification treatment method of the ganoderma lucidum mycelia in the step (3) comprises the following steps:
A. adding 1/3 mass Ganoderma colonies into the suspension obtained in step (2), mixing uniformly, and dispersing in a 400r/min dispersion machine for 20min to obtain seed suspension;
B. mixing gelatin with 6 times of water to obtain gel, granulating the gel with a granulator to obtain 20-mesh gel particles, and coating gauze on the surfaces of the gel particles to obtain a fixed carrier;
C. inoculating the seed suspension into a liquid culture medium according to the inoculation amount of 10%, then adding a fixed carrier, placing the fixed carrier in a shaking table at 24 ℃ and 180r/min for culturing until ganoderma lucidum mycelia are gathered on the fixed carrier and grow to form stable mycelium pellets, and collecting the mycelium pellets;
D. washing the collected mycelium pellets with sterile water, removing gauze on the fixed carrier, and collecting mycelium on the gauze to obtain the purified ganoderma lucidum mycelium.
The Ganoderma lucidum colonies in step (4) are now the mass of the residual 2/3 of the Ganoderma lucidum colonies collected in step (1).
The liquid culture medium in the step C is as follows: 200mL of clear water, 10g of glucose, 0.1g of peptone, 0.8g of KH2PO40, 0.0 g of MgSO41, 10.1g of VB10, and 1.0g of yeast powder.
The mass ratio of the gelatin to the seed suspension is 0.1: 1, the mesh number of the gauze is 200 meshes.
Example 5
The embodiment provides a process for extracting effective components from a culture medium after lucid ganoderma is picked, which comprises the following steps:
(1) selecting a culture medium without impurity bacteria pollution after picking the lucid ganoderma, spraying water on the surface of the culture medium, placing the culture medium at 28 ℃ for 3 days under the sterile dark condition, collecting bacterial colonies on the surface of the culture medium, repeatedly culturing for 4 times in the way, and combining the bacterial colonies on the surface of the culture medium to obtain lucid ganoderma bacterial colonies;
(2) washing the culture medium obtained in the step (1) to obtain a washing solution, standing the washing solution for 1.5h, and removing bottom sediment to obtain a suspension containing ganoderma lucidum mycelia;
(3) separating and purifying the ganoderma lucidum mycelia in the suspension obtained in the step (2) to obtain purified ganoderma lucidum mycelia;
(4) and (3) combining the ganoderma lucidum colonies and the ganoderma lucidum mycelia purified in the step (3), placing the ganoderma lucidum colonies and the ganoderma lucidum mycelia purified in the step (3) into a grinder with the speed of 350r/min for grinding, sieving by a 55-mesh sieve, performing reflux extraction for 4 times at 103 ℃ by 10 times of water, combining extracting solutions, standing for 7h, removing bottom sediment, and taking supernatant to obtain the extracting solution of the effective components in the culture medium after picking the ganoderma lucidum.
The method for washing and treating the culture medium in the step (2) comprises the following steps:
s1, washing the culture medium obtained in the step (1) with water for 3 times, spreading the culture medium in an open container, and keeping the thickness of the culture medium in the container to be 1.1 cm;
s2, adding fine sand into the culture medium in the container, uniformly stirring, spraying castor oil into the culture medium by using a nozzle, uniformly stirring, transferring the materials into an elution column, eluting for 3 times by using 9 times of 65% ethanol solution, and merging the eluates;
s3, distilling the eluent obtained in the step S2 under reduced pressure to remove ethanol, then placing the eluent in a separation funnel, and collecting the lower layer liquid to obtain the washing liquid.
The mass ratio of the culture medium in the container to the fine sand and the castor oil is 1: 0.4: 0.75.
the elution column is an organic glass tube without a bottom, and a 35-mesh filter screen is arranged at the bottom of the organic glass tube.
The separation and purification treatment method of the ganoderma lucidum mycelia in the step (3) comprises the following steps:
A. adding 1/3 mass Ganoderma colonies into the suspension obtained in step (2), mixing uniformly, and dispersing in a dispersion machine at 450r/min for 25min to obtain seed suspension;
B. mixing gelatin with 7.5 times of water to obtain gel, granulating the gel with a granulator to obtain 30-mesh gel particles, and coating gauze on the surfaces of the gel particles to obtain a fixed carrier;
C. inoculating the seed suspension into a liquid culture medium according to the inoculation amount of 11%, then adding a fixed carrier, placing the fixed carrier at 25 ℃ for shake cultivation at 190r/min until ganoderma lucidum mycelia are gathered on the fixed carrier and grow to form stable mycelium pellets, and collecting the mycelium pellets;
D. washing the collected mycelium pellets with sterile water, removing gauze on the fixed carrier, and collecting mycelium on the gauze to obtain the purified ganoderma lucidum mycelium.
The ganoderma lucidum colonies in the step (4) are the mass of the residual 2/3 of the ganoderma lucidum colonies collected in the step (1).
The liquid culture medium in the step C is as follows: 200mL of clear water, 10g of glucose, 0.1g of peptone, 0.8g of KH2PO40, 0.0 g of MgSO41, 10.1g of VB10, and 1.0g of yeast powder.
The mass ratio of the gelatin to the seed suspension is 0.2: 1, the mesh number of the gauze is 250 meshes.
Example 6
The embodiment provides a process for extracting effective components from a culture medium after lucid ganoderma is picked, which comprises the following steps:
(1) selecting culture medium without contamination of infectious microbes after picking Ganoderma, spraying water on the surface, standing at 30 deg.C in sterile dark for 4 days, collecting bacterial colony on the surface of the culture medium, repeating the culture for 5 times, and mixing bacterial colonies on the surface of the culture medium to obtain Ganoderma bacterial colony;
(2) washing the culture medium obtained in the step (1) to obtain a washing solution, standing the washing solution for 2 hours, and removing bottom sediment to obtain a suspension containing ganoderma lucidum mycelia;
(3) separating and purifying the ganoderma lucidum mycelia in the suspension obtained in the step (2) to obtain purified ganoderma lucidum mycelia;
(4) and (3) combining the ganoderma lucidum colonies and the ganoderma lucidum mycelia purified in the step (3), placing the ganoderma lucidum colonies and the ganoderma lucidum mycelia in a grinder of 400r/min for grinding, sieving by a 60-mesh sieve, performing reflux extraction for 5 times at 105 ℃ by using 12 times of water, combining the extracting solutions, standing for 8h, removing bottom sediment, and taking supernatant to obtain the extracting solution of the effective components in the culture medium after picking the ganoderma lucidum.
The method for washing and treating the culture medium in the step (2) comprises the following steps:
s1, washing the culture medium obtained in the step (1) with water for 3 times, spreading the culture medium in an open container, and keeping the thickness of the culture medium in the container to be 1.2 cm;
s2, adding fine sand into the culture medium in the container, uniformly stirring, spraying castor oil into the culture medium by using a nozzle, uniformly stirring, transferring the materials into an elution column, eluting for 3 times by using 10 times of 70% ethanol solution, and merging the eluates;
s3, distilling the eluent obtained in the step S2 under reduced pressure to remove ethanol, then placing the eluent in a separation funnel, and collecting the lower-layer liquid to obtain a washing liquid;
the mass ratio of the culture medium in the container to the fine sand and the castor oil is 1: 0.5: 0.8.
the elution column is an organic glass tube without a bottom, and a 40-mesh filter screen is arranged at the bottom of the organic glass tube.
The separation and purification treatment method of the ganoderma lucidum mycelia in the step (3) comprises the following steps:
A. adding 1/3 mass Ganoderma colonies into the suspension obtained in step (2), mixing uniformly, and dispersing in a 500r/min dispersion machine for 30min to obtain seed suspension;
B. mixing gelatin with 9 times of water to obtain gel, granulating the gel with a granulator to obtain 40-mesh gel particles, and coating gauze on the surfaces of the gel particles to obtain a fixed carrier;
C. inoculating the seed suspension into a liquid culture medium according to the inoculation amount of 12%, then adding a fixed carrier, placing at 26 ℃ for shake cultivation at 200r/min until ganoderma lucidum mycelia are gathered on the fixed carrier and grow to form stable mycelium pellets, and collecting the mycelium pellets;
D. washing the collected mycelium pellets with sterile water, removing gauze on the fixed carrier, and collecting mycelium on the gauze to obtain the purified ganoderma lucidum mycelium.
The ganoderma lucidum colonies in the step (4) are the mass of the residual 2/3 of the ganoderma lucidum colonies collected in the step (1).
The liquid culture medium in the step C is as follows: 200mL of clear water, 10g of glucose, 0.1g of peptone, 0.8g of KH2PO40, 0.0 g of MgSO41, 10.1g of VB10, and 1.0g of yeast powder.
The mass ratio of the gelatin to the seed suspension is 0.3: 1, the mesh number of the gauze is 300 meshes.
Polysaccharide content determination of extract of effective components in culture medium after picking Ganoderma lucidum
Since the content of ganoderma triterpene in ganoderma mycelium state is very little and has no reference, only the content of crude polysaccharide is measured. The extract of the effective components in the culture medium obtained after picking the ganoderma lucidum in the examples 1-6 is taken as a sample, and the content of crude polysaccharide (625 nm) in the sample is determined by adopting an ultraviolet spectrophotometry, and the results are shown in the following table 1:
as can be seen from examples 4-6 in Table 1, the higher crude polysaccharides were detected in the extracts of the effective components in the culture medium after picking Ganoderma lucidum, indicating that the process of the present invention can be effectively used for extracting the effective components in the culture medium after picking Ganoderma lucidum.
As is clear from comparison between examples 1 to 3 and examples 4 to 6, more effective components can be extracted from the culture medium after picking up the Ganoderma lucidum by the method for washing the culture medium and the method for separating and purifying the Ganoderma lucidum mycelia of the present invention.
The invention has the beneficial effects that: the invention provides a process for extracting effective components from a culture medium after lucid ganoderma is picked, which comprises the steps of collecting lucid ganoderma hyphae in the culture medium after lucid ganoderma is picked, separating and purifying the lucid ganoderma hyphae, and extracting the effective components.
Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and not intended to limit the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or some technical features thereof can be replaced. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A process for extracting effective components from a culture medium after picking lucid ganoderma is characterized in that: the method comprises the following steps:
(1) selecting culture medium without contamination of infectious microbes after picking Ganoderma, spraying water on surface, placing at 25-30 deg.C under sterile dark condition for 2-4 days, collecting bacterial colony on the surface of the culture medium, repeatedly culturing for 3-5 times, and mixing bacterial colonies on the surface of the culture medium to obtain Ganoderma bacterial colony;
(2) washing the culture medium obtained in the step (1) to obtain a washing solution, standing the washing solution for 1-2h, and removing bottom sediment to obtain a suspension containing ganoderma lucidum mycelia;
(3) separating and purifying the ganoderma lucidum mycelia in the suspension obtained in the step (2) to obtain purified ganoderma lucidum mycelia;
(4) and (3) combining the ganoderma lucidum colonies and the ganoderma lucidum mycelia purified in the step (3), placing the ganoderma lucidum colonies and the ganoderma lucidum mycelia in a pulverizer with the speed of 300-400r/min for pulverizing, sieving by a 50-60-mesh sieve, performing reflux extraction for 3-5 times at 105 ℃ by using 8-12 times of water, combining the extracting solutions, standing for 6-8h, removing bottom sediment, and taking supernatant to obtain the extracting solution of the effective components in the culture medium after picking the ganoderma lucidum.
2. The process for extracting effective components from the culture medium after the ganoderma lucidum is picked according to claim 1, which is characterized in that: the method for washing and treating the culture medium in the step (2) comprises the following steps:
s1, washing the culture medium obtained in the step (1) with water for 2-3 times, spreading the culture medium in an open container, and keeping the thickness of the culture medium in the container to be 1-1.2 cm;
s2, adding fine sand into the culture medium in the container, uniformly stirring, spraying castor oil into the culture medium by using a nozzle, uniformly stirring, transferring the materials into an elution column, eluting for 2-3 times by using 8-10 times of 60-70% ethanol solution, and merging the eluates;
s3, distilling the eluent obtained in the step S2 under reduced pressure to remove ethanol, then placing the eluent in a separation funnel, and collecting the lower layer liquid to obtain the washing liquid.
3. The process for extracting effective components from the culture medium after the ganoderma lucidum is picked according to claim 2, which is characterized in that: the mass ratio of the culture medium to the fine sand and the castor oil in the container is 1: (0.3-0.5): (0.7-0.8).
4. The process for extracting effective components from the culture medium after the ganoderma lucidum is picked according to claim 2, which is characterized in that: the elution column is an organic glass tube without a bottom, and a 30-40 mesh filter screen is arranged at the bottom of the organic glass tube.
5. The process for extracting effective components from the culture medium after the ganoderma lucidum is picked according to claim 1, which is characterized in that: the separation and purification treatment method of the ganoderma lucidum mycelia in the step (3) comprises the following steps:
A. adding 1/3 mass Ganoderma colonies into the suspension obtained in step (2), mixing uniformly, and dispersing in a dispersion machine of 400-500r/min for 20-30min to obtain seed suspension;
B. mixing gelatin with 6-9 times of water to obtain gel, granulating the gel with a granulator to obtain 20-40 mesh gel particles, and coating gauze on the surface of the gel particles to obtain a fixed carrier;
C. inoculating the seed suspension into a liquid culture medium according to the inoculation amount of 10-12%, adding a fixed carrier, performing shake culture at 24-26 ℃ and 180-;
D. washing the collected mycelium pellets with sterile water, removing gauze on the fixed carrier, and collecting mycelium on the gauze to obtain the purified ganoderma lucidum mycelium.
6. The process for extracting effective components from the culture medium after the ganoderma lucidum is picked according to claim 5, wherein the process comprises the following steps: the colonies in step (4) are the mass 2/3 remaining from the colonies collected in step (1).
7. The process for extracting effective components from the culture medium after the ganoderma lucidum is picked according to claim 5, wherein the process comprises the following steps: the liquid culture medium in the step C is as follows: 200mL of clear water, 10g of glucose, 0.1g of peptone, 0.8g of KH2PO40, 0.0 g of MgSO41, 10.1g of VB10, and 1.0g of yeast powder.
8. The process for extracting effective components from the culture medium after the ganoderma lucidum is picked according to claim 5, wherein the process comprises the following steps: the mass ratio of the gelatin to the seed suspension is (0.1-0.3): 1, the mesh number of the gauze is 200 meshes and 300 meshes.
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