CN111647548B - Ganoderma lucidum mycelium culture medium for high-yield triterpene and culture method thereof - Google Patents
Ganoderma lucidum mycelium culture medium for high-yield triterpene and culture method thereof Download PDFInfo
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Abstract
The invention discloses a ganoderma lucidum mycelium culture medium for high-yield triterpene, which can provide nutrient substances required by the growth of ganoderma lucidum mycelium, promote the growth of ganoderma lucidum mycelium, increase the triterpene substances of the ganoderma lucidum mycelium and improve the tolerance of ganoderma lucidum to metal. The method is simple and effective, is easy to operate, can improve the production efficiency and shorten the culture time, and is favorable for the industrial production of the ganoderma lucidum mycelia with high triterpene yield.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a ganoderma lucidum mycelium culture medium for high-yield triterpene and a culture method thereof.
Background
Ganoderma (Ganoderma lucidum) belonging to Basidiomycetes, Polyporaceae, Ganoderma genus has various effective components, such as: polysaccharides, triterpenes, proteins, amino acid polypeptides, nucleosides, trace components and the like, wherein the ganoderma triterpenes are important components in ganoderma lucidum, have various effects of resisting virus, resisting tumor, resisting oxidation, neurasthenia, enhancing immunity, reducing hypertension, delaying aging and the like, and have extremely low toxicity.
The traditional cultivation of ganoderma lucidum is easily influenced by external factors such as environment, artificial planting mode and the like, and has long growth period, thus being difficult to realize large-scale industrial production. The liquid fermentation ganoderma lucidum mycelium is a culture mode which can ensure the increase of the quality, the quantity and the polysaccharide content of the mycelium and quickly generate a large amount of secondary metabolites by introducing sterile air into a shake flask or a fermentation tank under the control of a proper culture medium and culture parameters, and has the advantages of high yield, short period, low cost, suitability for industrial production and the like.
The ganoderma triterpene is used as a main biological active component of ganoderma, further research on the ganoderma triterpene is beneficial to searching effective components in the ganoderma and further exploring new activity and clarifying a pharmacological activity mechanism of the ganoderma triterpene, and how to increase the yield of the ganoderma triterpene by adjusting a fermentation culture medium and culture conditions is a technical problem which is urgently needed to be solved at present.
Disclosure of Invention
The invention aims to provide a ganoderma lucidum mycelium culture medium for high-yield triterpene, which can provide nutrient substances required by the growth of ganoderma lucidum mycelium, promote the growth of ganoderma lucidum mycelium, improve ganoderan and simultaneously increase the tolerance of ganoderma lucidum mycelium to metal.
The above object of the present invention is achieved by the following technical solutions:
a ganoderma lucidum mycelium culture medium for high-yield triterpene comprises a seed culture medium and a fermentation culture medium, wherein 1L of the seed culture medium comprises the following components: 1-5g of glucose, 2-5g of peptone, 0.5-8g of yeast powder, 0.1-1.0g of monopotassium phosphate, 0.01-0.25g of magnesium sulfate heptahydrate and vitamin B10.001-0.05g and the balance of water;
the fermentation culture medium is 1L culture medium composed of 10-30g of soluble starch, 5-15g of aureobasidium polysaccharide, 0.5-4g of N-dodecyl-N methyl-2 pyrrolidone bromide, 5-10g of peptone, 0.5-8g of yeast powder, 0.1-1.0g of monopotassium phosphate, 0.01-0.5g of magnesium sulfate heptahydrate, and vitamin B10.05-0.1g, 0.01-0.05g of heme peptide and the balance of water.
Preferably, the seed culture medium is 1L culture medium composed of the following components: 2g of glucose, 3g of peptone, 1.5g of yeast powder, 0.3g of monopotassium phosphate, 0.15g of magnesium sulfate heptahydrate, 10.03g of vitamin B and the balance of water;
the fermentation medium is 1L of culture medium which comprises 25g of soluble starch, 18g of aureobasidium pullulans, 2g of N-dodecyl-N-methyl-2 pyrrolidone bromide salt, 8g of peptone, 5g of yeast powder, 0.5g of monopotassium phosphate, 0.25g of magnesium sulfate heptahydrate, and vitamin B10.03g, 0.05g of heme peptide and the balance of water.
Experiments show that the yield of ganoderma triterpenes in ganoderma lucidum mycelia can be increased by taking soluble starch and aureobasidium pullulans as carbon sources and peptone as nitrogen sources; and the 13-hydroxyoctadecadienoic acid is added into the fermentation medium, and can generate combined action with the heme peptide, so that the yield of the ganoderma triterpene can be further promoted, the aureobasidium pullulans is added into the fermentation medium, so that the conversion rate of the ganoderma polysaccharide can be remarkably increased, the accumulation of extracellular polymers of the ganoderma is promoted, the tolerance to metal ions is improved, and meanwhile, the N-dodecyl-N methyl-2 pyrrolidone bromide salt can reduce the viscosity of the culture medium in the culture medium, promote the transfer of oxygen, prevent the growth stop of thalli caused by high viscosity of the culture medium, and further promote the synthesis of the ganoderma triterpene.
The invention also provides a method for culturing ganoderma lucidum mycelia with high yield of triterpenes and polysaccharides, which comprises the following steps:
s1, inoculating the ganoderma lucidum mycelia into a seed culture medium for culture to obtain a seed solution;
s2, inoculating the seed liquid into a fermentation tank filled with a fermentation culture medium for fermentation tank culture, adding 0.01-0.05g of 13-hydroxyoctadecadienoic acid into the fermentation culture medium during seed liquid inoculation, and filtering after fermentation to obtain the ganoderma lucidum mycelia.
Preferably, in step S1, the ganoderma lucidum mycelia are inoculated into the seed culture medium in an inoculation amount of 5-10%.
Preferably, the inoculation amount of inoculating the seed solution into the fermentation medium in the step S2 for fermentation culture is 10-15%.
Preferably, the conditions for inoculating the ganoderma lucidum mycelia into the seed culture medium in the step S1 are as follows: the temperature is 25-30 ℃, the rotating speed is 100-180r/min, the pressure is 0.03-0.05MPa, the air flow is 1:0.3-0.7v/v.min, and the dark culture is carried out for 2-5 days.
Preferably, the conditions for inoculating the ganoderma lucidum mycelia into the seed culture medium in the step S1 are as follows: the temperature is 25-30 ℃, the rotating speed is 80-180r/min, the pressure is 0.03-0.05MPa, the air flow is 1:0.5-1.8v/v.min, and the dark culture is carried out for 3-7 days.
13-hydroxy octadecadienoic acid is added into the culture medium, so that the dissolved oxygen concentration and the permeability of cell membranes in the culture medium can be greatly improved, the production capacity of strains cannot be influenced, and the process of exchanging substances between cells and the environment is changed, thereby promoting the synthesis of ganoderma triterpenoids from ganoderma mycelia; the addition of Aureobasidium pullulans in the fermentation medium can promote the formation of extracellular polymer of Ganoderma lucidum and increase the Fe content of Ganoderma lucidum2+And Se2+The tolerance of the ganoderma lucidum is improved, and the growth of ganoderma lucidum hyphae is promoted; wherein the aureobasidium pullulans and the soluble starch can act together to promote the transformation of the ganoderan.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, soluble starch and aureobasidium pullulans are used as carbon sources, peptone and heme peptide are used as nitrogen sources and used as fermentation culture media to culture ganoderma lucidum mycelia, and a proper amount of 13-hydroxyoctadecadienoic acid is added in the fermentation process, so that the yield of ganoderma lucidum triterpenoids in ganoderma lucidum mycelia can be greatly improved.
(2) The culture medium is added with the aureobasidium pullulans to culture the ganoderma lucidum mycelia, so that the tolerance of the ganoderma lucidum mycelia to metal ions can be greatly improved, and the growth of the ganoderma lucidum mycelia is promoted.
(3) The method is simple and effective, is easy to operate, can improve the production efficiency, and is favorable for the industrial production of high-yield triterpenoid ganoderma lucidum mycelia.
Detailed Description
The present invention will be described in further detail below.
The ganoderma lucidum strain is purchased from China general microbiological culture collection center with CCGMC No. 5.616.
Example 1
Seed culture medium: 1L of culture medium comprises 1g of glucose, 2.5g of peptone, 0.5g of yeast powder, 0.1g of monopotassium phosphate, 0.25g of magnesium sulfate heptahydrate, 10.001g of vitamin B and the balance of water;
fermentation medium: the 1L culture medium comprises 10g of soluble starch, 15g of aureobasidium pullulans, 0.5g of N-dodecyl-N methyl-2 pyrrolidone bromide salt, 5g of peptone, 0.5g of yeast powder, 0.5g of monopotassium phosphate, 0.01g of magnesium sulfate heptahydrate, 10.1g of vitamin B, 0.01g of heme peptide and the balance of water.
The method for culturing the ganoderma lucidum mycelia with high triterpene yield comprises the following steps:
s1, inoculating the ganoderma lucidum mycelia into a seed culture medium for culture to obtain a seed solution, wherein the inoculation amount is 6%; culturing at 25 deg.C, rotation speed of 100r/min, pressure of 0.03MPa, airflow rate of 1:0.3v/v.min in dark for 2 days; s2, inoculating the seed solution into a fermentation tank filled with a fermentation culture medium for fermentation tank culture, adding 0.01g of 13-hydroxy octadecadienoic acid into the fermentation culture medium during seed solution inoculation, wherein the inoculation amount is 15%, the temperature is 25 ℃, the rotating speed is 80r/min, the pressure is 0.035MPa, the airflow is 1:0.5v/v.min, dark culture is carried out for 3 days, and after fermentation is finished, filtration is carried out to obtain the ganoderma lucidum mycelia.
Example 2
Seed culture medium: the 1L culture medium comprises 5g of glucose, 3g of peptone, 8g of yeast powder, 1.0g of monopotassium phosphate, 0.01g of magnesium sulfate heptahydrate, 10.05g of vitamin B and the balance of water;
fermentation medium: the 1L culture medium comprises 30g of soluble starch, 5g of aureobasidium pullulans, 4g of N-dodecyl-N methyl-2 pyrrolidone bromide salt, 10g of peptone, 8g of yeast powder, 0.3g of monopotassium phosphate, 0.4g of magnesium sulfate heptahydrate, 10.05g of vitamin B, 0.03g of heme peptide and the balance of water.
The method for culturing the ganoderma lucidum mycelia with high triterpene yield comprises the following steps:
s1, inoculating the ganoderma lucidum mycelia into a seed culture medium for culture to obtain a seed solution, wherein the inoculation amount is 5%; culturing at 25 deg.C, rotation speed of 120r/min, pressure of 0.03MPa, airflow rate of 1:0.4v/v.min in dark for 2 days; s2, inoculating the seed solution into a fermentation tank filled with a fermentation culture medium for fermentation tank culture, adding 0.03g of 13-hydroxy octadecadienoic acid into the fermentation culture medium during seed solution inoculation, wherein the inoculation amount is 10%, the temperature is 25 ℃, the rotating speed is 80r/min, the pressure is 0.03MPa, the airflow is 1:0.6v/v.min, dark culture is carried out for 7 days, and after fermentation is finished, filtration is carried out to obtain the ganoderma lucidum mycelia.
Example 3
Seed culture medium: 1L of culture medium comprises 4g of glucose, 2g of peptone, 2g of yeast powder, 0.4g of monopotassium phosphate, 0.15g of magnesium sulfate heptahydrate, 10.04g of vitamin B and the balance of water;
fermentation medium: the 1L culture medium comprises 15g of soluble starch, 10g of aureobasidium pullulans, 1g of N-dodecyl-N methyl-2 pyrrolidone bromide salt, 9g of peptone, 2g of yeast powder, 0.4g of monopotassium phosphate, 0.4g of magnesium sulfate heptahydrate, 10.08g of vitamin B, 0.04g of heme peptide and the balance of water.
The method for culturing the ganoderma lucidum mycelia with high triterpene yield comprises the following steps:
s1, inoculating the ganoderma lucidum mycelia into a seed culture medium for culture to obtain a seed solution, wherein the inoculation amount is 6%; culturing at 25 deg.C, rotation speed of 180r/min, pressure of 0.03MPa, airflow rate of 1:0.5v/v.min in dark for 2 days; s2, inoculating the seed solution into a fermentation tank filled with a fermentation culture medium for fermentation tank culture, adding 0.04g of 13-hydroxy octadecadienoic acid into the fermentation culture medium during seed solution inoculation, wherein the inoculation amount is 12%, the temperature is 25 ℃, the rotating speed is 140r/min, the pressure is 0.04MPa, the airflow is 1:0.9v/v.min, dark culture is carried out for 4 days, and after fermentation is finished, filtration is carried out to obtain the ganoderma lucidum mycelia.
Example 4
Seed culture medium: 1L of the medium consisted of the following components: 3g of glucose, 5g of peptone, 4g of yeast powder, 0.6g of monopotassium phosphate, 0.20g of magnesium sulfate heptahydrate, 10.03g of vitamin B and the balance of water;
fermentation medium: the 1L culture medium comprises 18g of soluble starch, 11.5g of aureobasidium pullulans, 2g of N-dodecyl-N methyl-2 pyrrolidone bromide salt, 8g of peptone, 4g of yeast powder, 0.5g of potassium dihydrogen phosphate, 0.3g of magnesium sulfate heptahydrate, 10.075g of vitamin B, 0.03g of heme peptide and the balance of water.
The method for culturing the ganoderma lucidum mycelia with high triterpene yield comprises the following steps:
s1, inoculating the ganoderma lucidum mycelia into a seed culture medium for culture to obtain a seed solution, wherein the inoculation amount is 7%; culturing at 25 deg.C, rotation speed of 140r/min, pressure of 0.03MPa, airflow rate of 1:0.45v/v.min in dark for 2 days; s2, inoculating the seed solution into a fermentation tank filled with a fermentation culture medium for fermentation tank culture, adding 0.03g of 13-hydroxy octadecadienoic acid into the fermentation culture medium during seed solution inoculation, wherein the inoculation amount is 11%, the temperature is 25 ℃, the rotating speed is 120r/min, the pressure is 0.04MPa, the airflow is 1:1.2v/v.min, dark culture is carried out for 5 days, and after fermentation is finished, filtration is carried out to obtain the ganoderma lucidum mycelia.
Example 5
Seed culture medium: 1L of culture medium comprises 2g of glucose, 4g of peptone, 3.6g of yeast powder, 0.8g of monopotassium phosphate, 0.18g of magnesium sulfate heptahydrate, 10.02g of vitamin B and the balance of water;
fermentation medium: the 1L culture medium comprises 25g of soluble starch, 15g of aureobasidium pullulans, 3g of N-dodecyl-N methyl-2 pyrrolidone bromide salt, 6g of peptone, 6g of yeast powder, 1.0g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 10.06g of vitamin B, 0.02g of heme peptide and the balance of water.
The method for culturing the ganoderma lucidum mycelia with high triterpene yield comprises the following steps:
s1, inoculating the ganoderma lucidum mycelia into a seed culture medium for culture to obtain a seed solution, wherein the inoculation amount is 10%; culturing at 25 deg.C, rotation speed of 160r/min, pressure of 0.03MPa, airflow rate of 1:0.7v/v.min in dark for 2 days; s2, inoculating the seed solution into a fermentation tank filled with a fermentation culture medium for fermentation tank culture, adding 0.04g of 13-hydroxy octadecadienoic acid into the fermentation culture medium during seed solution inoculation, wherein the inoculation amount is 14%, the temperature is 25 ℃, the rotating speed is 180r/min, the pressure is 0.05MPa, the airflow is 1:1.8v/v.min, dark culture is carried out for 6 days, and after fermentation is finished, filtration is carried out to obtain the ganoderma lucidum mycelia.
Comparative example 1
The difference from example 4 is that the fermentation medium is not supplemented with heme peptide, and the mass of the deletion part is supplemented with corn oligopeptide.
Comparative example 2
The difference from example 4 is that the fermentation medium is not supplemented with N-dodecyl-N-methyl-2-pyrrolidone bromide, and the mass of the missing part is supplemented with Tween-20.
Comparative example 3
The difference from example 4 is that no pullulan was added to the fermentation medium and the mass of the missing part was made up with soluble starch.
Comparative example 4
The difference from example 4 is that the fermentation medium is not supplemented with aureobasidium pullulans, and the mass of the missing part is supplemented with a heme peptide.
Comparative example 5
The difference from example 4 is that the fermentation medium is not supplemented with soluble starch and the missing part of the mass is supplemented with pullulan.
Comparative example 6
The difference from example 4 is that during the fermentation, no 13-hydroxyoctadecadienoic acid is added during the inoculation of the seed liquid.
Test example 1
The medium in comparative examples 1 to 6 and step 2 of examples 1 to 5 was measured for the viscosity of the solution at 30 ℃ with a rotational viscometer, and the measurement results are shown in Table 1:
as shown in Table 1, the viscosities of examples 1 to 5 were all lower than 100 pas, with the viscosity being the lowest in example 4 and relatively high in comparative examples 1 to 6.
Test example 2 measurement of hyphal Biomass and intracellular triterpene content
Measurement of hyphal biomass: and (3) adding water into the fermented mycelia in comparative examples 1-6 and examples 1-5, uniformly mixing, performing suction filtration, separating the mycelia from the fermentation liquor, drying the obtained mycelia at 60 ℃ to constant weight, and calculating the biomass of each strain (calculated by dry weight g of the mycelia per 100mL of the fermentation liquor).
Determination of the yield of intracellular triterpenes: the fermented ganoderma lucidum mycelia of examples 1-6 and comparative examples 1-7 were washed with 95% (v/v) ethanol for 2 times, centrifuged, and mycelia were collected, and an appropriate amount of mycelia was taken and added to 95% (v/v) ethanol at 1g/50mL and disrupted with a cell disrupter (1 min. times.4), extracted at 60 ℃ for 1h at 400W, and repeated once, and the supernatants were combined and fixed to volume to obtain solutions to be tested of examples 1-6 and comparative examples 1-7. The content of the triterpene is determined by adopting a vanillin perchloric acid method.
Vanillin-perchloric acid assay method: respectively putting 0.1mL of the solution to be detected of the examples 1-6 and the comparative examples 1-7 in a test tube, heating at 70 ℃ to volatilize the solvent, adding 0.2mL of 5% (m/v) vanillin glacial acetic acid solution and 0.5mL of perchloric acid, uniformly mixing, putting in a water bath at 60 ℃, preserving heat for 20min, taking out, putting in cold water for 10min, finally adding 5mL of glacial acetic acid, and determining the light absorption value at 550 nm.
The measurement results of the hyphal biomass and the intracellular triterpene content are shown in Table 1.
TABLE 1 measurement of hyphal biomass and intracellular triterpene content
| Hypha biomass g/100mL | Yield of intracellular triterpene mg/g | |
| Example 1 | 6.75 | 37.11 |
| Example 2 | 5.94 | 28.97 |
| Example 3 | 6.44 | 31.45 |
| Example 4 | 7.36 | 48.78 |
| Example 5 | 6.56 | 32.47 |
| Comparative example 1 | 5.01 | 19.61 |
| Comparative example 2 | 4.11 | 15.62 |
| Comparative example 3 | 5.89 | 17.32 |
| Comparative example 4 | 5.09 | 16.13 |
| Comparative example 5 | 4.39 | 15.51 |
| Comparative example 6 | 4.86 | 13.47 |
As shown in Table 2, the present example can significantly improve the biomass of Ganoderma lucidum mycelia and the yield of Ganoderma lucidum polysaccharide, wherein the biomass of Ganoderma lucidum mycelia reaches 5.94-7.36g/100mL, and the yield of Ganoderma intracellular triterpene reaches 31.4-48.78mg/g, wherein example 5 is the best example.
Test example 2 measurement of Ganoderma lucidum hypha Biomass under Metal stress
Slowly shaking Ganoderma mycelium obtained in example 5 and comparative examples 1-7, filtering, and packaging into 500mL triangular flasks with 200mL each, respectively inoculating 800mg/L Fe2+Solution and Se at a final concentration of 100mg/L2+Continuously culturing the solution for three days, accurately measuring 100ml of mycelium fermentation liquid in a conical flask, adding water into the mycelium, mixing, vacuum filtering, separating mycelium and fermentation liquid according to the amount of wet myceliumFor biomass, the results are shown in Table 2.
TABLE 2 determination of Ganoderma mycelium biomass under metal stress
As shown in Table 2, this example can significantly improve the Fe content in the Ganoderma mycelium2+And Se2+Tolerance of (3), Fe2+Under the stress, the biomass of ganoderma lucidum hypha reaches 7.21-10.88g/100mL, and Se is2+Under the stress, the biomass of ganoderma lucidum mycelia reaches 7.55-9.78g/100mL, wherein example 4 is the best example.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. All cooked
Those skilled in the art can modify and change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (6)
1. A ganoderma lucidum mycelium culture medium for high-yield triterpene comprises a seed culture medium and a fermentation culture medium, and is characterized in that the seed culture medium is characterized in that 1L of the culture medium consists of the following components: 1-5g of glucose, 2-5g of peptone, 0.5-8g of yeast powder, 0.1-1.0g of monopotassium phosphate, 0.01-0.25g of magnesium sulfate heptahydrate and vitamin B10.001-0.05g and the balance of water;
the fermentation culture medium is 1L culture medium composed of 10-30g of soluble starch, 5-15g of aureobasidium polysaccharide, 0.5-4g of N-dodecyl-N methyl-2 pyrrolidone bromide, 5-10g of peptone, 0.5-8g of yeast powder, 0.1-1.0g of monopotassium phosphate, 0.01-0.5g of magnesium sulfate heptahydrate, and vitamin B10.05-0.1g, 0.01-0.05g of heme peptide and the balance of water.
2. A method for culturing ganoderma lucidum mycelia with high triterpene yield is characterized by comprising the following steps:
s1, inoculating the ganoderma lucidum mycelia into a seed culture medium for culture to obtain a seed solution;
s2, inoculating the seed liquid into a fermentation tank filled with a fermentation culture medium for fermentation tank culture, adding 0.01-0.05g of 13-hydroxyoctadecadienoic acid into the fermentation culture medium during seed liquid inoculation, and filtering after fermentation to obtain ganoderma lucidum mycelia;
the seed culture medium is characterized in that 1L of the culture medium consists of the following components: 1-5g of glucose, 2-5g of peptone, 0.5-8g of yeast powder, 0.1-1.0g of monopotassium phosphate, 0.01-0.25g of magnesium sulfate heptahydrate and vitamin B10.001-0.05g and the balance of water;
the fermentation culture medium is 1L culture medium composed of 10-30g of soluble starch, 5-15g of aureobasidium polysaccharide, 0.5-4g of N-dodecyl-N methyl-2 pyrrolidone bromide, 5-10g of peptone, 0.5-8g of yeast powder, 0.1-1.0g of monopotassium phosphate, 0.01-0.5g of magnesium sulfate heptahydrate, and vitamin B10.05-0.1g, 0.01-0.05g of heme peptide and the balance of water.
3. The method according to claim 2, wherein the Ganoderma lucidum mycelia are inoculated into the seed medium in the step S1 in an amount of 5-10%.
4. The culture method according to claim 2, wherein the inoculation amount of the seed solution into the fermentation medium for fermentation culture in step S2 is 10-15%.
5. The culture method according to claim 2, wherein the conditions for inoculating Ganoderma lucidum mycelia into the seed culture medium in step S1 are as follows: the temperature is 25-30 ℃, the rotating speed is 100-180r/min, the pressure is 0.03-0.05MPa, the air flow is 1:0.3-0.7v/v.min, and the dark culture is carried out for 2-5 days.
6. The culture method according to claim 2, wherein the conditions for inoculating Ganoderma lucidum mycelia into the seed culture medium in step S1 are as follows: the temperature is 25-30 ℃, the rotating speed is 80-180r/min, the pressure is 0.03-0.05MPa, the air flow is 1:0.5-1.8v/v.min, and the dark culture is carried out for 3-7 days.
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