CN111662862A - Application of edible vegetable oil in improving content of poria triterpene in poria mycelium cells - Google Patents
Application of edible vegetable oil in improving content of poria triterpene in poria mycelium cells Download PDFInfo
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Abstract
The invention discloses application of edible vegetable oil, which is characterized by being applied to improving the content of tuckahoe triterpenes in tuckahoe mycelia cells. According to the invention, edible vegetable oil is added into the culture medium of the fully-grown tuckahoe mycelium, so that the content of tuckahoe triterpenoid in the tuckahoe mycelium cells can be obviously improved. The preferable scheme can even improve the content of the tuckahoe triterpenes by more than 4 times. The edible vegetable oil has edible safety, and can induce the synthesis of triterpene in trace amount. The invention uses edible vegetable oil as an inducer to realize high-level expression of the content of the triterpenoids in the tuckahoe cells.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of edible vegetable oil in improving the content of tuckahoe triterpenoid in tuckahoe mycelia.
Background
Poria cocos (Schw.) Wolf) is a large medicinal fungus belonging to the phylum Eumycota, the class Basidiomycetes, the order Polyporales, the family Polyporaceae, the genus Polyporus, and has been used for thousands of years in China. Modern science has proved that the chemical components of tuckahoe include polysaccharides, triterpenes, sterols, alkaloid amino acids and the like, wherein one of the most important medicinal components is tuckahoe triterpenes which have the effects of resisting tumors, protecting the liver, regulating the immunity and the like. The improvement of the triterpene content of the tuckahoe plays an important role in the wide application of the tuckahoe.
The current research shows that methyl jasmonate, acetic acid, phenobarbital, miconazole, aspirin, copper ions, manganese ions and the like can induce the synthesis of the tuckahoe triterpenes. These efforts provide good materials and phenotypes for the study of the regulatory mechanisms of the biosynthesis of poria triterpene. However, if the tuckahoe is taken as a medicine-food homologous traditional Chinese medicine and is expected to promote the wide use in the medical field, the safety and the economical efficiency of fermentation must be considered. Methyl jasmonate is used as a plant hormone, and has not been practically applied due to the existence of health safety hidden troubles; phenobarbital and miconazole are central neuroleptics, aspirin is an antipyretic analgesic, and these drugs may remain to have adverse effects on healthy people; copper ions and manganese ions are heavy metals and may cause poisoning of the human body. Therefore, there is a need to develop a method, conditions and inducer for improving the fermentation efficiency of Poria cocos liquid, and increasing the triterpene compound content of Poria cocos in the mycelium stage.
Disclosure of Invention
Aiming at the defects or improvement requirements of the prior art, the invention provides the application of edible vegetable oil in improving the content of the tuckahoe triterpenoids in tuckahoe mycelia, and aims to obtain an obvious effect of inducing the expression of the tuckahoe triterpenoids by using the edible vegetable oil with food safety level as an inducer for inducing the increase of the content of the tuckahoe triterpenoids in liquid fermentation tuckahoe mycelia packages through low-dose addition, thereby solving the technical problems of potential safety hazard caused by the residue of the existing inducer for improving the content of the tuckahoe triterpenoids in the tuckahoe mycelia.
To achieve the above objects, according to one aspect of the present invention, there is provided an edible vegetable oil for increasing the intracellular triterpene content of Poria cocos among mycelia of Poria cocos.
Preferably, the edible vegetable oil is applied to improve the intracellular tuckahoe triterpene content of tuckahoe mycelium liquid fermentation.
Preferably, the edible vegetable oil has an iodine value of more than 50g/100g, preferably 50 to 110g/100 g; the saponification value of the edible vegetable oil is more than 180mgKOH/g, preferably 180 to 220 mgKOH/g; the edible vegetable oil is preferably olive oil, peanut oil, rapeseed oil, palm oil, soybean oil, and/or coix seed oil, more preferably olive oil.
Preferably, the edible vegetable oil is used, wherein the poria cocos mycelium liquid fermentation comprises a growth stage and an induction stage;
in the growth stage, the poria cocos mycelia fully grow until the poria cocos mycelia are distributed in a flocculent manner and the mycelia are vigorous;
in the induction stage, edible vegetable oil is added into the fermentation liquor to induce the poria cocos mycelium to improve the content of the intracellular poria cocos triterpenes.
Preferably, the edible vegetable oil is used, wherein the induction stage is added with 1 to 4 percent of edible vegetable oil according to the volume proportion.
Preferably, the edible vegetable oil is added between the beginning of the induction phase and 96 hours, the fermentation temperature is 28-30 ℃, the rotation speed is 120-150r/min, the fermentation time is 4-12 days, the density of the mycelium is increased, and the fermentation is finished before the mycelium is browned.
Preferably, the edible vegetable oil is used, wherein the growth stage is to inoculate tuckahoe liquid strain in the culture medium, and the tuckahoe liquid strain is cultured for 5 to 6 days at the temperature of 28 to 30 ℃ and the rotating speed of 120-150 r/min.
Preferably, said edible vegetable oil is used, wherein said growth phase is inoculated with liquid poria cocos strain in the culture medium at 4% to 8% of the volume of the culture medium.
Preferably, the edible vegetable oil is used, and the poria cocos liquid strain is prepared according to the following method:
(1) activating the tuckahoe mycelium by using a PDA liquid culture medium at the temperature of 28-30 ℃ and the rotating speed of 120-150r/min for 6-7 days to obtain the activated tuckahoe mycelium;
(2) inoculating the activated Poria cocos mycelium obtained in the step (1) into the Poria cocos seed culture medium according to 10% of the culture volume of the Poria cocos seeds, and culturing at the temperature of 28-30 ℃ and the rotation speed of 120-150r/min for 5-6 days to obtain the Poria cocos liquid strain.
Preferably, the edible vegetable oil is prepared by using a PDA culture medium as the poria cocos seed culture medium according to the following method:
cutting 200 g of potato into small pieces, boiling for 20-30 min, filtering eight layers of gauze, adding 20 g of glucose into the filtrate, fixing the volume to 1L, and sterilizing for 20-25 min at 121 ℃ by high-pressure steam.
Preferably, the edible vegetable oil is prepared by the following method, wherein the culture medium of the growth stage is CYM culture medium:
35 g of glucose, 5g of peptone, 5g of yeast powder, 0.883 g of monopotassium phosphate and 0.5 g of magnesium sulfate heptahydrate are added to the mixture, the volume is adjusted to 1 liter by using distilled water, and the mixture is sterilized for 20 to 25 minutes at 121 ℃ by using high-pressure steam.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
according to the invention, edible vegetable oil is added into the culture medium of the fully-grown tuckahoe mycelium, so that the content of tuckahoe triterpenoid in the tuckahoe mycelium cells can be obviously improved. The preferable scheme can even improve the content of the tuckahoe triterpenes by more than 4 times. The edible vegetable oil has edible safety, and especially the synthesis of triterpene can be induced by trace amount of olive oil. The invention uses edible vegetable oil as an inducer to realize high-level expression of the content of the triterpenoids in the tuckahoe cells. The edible vegetable oil is safe and efficient when used as an inducer, can induce the synthesis of triterpene in trace amount, has edible safety, and can obviously improve the triterpene content of the intracellular tuckahoe.
Drawings
FIG. 1 is a graph showing the results of increasing the content of Poria triterpene in the Poria cocos mycelial bag using various kinds of edible vegetable oils as provided in example 1;
FIG. 2 is a graph showing the results of increasing the content of triterpene from Poria cocos in Poria cocos mycelium using different concentrations of olive oil as provided in example 2;
FIG. 3 is a graph showing the results of increasing the intracellular triterpene content of Poria cocos in Poria cocos mycelium at different treatment times using olive oil as provided in example 3;
FIG. 4 is a graph showing the results of increasing the intracellular triterpene content of Poria cocos in Poria cocos mycelium with olive oil at different fermentation periods as provided in example 4;
FIG. 5 is a graph showing the results of the triterpene content of fermented Poria cocos (Poria cocos) mycelia from Poria cocos (Poria cocos) mycelia in different inoculation amounts as provided in the comparative example.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The edible vegetable oil provided by the invention is applied to improving the intracellular poria triterpene content of the poria cocos mycelium, and is preferably applied to improving the intracellular poria cocos triterpene content of the poria cocos mycelium liquid fermentation; the method comprises the following specific steps:
in the growth stage, the poria cocos mycelia fully grow until the poria cocos mycelia are distributed in a flocculent manner and the mycelia are vigorous; inoculating Poria cocos liquid strain in culture medium, wherein the inoculation amount is preferably 4-8% of the volume of the culture medium, and culturing at 28-30 deg.C, preferably 28 deg.C, at rotation speed of 120-150r/min for 5-6 days, preferably 5 days. The culture medium for the growth phase is preferably CYM culture medium, and is prepared according to the following method:
35 g of glucose, 5g of peptone, 5g of yeast powder, 0.883 g of monopotassium phosphate and 0.5 g of magnesium sulfate heptahydrate are added to the mixture, the volume is adjusted to 1 liter by using distilled water, and the mixture is sterilized for 20 to 25 minutes at 121 ℃ by using high-pressure steam.
The poria cocos liquid strain is prepared by the following method:
(1) activating Poria cocos mycelium with PDA liquid culture medium at 28-30 deg.C and rotation speed of 120-150r/min for 6-7 days, preferably for 7 days to obtain activated Poria cocos mycelium;
(2) inoculating the activated Poria cocos mycelium obtained in the step (1) into the Poria cocos seed culture medium according to 10% of the culture volume of the Poria cocos seed, and culturing at the temperature of 28-30 ℃ and the rotation speed of 120-150r/min for 5-6 days, preferably 5 days, to obtain the Poria cocos liquid strain. The poria cocos seed culture medium is a PDA culture medium and is prepared according to the following method: cutting 200 g of potato into small pieces, boiling for 20-30 min, filtering eight layers of gauze, adding 20 g of glucose into the filtrate, fixing the volume to 1L, and sterilizing for 20-25 min at 121 ℃ by high-pressure steam.
And an induction stage, adding edible vegetable oil into the fermentation liquor to induce the poria cocos mycelium to improve the content of the intracellular poria cocos triterpenes. Edible vegetable oil is added into the induction stage according to the mass ratio of 1-4%, and preferably the mass ratio is 2%; edible vegetable oil is added between the beginning and 96 hours, preferably at the beginning of the induction phase, the fermentation temperature is between 28 and 30 ℃, preferably 28 ℃, the rotating speed is 120-150r/min, preferably 150r/min, the fermentation time is 4 to 12 days, the density of the mycelium is increased, and the fermentation is finished before the mycelium is browned.
The edible vegetable oil has edible safety, and can induce the synthesis of triterpene in trace amount. The invention uses edible vegetable oil as an inducer to realize high-level expression of the content of the triterpenoids in the tuckahoe cells. The iodine value of the edible vegetable oil is more than 50g/100g, preferably 50 to 110g/100 g; the saponification value of the edible vegetable oil is more than 180mgKOH/g, preferably 180 to 220 mgKOH/g; the edible vegetable oil is preferably olive oil, peanut oil, rapeseed oil, palm oil, soybean oil, and/or coix seed oil, more preferably olive oil. The iodine value of the oil indicates the degree of unsaturation, the saponification value is related to the molecular weight of the oil, and the two values influence the oil to participate in the intracellular secondary metabolism of the poria cocos mycelium.
The following are examples:
example 1
Preparing poria cocos liquid strains:
(1) activating Poria cocos mycelium with PDA liquid culture medium at 28-30 deg.C and rotation speed of 120-.
(2) Inoculating 10% (v: v) of Poria cocos mycelium in (1) into Poria cocos seed culture medium, and culturing at 28-30 deg.C and rotation speed of 120-.
And (3) growth stage culture:
inoculating Poria cocos liquid strain (2) into CYM culture medium, wherein the inoculation amount of the seed liquid is 1%, culturing for 3 days at 28-30 ℃ and at the rotation speed of 120-.
And (3) an induction culture stage:
after the fermentation in the growth stage, 1% (v/v) of 6 edible vegetable oils, i.e., peanut oil, rapeseed oil, palm oil, soybean oil, olive oil, and coix seed oil, were added under aseptic conditions as treatment groups (inoculation amount 1%, fermentation for 6 days) and control (no oil, inoculation amount 1%, fermentation for 6 days). The second stage fermentation was continued with three replicates of each treatment. The iodine value of 6 vegetable oils is determined by the Welch method, and the saponification value of 6 plants is determined according to the determination of animal and vegetable oil saponification value (GB/T5534-2008).
After fermentation, collecting mycelium pellets by using a Buchner funnel, centrifugally settling the fermentation liquor for 3min at 3000r/min, separating thalli from the fermentation liquor, removing vegetable oil suspended on the upper layer, filtering by using four layers of medical gauze on the Buchner funnel, repeatedly washing by using deionized water for three times, centrifugally settling for 3min again, separating out mycelium by using the Buchner funnel again, repeatedly washing by using the deionized water until the mycelium is colorless, drying the obtained mycelium in a 60 ℃ oven to constant weight, weighing to obtain biomass, and measuring the content of the poria cocos triterpenes by using a spectrophotometry. Taking 0.05g of oven-dried mycelium powder, adding 5mL of 50% (v/v) ethanol, and ultrasonically extracting for 2h (repeating once), each time 5mL of 50% (v/v) ethanol. Centrifuging to obtain supernatant, mixing the two supernatants with total volume of 10mL, placing in rotary evaporator, and vacuum evaporating at 50 deg.C. The residue after evaporation was resuspended in 3mL of ddH2O and extracted twice with 5mL of chloroform; the chloroform layers were combined and dried in a total volume of 10mL under vacuum at 45 ℃ and then dissolved in 3mL of methanol. The detection result is shown in figure 1, the content is obviously increased compared with the comparative example, and the increase percentage of the tuckahoe triterpene content is shown in table 1.
TABLE 6 Effect of second stage addition of different oils optimized on Poria triterpene
Example 2
Preparing poria cocos liquid strains:
(1) activating Poria cocos mycelium with PDA liquid culture medium at 28-30 deg.C and rotation speed of 120-.
(2) Inoculating 10% (v: v) of Poria cocos mycelium in (1) into Poria cocos seed culture medium, and culturing at 28-30 deg.C and rotation speed of 120-.
And (3) growth stage culture:
inoculating Poria cocos liquid strain into CYM culture medium, wherein the inoculation amount of the seed liquid is 1%, culturing for 3 days at 28-30 ℃ and at the rotation speed of 120-.
And (3) culture in an induction stage:
after the growth stage fermentation is finished, respectively adding 1%, 2%, 3% and 4% of olive oil into the fermentation liquor under aseptic condition, and continuing to perform the second stage fermentation, wherein each treatment has three repeated parallel samples; the temperature is 28-30 ℃, the rotating speed is 120-150r/min, the mycelium density is increased after 6 days of culture, and the fermentation is finished.
After fermentation, collecting mycelium pellets by using a Buchner funnel, centrifugally settling the fermentation liquor for 3min at 3000r/min, separating thalli from the fermentation liquor, removing vegetable oil suspended on the upper layer, filtering by using four layers of medical gauze on the Buchner funnel, repeatedly washing by using deionized water for three times, centrifugally settling for 3min again, separating out mycelium by using the Buchner funnel again, repeatedly washing by using the deionized water until the mycelium is colorless, drying the obtained mycelium in a 60 ℃ oven to constant weight, weighing to obtain biomass, and measuring the content of the poria cocos triterpenes by using a spectrophotometry. Taking 0.05g of oven-dried mycelium powder, adding 5mL of 50% (v/v) ethanol, and ultrasonically extracting for 2h (repeating once), each time 5mL of 50% (v/v) ethanol. Centrifuging to obtain supernatant, mixing the two supernatants with total volume of 10mL, placing in rotary evaporator, and vacuum evaporating at 50 deg.C. Using 3mL ddH2O resuspending the residue after evaporation to dryness, and extracting twice with 5mL of chloroform; the chloroform layers were combined and dried in a total volume of 10mL under vacuum at 45 ℃ and then dissolved in 3mL of methanol. The results are shown in FIG. 2. The content is obviously increased compared with the comparative example, and the increase percentage of the tuckahoe triterpene content is shown in a table 2.
TABLE 2 Effect of different concentrations of Olive oil on Poria growth and Poria triterpene biosynthesis in liquid fermentation
| Olive oil concentration at induction stage% | Poria triterpene content mg/100ml | The content of the tuckahoe triterpenes is improved by |
| 0 | 18.03 | - |
| 1 | 34.06 | 88.85 |
| 2 | 42.01 | 132.95 |
| 3 | 41.58 | 130.55 |
| 4 | 31.28 | 73.48 |
Example 3
Preparing poria cocos liquid strains:
(1) activating Poria cocos mycelium with PDA liquid culture medium at 28-30 deg.C and rotation speed of 120-.
(2) Inoculating 10% (v: v) of Poria cocos mycelium in (1) into Poria cocos seed culture medium, and culturing at 28-30 deg.C and rotation speed of 120-.
And (3) growth stage culture:
inoculating Poria cocos liquid strain (2) into CYM culture medium, culturing for 3 days at the temperature of 28-30 ℃ and the rotation speed of 120-.
And (3) culture in an induction stage:
after the end of the growth phase fermentation, the liquid fermentation started under aseptic conditions at 0h, 24h, 48h, 72h, 96h as the time point for 2% olive oil addition. Continuing to perform the second stage fermentation, wherein each treatment comprises three repeated parallel samples; the temperature is 28-30 ℃, the rotating speed is 120-150r/min, the mycelium density is increased after 6 days of culture, and the fermentation is finished.
After fermentation, collecting mycelium pellets by using a Buchner funnel, centrifugally settling the fermentation liquor for 3min at 3000r/min, separating thalli from the fermentation liquor, removing vegetable oil suspended on the upper layer, filtering by using four layers of medical gauze on the Buchner funnel, repeatedly washing by using deionized water for three times, centrifugally settling for 3min again, separating out mycelium by using the Buchner funnel again, repeatedly washing by using the deionized water until the mycelium is colorless, drying the obtained mycelium in a 60 ℃ oven to constant weight, weighing to obtain biomass, and measuring the content of the poria cocos triterpenes by using a spectrophotometry. Taking 0.05g of oven-dried mycelium powder, adding 5mL of 50% (v/v) ethanol, and ultrasonically extracting for 2h (repeating once), each time 5mL of 50% (v/v) ethanol. Centrifuging to obtain supernatant, mixing the two supernatants with total volume of 10mL, placing in rotary evaporator, and vacuum evaporating at 50 deg.C. The residue after evaporation was resuspended in 3mL of ddH2O and extracted twice with 5mL of chloroform; the chloroform layers were combined and dried in a total volume of 10mL under vacuum at 45 ℃ and then dissolved in 3mL of methanol. The results are shown in FIG. 3. The content is obviously increased compared with the comparative example, and the increase percentage of the tuckahoe triterpene content is shown in a table 3.
TABLE 3 Effect of different Olive oil addition times on Poria triterpene biosynthesis in the second stage
| Adding oleum Olivarum for hr | Poria triterpene content mg/100mL | The content of the tuckahoe triterpenes is improved by percentage |
| - | 16.38 | - |
| 0 | 40.94 | 149.99 |
| 24 | 36.71 | 124.17 |
| 48 | 31.30 | 91.12 |
| 72 | 29.58 | 80.63 |
| 96 | 28.61 | 74.72 |
Example 4
Preparing poria cocos liquid strains:
(1) activating Poria cocos mycelium with PDA liquid culture medium at 28-30 deg.C and rotation speed of 120-.
(2) Inoculating 10% (v: v) of Poria cocos mycelium in (1) into Poria cocos seed culture medium, and culturing at 28-30 deg.C and rotation speed of 120-.
And (3) growth stage culture:
inoculating Poria cocos liquid strain (2) into CYM culture medium, culturing at 28-30 deg.C and rotation speed of 120-.
And (3) culture in an induction stage:
after fermentation in the growth stage is finished, two groups of treatments are set under the aseptic condition, wherein one group is added with 2 percent of olive oil, and the other group is added on the 0 th day of fermentation, and the inoculation amount of the seed liquid is 8 percent; one group was treated without oil as a control (i.e. no olive oil added, seed inoculum 8%). Setting 6 fermentation time gradients of 2, 4, 6, 8, 10 and 12 days. The second stage fermentation was continued with three replicates per treatment.
After fermentation, collecting mycelium pellets by using a Buchner funnel, centrifugally settling the fermentation liquor for 3min at 3000r/min, separating thalli from the fermentation liquor, removing vegetable oil suspended on the upper layer, filtering by using four layers of medical gauze on the Buchner funnel, repeatedly washing by using deionized water for three times, centrifugally settling for 3min again, separating out mycelium by using the Buchner funnel again, repeatedly washing by using the deionized water until the mycelium is colorless, drying the obtained mycelium in a 60 ℃ oven to constant weight, weighing to obtain biomass, and measuring the content of the poria cocos triterpenes by using a spectrophotometry. Taking 0.05g of oven-dried mycelium powder, adding 5mL of 50% (v/v) ethanol, and ultrasonically extracting for 2h (repeating once), each time 5mL of 50% (v/v) ethanol. Centrifuging to obtain supernatant, mixing the two supernatants with total volume of 10mL, placing in rotary evaporator, and vacuum evaporating at 50 deg.C. The residue after evaporation was resuspended in 3mL of ddH2O and extracted twice with 5mL of chloroform; the chloroform layers were combined and dried in a total volume of 10mL under vacuum at 45 ℃ and then dissolved in 3mL of methanol. The results are shown in FIG. 4. At the same inoculation amount of 8%, the content of the added olive oil is obviously increased compared with the comparative example, and the increase percentage of the tuckahoe triterpene content is shown in a table 4.
TABLE 4 optimization of the process of inducing increase in triterpene yield from Poria by liquid fermentation with olive oil addition in the second stage
Example 6
Preparing poria cocos liquid strains:
(1) activating Poria cocos mycelium with PDA liquid culture medium at 28-30 deg.C and rotation speed of 120-.
(2) Inoculating 10% (v: v) of Poria cocos mycelium in (1) into Poria cocos seed culture medium, and culturing at 28-30 deg.C and rotation speed of 120-.
And (3) growth stage culture:
inoculating Poria cocos liquid strain (2) into CYM culture medium, culturing at 28-30 deg.C and rotation speed of 120-.
And (3) an induction culture stage:
after the fermentation in the growth period is finished, the group is optimized under the aseptic condition (the addition time of the olive oil is 0h of the fermentation, the inoculation amount of the seed liquid is 8%, the fermentation time is 8 days), and the group is compared (the group is 1% without oil, and the fermentation time is 6 days). The second stage fermentation was continued with three replicates of each treatment.
After fermentation, collecting mycelium pellets by using a Buchner funnel, centrifugally settling the fermentation liquor for 3min at 3000r/min, separating thalli from the fermentation liquor, removing vegetable oil suspended on the upper layer, filtering by using four layers of medical gauze on the Buchner funnel, repeatedly washing by using deionized water for three times, centrifugally settling for 3min again, separating out mycelium by using the Buchner funnel again, repeatedly washing by using the deionized water until the mycelium is colorless, drying the obtained mycelium in a 60 ℃ oven to constant weight, weighing to obtain biomass, and measuring the content of the poria cocos triterpenes by using a spectrophotometry. Taking 0.05g of oven-dried mycelium powder, adding 5mL of 50% (v/v) ethanol, and ultrasonically extracting for 2h (repeating once), each time 5mL of 50% (v/v) ethanol. Centrifuging to obtain supernatant, mixing the two supernatants with total volume of 10mL, placing in rotary evaporator, and vacuum evaporating at 50 deg.C. The residue after evaporation was resuspended in 3mL of ddH2O and extracted twice with 5mL of chloroform; the chloroform layers were combined and dried in a total volume of 10mL under vacuum at 45 ℃ and then dissolved in 3mL of methanol. The content is obviously increased compared with the comparative example, and the increase percentage of the tuckahoe triterpene content is shown in a table 5.
TABLE 5 Effect of second stage olive oil addition optimization on Poria triterpene
Comparative example 4
Preparing poria cocos liquid strains:
(1) activating Poria cocos mycelium with PDA liquid culture medium at 28-30 deg.C and rotation speed of 120-.
(2) Inoculating 10% (v: v) of Poria cocos mycelium in (1) into Poria cocos seed culture medium, and culturing at 28-30 deg.C and rotation speed of 120-.
And (3) growth and culture stage:
inoculating Poria cocos liquid strain (2) into a CYM culture medium, determining the optimal inoculation amount in a fermentation culture medium (without oil), and setting 6 seed liquid inoculation amount gradients of 1%, 2%, 4%, 6%, 8% and 10% (v/v). The second stage fermentation was continued with three replicates per treatment. Culturing at 28-30 deg.C and rotation speed of 120-. Continuing to perform the second stage fermentation at 28-30 deg.C and rotation speed of 120-.
After fermentation, collecting mycelium pellets by using a Buchner funnel, centrifugally settling the fermentation liquor for 3min at 3000r/min, separating thalli from the fermentation liquor, filtering the mycelium pellets by using four layers of medical gauze on the Buchner funnel, repeatedly washing the mycelium pellets for three times by using deionized water, centrifugally settling for 3min once again, separating the mycelium pellets by using the Buchner funnel again, repeatedly washing the mycelium pellets by using the deionized water until the mycelium pellets are colorless, drying the mycelium pellets in an oven at 60 ℃ until the mycelium pellets are constant in weight, weighing the mycelium pellets to obtain biomass, and measuring the triterpene content of the poria cocos by using a spectrophotometry. Taking 0.05g of oven-dried mycelium powder, adding 5mL of 50% (v/v) ethanol, and ultrasonically extracting for 2h (repeating once), each time 5mL of 50% (v/v) ethanol. Centrifuging to obtain supernatant, mixing the two supernatants with total volume of 10mL, placing in rotary evaporator, and vacuum evaporating at 50 deg.C. The residue after evaporation was resuspended in 3mL ddh2O and extracted twice with 5mL chloroform; the chloroform layers were combined and dried in a total volume of 10mL under vacuum at 45 ℃ and then dissolved in 3mL of methanol. The results are shown in FIG. 5. The increase was most pronounced at 8% inoculum size, with the percentage increase in poria triterpene content shown in table 6.
TABLE 6 Effect of inoculum size on Poria triterpene
| The second stage bacterial liquid inoculation amount% | Poria triterpene content mg/ |
| 1 | 18.03 |
| 2 | 17.65 |
| 4 | 20.34 |
| 6 | 22.47 |
| 8 | 24.12 |
| 10 | 22.34 |
As can be seen from Table 6, the amount of tuckahoe triterpene increased with increasing inoculum size, but even with high inoculation (8% inoculum size), the amount of tuckahoe triterpene remained at a low level. In the embodiment of adding the inducer, even if the inoculation amount is only 1 percent, the triterpene content of the tuckahoe is still obviously improved.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. An application of edible vegetable oil is characterized in that the edible vegetable oil is applied to increase the content of tuckahoe triterpenes in tuckahoe mycelia cells.
2. The use of an edible vegetable oil according to claim 1, for increasing the intracellular poria triterpene content of liquid fermentation of poria cocos mycelium.
3. Use of an edible vegetable oil according to any of claims 1 or 2, wherein the edible vegetable oil has an iodine value of above 50g/100g, preferably between 50 and 110g/100 g; the saponification value of the edible vegetable oil is more than 180mgKOH/g, preferably 180 to 220 mgKOH/g; the edible vegetable oil is preferably olive oil, peanut oil, rapeseed oil, palm oil, soybean oil, and/or coix seed oil, more preferably olive oil.
4. Use of an edible vegetable oil according to any of claims 1 to 3, wherein the liquid fermentation of Poria cocos mycelium comprises a growth phase and an induction phase;
in the growth stage, the poria cocos mycelia fully grow until the poria cocos mycelia are distributed in a flocculent shape;
in the induction stage, edible vegetable oil is added into the fermentation liquor to induce the poria cocos mycelium to improve the content of the intracellular poria cocos triterpenes.
5. The use of an edible vegetable oil according to claim 4, wherein the induction phase is added at a volume ratio of 1% to 4% edible vegetable oil.
6. The use of an edible vegetable oil as claimed in claim 4, wherein the edible vegetable oil is added between the start of the induction phase and 96 hours, the fermentation temperature is 28-30 ℃, the rotation speed is 120-150r/min, until the mycelium density is increased, and the fermentation is ended before the mycelium is browned.
7. The use of the edible vegetable oil as claimed in claim 4, wherein the liquid Poria cocos strain is inoculated in the culture medium during the growth stage, and cultured at a temperature of 28-30 ℃ and a rotation speed of 120-150r/min for 5-6 days.
8. The use of an edible vegetable oil according to claim 4, wherein the growth phase is inoculated with a liquid strain of Poria cocos in the culture medium in an amount of 4% to 8% by volume of the culture medium.
9. The use of edible vegetable oil according to claim 8, wherein said liquid Poria cocos strain is prepared by the following method:
(1) activating the tuckahoe mycelium by using a PDA liquid culture medium at the temperature of 28-30 ℃ and the rotating speed of 120-150r/min for 6-7 days to obtain the activated tuckahoe mycelium;
(2) inoculating the activated tuckahoe mycelium obtained in the step (1) into a tuckahoe seed culture medium according to 10 percent of the culture volume of tuckahoe seeds, and culturing for 5 to 6 days at the temperature of 28 to 30 ℃ and the rotating speed of 120-150r/min to obtain tuckahoe liquid strains.
10. The use of edible vegetable oil according to claim 9, wherein the poria cocos seed culture medium is a PDA culture medium prepared as follows: cutting 200 g of potato into small pieces, boiling for 20-30 min, filtering eight layers of gauze, adding 20 g of glucose into the filtrate, fixing the volume to 1L, and sterilizing for 20-25 min at 121 ℃ by high-pressure steam.
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