CN114854662A - Method for increasing content of ganoderma lucidum low-polarity ganoderma lucidum triterpenes - Google Patents
Method for increasing content of ganoderma lucidum low-polarity ganoderma lucidum triterpenes Download PDFInfo
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Abstract
The invention discloses a method for improving the content of ganoderma lucidum triterpene with low polarity, which is to add citric acid into a ganoderma lucidum liquid fermentation culture medium for treatment to improve the content of ganoderma lucidum triterpene compounds with low polarity in ganoderma lucidum. The addition of a proper amount of citric acid into the ganoderma lucidum liquid fermentation medium can increase the total ganoderma lucidum triterpene content in the wild type strain by about 24.68%. And the proportion of the low-polarity ganoderma triterpene compound content in the total ganoderma triterpene content after 42min in the strain is also effectively improved. When the treatment concentration of citric acid is 400mM and the treatment time is 48h, in the wild type, HPLC results show that the proportion of the low-polarity ganoderma triterpene compounds in the total ganoderma triterpene compounds after 42min is increased, and the proportion of the WT low-polarity ganoderma triterpene compounds is increased by 13.78%. The method has obvious effect of improving the content of the ganoderma lucidum low-polarity ganoderma lucidum triterpene.
Description
Technical Field
The invention belongs to the technical field of fungus cultivation, and relates to a method for improving the content of low-polarity ganoderma lucidum triterpene compounds in ganoderma lucidum, in particular to application of citric acid in improving the content of low-polarity ganoderma lucidum triterpene compounds in total ganoderma lucidum triterpene of a ganoderma lucidum strain.
Relevant background
Ganoderma lucidum (Ganoderma lucidum) P.Karst) is a traditional medicinal fungus in China, and can synthesize a large amount of bioactive compounds, such as triterpene, polysaccharide, nucleotide, sterol steroid, etc. Wherein, ganoderma triterpene (GA) substances play an important role in the pharmacological aspect of ganoderma, so the content of the substances is one of the important indexes for measuring the quality of ganoderma. Research shows that various ganoderma triterpene compounds have important medicinal effects of resisting HIV virus, resisting tumor, protecting liver, expelling toxin, reducing cholesterol content in vivo and the like, and are natural organic compounds with multiple effects. Therefore, increasing the yield of ganoderma triterpene compounds will further increase the medicinal potential and commercial value of ganoderma. Meanwhile, the ganoderma triterpenoids with different polarities or types have different functions, and the ganoderma triterpenoids with corresponding functions obtained according to the requirements can further reduce the culture cost and improve the product value. The existing research shows that the low-polarity ganoderma triterpene compound monomers such as ganodermanone A, ganodermanol F and the like have obvious antitumor activity in vitro. In the inhibition effect on the proliferation of tumor cells SW620 and L1210, the low-polarity triterpene part is found to be similar to the IC50 of the total triterpene, which indicates that the low-polarity triterpene compound has remarkable in vitro anti-tumor and anti-inflammatory activities. In addition, the lower polarity of ganoderic acid F, ganoderic alcohol B and ganoderic acid DM also have a better inhibition effect on the respiratory burst of macrophage RAW 264.7. Therefore, the method has obvious economic value for improving the content of the low-polarity ganoderma triterpene.
The invention provides a method for improving the content of low-polarity ganoderma triterpene compounds in ganoderma lucidum, and aims to solve the technical problem.
Disclosure of Invention
The invention aims to provide application of citric acid in improving the content of low-polarity ganoderma triterpene compounds in ganoderma lucidum. The influence of citric acid on the content of different types of ganoderma triterpene is disclosed.
The invention also aims to provide a method for improving the content of the low-polarity ganoderma lucidum triterpene compound in the ganoderma lucidum.
In order to realize the purpose of the invention, the technical scheme is as follows:
application of citric acid in improving the content of low-polarity ganoderma triterpene compounds in ganoderma lucidum is provided. As a preferred embodiment: adding citric acid into Ganoderma liquid fermentation culture medium for treatment to increase the content of low-polarity Ganoderma triterpene compounds in Ganoderma total Ganoderma triterpene.
Further preferred is: the addition concentration of the citric acid in the ganoderma lucidum liquid fermentation culture medium is as follows: 400-800 mM; most preferably: 400 mM.
As a preferred embodiment: the treatment time was 48 h.
The ganoderma lucidum liquid fermentation culture medium is a CYM liquid culture medium.
A method for increasing the content of low-polarity Ganoderma triterpene compounds in Ganoderma comprises adding citric acid into Ganoderma liquid fermentation culture medium for treatment to increase the content of low-polarity Ganoderma triterpene compounds in Ganoderma.
In the method, the addition amount of the citric acid in the ganoderma lucidum liquid fermentation culture medium is as follows: 400-800 mM; preferably: 400 mM. The treatment time was 48 h.
In the method, citric acid is prepared into a citric acid solution, and then the citric acid solution is added into the ganoderma lucidum liquid fermentation culture medium after being sterilized by a high-pressure steam sterilization method.
The operation process of the method specifically comprises the following steps:
(1) activating strains: inoculating Ganoderma strain stored in PDA glycerol seed-preserving tube into CYM solid culture medium, culturing and activating in 28 deg.C incubator, transferring with puncher when mycelium grows to certain size, and transferring twice.
(2) Preparing a seed solution: and punching 6 small holes on the activated ganoderma lucidum by using a puncher, inoculating the ganoderma lucidum into a CYM liquid culture medium, and culturing in a shaking table at the speed of 150rpm and the temperature of 28 ℃.
(3) Fermentation treatment: crushing the seed liquid, inoculating the crushed seed liquid into a CYM liquid culture medium for fermentation, and adding a citric acid solution for treatment.
According to the weight percentage, the formula of the CYM liquid culture medium is as follows: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO 4 ·7H 2 O 0.05％、KH 2 PO 4 0.46 percent, and the rest water is less than 100 percent.
Activating strain, culturing in 28 deg.C incubator for 14-20d, culturing seed liquid in 28 deg.C shaking table for 7d, fermenting, culturing in 28 deg.C shaking table for 7d, and adding citric acid solution for 48 hr.
The invention has the beneficial effects that:
the content of ganoderma triterpene is one of important indexes for measuring the quality of ganoderma, and the content of ganoderma triterpene of different species can be influenced by adding citric acid into a liquid culture medium of ganoderma strains. When the treatment concentration of the citric acid is 400mM and the treatment time is 48h, the total content of the ganoderma triterpene is improved to a certain degree. The ganoderma triterpene is not only a substance, but also comprises various different types of bioactive substances, and the content and the type of the substances of the triterpene are different from those of the substances of the triterpene in an untreated group by analyzing the content and the type of the citric acid treatment by an HPLC method, so that the result shows that the total triterpene content in the wild ganoderma species is improved by about 24.68 percent, and the content of high-polarity and medium-polarity ganoderma acid represented by the peak area before 42min in the HPLC result is improved by about 11.12 percent, and the difference is not obvious. However, the peak area of the low-polarity ganoderma triterpene compound content accounting for the total ganoderma triterpene compound content after 42min in the HPLC process is increased remarkably, and the increase amplitude is about 13.78%.
Description of the drawings:
FIG. 1 shows the total triterpene content of Ganoderma treated with citric acid at different concentrations.
FIG. 2 shows total ganoderic triterpene content of citric acid treated and untreated ganoderma lucidum.
FIG. 3 shows fingerprints of citric acid-treated and untreated ganoderma triterpene.
FIG. 4 shows the ratio of the peak area of the high-polarity ganoderma triterpene to the total peak area.
FIG. 5 shows the ratio of the peak area of low polarity ganoderma triterpene to the total peak area.
The specific implementation mode is as follows:
the present invention is further illustrated in detail below with reference to examples:
example 1
(1) Activating strains: inoculating Ganoderma strain (strain number: ACCC53264) stored in glycerol PDA seed-preserving tube into CYM solid culture medium, activating in 28 deg.C incubator, punching hole on the edge when bacterial colony grows over the whole plate, transferring to CYM solid culture medium, and co-transferring twice.
(2) Preparing a seed solution: punching 6 small holes with diameter of 5mm on the activated Ganoderma strain colony edge with a puncher, inoculating into CYM liquid culture medium with inoculating needle, and culturing in shaker at 28 deg.C and rotation speed of 150r/min to prepare seed solution, and culturing for 7 days.
(3) Fermentation treatment: crushing the fermented ganoderma lucidum hypha seed liquid by using a crusher, then inoculating the crushed ganoderma lucidum hypha seed liquid into 100mL of CYM liquid culture medium, inoculating 5% of the crushed ganoderma lucidum hypha seed liquid into each bottle according to the volume percentage content, and fermenting in a shaking table at the temperature of 28 ℃ and the rotating speed of 150 r/min.
(4) After fermenting for 5 days, adding 400mM, 800mM and 1200mM citric acid (the citric acid is prepared into a mother solution with 5mol/L and is added according to a proportion) respectively for treatment, and then continuing fermentation culture for 48 hours; the same volume of sterile water was added as a control.
(5) Separating and collecting mycelium and extracellular filtrate of treated and control groups by a filter screen, drying the mycelium and grinding the mycelium into powder.
(6) And (3) measuring the content of the triterpene: accurately weighing 0.03g of mycelium dry powder, adding 1.5mL of 95% ethanol, carrying out ultrasonic crushing for 2h, mixing uniformly or intermittently, centrifuging (4000rpm, 10min), sucking 100 mu L of supernatant, adding 200 mu L of vanillin and 500 mu L of perchloric acid, mixing uniformly in a water bath (60 ℃, 20min), cooling for 10min, and detecting absorbance when the lambda is 550nm for a dot plate (32 mu L of sample per well and 200 mu L of glacial acetic acid).
Calculating the total triterpene content according to the OD value, wherein the calculation formula is as follows:
wherein x is the average OD value.
According to the weight percentage, the formula of the CYM liquid culture medium is as follows: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO 4 ·7H 2 O 0.05％、KH 2 PO 4 0.46 percent, and the balance of water to make up 100 percent.
The PDA solid culture medium comprises the following components in percentage by weight: 1L of culture medium contains 200g of peeled potato, 20g of glucose and 20g of agar; the preparation thereof is well known to those skilled in the art.
FIG. 1 shows the total triterpene content of Ganoderma treated with citric acid at different concentrations, and the results show that the total triterpene content of Ganoderma treated with citric acid is significantly influenced, and the total triterpene content of Ganoderma treated with citric acid at 400mM is highest.
Example 2
(1) Activating strains: inoculating Ganoderma strain (strain number: ACCC53264) stored in glycerol PDA seed-preserving tube into CYM solid culture medium, activating in 28 deg.C incubator, punching hole on the edge when bacterial colony grows over the whole plate, transferring to CYM solid culture medium, and co-transferring twice.
(2) Preparing a seed solution: punching 6 small holes with diameter of 5mm on the activated Ganoderma strain colony edge with a puncher, inoculating into CYM liquid culture medium with inoculating needle, and culturing in shaker at 28 deg.C and rotation speed of 150r/min to prepare seed solution, and culturing for 7 days.
(3) Fermentation treatment: crushing the fermented ganoderma lucidum hypha seed liquid by using a crusher, then inoculating the crushed ganoderma lucidum hypha seed liquid into 100mL of CYM liquid culture medium, inoculating 5% of the crushed ganoderma lucidum hypha seed liquid into each bottle according to the volume percentage content, and fermenting in a shaking table at the temperature of 28 ℃ and the rotating speed of 150 r/min.
(4) After fermenting for 5 days, adding 400mM citric acid for treatment, and continuing to ferment and culture for 48 h; the same volume of sterile water was added as a control.
(5) Separating and collecting mycelium and extracellular filtrate of treated and control groups by a filter screen, drying the mycelium and grinding the mycelium into powder.
(6) And (3) measuring the content of the triterpene: accurately weighing 0.03g of mycelium dry powder, adding 1.5mL of 95% ethanol, carrying out ultrasonic crushing for 2h, mixing uniformly or intermittently, centrifuging (4000rpm, 10min), sucking 100 mu L of supernatant, adding 200 mu L of vanillin and 500 mu L of perchloric acid, mixing uniformly in a water bath (60 ℃, 20min), cooling for 10min, and detecting absorbance when the lambda is 550nm for a dot plate (32 mu L of sample per well and 200 mu L of glacial acetic acid).
Calculating the total triterpene content according to the OD value, wherein the calculation formula is as follows:
wherein x is the average OD value.
The formulation of the medium was the same as in example 1.
According to the experimental result of the embodiment 1, the citric acid treatment concentration with the best effect is selected for treatment and the effect is verified, and the verification result is shown in fig. 2.
Example 3: analysis of triterpene species and content by HPLC
The experimental procedures of steps (1) to (5) were the same as in example 2.
(6) Taking 0.1g of mycelium dry powder to a 10mL centrifuge tube, adding 2mL of ethyl acetate to carry out water bath ultrasound for 2h, centrifuging (4000g, 10min), taking supernatant, drying, adding 600 μ L of methanol, shaking for dissolution, and filtering through a membrane (0.22 μm).
(7) A chromatographic column: shim-pack GIST C185 μm (4.6 I.D.. times.250 mm), flow rate 1mL/min, column temperature 30 ℃, detector wavelength 252nm, sample size 5 μ L. Gradient elution was performed with acetonitrile (B) and an aqueous solution (a) containing acetic acid (0.01%), the volume ratio of water to acetic acid in a being 100: 0.01, gradient elution procedure B pump: 20-26.5%, 8 min; 2.5-65% for 12 min; 65-75% for 10 min; 75-100% for 20 min; gradient elution is carried out for 100%, 16min, 100-20%, 2min, 20%, 4min for 72 min.
(8) The data analysis is carried out by using traditional Chinese medicine chromatogram fingerprint similarity evaluation system software (2012 edition) released by the national pharmacopoeia committee, and an averaging method is adopted in a comparison map generation mode. And performing variance analysis and multiple comparisons on the peak area data to express the content of the ganoderic acid with different polarities.
The formulation of the medium was the same as in example 1.
The fingerprint of citric acid-treated and untreated ganoderma triterpene is shown in figure 3. The ratio of the peak area of the high-polarity Ganoderma triterpene to the total peak area is shown in FIG. 4. The ratio of the peak area of the low-polarity Ganoderma triterpene to the total peak area is shown in FIG. 5.
The experimental result shows that when the citric acid treatment concentration is 400mM and the treatment time is 48h, the total content of ganoderma triterpene is improved to a certain extent. The ganoderma triterpene is not only a substance, but also comprises various different types of bioactive substances, and the content and the type of the substances of the triterpene are different from those of the substances of the triterpene in an untreated group by analyzing the content and the type of the citric acid treatment by an HPLC method, so that the result shows that the total triterpene content in the wild ganoderma species is improved by about 24.68 percent, and the content of high-polarity and medium-polarity ganoderma acid represented by the peak area before 42min in the HPLC result is improved by about 11.12 percent, and the difference is not obvious. However, the peak area of the low-polarity ganoderma triterpene compound content accounting for the total ganoderma triterpene compound content after 42min in the HPLC process is increased remarkably, and the increase amplitude is about 13.78%.
Claims (8)
1. Application of citric acid in improving the content of low-polarity ganoderma triterpene compounds in ganoderma lucidum is provided.
2. Use according to claim 1, characterized in that: adding citric acid into Ganoderma liquid fermentation culture medium for treatment to increase the content of low-polarity Ganoderma triterpene compounds in Ganoderma total Ganoderma triterpene.
3. Use according to claim 2, characterized in that: the addition concentration of the citric acid in the ganoderma lucidum liquid fermentation culture medium is as follows: 400-800 mM; preferably: 400 mM.
4. Use according to claim 2, characterized in that the time of the treatment is 48 h.
5. A method for improving the content of low-polarity ganoderma triterpene compounds in ganoderma lucidum is characterized by comprising the following steps: adding citric acid into Ganoderma liquid fermentation culture medium for treatment to increase the content of low-polarity Ganoderma triterpene compounds in Ganoderma.
6. The method according to claim 5, wherein the citric acid is added in the ganoderma lucidum liquid fermentation medium in an amount of 400 mM-800 mM, preferably: 400 mM.
7. The method of claim 5, wherein the processing time is 48 hours.
8. The method according to claim 5, wherein the citric acid is formulated into a citric acid solution, and then added to the ganoderma lucidum liquid fermentation medium after being sterilized by autoclaving.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102488719A (en) * | 2011-12-25 | 2012-06-13 | 南京农业大学 | Method for improving triterpene output of Ganoderma lucidum liquid fermented mycelia |
| CN104017852A (en) * | 2014-05-30 | 2014-09-03 | 上海市农业科学院 | Method for improving content of ganoderma triterpenes in ganoderma liquid deep fermentation mycelium |
| CN105106250A (en) * | 2015-07-17 | 2015-12-02 | 成都大学 | Mythic Fungus total triterpene composition and preparation methods thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102488719A (en) * | 2011-12-25 | 2012-06-13 | 南京农业大学 | Method for improving triterpene output of Ganoderma lucidum liquid fermented mycelia |
| CN104017852A (en) * | 2014-05-30 | 2014-09-03 | 上海市农业科学院 | Method for improving content of ganoderma triterpenes in ganoderma liquid deep fermentation mycelium |
| CN105106250A (en) * | 2015-07-17 | 2015-12-02 | 成都大学 | Mythic Fungus total triterpene composition and preparation methods thereof |
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| Title |
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| SHIZHONG ZHENG 等: "Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities", 《PLOS ONE》 * |
| 朱静 等: "灵芝三萜生物合成的研究进展", 《南京农业大学学报》 * |
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