CN114854662B - Method for improving low-polarity ganoderma lucidum triterpene content of ganoderma lucidum - Google Patents
Method for improving low-polarity ganoderma lucidum triterpene content of ganoderma lucidum Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
Abstract
The invention discloses a method for improving the content of low-polarity ganoderma lucidum triterpene in ganoderma lucidum. The addition of a proper amount of citric acid to the ganoderma lucidum liquid fermentation medium can increase the total ganoderma lucidum triterpene content in the wild type strain by about 24.68 percent. And the ratio of the low-polarity ganoderma lucidum triterpene compound content to the total ganoderma lucidum triterpene content in the strain after 42min is also effectively improved. When the citric acid treatment concentration is 400mM and the treatment time is 48 hours, in the wild type, the HPLC result shows that the proportion of the low-polarity ganoderma lucidum triterpene after 42 minutes to the total ganoderma lucidum triterpene is improved, and the proportion of the WT low-polarity ganoderma lucidum triterpene compound is improved by 13.78 percent. The method has remarkable effect of improving the content of the low-polarity ganoderma lucidum triterpenes.
Description
Technical Field
The invention belongs to the technical field of fungus cultivation, relates to a method for improving the content of low-polarity ganoderma lucidum triterpene compounds in ganoderma lucidum, and particularly relates to application of citric acid in improving the content of low-polarity ganoderma lucidum triterpene compounds in ganoderma lucidum strain total ganoderma lucidum triterpene compounds.
Related background
Ganoderma lucidum (Ganoderma lucidum (Curtis) P.Karst) is a traditional medicinal fungus in China, and can synthesize a large amount of bioactive compounds such as triterpenes, polysaccharides, nucleotides, sterol steroids and the like. Wherein, the ganoderma triterpene (GA) substance plays an important role in the pharmacology of ganoderma lucidum, so the content thereof is one of important indexes for measuring the quality of ganoderma lucidum. Researches show that various ganoderma lucidum triterpene compounds have important medicinal effects of resisting HIV virus, resisting tumor, protecting liver, expelling toxin, reducing cholesterol content in a body and the like, and are natural organic compounds with multiple effects. Thus, increasing the yield of ganoderma triterpene compounds would further increase the pharmaceutical potential and commercial value of ganoderma lucidum. Meanwhile, the ganoderma lucidum triterpenes with different polarities or types have different effects, and the ganoderma lucidum triterpenes with corresponding functions can be obtained according to requirements, so that the culture cost can be further reduced, and the product value can be improved. Studies have shown that low polarity ganoderic compounds such as ganoderic ketone a, ganoderic alcohol a and ganoderic alcohol F have significant anti-tumor activity in vitro. In the inhibition of proliferation of tumor cells SW620 and L1210, the low polarity triterpene moiety was found to be similar to the IC50 of total triterpenes, indicating that the low polarity triterpene compounds have significant anti-tumor and anti-inflammatory activity in vitro. In addition, ganoderic acid F, ganoderic alcohol B and ganoderic acid DM with low polarity also have good inhibition effect on respiratory burst of macrophage RAW 264.7. Therefore, the method has remarkable economic value for improving the triterpene content of the low-polarity ganoderma lucidum.
The invention provides a method for improving the content of low-polarity ganoderma lucidum triterpene compounds in ganoderma lucidum, which aims to solve the technical problem.
Disclosure of Invention
The invention aims to provide an application of citric acid in improving the content of low-polarity ganoderma lucidum triterpene compounds in ganoderma lucidum. The effect of citric acid on the triterpene content of different types of ganoderma lucidum is disclosed.
Another object of the present invention is to provide a method for increasing the content of low-polarity ganoderma lucidum triterpene compounds in ganoderma lucidum.
In order to achieve the aim of the invention, the technical scheme is as follows:
the application of citric acid in improving the content of low-polarity Ganoderma triterpene compounds in Ganoderma is provided. As a preferred embodiment: adding citric acid into Ganoderma liquid fermentation medium for processing 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 medium is as follows: 400-800 mM; most preferably: 400mM.
As a preferred embodiment: the treatment time was 48 hours.
The ganoderma lucidum liquid fermentation medium is CYM liquid medium.
A method for increasing the content of low-polarity Ganoderma triterpene compounds in Ganoderma comprises adding citric acid into Ganoderma liquid fermentation medium for processing 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 medium is as follows: 400-800 mM; preferably, it is: 400mM. The treatment time was 48 hours.
In the method, the citric acid is prepared into a citric acid solution, and then the citric acid solution is sterilized by a high-pressure steam sterilization method and added into the ganoderma lucidum liquid fermentation medium.
The operation process of the method specifically comprises the following steps:
(1) Activating strains: inoculating Ganoderma strain stored in PDA glycerol seed holding tube into CYM solid culture medium, culturing and activating in 28 deg.C incubator, transferring with puncher when mycelium grows to a certain size, and transferring twice.
(2) Seed liquid preparation: and (3) punching 6 small holes on the activated ganoderma lucidum by using a puncher, inoculating the ganoderma lucidum into a CYM liquid culture medium, and placing the ganoderma lucidum into a shaking table at 150rpm and 28 ℃ for culture.
(3) Fermentation treatment: pulverizing the seed liquid, inoculating into CYM liquid culture medium, fermenting, and adding citric acid solution for treatment.
The formula of the CYM liquid culture medium comprises the following components in percentage by weight: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, mgSO 4 ·7H 2 O 0.05％、KH 2 PO 4 0.46% and the rest of water is less than 100%.
The strain is activated and cultured in a 28 ℃ incubator for 14-20d, the seed solution is prepared and cultured in a 28 ℃ shaking table for 7d, the fermentation treatment is performed and cultured in the 28 ℃ shaking table for 7d, and finally the citric acid solution is added for 48h for treatment.
The invention has the beneficial effects that:
the content of the triterpene of the ganoderma lucidum is one of important indexes for measuring the quality of the ganoderma lucidum, and the content of the triterpene of the ganoderma lucidum with different polarities can be influenced by adding citric acid into a liquid culture medium of ganoderma lucidum strains. When the citric acid treatment concentration is 400mM and the treatment time is 48 hours, the total content of the ganoderma lucidum triterpenes is improved to a certain extent. The ganoderma lucidum triterpene is not only a substance, but also contains a plurality of different bioactive substances, the content and the type of the triterpene substances in the citric acid treatment group and the untreated group are different through an HPLC method, the result shows that the total triterpene content in the wild ganoderma lucidum strain is improved by about 24.68 percent, the content of the high-polarity ganoderic acid and the medium-polarity ganoderic acid which are expressed by peak areas before 42min in the HPLC result is improved by about 11.12 percent, and the difference is not obvious. But the peak area of the low-polarity ganoderma lucidum triterpene compound content accounting for the total ganoderma lucidum triterpene compound content in the HPLC process after 42min is obviously increased, and the increase range is about 13.78 percent.
Description of the drawings:
FIG. 1 shows the total triterpene content of Ganoderma lucidum treated with citric acid at different concentrations.
FIG. 2 shows the total triterpene content of the citric acid treated and untreated ganoderma lucidum.
FIG. 3 shows the fingerprint of citric acid treated and untreated Ganoderma lucidum triterpenes.
FIG. 4 shows the ratio of the peak area to the total peak area of the high polarity ganoderma lucidum triterpene.
FIG. 5 shows the ratio of the area of the low polarity ganoderma lucidum triterpene peak to the total peak area.
The specific embodiment is as follows:
the invention is further illustrated by the following examples:
example 1
(1) Activating strains: inoculating Ganoderma strain (strain number: ACCC 53264) stored in glycerol PDA seed-retaining tube into CYM solid culture medium, activating in 28 deg.C incubator, and inoculating to CYM solid culture medium twice with hole puncher when colony grows over whole plate.
(2) Seed liquid preparation: 6 small holes with the diameter of 5mm are punched on the edge of the colony of the activated ganoderma lucidum strain, inoculated into a CYM liquid culture medium by an inoculating needle, and cultured in a shaking table at the temperature of 28 ℃ and the rotating speed of 150r/min to prepare seed liquid, and the seed liquid is cultured for 7 days.
(3) Fermentation treatment: crushing the fermented ganoderma lucidum mycelium seed liquid by a crusher, then inoculating into 100mL of CYM liquid culture medium, inoculating 5% of the liquid into each bottle according to the volume percentage, and putting into a shaking table at the temperature of 28 ℃ and the rotating speed of 150r/min for fermentation.
(4) After fermenting for 5 days, 400mM, 800mM and 1200mM of citric acid (the mother solution of which the citric acid is prepared into 5mol/L is added in proportion) are respectively added for treatment, and then fermentation culture is continued for 48 hours; the same volume of sterile water was added as a control.
(5) Separating and collecting treated mycelium and extracellular filtrate of control group by filter screen, oven drying mycelium, and grinding into powder.
(6) Triterpene content determination: accurately weighing 0.03g of mycelium dry powder, adding 1.5mL of 95% ethanol, carrying out ultrasonic crushing for 2 hours, occasionally mixing, sucking 100 mu L of supernatant by centrifugation (4000 rpm,10 min), adding 200 mu L of vanillin and 500 mu L of perchloric acid, mixing in a water bath (60 ℃ C., 20 min), cooling a 10-min spot plate (32 mu L of sample per hole+200 mu L of glacial acetic acid) and detecting absorbance at lambda=550 nm.
And calculating the total triterpene content according to the OD value, wherein the calculation formula is as follows:
where x is the average OD value.
The CY is calculated by weight percentThe formula of the M 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%, the balance being water to make up 100%.
The formula of the PDA solid culture medium is as follows: 1L of culture medium contains peeled potato 200g, glucose 20g and agar 20g; the preparation methods are well known to those skilled in the art.
FIG. 1 shows that the total ganoderma lucidum triterpene content is treated by citric acid with different concentrations, and the result shows that the treatment concentration of the citric acid significantly influences the total ganoderma lucidum triterpene content, and the total ganoderma lucidum triterpene content is highest when the total ganoderma lucidum triterpene content is treated at 400mM.
Example 2
(1) Activating strains: inoculating Ganoderma strain (strain number: ACCC 53264) stored in glycerol PDA seed-retaining tube into CYM solid culture medium, activating in 28 deg.C incubator, and inoculating to CYM solid culture medium twice with hole puncher when colony grows over whole plate.
(2) Seed liquid preparation: 6 small holes with the diameter of 5mm are punched on the edge of the colony of the activated ganoderma lucidum strain, inoculated into a CYM liquid culture medium by an inoculating needle, and cultured in a shaking table at the temperature of 28 ℃ and the rotating speed of 150r/min to prepare seed liquid, and the seed liquid is cultured for 7 days.
(3) Fermentation treatment: crushing the fermented ganoderma lucidum mycelium seed liquid by a crusher, then inoculating into 100mL of CYM liquid culture medium, inoculating 5% of the liquid into each bottle according to the volume percentage, and putting into a shaking table at the temperature of 28 ℃ and the rotating speed of 150r/min for fermentation.
(4) Fermenting for 5 days, adding 400mM citric acid, and continuing fermentation culture for 48 hours; the same volume of sterile water was added as a control.
(5) Separating and collecting treated mycelium and extracellular filtrate of control group by filter screen, oven drying mycelium, and grinding into powder.
(6) Triterpene content determination: accurately weighing 0.03g of mycelium dry powder, adding 1.5mL of 95% ethanol, carrying out ultrasonic crushing for 2 hours, occasionally mixing, sucking 100 mu L of supernatant by centrifugation (4000 rpm,10 min), adding 200 mu L of vanillin and 500 mu L of perchloric acid, mixing in a water bath (60 ℃ C., 20 min), cooling a 10-min spot plate (32 mu L of sample per hole+200 mu L of glacial acetic acid) and detecting absorbance at lambda=550 nm.
And calculating the total triterpene content according to the OD value, wherein the calculation formula is as follows:
where x is the average OD value.
The formulation of the medium was the same as in example 1.
According to the experimental result of example 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: HPLC analysis of triterpene type and content
The experimental procedures of steps (1) to (5) were the same as in example 2.
(6) Taking mycelium dry powder 0.1g into a 10mL centrifuge tube, adding 2mL ethyl acetate into the centrifuge tube, carrying out water bath ultrasonic treatment for 2h, centrifuging (4000 g,10 min), taking supernatant, drying, adding 600 mu L of methanol, shaking and dissolving, and filtering through a membrane (0.22 mu m).
(7) Chromatographic column: the Shim-pack GIST C18 μm (4.6 I.D.×250 mm) flow rate was 1mL/min, column temperature was 30 ℃, detector wavelength was 252nm, and sample injection amount was 5. Mu.L. Gradient elution was carried out 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%,8min;2.5-65%,12min;65-75%,10min;75-100%,20min;100%,16min,100-20%,2min,20%,4min gradient elution lasting for a total of 72min.
(8) And analyzing data by using traditional Chinese medicine chromatographic fingerprint similarity evaluation system software (2012 edition) released by the national formulary committee, wherein a mean value method is adopted for comparison pattern generation mode. Analysis of variance and multiple comparison were performed on the peak area data to represent ganoderic acid content of different polarities.
The formulation of the medium was the same as in example 1.
The citric acid treated and untreated Ganoderma triterpene fingerprint is shown in figure 3. The ratio of the area of the high polarity ganoderma lucidum triterpene peak to the total peak area is shown in figure 4. The ratio of the area of the low polarity ganoderma lucidum triterpene peak to the total peak area is shown in figure 5.
Experimental results show that when the citric acid treatment concentration is 400mM and the treatment time is 48 hours, the total content of the ganoderma lucidum triterpenes is improved to a certain extent. The ganoderma lucidum triterpene is not only a substance, but also contains a plurality of different bioactive substances, the content and the type of the triterpene substances in the citric acid treatment group and the untreated group are different through an HPLC method, the result shows that the total triterpene content in the wild ganoderma lucidum strain is improved by about 24.68 percent, the content of the high-polarity ganoderic acid and the medium-polarity ganoderic acid which are expressed by peak areas before 42min in the HPLC result is improved by about 11.12 percent, and the difference is not obvious. But the peak area of the low-polarity ganoderma lucidum triterpene compound content accounting for the total ganoderma lucidum triterpene compound content in the HPLC process after 42min is obviously increased, and the increase range is about 13.78 percent.
Claims (7)
1. The application of citric acid in improving the total content of low-polarity ganoderma lucidum triterpene compounds in ganoderma lucidum is characterized in that: adding citric acid into Ganoderma liquid fermentation medium for processing to increase total content of low polarity Ganoderma triterpene compounds in Ganoderma total triterpene; the addition concentration of the citric acid in the ganoderma lucidum liquid fermentation medium is as follows: 400-800 mM.
2. The use according to claim 1, characterized in that: the addition concentration of the citric acid in the ganoderma lucidum liquid fermentation medium is as follows: 400mM.
3. The use according to claim 1, wherein the treatment is for a time of 48h.
4. A method for improving the total content of low-polarity ganoderma lucidum triterpene compounds in ganoderma lucidum is characterized by comprising the following steps: adding citric acid into Ganoderma liquid fermentation medium for processing to increase total content of low polarity Ganoderma triterpene compounds in Ganoderma; the addition amount of the citric acid in the ganoderma lucidum liquid fermentation medium is 400 mM-800 mM.
5. The method according to claim 4, wherein the citric acid is added to the ganoderma lucidum liquid fermentation medium in an amount of: 400mM.
6. The method of claim 4, wherein the time of the treatment is 48h.
7. The method of claim 4, wherein the citric acid is formulated into a citric acid solution and then sterilized by autoclaving and added to the ganoderma lucidum liquid fermentation medium.
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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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| Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities;Shizhong Zheng 等;《PLOS ONE》;第1-16页 * |
| 灵芝三萜生物合成的研究进展;朱静 等;《南京农业大学学报》;第981-989页 * |
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