Improved fermentation liquid culture medium for increasing content of ganoderma applanatum triterpenoid

Abstract

The invention discloses an improved fermentation liquid culture medium for increasing the content of ganoderma applanatum triterpenoids, and relates to the field of microbial fermentation. According to the culture medium, a carbon source in a basic culture medium is improved into a corn flour extracting solution, a nitrogen source is improved into ammonium chloride, and when the fermentation culture medium is prepared by taking the corn flour extracting solution as the carbon source and the ammonium chloride as the nitrogen source and is used for fermenting ganoderma lucidum, the effect of increasing the content of ganoderma applanatum triterpenoid is remarkable.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation, and specifically discloses an improved fermentation liquid culture medium for increasing the content of genus lingulata triterpenes by changing the carbon and nitrogen sources in the basal culture medium. Background Technology

[0002] Ganoderma applanatum (Pers. ex Wallr) Pat., is a fungus belonging to the Polyporaceae family. It is distributed throughout China and is a globally widespread species. It possesses anti-inflammatory and anti-cancer properties. It can synthesize a large number of bioactive compounds, such as triterpenes and polysaccharides. Triterpenes play a crucial role in the pharmacology of Ganoderma, therefore their content is one of the important indicators for evaluating the quality of Ganoderma. Studies have shown that various Ganoderma triterpenoids have important medicinal effects such as anti-HIV, anti-tumor, liver protection and detoxification, and lowering cholesterol levels, making them multifunctional natural organic compounds. Therefore, increasing the yield of Ganoderma triterpenoids will further enhance the medicinal potential and commercial value of Ganoderma. Because liquid fermentation of Ganoderma applanatum has advantages such as a short culture cycle, easy control of fermentation conditions, and stable yield and quality, developing Ganoderma applanatum products using fermented products is a trend. Research on liquid fermentation conditions of Ganoderma applanatum mainly focuses on fermentation conditions and the addition of exogenous compounds.

[0003] The present invention addresses the technical problem of providing a method for increasing the content of triterpenoid compounds in tree tongue. Summary of the Invention

[0004] The purpose of this invention is to improve the application of carbon and nitrogen sources in basal culture media to increase the content of triterpenoids in *Gynostemma pentaphyllum*. The effects of different carbon and nitrogen sources on the content of triterpenoids in *Gynostemma pentaphyllum* are revealed.

[0005] Another object of the present invention is to provide an improved fermentation liquid culture medium for increasing the content of triterpenoid compounds in tree syrup, the culture medium comprising corn flour extract, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1;

[0006] A further preferred embodiment: the carbon-to-nitrogen ratio of the culture medium is 1:1;

[0007] The modified fermentation medium contains 20.00g of corn flour extract, 20.00g of ammonium chloride, 3.00g of potassium dihydrogen phosphate, 1.50g of magnesium sulfate, and 0.05g of vitamin B1 per 1L of medium.

[0008] As a preferred embodiment, the fermentation conditions of the culture medium include: a temperature of 26°C, a natural pH, and a fermentation time of 7 days.

[0009] The base culture medium ingredients are glucose, proteose peptone, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1;

[0010] In the method, the corn powder and water are mixed at a mass ratio of 3:100, then boiled for 0.5 hours, filtered with gauze, and the obtained filtrate is the corn powder extract.

[0011] The operation process of the method specifically includes the following steps:

[0012] (1) Strain activation: the preserved Phellinus sp. is inoculated into PDA solid culture medium, and cultured in a 25 DEG C constant temperature incubator in the dark until the mycelium grows to a certain size, then transferred with a puncher for a total of two times.

[0013] (2) Seed liquid preparation: the activated Phellinus sp. is inoculated into seed culture medium with a puncher, and cultured in a 120 rpm, 28 DEG C shaker.

[0014] (3) Fermentation treatment: the seed liquid is crushed, then inoculated into modified fermentation liquid medium for fermentation.

[0015] The formula of each 1L seed culture medium is: peeled potato 20.00 g, glucose 20.00 g, proteose peptone 18.00 g, potassium dihydrogen phosphate 3.00 g, magnesium sulfate 1.50 g, and vitamin B1 0.05 g.

[0016] The strain activation is cultured in a 25 DEG C incubator for 14-20 days, the seed liquid preparation is cultured in a 26 DEG C shaker for 7 days, and the fermentation treatment is cultured in a 26 DEG C shaker for 7 days.

[0017] The beneficial effects of the application are:

[0018] The triterpene content is one of important indexes for measuring the quality of Ganoderma lucidum, and the carbon and nitrogen sources in the modified base culture medium have an influence on the triterpene content of Phellinus. When the carbon source is corn powder extract and the nitrogen source is ammonium chloride, and the carbon-nitrogen ratio is 1:1, the total triterpene content of Phellinus is improved to a certain extent. The triterpene of Phellinus is not a single substance, but contains various kinds of bioactive substances. The different triterpene contents cultured in the base culture medium and the modified fermentation liquid medium are analyzed by the vanillin-glacial acetic acid method, and the results show that the total triterpene content is improved by using the modified fermentation liquid medium. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The total triterpene content of Phellinus treated by different carbon sources.

[0020] Figure 2 The total triterpene content of Phellinus treated by different nitrogen sources.

[0021] Figure 3The total triterpenoids content of G. abdita was treated by different concentrations of corn powder.

[0022] Figure 4 The total triterpenoids content of G. abdita was treated by different concentrations of NH4Cl. DETAILED DESCRIPTION

[0023] The application is further described in detail below with examples:

[0024] (1) Strain activation: The G. abdita strain stored in 4℃ refrigerator was inoculated into PDA solid medium, and cultured in a 25℃ constant temperature incubator in the dark. When the mycelium grew over the whole plate, puncher was used to punch the edge and transfer to PDA solid medium, and the transfer was repeated twice.

[0025] (2) Seed liquid preparation: The activated G. abdita strain colony edge was punched with puncher to form 6 small holes with a diameter of 4mm, and inoculated into seed medium with inoculation needle. The seed liquid was prepared by culturing in a 26℃ shaker at a speed of 120r / min for 7d.

[0026] (3) Fermentation treatment: The fermented G. abdita mycelium seed liquid was crushed with homogenizer, and then inoculated into 100mL modified fermentation liquid medium in a 250mL triangular flask. The inoculation amount was 4% according to volume and percentage content, and the flask was placed in a 26℃ shaker at a speed of 120r / min for 7d.

[0027] (4) Control treatment: The modified fermentation liquid medium in (3) was replaced with basic medium, and other treatments were the same.

[0028] (5) The treated and control group mycelium and extracellular filtrate were separated and collected by filter screen, and the mycelium was dried and ground into powder.

[0029] (6) Triterpenoids content determination:

[0030] The triterpenoid content is calculated by vanillin-glacial acetic acid coloration method, 0.1 g of betulin standard is accurately weighed, 80% ethanol is added according to the solid-liquid ratio of 1:20, and a standard solution is obtained after mixing, 0, 200, 400, 600, 800 and 1000 muL of the standard solution are taken into 10 mL centrifuge tubes, ultrasonic extraction is carried out for 3 times under the condition of 220V 50Hz and 70 DEG C for 30 min, and centrifugation is carried out at 12000 r / min for 10 min. After 200 uL of supernatant is taken into 10 mL centrifuge tubes, 200 muL of 5% vanillin-glacial acetic acid (freshly prepared) and 800 muL of perchloric acid are added, and the reaction is carried out in a 70 DEG C water bath for 20 min, then the solution is cooled by flowing water, and the volume is adjusted to 5 mL by adding ethyl acetate, and then the solution is shaken uniformly, and the blank control is carried out without adding sample solution, and the absorbance value is read at a wavelength of 551 nm. The mass of betulin is taken as the abscissa, and the absorbance value is taken as the ordinate, and a standard curve is drawn, and a regression equation is obtained. The dried mycelium is ground into powder instead of betulin, and the above steps are repeated, and the total triterpenoid content of the fungus is calculated according to the standard curve.

[0031] The formula of the PDA solid culture medium is: 200 g of peeled potato, 20 g of glucose and 20 g of agar in 1 L of culture medium; the formula of the seed culture medium is: 20.00 g of peeled potato, 20.00 g of glucose, 18.00 g of peptone, 3.00 g of potassium dihydrogen phosphate, 1.50 g of magnesium sulfate and 0.05 g of vitamin B1 in 1 L of culture medium.

[0032] The above detailed description of the preferred embodiments of the present application, however, the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application, and fall within the protection scope of the present application.

Claims

1. A tree tongue fermentation liquid culture medium, characterized in that, The culture medium contains a carbon source and a nitrogen source; corn flour extract is used as the carbon source and ammonium chloride is used as the nitrogen source. In addition to carbon and nitrogen sources, the culture medium also includes potassium dihydrogen phosphate, magnesium sulfate, vitamin B1, and sterile water.

2. The culture medium according to claim 1, wherein, The carbon-nitrogen ratio is 1:1, that is, corn flour extract: ammonium chloride (m:m) = 1:

1.

3. The culture medium according to claim 1, wherein, The preparation method of the corn flour extract includes: corn flour (g) and water (mL) = 3:100, then boiling for 0.5 hours, filtering with gauze, and the resulting filtrate is the corn flour extract.

4. The culture medium according to claim 1, wherein, Ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1 were of analytical grade.

5. The culture medium according to claim 1, wherein, Each 1L of culture medium contains 20.00g of corn flour extract, 20.00g of ammonium chloride, 3.00g of potassium dihydrogen phosphate, 1.50g of magnesium sulfate, and 0.05g of vitamin B1.

6. The culture medium according to claims 1-5, wherein, The fermentation conditions included a temperature of 26°C, a natural pH, and a time of 7 days.

7. The method according to claim 6, wherein, The tree tongue was inoculated with seed liquid at a volume of 4%.

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