CN113046407B - A method for large-scale production of Ganoderma lucidum triterpenes by liquid fermentation - Google Patents

Abstract

The invention discloses a method for large-scale production of Ganoderma lucidum triterpenes by liquid fermentation. The method comprises the following steps: (1) fermentation of Ganoderma lucidum mycelium is divided into two stages: the first stage of fermentation (0-5d): control The aeration ratio of the fermenter is 0.8-1.5vvm, the temperature is 26°C, and the stirring speed is 0-100r/min; the second stage of fermentation (5d-fermentation ends): increase the aeration ratio of the fermenter to 2.0-3.0vvm, and the temperature is 26°C, The stirring speed is 0-100r/min, until the fermentation liquid is all dried up, and the solids are collected when there is no moisture; that is, the required Ganoderma lucidum mycelium solids; (2) the solids collected by fermentation are extracted and measured by Ganoderma lucidum triterpenes; Among them, the extraction of Ganoderma lucidum triterpenes adopts the ethanol extraction method. The liquid fermentation control process of Ganoderma lucidum triterpenes provided by the invention is simple and efficient, and the yield of Ganoderma lucidum triterpenes in the Ganoderma lucidum mycelium produced by the method of the invention is obviously improved.

Description

A method for large-scale production of ganoderma lucidum triterpenes by liquid fermentation

technical field

The invention relates to the field of edible fungus production, in particular to a method for large-scale production of ganoderma lucidum triterpenes by liquid fermentation.

Background technique

Ganoderma lucidum belongs to Basidiomycetes, one of the most famous medicinal fungi in the world, and has been widely used as traditional Chinese medicine for thousands of years. Ganoderma lucidum triterpenoids are an important class of active substances in the secondary metabolism of Ganoderma lucidum. It has pharmacological activities such as anti-aging, anti-virus, anti-tumor, anti-inflammation, lowering blood pressure, lowering blood sugar, lowering blood fat, and immune regulation.

Ganoderma lucidum triterpenoids can be obtained not only from fruiting bodies and spores of Ganoderma lucidum produced by cultivation, but also from mycelium or fermentation broth produced by fermentation. Because cultivating fruiting bodies is time-consuming and the cultivation process and environmental conditions are not easy to control, it is difficult to meet the demands of the social market for its quality. The liquid submerged fermentation method has the advantages of high equipment utilization rate, stable product quality, and easy automatic control, which makes it possible to obtain a large amount of Ganoderma lucidum triterpenoids. The submerged fermentation of Ganoderma lucidum, as an efficient method for producing triterpenoids of Ganoderma lucidum, has attracted extensive attention from scholars at home and abroad. The low yield of triterpenes in traditional liquid submerged fermentation has always been a bottleneck problem restricting the development of this industry, which has brought great obstacles to large-scale application.

How to further increase the output of Ganoderma lucidum triterpenoids to reduce production costs and improve economic benefits has become a problem to be solved to accelerate the industrialization process of Ganoderma lucidum products.

Contents of the invention

The object of the present invention is at first to provide a kind of method of large-scale production of ganoderma lucidum triterpenes by liquid fermentation, the method comprises the steps:

(1) The fermentation of Ganoderma lucidum mycelium is divided into two stages:

The first stage of fermentation (0-5d):

Control the aeration ratio of the fermenter to 0.8-1.5vvm (preferably 1.0vvm), the temperature is 26°C, and the stirring speed is 0-100r/min;

The second stage of fermentation (5d-end of fermentation):

Increase the aeration ratio of the fermenter to 2.0-3.0vvm (preferably 3.0vvm), the temperature is 26°C, the stirring speed is 0-100r/min, until the fermentation broth is completely dry, and the solids are collected when there is no water; that is the required Ganoderma lucidum mycelium solids;

(2) Carrying out Ganoderma lucidum triterpenes extraction to the solid matter collected by fermentation and measuring content;

The extraction of Ganoderma lucidum triterpenoids can adopt the conventional ethanol extraction method, such as: after grinding the fermented Ganoderma lucidum mycelium solid into powder, add 50ml of 95% ethanol to 1g of mycelium solid to extract for 14 hours and then filter , the supernatant is the Ganoderma lucidum triterpene solution.

The present invention also provides a method for large-scale production of ganoderma lucidum mycelium by liquid fermentation, the method comprising the following steps:

(1) The first stage of fermentation (0-5d):

Control the aeration ratio of the fermenter to 0.8-1.5vvm (preferably 1.0vvm), the temperature is 26°C, and the stirring speed is 100r/min;

(2) The second stage of fermentation (5d-end of fermentation):

Increase the aeration ratio of the fermenter to 2.0-3.0vvm (preferably 3.0vvm), the temperature is 26°C, the stirring speed is 100r/min, until the fermentation broth is completely dry, and the solids are collected when there is no moisture. That is the required ganoderma mycelium solid.

Beneficial effect

Beneficial effects of the present invention: the Ganoderma lucidum mycelia produced by the method of the present invention can effectively increase the yield of Ganoderma lucidum triterpenes. The ganoderma triterpene liquid-state fermentation control process provided by the invention is simple and efficient, has important industrial application value, and has certain guiding significance for the liquid-state submerged fermentation production of other edible and medicinal fungi.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Ganoderma lucidum strain: Ganoderma lucidum G0119 (provided and preserved by the Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, preservation number: Hunong Lingzhi No.1), which has been published in [Biotechnology and BioprocessEngineering 2014 Volume 19 Issue 4 727-732].

The raw materials required in the following medium are all common commercially available products.

Slant medium (g/L): 39g of potato dextrose agar dissolved in 1L of deionized water and sterilized.

The components of the seed medium are: yeast powder 2g/L, anhydrous glucose 25g/L, magnesium sulfate heptahydrate 1.5g/L, potassium dihydrogen phosphate 3.0g/L, natural pH.

The fermentation medium is: yeast powder 4g/L, anhydrous glucose 20g/L, magnesium sulfate heptahydrate 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH.

Cultivation method: Activate the preserved strains on the slant medium for 7 days, pick 4 pieces of mycelium and insert them into the seed medium, at a temperature of 26°C, with a shaker speed of 150r/min, and shake the flask for 10 days. The cultivated seed solution is inserted into the fermentation medium in the fermenter with a 10% inoculation amount to carry out subsequent fermentation culture. Wherein the liquid volume of the fermentation tank is 70% of the total volume.

Wherein the extraction and determination method of Ganoderma lucidum triterpenes are:

Grind the ganoderma lucidum mycelium solid matter obtained by fermentation into powder, add 1g of mycelium solid matter into 50ml of 95% (weight percent) ethanol for leaching for 14 hours, and then filter, and the supernatant is the ganoderma lucidum triterpene solution to be tested .

Add 0.1ml of Ganoderma lucidum triterpene solution to be tested in the test tube (three repetitions), add 0.2ml of 5% (weight percent) vanillin glacial acetic acid solution, 0.5ml of perchloric acid, mix thoroughly, and place in a 60°C water bath Incubate in medium for 20 minutes, take it out and immediately cool to room temperature, then add 5ml of glacial acetic acid and shake well, then measure the absorbance of the sample at 550nm, the triterpene content in the solution to be tested can be calculated according to the standard curve.

Embodiment one

The method for producing Ganoderma lucidum triterpenes by liquid state fermentation in 5L fermenter:

The first stage of fermentation (0-5d): control the aeration ratio of the fermenter to 1.0vvm, the temperature at 26°C, and the stirring speed at 100r/min; the second stage of fermentation (5d-12d): increase the aeration ratio of the fermenter to 1.0vvm, and the temperature At 26°C, the stirring speed was 100r/min, until the fermentation was completed on the 12th day, and the solids were collected after centrifugation. The solids were extracted according to the above method and the content of Ganoderma lucidum triterpenes was determined.

As shown in Table 1, the yield of Ganoderma lucidum triterpenes was determined to be (0.21±0.02) g/L.

Embodiment two

The method for producing Ganoderma lucidum triterpenes by liquid state fermentation in 5L fermenter:

The first stage of fermentation (0-5d): control the aeration ratio of the fermenter to 1.0vvm, the temperature at 26°C, and the stirring speed at 100r/min; the second stage of fermentation (5d-end of fermentation): increase the aeration ratio of the fermenter to 3.0vvm, The temperature is 26°C, the stirring speed is 100r/min, until the fermentation liquid is completely dry, and the solid matter is collected when there is no water. The solids were extracted according to the above method and the content of Ganoderma lucidum triterpenes was determined.

As shown in Table 1, the yield of ganoderma lucidum triterpenoids was determined to be (1.20±0.08) g/L, which was 471% higher than that of the control.

Embodiment three

The method for producing Ganoderma lucidum triterpenes by liquid fermentation in 30L fermenter:

The first stage of fermentation (0-5d): control the aeration ratio of the fermenter to 1.0vvm, the temperature at 26°C, and the stirring speed at 100r/min; the second stage of fermentation (5d-end of fermentation): increase the aeration ratio of the fermenter to 3.0vvm, The temperature is 26°C, the stirring speed is 100r/min, until the fermentation liquid is completely dry, and the solid matter is collected when there is no water. The solids were extracted according to the above method and the content of Ganoderma lucidum triterpenes was determined.

As shown in Table 1, the yield of Ganoderma lucidum triterpenes was determined to be (1.16±0.10) g/L, which was 452% higher than that of the control.

Embodiment Four

The method for producing ganoderma lucidum triterpenes by liquid state fermentation in 50L fermenter:

The first stage of fermentation (0-5d): control the aeration ratio of the fermenter to 1.0vvm, the temperature at 26°C, and the stirring speed at 100r/min; the second stage of fermentation (5d-end of fermentation): increase the aeration ratio of the fermenter to 3.0vvm, The temperature is 26°C, the stirring speed is 100r/min, until the fermentation liquid is completely dry, and the solid matter is collected when there is no water. The solids were extracted according to the above method and the content of Ganoderma lucidum triterpenes was determined.

As shown in Table 1, the yield of ganoderma lucidum triterpenoids was determined to be (1.12±0.11) g/L, which was 433% higher than that of the control.

Embodiment five

The method for producing ganoderma lucidum triterpenes by liquid state fermentation in 500L fermenter:

The first stage of fermentation (0-5d): control the aeration ratio of the fermenter to 1.0vvm, the temperature at 26°C, and the stirring speed at 100r/min; the second stage of fermentation (5d-end of fermentation): increase the aeration ratio of the fermenter to 3.0vvm, The temperature is 26°C, the stirring speed is 100r/min, until the fermentation liquid is completely dry, and the solid matter is collected when there is no water. The solids were extracted according to the above method and the content of Ganoderma lucidum triterpenes was determined.

As shown in Table 1, the yield of Ganoderma lucidum triterpenoids was determined to be (1.08±0.04) g/L, which was 414% higher than that of the control.

The detection result comparison of each embodiment Ganoderma lucidum triterpenes of table 1

serial number Fermentation time (d) Ganoderma lucidum triterpenes yield (g/L) Ratio of improvement (%) compared to the control example Embodiment one 12 0.21±0.02 - Embodiment two 12 1.20±0.08 471 Embodiment three 11 1.16±0.10 452 Embodiment Four 13 1.12±0.11 433 Embodiment five 10 1.08±0.04 414

Claims (4)

1. A method for producing ganoderma triterpene in large scale by liquid state fermentation is characterized by comprising the following steps:

(1) The fermentation of the ganoderma lucidum mycelia is divided into two stages:

first stage of fermentation, 0-5d:

controlling the aeration ratio of the fermentation tank to be 0.8-1.5vvm, the temperature to be 26 ℃, and the stirring speed to be 0-100r/min;

second stage of fermentation, 5 d-end of fermentation:

increasing the aeration ratio of the fermentation tank to 2.0-3.0vvm at 26 deg.C, stirring at 0-100r/min until the fermentation liquid is completely dried, and collecting solid when no water exists; obtaining the required solid ganoderma lucidum mycelium;

(2) Extracting and measuring ganoderma triterpene in solid obtained by fermentation;

wherein the extraction of the ganoderma triterpene adopts an ethanol extraction method.

2. The method for large-scale production of ganoderma triterpene through liquid fermentation according to claim 1, wherein the fermentation of ganoderma lucidum mycelia in the step (1) is divided into two stages:

first stage of fermentation, 0-5d:

controlling the aeration ratio of the fermentation tank to be 1.0vvm, the temperature to be 26 ℃, and the stirring speed to be 0-100r/min;

second stage of fermentation, 5 d-end of fermentation:

increasing the aeration ratio of the fermentation tank to 3.0vvm at 26 deg.C, stirring at 0-100r/min until the fermentation liquid is completely dried, and collecting solid substance when no water exists; the required solid ganoderma lucidum mycelia is obtained.

3. A method for producing ganoderma lucidum mycelia in a large scale by liquid fermentation comprises the following steps:

(1) First stage of fermentation, 0-5d:

controlling the aeration ratio of the fermentation tank to be 0.8-1.5vvm, the temperature to be 26 ℃, and the stirring speed to be 100r/min;

(2) Second stage of fermentation, 5 d-end of fermentation:

and (3) increasing the aeration ratio of the fermentation tank to 2.0-3.0vvm at 26 ℃, stirring at a rotating speed of 100r/min until the fermentation liquor is completely dried, and collecting solid substances when no water exists, namely the required ganoderma lucidum mycelium solid substances.

4. The method for large-scale production of ganoderma lucidum mycelia through liquid fermentation according to claim 3, wherein the method comprises the following steps:

(1) First stage of fermentation, 0-5d:

controlling the aeration ratio of the fermentation tank to be 1.0vvm, the temperature to be 26 ℃, and the stirring speed to be 100r/min;

(2) Second stage of fermentation, 5 d-end of fermentation:

and (3) increasing the aeration ratio of the fermentation tank to 3.0vvm, controlling the temperature to be 26 ℃, and stirring at a rotating speed of 100r/min until the fermentation liquor is completely dried, and collecting a solid substance when no water exists, namely the required solid substance of the ganoderma lucidum mycelia.