CN113046407B - Method for producing ganoderma triterpene in large scale through liquid state fermentation - Google Patents
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Abstract
The invention discloses a method for producing ganoderma triterpene in a large scale by liquid state fermentation, which comprises the following steps: (1) fermenting ganoderma lucidum mycelia, which comprises two stages: first stage of fermentation (0-5 d): 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 a rotation speed of 0-100r/min until the fermentation liquid is completely dried, and collecting solid substance 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. The liquid fermentation control process of the ganoderma triterpene provided by the invention is simple and efficient, and the yield of the ganoderma triterpene in the ganoderma mycelium produced by the method is obviously improved.
Description
Technical Field
The invention relates to the field of edible fungus production, in particular to a method for producing ganoderma triterpene in a large scale by liquid state fermentation.
Background
Ganoderma lucidum belongs to basidiomycetes, is one of the most famous medicinal fungi in the world, and has been widely used as a traditional Chinese medicine for thousands of years. Ganoderma triterpenes are important active substances in Ganoderma secondary metabolism. Has the pharmacological activities of resisting aging, virus and tumor, resisting inflammation, lowering blood pressure, reducing blood sugar, reducing blood lipid, regulating immunity, etc.
The Ganoderma triterpene can be obtained from fruiting body and Ganoderma spore produced by cultivation, and also can be obtained from mycelium or fermentation liquid produced by fermentation. The cultivation of the fruit body is time-consuming, and the cultivation process and the environmental conditions are not easy to control, so that the quality requirement of the social market is difficult to meet. The liquid submerged fermentation mode has the advantages of high equipment utilization rate, stable product quality, convenient automatic control and the like, so that the mass acquisition of ganoderma lucidum triterpenoids is possible. Ganoderma lucidum liquid submerged fermentation has received wide attention from scholars at home and abroad as a method for efficiently producing ganoderma lucidum triterpenes. The low triterpene yield in the traditional liquid submerged fermentation is always a bottleneck problem which restricts the development of the industry, and brings great obstruction to large-scale application.
How to further improve the yield of ganoderma triterpene so as to reduce production cost and improve economic benefit becomes a problem to be solved for accelerating the industrialization process of ganoderma products.
Disclosure of Invention
The invention aims to provide a method for producing ganoderma triterpene in a large scale by liquid state fermentation, which comprises the following steps:
(1) The fermentation of the ganoderma lucidum mycelia is divided into two stages:
first stage of fermentation (0-5 d):
controlling the aeration ratio of the fermentation tank to be 0.8-1.5vvm (preferably 1.0 vvm), 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 (preferably 3.0 vvm), controlling the temperature at 26 deg.C, stirring at a rotation speed of 0-100r/min, and collecting solid when the fermentation liquid is dried and no water exists; obtaining the required solid ganoderma lucidum mycelium;
(2) Extracting ganoderma triterpenoids from the solid matters collected by fermentation and measuring the content of the ganoderma triterpenoids;
the extraction of ganoderma triterpene can adopt a conventional ethanol extraction method, such as: grinding the fermented solid ganoderma mycelia into powder, adding 50ml 95% ethanol into 1g solid ganoderma mycelia, extracting for 14 hr, and filtering to obtain the supernatant as ganoderma triterpene solution.
The invention also provides a method for producing ganoderma lucidum mycelia in a large scale by liquid fermentation, which comprises the following steps:
(1) First stage of fermentation (0-5 d):
controlling the aeration ratio of the fermentation tank to be 0.8-1.5vvm (preferably 1.0 vvm), the temperature to be 26 ℃, and the stirring speed to be 100r/min;
(2) Second stage of fermentation (5 d-end of fermentation):
increasing the aeration ratio of the fermentation tank to 2.0-3.0vvm (preferably 3.0 vvm), controlling the temperature at 26 ℃, and stirring at a rotating speed of 100r/min until the fermentation liquor is completely dried and the solid is collected when no water exists. The required solid ganoderma lucidum mycelia is obtained.
Advantageous effects
The invention has the beneficial effects that: the ganoderma lucidum mycelia produced by the method can effectively improve 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 liquid state submerged fermentation production of other edible and medicinal fungi.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
The ganoderma lucidum strain: ganoderma lucidum G0119 (provided and deposited by the institute of edible fungi, national agrology of Shanghai, accession number: hunong Lingzhi No. 1) has been disclosed [ Biotechnology and Bioprocess Engineering 2014, vol.19, no. 4, 727-732 ].
The raw materials required in the following culture media are all common commercial products.
Slant medium (g/L): dissolving 39g of potato dextrose agar in 1L of deionized water, and sterilizing.
The seed culture medium comprises the following components: 2g/L of yeast powder, 25g/L of anhydrous glucose, 1.5g/L of magnesium sulfate heptahydrate, 3.0g/L of monopotassium phosphate and natural pH.
The fermentation medium is as follows: 4g/L of yeast powder, 20g/L of anhydrous glucose, 1.5g/L of magnesium sulfate heptahydrate, 1.5g/L of monopotassium phosphate and natural pH.
The culture method comprises the following steps: activating the preserved strain on a slant culture medium for 7 days, selecting 4 mycelia, inoculating into a seed culture medium, culturing at 26 deg.C and 150r/min in a shaking table, and shaking for 10 days. Inoculating the cultured seed liquid into a fermentation medium in a fermentation tank in an inoculation amount of 10%, and performing subsequent fermentation culture. Wherein the liquid loading of the fermentation tank is 70% of the total volume.
The extraction and determination method of the ganoderma triterpene comprises the following steps:
grinding the fermented ganoderma lucidum mycelium solid into powder, adding 1g of mycelium solid into 50ml of 95% (weight percentage) ethanol, extracting for 14 hours, and filtering to obtain supernatant, namely the ganoderma lucidum triterpene solution to be detected.
Adding 0.1ml of ganoderma triterpene solution to be detected into a test tube (three times are set), adding 0.2ml of vanillin glacial acetic acid solution and 0.5ml of perchloric acid in a weight percentage of 5 percent, fully and uniformly mixing, keeping the temperature in a water bath at 60 ℃ for 20 minutes, taking out, immediately cooling to room temperature, then adding 5ml of glacial acetic acid, shaking uniformly, and detecting the absorbance of a sample at 550nm, wherein the triterpene content in the solution to be detected can be calculated according to a standard curve.
Example one
The method for producing the ganoderma triterpene by liquid fermentation in a 5L fermentation tank comprises the following steps:
first stage of fermentation (0-5 d): 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; second stage of fermentation (5 d-12 d): increasing the aeration ratio of the fermentation tank to 1.0vvm at 26 ℃, stirring at a rotating speed of 100r/min until the 12 th fermentation is finished, and centrifuging to collect solid matters. The content of Ganoderma triterpene in the solid is extracted and determined by the above method.
As shown in Table 1, the yield of ganoderma triterpene was determined to be (0.21. + -. 0.02) g/L.
Example two
A method for producing ganoderma triterpene by liquid fermentation in a 5L fermentation tank comprises the following steps:
first stage of fermentation (0-5 d): 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; 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 the rotating speed of 100r/min until the fermentation liquor is completely dried up and the solid is collected when no water exists. The content of Ganoderma triterpene in the solid is extracted and determined by the above method.
As shown in Table 1, the yield of the ganoderma triterpene is determined to be (1.20 +/-0.08) g/L, which is improved by 471 percent compared with the control.
EXAMPLE III
A method for producing ganoderma triterpene by liquid fermentation in a 30L fermentation tank comprises the following steps:
first stage of fermentation (0-5 d): 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; 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 solid matters are collected when no water exists. The content of Ganoderma triterpene in the solid is extracted and determined by the above method.
As shown in Table 1, the yield of the ganoderma triterpene is determined to be (1.16 +/-0.10) g/L, which is 452% higher than that of the control.
Example four
A method for producing ganoderma triterpene by liquid fermentation in a 50L fermentation tank comprises the following steps:
first stage of fermentation (0-5 d): 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; 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 solid matters are collected when no water exists. The content of Ganoderma triterpene in the solid is extracted and determined by the above method.
As shown in Table 1, the yield of the ganoderma triterpene is measured to be (1.12 +/-0.11) g/L, which is 433 percent higher than that of the control.
EXAMPLE five
The method for producing the ganoderma triterpene by liquid fermentation in a 500L fermentation tank comprises the following steps:
first stage of fermentation (0-5 d): 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; 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 the rotating speed of 100r/min until the fermentation liquor is completely dried up and the solid is collected when no water exists. The content of Ganoderma triterpene in the solid is extracted and determined by the above method.
As shown in Table 1, the yield of the ganoderma triterpene is determined to be (1.08 +/-0.04) g/L, which is 414% higher than that of the control.
TABLE 1 comparison of the results of the detection of Ganoderma triterpene in each example
| Serial number | Fermentation time (d) | Ganoderma triterpene output (g/L) | Ratio of increase (%) |
| Example one | 12 | 0.21±0.02 | - |
| Example two | 12 | 1.20±0.08 | 471 |
| EXAMPLE III | 11 | 1.16±0.10 | 452 |
| Example four | 13 | 1.12±0.11 | 433 |
| EXAMPLE 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.
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