CN110172492A - A kind of method of liquid submerged fermentation production ganodenic acid - Google Patents
A kind of method of liquid submerged fermentation production ganodenic acid Download PDFInfo
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- CN110172492A CN110172492A CN201910512095.8A CN201910512095A CN110172492A CN 110172492 A CN110172492 A CN 110172492A CN 201910512095 A CN201910512095 A CN 201910512095A CN 110172492 A CN110172492 A CN 110172492A
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Abstract
The invention discloses a kind of methods of liquid submerged fermentation production ganodenic acid, it include: that (1) takes the ganoderma lucidum mycelium after activating in slant medium, picking mycelia block accesses in seed culture medium, it is cultivated 8-12 days under 25-30 DEG C, 100-180r/min after inoculation, obtains seed culture fluid；(2) by cultured seed culture fluid, according in the inoculum concentration access fermentation medium of 8vol%-12vol%, it is cultivated 7-10 days under 25-30 DEG C, 100-180r/min after inoculation, the sodium taurocholate of 0.8g/L-1.5g/L is added during fermented and cultured, fermentation ends obtain ganoderma lucidum mycelium, and extract the ganodenic acid in mycelium.Method and process of the invention is simple, effective, there is application value in industrialized production, can successfully realize the purpose for significantly improving expression product content, shorten fermentation period, production efficiency is improved, and there is certain guidance to other fungal mycelium optimizing fermentations and edify meaning.
Description
Technical field
The invention belongs to glossy ganoderma fermentation field, in particular to a kind of method of liquid submerged fermentation production ganodenic acid.
Background technique
Ganoderma lucidum (Ganoderma lucidum) is Basidiomycetes, Polyporaceae, ganoderma lucidum Pseudomonas fungi, is that China is most famous
High medicinal fungi.The crucial effective component of ganoderma lucidum is ganoderma lucidum polysaccharide and ganodenic acid.Wherein ganodenic acid stops with anti-inflammatory
Bitterly, calmness, removing toxic substances, liver protection, AIDS virus resisting, antitumor, hepatoprotective effect inhibit histamine release, angiotensin converting enzyme
Etc. functions, be ganoderma lucidum play drug effect important material base.
Triterpene compound is main secondary metabolite in ganoderma lucidum, and in recent years, domestic and foreign scholars grind for ganoderma lucidum
Study carefully very extensive.The mode of ganodenic acid is obtained mainly from three kinds of fructification, conidia powder and liquid state fermentation modes, due to sub real
There are certain drawbacks for the reasons such as body cultivation period is long, and product is unstable, and triterpene content is less in conidia powder, by liquid state fermentation side
Formula, which obtains triterpene, becomes a kind of novel means.But only by optimization liquid fermentation medium and technological condition for fermentation (such as temperature
Degree, pH value, revolving speed and ventilatory capacity) it is difficult to further increase the yield of ganodenic acid, therefore many scholars have also carried out by adding
Additives matter (such as metal ion, Chinese medical extract, fatty acid, precursor substance) is added to promote grinding for ganodenic acid synthesis
Study carefully.
Current study show that methyl jasmonate, phenobarbital, aspirin, copper ion and calcium ion can induce spirit
The synthesis of sesame triterpene.These work provide good material and phenotype to study the regulatory mechanism of ganodenic acid biosynthesis.But
It is ganodenic acid fermented product as food and drug, it is necessary to consider the safety and economy of its fermentation.Methyl jasmonate sheet
Body is expensive, and phenobarbital is a kind of generality central nervous depressant, and aspirin is a kind of analgesic-antipyretic, copper ion
It is a heavy metal species.
Therefore, it is necessary to develop a kind of method of new production ganodenic acid.
Summary of the invention
The purpose of the present invention is to provide a kind of method of liquid submerged fermentation production ganodenic acid, this method includes as follows
Step:
(1) ganoderma lucidum mycelium after activating in slant medium is taken, picking mycelia block accesses in seed culture medium, inoculation
It is cultivated 8-12 days under 25-30 DEG C, 100-180r/min afterwards, obtains seed culture fluid；
(2) it by cultured seed culture fluid, is accessed in fermentation medium according to the inoculum concentration of 8-12vol%, after inoculation
It is cultivated under 25-30 DEG C, 100-180r/min 7-10 days, adds the sodium taurocholate of 0.8-1.5g/L, fermentation knot during the cultivation process
Beam obtains ganoderma lucidum mycelium, and extracts the ganodenic acid in mycelium；
Slant medium in the step (1) are as follows: 39g potato dextrose agar is dissolved in 1L deionized water after sterilizing
To obtain the final product.
The component of seed culture medium in the step (1) are as follows: DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, seven water sulfuric acid
Magnesium 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH.
The component of fermentation medium in the step (2) are as follows: DEXTROSE ANHYDROUS 30g/L, yeast powder 3g/L, seven water sulfuric acid
Magnesium 2.0g/L, potassium dihydrogen phosphate 2.0g/L, natural pH.
Fermented and cultured 0h in the step (2), for 24 hours, 40h or 48h add 0.8g/L-1.5g/L sodium taurocholate.
The extracting method of ganodenic acid in the step (2) are as follows: extract Ganoderma lucidum mycelium with the ethanol water of 90%-95%
Body obtains ganodenic acid.
Beneficial effect
The method of liquid submerged fermentation production ganodenic acid of the invention, the sodium taurocholate of addition be aliment security level it is other and
It is cheap.Present invention process is simple, effective, can be applied in the industrialized production of ganodenic acid, has in industrialized production
There is application value, can successfully realize the purpose for significantly improving expression product yield, shorten fermentation period, improves production effect
Rate, and there is certain guidance to other fungal mycelium optimizing fermentations and edify meaning.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Ganoderma strain: No. 1 Ganoderma lucidum of Shanghai agriculture ganoderma lucidum, in [Biotechnology and
Bioprocess Engineering the 4th phase 727-732 of volume 19 in 2014] it is open.
Embodiment 1 (control group)
(1) take that (potato dextrose agar (U.S. company BD production) powder 39g is dissolved in 1L deionization in slant medium
Inclined-plane ganoderma lucidum mycelium in after sterilizing in water to obtain the final product) after activation, the mycelia block access 100mL seed training of picking soybean grain size
It supports in base (DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, epsom salt 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH),
In 26 DEG C after inoculation, carried out fermented and cultured 10 days under 150r/min；
(2) by cultured seed culture fluid, fermentation medium (DEXTROSE ANHYDROUS is accessed according to the inoculum concentration of 10vol%
30g/L, yeast powder 3g/L, epsom salt 2.0g/L, potassium dihydrogen phosphate 2.0g/L, natural pH) in, in 26 DEG C after inoculation,
8 days acquisition ganoderma lucidum myceliums are cultivated under 150r/min.
The wherein extracting method and measuring method of ganodenic acid are as follows:
The ganoderma lucidum mycelium that fermentation obtains is pulverized last, the ethanol water of 50ml 95% is added in 1g mycelium
Middle room temperature filters after extracting 14 hours, and supernatant is testing sample solution.
Add testing sample solution 0.1ml (to set three repetitions) in test tube, then plus 5% vanillic aldehyde glacial acetic acid solution 0.2ml,
Perchloric acid 0.5ml after mixing well, keeps the temperature 20 minutes in 60 DEG C of water-baths, is cooled to room temperature after taking-up, then adds immediately
Glacial acetic acid 5ml measures the absorbance of sample at 550nm after shaking up, the triterpene content of sample can according to calculating on standard curve
?.
Measurement ganodenic acid content is (0.214 ± 0.068) g/L.
Embodiment 2
(1) take that (potato dextrose agar (U.S. company BD production) powder 39g is dissolved in 1L deionization in slant medium
Inclined-plane ganoderma lucidum mycelium in after sterilizing in water to obtain the final product) after activation, the mycelia block access 100mL seed training of picking soybean grain size
It supports in base (DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, epsom salt 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH),
In 26 DEG C after inoculation, carried out fermented and cultured 10 days under 150r/min；
(2) by cultured seed culture fluid, fermentation medium (DEXTROSE ANHYDROUS is accessed according to the inoculum concentration of 10vol%
30g/L, yeast powder 3g/L, epsom salt 2.0g/L, potassium dihydrogen phosphate 2.0g/L, natural pH) in, in 26 DEG C after inoculation,
8 days acquisition ganoderma lucidum myceliums are cultivated under 150r/min.
Using the method for fermentation 0h addition 1.5g/L sodium taurocholate in fermentation process described in step (2).
The extracting method and measuring method of ganodenic acid with embodiment 1, measure ganodenic acid content be (0.681 ±
0.014)g/L。
Embodiment 3
(1) take that (potato dextrose agar (U.S. company BD production) powder 39g is dissolved in 1L deionization in slant medium
Inclined-plane ganoderma lucidum mycelium in after sterilizing in water to obtain the final product) after activation, the mycelia block access 100mL seed training of picking soybean grain size
It supports in base (DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, epsom salt 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH),
In 26 DEG C after inoculation, carried out fermented and cultured 10 days under 150r/min；
(2) by cultured seed culture fluid, fermentation medium (DEXTROSE ANHYDROUS is accessed according to the inoculum concentration of 10vol%
30g/L, yeast powder 3g/L, epsom salt 2.0g/L, potassium dihydrogen phosphate 2.0g/L, natural pH) in, in 26 DEG C after inoculation,
8 days acquisition ganoderma lucidum myceliums are cultivated under 150r/min.
Add the method for 1.5g/L sodium taurocholate in fermentation process described in step (2) for 24 hours using fermentation the.
The extracting method and measuring method of ganodenic acid with embodiment 1, measure ganodenic acid content be (0.554 ±
0.099)g/L。
Embodiment 4
(1) take that (potato dextrose agar (U.S. company BD production) powder 39g is dissolved in 1L deionization in slant medium
Inclined-plane ganoderma lucidum mycelium in after sterilizing in water to obtain the final product) after activation, the mycelia block access 100mL seed training of picking soybean grain size
It supports in base (DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, epsom salt 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH),
In 26 DEG C after inoculation, carried out fermented and cultured 10 days under 150r/min；
(2) by cultured seed culture fluid, fermentation medium (DEXTROSE ANHYDROUS is accessed according to the inoculum concentration of 10vol%
30g/L, yeast powder 3g/L, epsom salt 2.0g/L, potassium dihydrogen phosphate 2.0g/L, natural pH) in, in 26 DEG C after inoculation,
8 days acquisition ganoderma lucidum myceliums are cultivated under 150r/min.
Using the method for fermentation 40h addition 1.5g/L sodium taurocholate in fermentation process described in step (2).
The extracting method and measuring method of ganodenic acid with embodiment 1, measure ganodenic acid content be (0.715 ±
0.027)g/L。
Embodiment 5
(1) take that (potato dextrose agar (U.S. company BD production) powder 39g is dissolved in 1L deionization in slant medium
Inclined-plane ganoderma lucidum mycelium in after sterilizing in water to obtain the final product) after activation, the mycelia block access 100mL seed training of picking soybean grain size
It supports in base (DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, epsom salt 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH),
In 26 DEG C after inoculation, carried out fermented and cultured 10 days under 150r/min；
(2) by cultured seed culture fluid, fermentation medium (DEXTROSE ANHYDROUS is accessed according to the inoculum concentration of 10vol%
30g/L, yeast powder 3g/L, epsom salt 2.0g/L, potassium dihydrogen phosphate 2.0g/L, natural pH) in, in 26 DEG C after inoculation,
8 days acquisition ganoderma lucidum myceliums are cultivated under 150r/min.
Using the method for fermentation 48h addition 1.5g/L sodium taurocholate in fermentation process described in step (2).
The extracting method and measuring method of ganodenic acid with embodiment 1, measure ganodenic acid content be (0.678 ±
0.032)g/L。
Embodiment 6
(1) take that (potato dextrose agar (U.S. company BD production) powder 39g is dissolved in 1L deionization in slant medium
Inclined-plane ganoderma lucidum mycelium in after sterilizing in water to obtain the final product) after activation, the mycelia block access 100mL seed training of picking soybean grain size
It supports in base (DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, epsom salt 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH),
In 26 DEG C after inoculation, carried out fermented and cultured 10 days under 150r/min；
(2) by cultured seed culture fluid, fermentation medium (DEXTROSE ANHYDROUS is accessed according to the inoculum concentration of 10vol%
30g/L, yeast powder 3g/L, epsom salt 2.0g/L, potassium dihydrogen phosphate 2.0g/L, natural pH) in, in 26 DEG C after inoculation,
8 days acquisition ganoderma lucidum myceliums are cultivated under 150r/min.
Using the method for fermentation 40h addition 1.2g/L sodium taurocholate in fermentation process described in step (2).
The extracting method and measuring method of ganodenic acid with embodiment 1, measure ganodenic acid content be (0.405 ±
0.041)g/L。
Embodiment 7
(1) take that (potato dextrose agar (U.S. company BD production) powder 39g is dissolved in 1L deionization in slant medium
Inclined-plane ganoderma lucidum mycelium in after sterilizing in water to obtain the final product) after activation, the mycelia block access 100mL seed training of picking soybean grain size
It supports in base (DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, epsom salt 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH),
In 26 DEG C after inoculation, carried out fermented and cultured 10 days under 150r/min；
(2) by cultured seed culture fluid, fermentation medium (DEXTROSE ANHYDROUS is accessed according to the inoculum concentration of 10vol%
30g/L, yeast powder 3g/L, epsom salt 2.0g/L, potassium dihydrogen phosphate 2.0g/L, natural pH) in, in 26 DEG C after inoculation,
8 days acquisition ganoderma lucidum myceliums are cultivated under 150r/min.
Using the method for fermentation 40h addition 1.0g/L sodium taurocholate in fermentation process described in step (2).
The extracting method and measuring method of ganodenic acid with embodiment 1, measure ganodenic acid content be (0.322 ±
0.010)g/L。
Embodiment 8
(1) take that (potato dextrose agar (U.S. company BD production) powder 39g is dissolved in 1L deionization in slant medium
Inclined-plane ganoderma lucidum mycelium in after sterilizing in water to obtain the final product) after activation, the mycelia block access 100mL seed training of picking soybean grain size
It supports in base (DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, epsom salt 1.5g/L, potassium dihydrogen phosphate 1.5g/L, natural pH),
In 26 DEG C after inoculation, carried out fermented and cultured 10 days under 150r/min；
(2) by cultured seed culture fluid, fermentation medium (DEXTROSE ANHYDROUS is accessed according to the inoculum concentration of 10vol%
30g/L, yeast powder 3g/L, epsom salt 2.0g/L, potassium dihydrogen phosphate 2.0g/L, natural pH) in, in 26 DEG C after inoculation,
8 days acquisition ganoderma lucidum myceliums are cultivated under 150r/min.
Using the method for fermentation 40h addition 0.8g/L sodium taurocholate in fermentation process described in step (2).
The extracting method and measuring method of ganodenic acid with embodiment 1, measure ganodenic acid content be (0.298 ±
0.033)g/L。
Claims (6)
1. a kind of method of liquid submerged fermentation production ganodenic acid, it is characterised in that this method comprises the following steps:
(1) take in slant medium activate after ganoderma lucidum mycelium, picking mycelia block access seed culture medium in, after inoculation in
25-30 DEG C, cultivate 8-12 days under 100-180r/min, obtain seed culture fluid；
(2) it by cultured seed culture fluid, is accessed in fermentation medium according to the inoculum concentration of 8vol%-12vol%, after inoculation
It is cultivated 7-10 days under 25-30 DEG C, 100-180r/min, the sodium taurocholate of 0.8g/L-1.5g/L is added during fermented and cultured,
Fermentation ends obtain ganoderma lucidum mycelium, and extract the ganodenic acid in mycelium.
2. a kind of method of liquid submerged fermentation production ganodenic acid according to claim 1, it is characterised in that: the step
Suddenly the sodium taurocholate of 1.5g/L is added in (2) during fermented and cultured.
3. a kind of method of liquid submerged fermentation production ganodenic acid according to claim 1, it is characterised in that: the step
Suddenly fermented and cultured 0h in (2), for 24 hours, 40h or 48h add 0.8g/L-1.5g/L sodium taurocholate.
4. a kind of method of liquid submerged fermentation production ganodenic acid according to claim 1, it is characterised in that: the step
Suddenly the component of the seed culture medium in (1) are as follows: DEXTROSE ANHYDROUS 30g/L, yeast powder 1g/L, epsom salt 1.5g/L, phosphoric acid
Potassium dihydrogen 1.5g/L, natural pH.
5. a kind of method of liquid submerged fermentation production ganodenic acid according to claim 1, it is characterised in that: the step
Suddenly the component of the fermentation medium in (2) are as follows: DEXTROSE ANHYDROUS 30g/L, yeast powder 3g/L, epsom salt 2.0g/L, phosphoric acid
Potassium dihydrogen 2.0g/L, natural pH.
6. a kind of method of liquid submerged fermentation production ganodenic acid according to claim 1, it is characterised in that: the step
Suddenly in (2) ganodenic acid extracting method are as follows: extract ganoderma lucidum mycelium with the ethanol water of 90%-95% and obtain ganoderma lucidum three
Terpene.
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| CN111607525A (en) * | 2020-05-29 | 2020-09-01 | 广州颜如玉生物科技有限公司 | Ganoderma lucidum mycelium culture medium and culture method thereof |
| CN111647548A (en) * | 2020-05-29 | 2020-09-11 | 广州颜如玉生物科技有限公司 | Ganoderma lucidum mycelium culture medium for high-yield triterpene and culture method thereof |
| CN112522114A (en) * | 2020-12-10 | 2021-03-19 | 上海应用技术大学 | Cordyceps militaris mushroom residue extracting solution, lucid ganoderma fermentation product, preparation method and application thereof |
| CN113046407A (en) * | 2021-03-30 | 2021-06-29 | 上海市农业科学院 | Method for producing ganoderma triterpene in large scale through liquid state fermentation |
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