CN105907653B - A kind of liquid submerged fermentation method of ganoderma lucidum - Google Patents
A kind of liquid submerged fermentation method of ganoderma lucidum Download PDFInfo
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- CN105907653B CN105907653B CN201610524014.2A CN201610524014A CN105907653B CN 105907653 B CN105907653 B CN 105907653B CN 201610524014 A CN201610524014 A CN 201610524014A CN 105907653 B CN105907653 B CN 105907653B
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Abstract
The invention discloses a kind of liquid submerged fermentation method of ganoderma lucidum, ganoderma lucidum seed liquor is inoculated in fermentor and carries out fermented and cultured by the present invention, when fermentation is to 22-26h, adds the n-dodecane of 20-30mL/L；Start the glucose for adding 1000g/L by index feed profile stream when fermentation is to 34-38h, until 84-88h stream plus terminating, wherein exponential fed-batch specific growth rate is 0.053h‑1.The present invention adds certain nutriment during ganoderma lucidum mycelium liquid submerged fermentation, promotes ganoderma lucidum mycelium growth, improves biomass, to improve the yield of ganodenic acid, lays the foundation to produce ganodenic acid for industrialized economy.
Description
Technical field
The present invention relates to fermentation engineering fields, more particularly to a kind of liquid submerged fermentation method of ganoderma lucidum.
Background technique
Ganoderma lucidum (Ganoderma lucidum) is Basidiomycetes Polyporaceae Ganoderma fungi, is rare medicinal in China
One of fungi.First recorded in Han dynasty Shennong's Herbal, has and strengthen the body resistance to consolidate the constitution, strengthening by means of tonics, promote longevity, maintain organism balance etc.
Effect.In recent years, ganoderma lucidum obtains the further investigation of country in Southeast Asia and many scholars of America & Canada.Ganoderma lucidum polysaccharide and spirit
Sesame triterpene is the main active constituent of ganoderma lucidum.Ganoderma lucidum triterpene compounds have bitter taste mostly, and power is with kind, the place of production, production
Condition and it is different.Modern pharmacological research proves that ganodenic acid has antitumor, protection liver, anti-HIV-1 and HIV-1 protease activity
Property, antihistaminicum release, anti-aging, antimicrobial, reducing blood lipid, it is hypoglycemic the effects of.
At this stage, the yield of ganoderma lucidum thallus and its crucial effective component becomes the bottleneck for hindering ganoderma lucidum product application.Due to
Liquid deep layer fermenting technology production ganoderma lucidum has the characteristics that with short production cycle, at present has become acquisition ganoderma lucidum and its active material
Most effectual way.In recent years, the liquid fermentation technology of ganoderma lucidum has made great progress, but the liquid fermentation process of ganoderma lucidum at present
In, it is using the regulation method of liquid submerged fermentation in batches, due to ganoderma lucidum mycelium growth and ganoderma lucidum mostly on zymotechnique
There are the competitions in carbon source and energy for the synthesis of triterpene, only realize that mycelial High Density Cultivation may not be able to improve ganoderma lucidum three
The production efficiency of terpene.The content of ganodenic acid is not high in the ganoderma lucidum mycelium obtained using the method.
How the yield of ganoderma lucidum mycelium or crucial effective component is further increased to reduce production cost, improve economic effect
Benefit, oneself, which becomes, accelerates ganoderma lucidum product industrialization process letter problem to be solved.
Summary of the invention
The present invention provides a kind of liquid submerged fermentation methods of ganoderma lucidum, and this method comprises the following steps:
By the seed liquor of ganoderma lucidum with the inoculum concentration of 10% (v/v), it is inoculated in the fermentor equipped with fermentation medium, entirely
Fermentation process cultivates 140-150h under 25-28 DEG C, 80-100r/min, ventilation ratio 1:0.5-1:1；
Wherein when fermentation is to 22-26h, the n-dodecane of 20-30mL/L is added；Start when fermentation is to 34-38h by finger
Number feed profile stream adds the glucose of 1000g/L, and exponential fed-batch specific growth rate is 0.053h-1, until 84-88h stream plus end.
Wherein the seed liquor is to obtain lucidum strain by following method culture:
Picking strain after 26 DEG C of culture 10-15d, takes fungus block to be inoculated in equipped with seed culture medium in slant medium
In culture bottle, in 25-28 DEG C on shaking table, 150r/min culture 8-12d obtains primary seed solution；Again by cultured first order seed
Liquid is inoculated in the culture bottle equipped with seed culture medium with the inoculum concentration of 10% (v/v), in 25-28 DEG C on shaking table,
150r/min culture 5-8d obtains secondary seed solution.
Wherein, slant medium is PDA culture medium: being purchased from U.S. company BD, the ratio of 1000mL distilled water is dissolved according to 39g
Example is prepared, spare after 121 DEG C of sterilizing 20min.
Wherein, seed culture medium is PDB culture medium: being purchased from U.S. company BD, the ratio of 1000mL distilled water is dissolved according to 24g
Example is prepared, spare after 121 DEG C of sterilizing 20min.
Wherein, the component of fermentation medium are as follows: glucose 20g/L, yeast powder 3g/L, potassium dihydrogen phosphate 2g/L, seven water sulphur
Sour magnesium 2g/L, surplus are water, spare after adjusting 5.5,121 DEG C of sterilizing 25min of pH.
Wherein, the n-dodecane of addition is through 0.22 micron of micro-filtrate membrane filtration degerming or through 121 DEG C of rears high-temperature sterilization 30min
It can be used；
Wherein, the ventilation ratio in fermented and cultured is 1:0.5-1:1.
A kind of liquid submerged fermentation method of ganoderma lucidum of the invention, machine adds certain nutriment in due course, promotes
Ganoderma lucidum mycelium growth, improves biomass, to improve the yield of ganodenic acid.Method of the present invention is a kind of more adjunction
Nearly industrial production scale is established more with the production method of industrial prospect to produce ganodenic acid for industrialized economy
Fixed basis.Method provided by the invention is the advantage is that during the whole culture process, dissolved oxygen concentration is appropriate in fermentation system, carbon
Source concentration of glucose is lower, and fermentation ends are depleted substantially, while reducing feedback inhibition, make yield of ganoderma lucidum triterpenoid have compared with
It is greatly improved.
A kind of liquid submerged fermentation method of ganoderma lucidum of the invention, using the control technique of feed supplement, this method is simple, has
Effect, yield of ganoderma lucidum triterpenoid are significantly improved, can be applied in the industrialized production of ganodenic acid, have in industrialized production
Application value, 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.
Strain: ganoderma strain is No. 1 Ganoderma lucidum of Shanghai agriculture ganoderma lucidum, Academy of Agricultural Sciences, Shanghai City.
Slant medium is PDA culture medium: being purchased from U.S. company BD, matches according to the ratio that 39g is dissolved in 1000mL distilled water
It makes, it is spare after 121 DEG C of sterilizing 20min
The component of seed culture medium are as follows: it is purchased from U.S. company BD, is prepared according to the ratio that 24g is dissolved in 1000mL distilled water,
It is spare after 121 DEG C of sterilizing 20min.
The component of fermentation medium are as follows: glucose 20g/L, yeast powder 3g/L, potassium dihydrogen phosphate 2g/L, epsom salt
It is spare after 2g/L, adjusting 5.5,121 DEG C of sterilizing 25min of pH.
Embodiment 1:
The strain of picking cryovial preservation after 26 DEG C of culture 15d, takes 3 pieces of about 0.2cm in slant medium2Size
Fungus block is inoculated in triangular flask, and in 26 DEG C on shaking table, 150r/min culture 12d obtains seed liquor.
Seed liquor is inoculated in the 50L fermentor equipped with 35L fermentation medium, in ventilation ratio 1:1,26 DEG C, 100r/
Cultivated under min, when fermentation to for 24 hours when, add the n-dodecane of 25mL/L；Start when fermentation is to 36h by index feed profile
The glucose of stream plus 1000g/L harvests mycelium until 86h stream plus end, whole process culture terminate to ferment to 145h.Wherein,
The flow rate F of glucose is calculated according to (1) formula.
Wherein, F is flow rate (L/h), μsetFor mycelial average specific growth rate (h-1), V0For Preliminary fermentation liquid
Volume (L), X0For initial mycelia bulk concentration (g/L), Yx/sIt is mycelium to glucose yield (g/g), SiIt is dense for stream plus glucose
It spends (g/L), S is concentration of glucose (g/L) in fermentor.Ganoderma Lucidum is obtained according to the data of ganoderma lucidum mycelium liquid submerged fermentation
The average specific growth rate μ of filamentset=0.053h-1, V0=35L, X0=0.36g/L, Yx/s=1.084g/g, Si=1000g/
L, S=20g/L substitute into data and calculate to obtain flow rate F=6.28 × 10-4× exp (0.053t), by the wriggling for being coupled to computer
The flow rate F of pump control glucose.
Ganoderma lucidum mycelium content 17.68g/L is measured after fermentation 145h, the yield of ganoderma lucidum triterpenoid of extraction is 4.58mg/
100mg mycelium.
The wherein measuring method of ganodenic acid are as follows:
The ganoderma lucidum mycelium that fermentation obtains is pulverized last, 1g mycelium is added in the ethyl alcohol of 50ml 95% and extracts
It is filtered after 14 hours, 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
?.
Embodiment 2:
The strain of picking cryovial preservation after 26 DEG C of culture 15d, takes 3 pieces of about 0.2cm in slant medium2Size
Fungus block is inoculated in triangular flask, and in 26 DEG C on shaking table, 150r/min culture 12d obtains seed liquor.
Seed liquor is inoculated in the 50L fermentor equipped with 35L fermentation medium, in ventilation ratio 1:1,26 DEG C, 100r/
Cultivated under min, when fermentation to for 24 hours when, add the n-dodecane of 25mL/L, whole process culture terminates to ferment to 145h, harvest
Mycelium.
Ganoderma lucidum mycelium content 17.68g/L is measured after fermentation 145h, the yield of ganoderma lucidum triterpenoid of extraction is 1.85mg/
100mg mycelium.(measuring method is with embodiment 1).
Embodiment 3:
The strain of picking cryovial preservation after 26 DEG C of culture 15d, takes 3 pieces of about 0.2cm in slant medium2Size
Fungus block is inoculated in triangular flask, and in 26 DEG C on shaking table, 150r/min culture 12d obtains seed liquor.
Seed liquor is inoculated in the 50L fermentor equipped with 35L fermentation medium, in ventilation ratio 1:1,26 DEG C, 100r/
It is cultivated under min, starts the glucose for adding 1000g/L by index feed profile stream when fermentation is to 36h, until 86h stream plus terminate, it is whole
A process culture terminates to ferment to 145h, harvests mycelium.Wherein, the flow rate F of glucose is calculated according to embodiment 1.
Ganoderma lucidum mycelium content 17.68g/L is measured after fermentation 145h, the yield of ganoderma lucidum triterpenoid of extraction is 2.14mg/
100mg.(measuring method is with embodiment 1).
Embodiment 4:
The strain of picking cryovial preservation after 26 DEG C of culture 15d, takes 3 pieces of about 0.2cm in slant medium2Size
Fungus block is inoculated in triangular flask, and in 26 DEG C on shaking table, 150r/min culture 12d obtains seed liquor.
Seed liquor is inoculated in the 50L fermentor equipped with 35L culture medium, in ventilation ratio 1:1,26 DEG C, under 100r/min
Culture, whole process culture terminate to ferment to 145h, harvest mycelium.
Ganoderma lucidum mycelium content 17.68g/L is measured after fermentation 145h, the yield of ganoderma lucidum triterpenoid of extraction is 0.72mg/
100mg.(measuring method is with embodiment 1).
Claims (1)
1. a kind of method that feed supplement liquid submerged fermentation improves yield of ganoderma lucidum triterpenoid, it is characterised in that this method includes following step
It is rapid:
The seed liquor of ganoderma lucidum is inoculated in the fermentor equipped with fermentation medium, entire fermentation process is at 25-28 DEG C, 80-
140-150h is cultivated under 100r/min, ventilation ratio 1:0.5-1:1；
Wherein when fermentation is to 22-26h, the n-dodecane of 20-30mL/L is added；Start to mend by index when fermentation is to 34-38h
Material mode stream adds the glucose of 1000g/L, and exponential fed-batch specific growth rate is 0.053h-1, until 84-88h stream plus end.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1316519A (en) * | 2001-03-20 | 2001-10-10 | 华东理工大学 | Process for preparing ganoderic polyose and ganoderic acid by fermentation during which raw materials are supplemented |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1316519A (en) * | 2001-03-20 | 2001-10-10 | 华东理工大学 | Process for preparing ganoderic polyose and ganoderic acid by fermentation during which raw materials are supplemented |
Non-Patent Citations (2)
| Title |
|---|
| 氧载体正十二烷对灵芝三萜液态深层发酵的影响;冯杰等;《食用菌学报》;20160630;第23卷(第2期);摘要 * |
| 灵芝三萜类化合物研究进展;李国华等;《中草药》;20150630;第46卷(第12期);1858-1862 * |
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