CN106754428A - A kind of Poria mycelium liquid fermentation method - Google Patents
A kind of Poria mycelium liquid fermentation method Download PDFInfo
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Abstract
The invention discloses a kind of Poria mycelium liquid fermentation method, comprise the following steps：Amplification Culture stage, the Poria mycelium liquid fermentation system at 28 DEG C to 30 DEG C is cultivated 5 to 7 days, daily with using high temperature or Poria mycelium liquid fermentation system 1 to 3 hour described in low-temperature treatment.The present invention is due to simulation Poria cocos field grown environment, carry out high/low temperature fermented and cultured, realize the high level accumulation of Poria cocos intracellular triterpene content, can be on the premise of it need not add any allogenic material, inexpensively, it is safe, easy to operate, intracellular fuling triterpene content is remarkably improved, so that for industrialized production fuling triterpene lays the foundation.
Description
Technical field
The invention belongs to biological technical field, more particularly, to a kind of Poria mycelium liquid fermentation method.
Background technology
Poria cocos has high medical value, its active ingredient：It is many Poria cocos including β-pachyman (β-Pachyman)
Sugar, pachymose and triterpene compound pachymic acid (Tumulasie acid), loose Siberian cocklebur acid (Pmicoic acid).Current Poria cocos has
The acquisition of composition is imitated, Poria cocos cultivation and wild collection is still relied primarily on.Because Poria cocos growth cycle is more long, yield is small,
Therefore, it is difficult to a large amount of productions.
And at present to Poria mycelium liquid fermentation method, it is concentrated mainly on the production of pachymaran, pachymose.To mycelia
The research report of the extraction of body tunning medicinal ingredient, such as pachymaran, purifying, detection, and its pharmacotoxicological effect is more,
These work provide good material and phenotype to study the regulatory mechanism of pachymic acid biosynthesis.But these Poria cocos mycelia
Body zymotechnique does not have obvious effect for improving fuling triterpene content and yield.
But almost do not have for the report that Poria cocos liquid fermentation condition, especially cultivation temperature carry out Study on thinning.Simultaneously
In view of Poria cocos as a kind of integration of drinking and medicinal herbs class Chinese medicine, if it is desired to promote it if medical field is widely used, it is necessary to consider
To the security and economy of fermentation.Therefore, it is necessary to the more preferable fermentation process of development effectiveness and condition, improve Poria cocos and exist
Mycelium stage triterpene compound yield.
The content of the invention
For the disadvantages described above or Improvement requirement of prior art, the invention provides a kind of Poria cocos hypha fermentation method body
, the temperature change its object is to pass through to simulate field stimulates induction Poria cocos synthesis fuling triterpene, improves fuling triterpene tiredization
The yield of compound, thus solves prior art fuling triterpene yield relatively low, it is impossible to the technical problem of large-scale production.
To achieve the above object, according to one aspect of the present invention, there is provided a kind of Poria mycelium liquid fermentation method,
It is characterised in that it includes seed growth phase and Amplification Culture stage；
The Amplification Culture stage, the Poria mycelium liquid fermentation system at 28 DEG C to 30 DEG C, culture 5 to 7 days, daily
With using high temperature or Poria mycelium liquid fermentation system 1 to 3 hour described in low-temperature treatment.
Preferably, the Poria mycelium liquid fermentation method, its described high temperature is between 32 DEG C to 36 DEG C；The low temperature
Between 0 DEG C to 8 DEG C.
Preferably, the Poria mycelium liquid fermentation method, it uses high temperature or low-temperature treatment Poria mycelium daily
Fermentation system 2 hours.
Preferably, the Poria mycelium liquid fermentation method, its described Poria cocos mycelia extract body fermentation system expands training
The stage of supporting culture medium is CYM culture mediums.
Preferably, the Poria mycelium liquid fermentation method, its seed liquor cultivation stage is using multistage seed fermentation training
Support.
Preferably, the Poria mycelium liquid fermentation method, its described seed liquor cultivation stage is comprised the following steps that：
(1) Poria mycelium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min trainings are activated using PDA liquid medium
Support 5 to 7 days, obtain primary seed solution；
(2) primary seed solution that will be obtained in step (1) is according to inoculum concentration volume ratio 1:5, it is inoculated in the first Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 5 days, obtains secondary seed solution；
(3) secondary seed solution that will be obtained in step (2) is according to inoculum concentration volume ratio 1:10, it is inoculated in the second Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 2 days, obtains three-level seed liquor；
(4) the three-level seed liquor that will be obtained in step (3) is according to inoculum concentration volume ratio 1:10, it is inoculated in CYM culture mediums,
28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 1 day, obtains the Poria mycelium liquid fermentation system.
Preferably, the Poria mycelium liquid fermentation method, its every liter PDA liquid medium contains glucose
20g and murphy juice 200g.
Preferably, the Poria mycelium liquid fermentation method, its every liter the first Poria cocos seed culture medium contains Portugal
Grape sugar 35g, peptone 5g, yeast extract 2.5g, potassium dihydrogen phosphate 0.883g, epsom salt 0.5g and vitamin B1
0.05ml。
Preferably, the Poria mycelium liquid fermentation method, its every liter the second Poria cocos seed culture medium contains Portugal
Grape sugar 35g, peptone 5g, yeast extract 5g, potassium dihydrogen phosphate 1.0g, seven hypophosphite monohydrate potassium dihydrogen 0.5g and vitamin B1
0.05ml。
Preferably, the Poria mycelium liquid fermentation method, its every liter CYM culture medium contain 35 grams of glucose,
0.05 gram of 5 grams of peptone, 5 grams of dusty yeast, 0.883 gram of potassium dihydrogen phosphate, 0.5 gram of bitter salt and vitamin B1.
In general, by the contemplated above technical scheme of the present invention compared with prior art, can obtain down and show
Beneficial effect：
Poria cocos field grown environment be unable to do without the influence of temperature, at present for temperature study limitation in incubated, and
It is not directed to simulate influence of the high/low temperature change of wild environment to Poria mycelium fermentation intracellular fuling triterpene accumulation.The present invention is logical
Simulation Poria cocos field grown environment is crossed, high/low temperature fermented and cultured is carried out, the high level accumulation of Poria cocos intracellular triterpene content is realized.
Can be cheap, safe, easy to operate on the premise of it need not add any allogenic material, it is remarkably improved intracellular fuling triterpene and contains
Amount, so that for industrialized production fuling triterpene lays the foundation.
Brief description of the drawings
Fig. 1 is that the embodiment of the present invention and comparative example fuling triterpene content improve result figure.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as additionally, technical characteristic involved in invention described below each implementation method
Not constituting conflict each other can just be mutually combined.
Poria mycelium liquid fermentation method of the invention, comprises the following steps：
Seed growth phase：Using multistage seed culture so that Poria mycelium can fully grow formation grow fine it is suitable
Should be able to the strong Poria mycelium liquid fermentation system of power, adapt to the intermittent warming of second stage, concrete operation step is as follows：
(1) Poria mycelium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min trainings are activated using PDA liquid medium
Support 5 to 7 days, obtain primary seed solution；
Wherein, every liter of PDA liquid medium contains glucose 20g and murphy juice 200g, 121 DEG C of sterilizings 20 to 30
Minute.
(2) primary seed solution that will be obtained in step (1) is according to inoculum concentration volume ratio 1:5, it is inoculated in the first Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 5 days, obtains secondary seed solution；
Every liter of the first Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 2.5g, biphosphate
Potassium 0.883g, epsom salt 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(3) secondary seed solution that will be obtained in step (2) is according to inoculum concentration volume ratio 1:10, it is inoculated in the second Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 2 days, obtains three-level seed liquor；
Every liter of the second Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 5g, potassium dihydrogen phosphate
1.0g, seven hypophosphite monohydrate potassium dihydrogen 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(4) the three-level seed liquor that will be obtained in step (3) is according to inoculum concentration volume ratio 1:10, it is inoculated in CYM culture mediums,
28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 1 day, obtains the Poria mycelium liquid fermentation system.
Every liter of CYM culture medium contains 35 grams of glucose, 5 grams of peptone, 5 grams of dusty yeast, potassium dihydrogen phosphate 0.883
Gram, 0.05 gram of 0.5 gram of bitter salt and vitamin B1, mentioned component distilled water constant volume to 1L, 121 DEG C of sterilizings 20 to
30 minutes.
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process is as follows：
The Poria mycelium liquid fermentation system at 28 DEG C to 30 DEG C, cultivates 5 to 7 days, preferably 6 days, high with using daily
Temperature or Poria mycelium liquid fermentation system 1 to 3 hour, preferably 2 hours described in low-temperature treatment.For example, 2 hours in 0 DEG C of condition
Lower fermentation, 22 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of ferment, then again under the conditions of 0 DEG C ferment 2 hours, so
Cyclic culture 144 hours.
High temperature or low-temperature treatment, need to consider effect of stimulation and Poria mycelium tolerance situation, be advisable with 1 to 3 hour, 2
Hour is preferred.It is experimentally confirmed that high-temperature process, temperature between 32 DEG C to 63 DEG C, preferably 36 DEG C；Low-temperature treatment, temperature at 0 DEG C extremely
Between 8 DEG C, preferably 4 DEG C, improve the effect of fuling triterpene content with obvious induction Poria mycelium, high-temperature process effect compared with
Low-temperature treatment is more preferably.
It is below embodiment：
Embodiment 1
A kind of method of the intracellular fuling triterpene content for improving Poria mycelium liquid fermentation, comprises the following steps：
Seed growth phase：Using multistage seed culture so that Poria mycelium can fully grow formation grow fine it is suitable
Should be able to the strong Poria mycelium liquid fermentation system of power, adapt to the intermittent warming of second stage, concrete operation step is as follows：
(1) Poria mycelium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min trainings are activated using PDA liquid medium
Support 7 days, obtain primary seed solution；
Wherein, every liter of PDA liquid medium contains glucose 20g and murphy juice 200g, 121 DEG C of sterilizings 20 to 30
Minute.
(2) primary seed solution that will be obtained in step (1) is according to inoculum concentration volume ratio 1:5, it is inoculated in the first Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 5 days, obtains secondary seed solution；
Every liter of the first Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 2.5g, biphosphate
Potassium 0.883g, epsom salt 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(3) secondary seed solution that will be obtained in step (2) is according to inoculum concentration volume ratio 1:10, it is inoculated in the second Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 2 days, obtains three-level seed liquor；
Every liter of the second Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 5g, potassium dihydrogen phosphate
1.0g, seven hypophosphite monohydrate potassium dihydrogen 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(4) the three-level seed liquor that will be obtained in step (3) is according to inoculum concentration volume ratio 1:10, it is inoculated in CYM culture mediums,
28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 1 day, obtains the Poria mycelium liquid fermentation system.
Every liter of CYM culture medium contains 35 grams of glucose, 5 grams of peptone, 5 grams of dusty yeast, potassium dihydrogen phosphate 0.883
Gram, 0.05 gram of 0.5 gram of bitter salt and vitamin B1, mentioned component distilled water constant volume to 1L, 121 DEG C of sterilizings 20 to
30 minutes.
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process is as follows：
2 hours under the conditions of 0 DEG C ferment, 22 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of ferment, then again 0
Under the conditions of DEG C ferment 2 hours after 22 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of continue ferment, such Cyclic culture 144
Hour.
Embodiment 2
A kind of method of the intracellular fuling triterpene content for improving Poria mycelium liquid fermentation, comprises the following steps：
Seed growth phase：Such as embodiment 1
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process is as follows：
2 hours under the conditions of 4 DEG C ferment, 22 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of ferment, then again 4
Fermented 2 hours under the conditions of DEG C, such Cyclic culture 144 hours.
Embodiment 3
A kind of method of the intracellular fuling triterpene content for improving Poria mycelium liquid fermentation, comprises the following steps：
Seed growth phase：Such as embodiment 1
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process is as follows：
2 hours under the conditions of 8 DEG C ferment, 22 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of ferment, then again 8
Fermented 2 hours under the conditions of DEG C, such Cyclic culture 144 hours.
Embodiment 4
A kind of method of the intracellular fuling triterpene content for improving Poria mycelium liquid fermentation, comprises the following steps：
Seed growth phase：Such as embodiment 1
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process is as follows：
2 hours under the conditions of 32 DEG C ferment, 22 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of ferment, then exist again
Fermented 2 hours under the conditions of 32 DEG C, such Cyclic culture 144 hours.
Embodiment 5
A kind of method of the intracellular fuling triterpene content for improving Poria mycelium liquid fermentation, comprises the following steps：
Seed growth phase：Such as embodiment 1
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process is as follows：
2 hours under the conditions of 36 DEG C ferment, 22 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of ferment, then exist again
Fermented 2 hours under the conditions of 36 DEG C, such Cyclic culture 144 hours.
Embodiment 6
A kind of method of the intracellular fuling triterpene content for improving Poria mycelium liquid fermentation, comprises the following steps：
Seed growth phase：Such as embodiment 1
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process is as follows：
3 hours under the conditions of 36 DEG C ferment, 21 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of ferment, then exist again
Fermented 3 hours under the conditions of 36 DEG C, such Cyclic culture 120 hours.
Embodiment 7
A kind of method of the intracellular fuling triterpene content for improving Poria mycelium liquid fermentation, comprises the following steps：
Seed growth phase：Such as embodiment 1
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process is as follows：
3 hours under the conditions of 36 DEG C ferment, 21 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of ferment, then exist again
Fermented 3 hours under the conditions of 36 DEG C, such Cyclic culture 168 hours.
Embodiment 8
A kind of method of the intracellular fuling triterpene content for improving Poria mycelium liquid fermentation, comprises the following steps：
Seed growth phase：Using multistage seed culture so that Poria mycelium can fully grow formation grow fine it is suitable
Should be able to the strong Poria mycelium liquid fermentation system of power, adapt to the intermittent warming of second stage, concrete operation step is as follows：
(1) Poria mycelium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min trainings are activated using PDA liquid medium
Support 5 days, obtain primary seed solution；
Wherein, every liter of PDA liquid medium contains glucose 20g and murphy juice 200g, 121 DEG C of sterilizings 20 to 30
Minute.
(2) primary seed solution that will be obtained in step (1) is according to inoculum concentration volume ratio 1:5, it is inoculated in the first Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 5 days, obtains secondary seed solution；
Every liter of the first Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 2.5g, biphosphate
Potassium 0.883g, epsom salt 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(3) secondary seed solution that will be obtained in step (2) is according to inoculum concentration volume ratio 1:10, it is inoculated in the second Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 2 days, obtains three-level seed liquor；
Every liter of the second Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 5g, potassium dihydrogen phosphate
1.0g, seven hypophosphite monohydrate potassium dihydrogen 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(4) the three-level seed liquor that will be obtained in step (3) is according to inoculum concentration volume ratio 1:10, it is inoculated in CYM culture mediums,
28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 1 day, obtains the Poria mycelium liquid fermentation system.
Every liter of CYM culture medium contains 35 grams of glucose, 5 grams of peptone, 5 grams of dusty yeast, potassium dihydrogen phosphate 0.883
Gram, 0.05 gram of 0.5 gram of bitter salt and vitamin B1, mentioned component distilled water constant volume to 1L, 121 DEG C of sterilizings 20 to
30 minutes.
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process such as embodiment 1.
Embodiment 9
A kind of method of the intracellular fuling triterpene content for improving Poria mycelium liquid fermentation, comprises the following steps：
Seed growth phase：Using multistage seed culture so that Poria mycelium can fully grow formation grow fine it is suitable
Should be able to the strong Poria mycelium liquid fermentation system of power, adapt to the intermittent warming of second stage, concrete operation step is as follows：
(1) Poria mycelium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min trainings are activated using PDA liquid medium
Support 6 days, obtain primary seed solution；
Wherein, every liter of PDA liquid medium contains glucose 20g and murphy juice 200g, 121 DEG C of sterilizings 20 to 30
Minute.
(2) primary seed solution that will be obtained in step (1) is according to inoculum concentration volume ratio 1:5, it is inoculated in the first Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 5 days, obtains secondary seed solution；
Every liter of the first Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 2.5g, biphosphate
Potassium 0.883g, epsom salt 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(3) secondary seed solution that will be obtained in step (2) is according to inoculum concentration volume ratio 1:10, it is inoculated in the second Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 2 days, obtains three-level seed liquor；
Every liter of the second Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 5g, potassium dihydrogen phosphate
1.0g, seven hypophosphite monohydrate potassium dihydrogen 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(4) the three-level seed liquor that will be obtained in step (3) is according to inoculum concentration volume ratio 1:10, it is inoculated in CYM culture mediums,
28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 1 day, obtains the Poria mycelium liquid fermentation system.
Every liter of CYM culture medium contains 35 grams of glucose, 5 grams of peptone, 5 grams of dusty yeast, potassium dihydrogen phosphate 0.883
Gram, 0.05 gram of 0.5 gram of bitter salt and vitamin B1, mentioned component distilled water constant volume to 1L, 121 DEG C of sterilizings 20 to
30 minutes.
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process such as embodiment 1.
Comparative example 1
Poria mycelium liquid fermentation method, comprises the following steps：
Seed growth phase：Using multistage seed culture so that Poria mycelium can fully grow formation grow fine it is suitable
Should be able to the strong Poria mycelium liquid fermentation system of power, adapt to the intermittent warming of second stage, concrete operation step is as follows：
(1) Poria mycelium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min trainings are activated using PDA liquid medium
Support 7 days, obtain primary seed solution；
Wherein, every liter of PDA liquid medium contains glucose 20g and murphy juice 200g, 121 DEG C of sterilizings 20 to 30
Minute.
(2) primary seed solution that will be obtained in step (1) is according to inoculum concentration volume ratio 1:5, it is inoculated in the first Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 5 days, obtains secondary seed solution；
Every liter of the first Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 2.5g, biphosphate
Potassium 0.883g, epsom salt 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(3) secondary seed solution that will be obtained in step (2) is according to inoculum concentration volume ratio 1:10, it is inoculated in the second Poria cocos seed
In culture medium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 2 days, obtains three-level seed liquor；
Every liter of the second Poria cocos seed culture medium contains glucose 35g, peptone 5g, yeast extract 5g, potassium dihydrogen phosphate
1.0g, seven hypophosphite monohydrate potassium dihydrogen 0.5g and vitamin B1 0.05ml, 121 DEG C sterilize 20 to 30 minutes.
(4) the three-level seed liquor that will be obtained in step (3) is according to inoculum concentration volume ratio 1:10, it is inoculated in CYM culture mediums,
28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 1 day, obtains the Poria mycelium liquid fermentation system.
Every liter of CYM culture medium contains 35 grams of glucose, 5 grams of peptone, 5 grams of dusty yeast, potassium dihydrogen phosphate 0.883
Gram, 0.05 gram of 0.5 gram of bitter salt and vitamin B1, mentioned component distilled water constant volume to 1L, 121 DEG C of sterilizings 20 to
30 minutes.
In the Amplification Culture stage, processed using constant temperature, 28 DEG C are cultivated 144 hours.
Embodiment 6
Poria mycelium liquid fermentation method, comprises the following steps：
Seed growth phase：Such as embodiment 1
In the Amplification Culture stage, using intermittent warming, by changing yeasting temperature, the high/low temperature for simulating wild environment becomes
Change the influence to Poria mycelium fermentation intracellular fuling triterpene accumulation to induce Poria mycelium to improve fuling triterpene content, specifically
Operating process is as follows：
2 hours under the conditions of 40 DEG C ferment, 22 hours be 28 DEG C to 30 DEG C in normal temperature under the conditions of ferment, then exist again
Fermented 2 hours under the conditions of 40 DEG C, such Cyclic culture 144 hours.
After embodiment 1 to 9 and the culture of comparative example 1,2 terminate, mycelium pellet is collected with Buchner funnel, and use distilled water flushing 5-
6 times, dried in 60 DEG C of baking ovens to constant weight, spectrophotometry fuling triterpene content.The hypha powder 0.1g of drying is taken, is placed in
95% ethanol 5ml is added in test tube, ultrasonication 40min (being repeated 3 times) in Ultrasonic Cell Disruptor merges supernatant, and 60 DEG C rotate,
Extracted three times with 5ml chloroforms after 3ml distillation aqueous suspensions, merge supernatant, dissolved with 5ml methyl alcohol after 45 DEG C of revolvings and preserved.Take extraction
Liquid 0.3ml in 10ml tool plug test tubes, 70 DEG C be evaporated after sequentially add 5% vanillic aldehyde-glacial acetic acid solution 0.3ml, perchloric acid
1ml, mixes, 70 DEG C of water-bath 25min, takes out cooled on ice 5min, adds 5ml glacial acetic acid, and mixing determines extinction at 550nm
Degree, determines fuling triterpene content.Testing result is shown in Table 1.Content increases obvious compared with comparative example, and fuling triterpene content increases by hundred
Ratio is divided to be shown in Table 1.
Table 1
As it will be easily appreciated by one skilled in the art that the foregoing is only presently preferred embodiments of the present invention, it is not used to
The limitation present invention, all any modification, equivalent and improvement made within the spirit and principles in the present invention etc., all should include
Within protection scope of the present invention.
Claims (10)
1. a kind of Poria mycelium liquid fermentation method, it is characterised in that including seed growth phase and Amplification Culture stage；
The Amplification Culture stage, the Poria mycelium liquid fermentation system at 28 DEG C to 30 DEG C is cultivated 5 to 7 days, daily with adopting
With Poria mycelium liquid fermentation system 1 to 3 hour described in high temperature or low-temperature treatment.
2. Poria mycelium liquid fermentation method as claimed in claim 1, it is characterised in that the high temperature is at 32 DEG C to 36 DEG C
Between；The low temperature is between 0 DEG C to 8 DEG C.
3. Poria mycelium liquid fermentation method as claimed in claim 1, it is characterised in that daily using high temperature or low temperature at
Reason Poria mycelium fermentation system 2 hours.
4. Poria mycelium liquid fermentation method as claimed in claim 1, it is characterised in that the Poria cocos mycelia extract body hair
Ferment system Amplification Culture stage culture medium is CYM culture mediums.
5. Poria mycelium liquid fermentation method as claimed in claim 1, it is characterised in that seed liquor cultivation stage is using more
Level seed fermentation culture.
6. Poria mycelium liquid fermentation method as claimed in claim 5, it is characterised in that the seed liquor cultivation stage tool
Body step is as follows：
(1) using PDA liquid medium activate Poria mycelium, 28 DEG C to 30 DEG C, shaking speed 120-150r/min culture 5 to
7 days, obtain primary seed solution；
(2) primary seed solution that will be obtained in step (1) is according to inoculum concentration volume ratio 1:5, it is inoculated in the first Poria cocos seed culture
In base, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 5 days, obtains secondary seed solution；
(3) secondary seed solution that will be obtained in step (2) is according to inoculum concentration volume ratio 1:10, it is inoculated in the second Poria cocos seed culture
In base, 28 DEG C to 30 DEG C, shaking speed 120-150r/min is cultivated 2 days, obtains three-level seed liquor；
(4) the three-level seed liquor that will be obtained in step (3) is according to inoculum concentration volume ratio 1:10, it is inoculated in CYM culture mediums, 28 DEG C
To 30 DEG C, shaking speed 120-150r/min is cultivated 1 day, obtains the Poria mycelium liquid fermentation system.
7. Poria mycelium liquid fermentation method as claimed in claim 6, it is characterised in that every liter of PDA Liquid Culture
Base contains glucose 20g and murphy juice 200g.
8. Poria mycelium liquid fermentation method as claimed in claim 6, it is characterised in that every liter of the first Poria cocos seed
Culture medium contain glucose 35g, peptone 5g, yeast extract 2.5g, potassium dihydrogen phosphate 0.883g, epsom salt 0.5g and
Vitamin B1 0.05ml.
9. Poria mycelium liquid fermentation method as claimed in claim 6, it is characterised in that every liter of the second Poria cocos seed
Culture medium contains glucose 35g, peptone 5g, yeast extract 5g, potassium dihydrogen phosphate 1.0g, seven hypophosphite monohydrate potassium dihydrogen 0.5g, with
And vitamin B1 0.05ml.
10. Poria mycelium liquid fermentation method as claimed in claim 5, it is characterised in that every liter of CYM culture medium contains
There are 35 grams of glucose, 5 grams of peptone, 5 grams of dusty yeast, 0.883 gram of potassium dihydrogen phosphate, 0.5 gram of bitter salt and dimension life
0.05 gram of plain B1.
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| CN111662862A (en) * | 2020-07-08 | 2020-09-15 | 华中农业大学 | Application of edible vegetable oil in improving content of poria triterpene in poria mycelium cells |
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2017
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Non-Patent Citations (1)
| Title |
|---|
| 林标声: "茯苓多糖的发酵、提取及其理化、结构性质鉴定的研究", 《河南大学硕士学位论文》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662862A (en) * | 2020-07-08 | 2020-09-15 | 华中农业大学 | Application of edible vegetable oil in improving content of poria triterpene in poria mycelium cells |
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