CN106978465A - A kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield - Google Patents
A kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield Download PDFInfo
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- CN106978465A CN106978465A CN201710222219.XA CN201710222219A CN106978465A CN 106978465 A CN106978465 A CN 106978465A CN 201710222219 A CN201710222219 A CN 201710222219A CN 106978465 A CN106978465 A CN 106978465A
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Abstract
The invention discloses a kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield, comprise the following steps：（1）The preparation of seed liquor；（2）Inoculation fermentation.The formula of seed culture medium and fermentation medium is optimized the present invention, raw material is easy to get, cost is low, and in the soya-bean oil for adding 2.5 ~ 3.5g/L before fermentation in the fermentation medium, fermentation parameter is optimized simultaneously improvement, method and step is simple, workable, fermentation period is short, and can significantly improve mycelium production and total triterpene yield simultaneously.
Description
Technical field
The present invention relates to a kind of utilization liquid submerged fermentation technology produce macro fungi active metabolite method, especially
It is to be related to a kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield.
Background technology
Inonotus obliquus is a kind of precious medicinal macro fungi being grown on silver birch, is mainly distributed on Russian the north,
The ground such as Northern Europe, China Heilungkiang, Jilin.Inonotus obliquus has considerably long make as traditional herbal medicine in the country such as Eastern Europe, Russia
Use history.Modern medicine study shows that Inonotus obliquus, which has, treats various cancer in digestive system, improves immunity, treatment glycosuria
Disease has preferable effect, while it also has anti-aging and antiviral effect.Long-term zoopery and clinical trial shows
Inonotus obliquus does not have any toxic side effect.The magical effect of Inonotus obliquus, no matter from economic value or social value, having must
Go to study, develop.
Fuscoporia obliqua polysaccharide and triterpene are its important functional components.Triterpenoid has antitumor, antiviral, anti-
The bioactivity such as scorching, raising immunity, with very high medicinal DEVELOPMENT PROSPECT.The Inonotus obliquus triterpenoid having now been found that
Up to tens kinds, be mostly tetracyclic triterpene and pentacyclic triterpene.And Inonotus obliquus triterpene is mainly separated from Wild sclerotium at present
, but wild Inonotus obliquus resource scarcity, and it is expensive.Artificial cultivation technique is still in conceptual phase, and Planting at present
Fructification is trained because growth cycle is short, active constituent content is low, is insufficient for its medicinal demand.Therefore, liquid submerged fermentation
Method becomes the focus of Inonotus obliquus functional component PRODUCTION TRAITS.
But current birch fuscoporia ferrugineo-fusca teng submerged fermentation research is concentrated mainly on using mycelium and polysaccharide to be sent out as target product
The optimization of ferment culture medium and zymotechnique.Chinese invention patent " a kind of birch fuscoporia ferrugineo-fusca teng submerged fermentation culture medium disclosed in 2011
And the submerged fermentation method of Inonotus obliquus " in (Publication No. CN102115350A), by optimizing fermentation medium and developing
Two benches zymotechnique, improves mycelium and intracellular, the yield of exocellular polysaccharide.Chinese invention patent " one disclosed in 2016
In the method for planting rapid deep zone liquid state fermentation production Inonotus obliquus bacterium powder " (Publication No. CN105670944A), one kind is developed
Semicontinuous fermentation process quick obtaining is rich in mycelial zymotic fluid, and zymotic fluid then is passed through into the isolated birch of micro-filtration film process
Brown pore fungi filament.Want to reach higher Inonotus obliquus total triterpene yield, fermentation mycelium yield and mycelium need to be improved simultaneously
In triterpene content.But generally all it is at present, that mycelium production is low in Inonotus obliquus triterpene fermentation research, triterpene content is high, but
Total triterpene contentses are not high.If any document report, addition methyl jasmonic acid ester and aliphatic acid improve triterpene content, but due to bacterium
Filament yield is not high, limits the further raising of total triterpene contentses, and addition methyl jasmonic acid ester and aliphatic acid cost compared with
It is high.
The content of the invention
The present invention is to be carried the problem of the Inonotus obliquus total triterpene fermentation yield for solving prior art is low there is provided one kind
The fermentation process of high Inonotus obliquus total triterpene fermentation yield, step is simple, and workable, fermentation period is short, and can significantly carry
High mycelium production and total triterpene contentses.
To achieve these goals, the present invention uses following technical scheme：
A kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield, comprises the following steps：
(1) preparation of seed liquor
Aseptically Inonotus obliquus is scraped from flat board, is inoculated into the shaking flask equipped with 100ml seed culture mediums, Yu Wen
Cultivated 3~5 days under the conditions of 28~30 DEG C of degree, 150~180rpm of speed of agitator, obtain primary seed solution；Primary seed solution is existed
Poured under aseptic condition in the medicine bottle equipped with bead that sterilized, concussion shakes up, Inonotus obliquus mycelium is broken up, trained with seed
Support on the basis of matrix product, inoculum concentration is linked into the shaking flask equipped with 1L seed culture mediums for 9~10% primary seed solution, in
Cultivated under the conditions of 28~30 DEG C of temperature, 150~180rpm of speed of agitator at least 2 days, obtain secondary seed solution.
(2) inoculation fermentation
It is 9~10% by inoculum concentration on the basis of fermentation medium volume after loading fermentation medium in fermentation tank and sterilizing
Secondary seed solution be aseptically inoculated into fermentation tank, while in the fermentation medium add 2.5~3.5g/L beans
Oil, in 28~30 DEG C of temperature, 150~180rpm of speed of agitator, throughput is to continuously ferment at least 9 days under the conditions of 5~7L/min,
Stop tank and complete whole fermentation process.The key point of the present invention is addition and its addition time, addition of soya-bean oil, only
Add and control its addition could improve mycelium production and total triterpene contentses in 2.5~3.5g/L before fermentation, otherwise
Effect is had a greatly reduced quality.
Preferably, in step (1), the formula of seed culture medium is：Glucose 35g/L, dusty yeast 7.5g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 1g/L, anhydrous magnesium sulfate 0.5g/L, pH 5.5~6.0.
Preferably, in step (2), fermentation medium volume accounts for the 60~70% of fermentation tank cumulative volume.
Preferably, the specific formula of the fermentation medium is：Glucose 45g/L, dusty yeast 10g/L, anhydrous slufuric acid
Magnesium 0.5g/L, potassium dihydrogen phosphate 1g/L, calcium chloride 0.4g/L, pH 5.5~6.0.
Preferably, in step (2), sterilising conditions are：Sterilize 30~40min at 120~125 DEG C.
Therefore, the present invention has the advantages that：The formula of seed culture medium and fermentation medium is optimized,
Raw material is easy to get, and cost is low, and in the soya-bean oil for adding 2.5~3.5g/L before fermentation in the fermentation medium, while to zymotechnique
Improvement is optimized in parameter, and method and step is simple, and workable, fermentation period is short, and can significantly improve mycelium production
With total triterpene yield.
Embodiment
Below by embodiment, the present invention will be further described.
The Inonotus obliquus bacterial strain (Inonotus obliquus) used in following examples and comparative example is preserved in Chinese woods
Industry Microbiological Culture Collection administrative center (strain encodes cfcc 60260).
Embodiment 1
(1) preparation of seed liquor
Aseptically Inonotus obliquus is scraped from flat board, is inoculated into the shaking flask equipped with 100ml seed culture mediums, Yu Wen
Cultivated 5 days under the conditions of 28 DEG C of degree, speed of agitator 150rpm, obtain primary seed solution；Primary seed solution is aseptically fallen
Enter to have sterilized in the medicine bottle equipped with bead, concussion shakes up, Inonotus obliquus mycelium is broken up, using seed culture medium volume as base
Standard, inoculum concentration is linked into the shaking flask equipped with 1L seed culture mediums for 9% primary seed solution, and in 28 DEG C of temperature, stirring turns
Cultivated under the conditions of fast 150rpm at least 2 days, obtain secondary seed solution；Wherein, the formula of seed culture medium is：Glucose 35g/L, ferment
Female powder 7.5g/L, potassium dihydrogen phosphate 1g/L, anhydrous magnesium sulfate 0.5g/L, pH 5.5~6.0
(2) inoculation fermentation
Load in fermentation tank after the fermentation medium for accounting for fermentation tank cumulative volume 60% and the 30min that sterilized at a temperature of 120 DEG C, with
On the basis of fermentation medium volume, inoculum concentration is aseptically inoculated into fermentation tank for 9% secondary seed solution, simultaneously
2.5g/L soya-bean oil is added in the fermentation medium, and in 28~30 DEG C of temperature, speed of agitator 150rpm, throughput is 5L/min bars
Continuously ferment under part at least 9 days, stop tank and complete whole fermentation process, the specific formula of wherein fermentation medium is：Glucose
45g/L, dusty yeast 10g/L, anhydrous magnesium sulfate 0.5g/L, potassium dihydrogen phosphate 1g/L, calcium chloride 0.4g/L, pH 5.5~6.0.
Sampling analysis mycelium production and total triterpene contentses after fermentation, as a result as shown in table 1.
Embodiment 2~3
Compared with Example 1, difference is embodiment 2~3：The addition of soya-bean oil is respectively 3.0g/L, 3.5g/L, and remaining is complete
It is identical.
Sampling analysis mycelium production and total triterpene contentses after fermentation, as a result as shown in table 1.
Comparative example 1~6
The difference of 1~comparative example of comparative example 6 and embodiment 1 is：The addition of soya-bean oil be respectively 0g/L, 0.5g/L,
1.0g/L, 1.5g/L, 2.0g/L, 4.0g/L, remaining is identical.Sampling analysis mycelium production and total triterpene contain after fermentation
Amount, as a result as shown in table 1.
Comparative example 7~9
The difference of comparative example 7~9 and embodiment 1 is：The addition time of soya-bean oil is respectively the 2nd after fermentation, 4,6 days, its
It is remaining identical.Sampling analysis mycelium production and total triterpene contentses after fermentation, as a result as shown in table 1.
The embodiment 1~3 of table 1 and the testing result of mycelium production and total triterpene contentses in comparative example 1~9
It is can be seen that by data in table 1 in the soya-bean oil for adding 2.5~3.5g/L before fermentation in the fermentation medium, can be simultaneously
Significantly improve mycelium production and total triterpene contentses.
Embodiment 4
Compared with Example 1, difference is the present embodiment：(1) primary seed solution condition of culture is：29 DEG C of temperature, stirring
Cultivated 4 days under the conditions of rotating speed 160rpm；Secondary seed solution condition of culture is：29 DEG C of temperature, speed of agitator 160rpm；(2) ferment
Process conditions are：Load in fermentation tank and account for the fermentation medium of fermentation tank cumulative volume 65% and sterilized at a temperature of 122 DEG C
After 35min, on the basis of fermentation medium volume, inoculum concentration is aseptically inoculated into for 9.5% secondary seed solution
In fermentation tank, in 29 DEG C of temperature, speed of agitator 160rpm, throughput is to continuously ferment at least 9 days under the conditions of 6L/min, and remaining is equal
It is identical with embodiment 1.
Embodiment 5
The present embodiment is compared with embodiment, and difference is：(1) primary seed solution condition of culture is：30 DEG C of temperature, stirring turns
Cultivated 3 days under the conditions of fast 180rpm；Secondary seed solution condition of culture is：30 DEG C of temperature, speed of agitator 180rpm；(2) fermentation work
Skill condition is：Load the fermentation medium and the 40min that sterilized at a temperature of 125 DEG C for accounting for fermentation tank cumulative volume 70% in fermentation tank
Afterwards, on the basis of fermentation medium volume, inoculum concentration is aseptically inoculated into fermentation tank for 10% secondary seed solution
In, in 30 DEG C of temperature, speed of agitator 180rpm, throughput is to continuously ferment at least 9 days under the conditions of 7L/min, remaining with implementation
Example 1 is identical.
Embodiment described above is a kind of preferably scheme of the present invention, not makees any formal to the present invention
Limitation, also has other variants and remodeling on the premise of without departing from the technical scheme described in claim.
Claims (5)
1. a kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield, it is characterised in that comprise the following steps：
（1）The preparation of seed liquor
Aseptically Inonotus obliquus is scraped from flat board, is inoculated into the shaking flask equipped with 100 ml seed culture mediums, in
Cultivated 3 ~ 5 days under the conditions of 28 ~ 30 DEG C of temperature, 150 ~ 180rpm of speed of agitator, obtain primary seed solution；By primary seed solution in nothing
Pour into the medicine bottle equipped with bead that sterilized, concussion shakes up, Inonotus obliquus mycelium is broken up, with seed culture under the conditions of bacterium
On the basis of matrix product, inoculum concentration is linked into the shaking flask equipped with 1L seed culture mediums for 9 ~ 10% primary seed solution, in temperature
28 ~ 30 DEG C, cultivate at least 2 days under the conditions of 150 ~ 180rpm of speed of agitator, obtain secondary seed solution；
（2）Inoculation fermentation
It is 9 ~ 10% by inoculum concentration on the basis of fermentation medium volume after loading fermentation medium in fermentation tank and sterilizing
Secondary seed solution is aseptically inoculated into fermentation tank, while 2.5 ~ 3.5g/L soya-bean oil is added in the fermentation medium,
In 28 ~ 30 DEG C of temperature, 150 ~ 180rpm of speed of agitator, throughput is to continuously ferment at least 9 days under the conditions of 5 ~ 7L/min, stops tank i.e.
Complete whole fermentation process.
2. a kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield according to claim 1, it is characterised in that
Step（1）In, the formula of seed culture medium is：The g/L of glucose 35, the g/L of dusty yeast 7.5, potassium dihydrogen phosphate 1 g/L are anhydrous
Magnesium sulfate 0.5 g/L, pH 5.5 ~ 6.0.
3. a kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield according to claim 1, it is characterised in that
Step（2）In, fermentation medium volume accounts for the 60 ~ 70% of fermentation tank cumulative volume.
4. a kind of fermentation process of raising Inonotus obliquus total triterpene fermentation yield according to claim 1 or 3, its feature exists
In the specific formula of the fermentation medium is：The g/L of glucose 45, the g/L of dusty yeast 10, the g/L of anhydrous magnesium sulfate 0.5, phosphorus
The g/L of acid dihydride potassium 1, calcium chloride 0.4 g/L, pH 5.5 ~ 6.0.
5. a kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield according to claim 1, it is characterised in that
Step（2）In, sterilising conditions are：Sterilize 30 ~ 40min at 120 ~ 125 DEG C.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107841523A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | Triterpene substance method is extracted from Inonotus obliquus using the induction of quorum sensing molecule |
| CN107841522A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | The method for extracting betulic acid from Inonotus obliquus using jasmonate induction |
| CN108715870A (en) * | 2018-04-19 | 2018-10-30 | 浙江理工大学 | A method of improving Inonotus obliquus fermenting and producing triterpene yield |
| CN109337953A (en) * | 2018-11-01 | 2019-02-15 | 黑龙江中医药大学 | A kind of inonotus obliquus D extract and preparation method thereof and detection method |
| CN110195083A (en) * | 2019-07-15 | 2019-09-03 | 江南大学 | A method of improving Inonotus obliquus liquid state fermentation Inonotus obliquus alcohol yield |
| WO2021017449A1 (en) | 2019-07-29 | 2021-02-04 | 浙江养生堂天然药物研究所有限公司 | Fermented brich juice, production method therefor and use thereof in composition for external application to skin |
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| CN1456670A (en) * | 2002-03-15 | 2003-11-19 | 林忠平 | Genes of resveratrol stilbene synthase in parthenocissus and their use |
| CN104099385A (en) * | 2014-07-15 | 2014-10-15 | 江苏阜丰生物科技有限公司 | Method for producing inonotus obliquus exopolysaccharides through submerged fermentation |
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| WO2003045161A1 (en) * | 2001-11-29 | 2003-06-05 | Nc Biotec Co., Ltd. | A preparation method of feed and feed additive using mushroom mycelium |
| CN1456670A (en) * | 2002-03-15 | 2003-11-19 | 林忠平 | Genes of resveratrol stilbene synthase in parthenocissus and their use |
| CN104099385A (en) * | 2014-07-15 | 2014-10-15 | 江苏阜丰生物科技有限公司 | Method for producing inonotus obliquus exopolysaccharides through submerged fermentation |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107841523A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | Triterpene substance method is extracted from Inonotus obliquus using the induction of quorum sensing molecule |
| CN107841522A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | The method for extracting betulic acid from Inonotus obliquus using jasmonate induction |
| CN108715870A (en) * | 2018-04-19 | 2018-10-30 | 浙江理工大学 | A method of improving Inonotus obliquus fermenting and producing triterpene yield |
| CN108715870B (en) * | 2018-04-19 | 2021-05-14 | 浙江理工大学 | Method for increasing yield of triterpene produced by fermentation of inonotus obliquus |
| CN109337953A (en) * | 2018-11-01 | 2019-02-15 | 黑龙江中医药大学 | A kind of inonotus obliquus D extract and preparation method thereof and detection method |
| CN109337953B (en) * | 2018-11-01 | 2021-11-26 | 黑龙江中医药大学 | Inonotus obliquus D extract and preparation method and detection method thereof |
| CN110195083A (en) * | 2019-07-15 | 2019-09-03 | 江南大学 | A method of improving Inonotus obliquus liquid state fermentation Inonotus obliquus alcohol yield |
| WO2021017449A1 (en) | 2019-07-29 | 2021-02-04 | 浙江养生堂天然药物研究所有限公司 | Fermented brich juice, production method therefor and use thereof in composition for external application to skin |
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