CN106119333A - A kind of method utilizing fatty acid-induced to extract betulic acid from Inonqqus obliquus - Google Patents
A kind of method utilizing fatty acid-induced to extract betulic acid from Inonqqus obliquus Download PDFInfo
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Abstract
The invention discloses a kind of method utilizing fatty acid-induced to extract betulic acid from Inonqqus obliquus, including: Inonqqus obliquus (Inonotus obliquus) CFCC 83414 of activation is accessed fluid medium, in 24 26 DEG C of fermentation culture 8 14 days, within 5th 8 day, fatty acid is added in fermentation, after having fermented, separating thallus and fermentation liquid, obtain betulic acid by post processing is isolated and purified from thalline and fermentation liquid.The present invention stimulates the growth of Inonqqus obliquus by adding fatty acid at ferment middle, and the method is without introducing engineering strain, external enzyme or xenobiontics, and post processing is simple, can be effectively improved the yield of betulic acid in Inonqqus obliquus.
Description
Technical field
The present invention relates to fermentable and metabolism field, particularly relate to one and utilize fatty acid-induced from Inonqqus obliquus
The method extracting betulic acid.
Background technology
Inonqqus obliquus (Inonotus obliquus) is the most rare and famous and precious a kind of medicinal fungi, belong to Eumycota,
Basidiomycotina, Hymenomycetes, non-brown Zoopagales, Polyporaceae, brown transverse hole fungus belong to.It is distributed mainly on Russia, Finland, Poland, day
The Northern Hemisphere north latitude 40 ° such as this Hokkaido～the area of 50 ° and the Greater Hinggan Mountains in Heilongjiang of China, Changbai mountain, Jilin one carry.From 16
Since century, the Inonqqus obliquus that is just widely used among the people of the country such as Russia, Poland, Finland to treat various difficult miscellaneous diseases, as
Various cancers, heart disease and diabetes etc..
Inonqqus obliquus is the domestomycetes being grown in frigid zone, causes the white rot of Betula platyphylla Suk., silvery birch, elm, Folium Et Cacumen Alni Japonicae.In wood
Inonqqus obliquus mycelia also will not freeze to death at subzero 40 DEG C, is the kind extremely resisted cold.
The morphological characteristic of Inonqqus obliquus: sclerotium presents warty (block of sterility), appearance grey black, has irregular ditch
Trace, internal yellow, stockless, diameter 25～40cm, dark, surface drastic crack, firmly, the most crisp time dry, part thickness 5mm, cot shape can be educated
Thin, crineous；Tube 3～10mm, the front end cracking of tube crisp, usual, the every mm in bacterium hole 6～8, circular, shallow white, become afterwards
Crineous；Bacterial context pin matter, has slight, ambiguous ring grain, and fresh (becoming clear) is khaki.Spore is that wealthy ellipticity is to ovum
Shape, smooth, 9～10 μ m 5.5～6.5 μm, there is bristle.
Inonqqus obliquus contains polysaccharide, triterpenes, Inonqqus obliquus alcohol, multiple oxidation triterpenoid compound, bolt bacterium sheep sour, multiple
Hair sterol type triterpenoid compound, folic acid derivatives, aromatic vanillic acid, syringic acid and γ hydroxy benzoic acid, also have been reported that
Claim separated go out tannin compound, steroid, alkaloid compound, melanin class, low molecule Polyphenols and lignin
Compound.Wherein, betulic acid, is a kind of very important natural active matter.
Betulic acid (Betulinic acid, BA), has another name called belulinic acid Betulinic acid, is a kind of pentacyclic triterpenoid.Extensively divide
It is distributed in various plants, such as rhamnaceous Semen Ziziphi Spinosae, the Japanese birch bark of Betulaceae, the Spina Gleditsiae of pulse family, rosaceous smooth bark wood
Melon, Araliaceae Radix Et Caulis Acanthopanacis Senticosi, Euphorbiaceae Bischofia polycarpa, Malvaceae Flos Hibisci Mutabilis etc..It is located away from the Fructus rhamni (Rhamnus davurica Pall.) being grown in eastern Africa the earliest
The bark of section aithullium.In China, the abundantest natural resources of betulic acid is Japanese birch bark.
Recent studies indicate that, betulic acid has many biological activitys, as antitumor, AntiHIV1 RT activity, antiinflammatory, antibacterial,
Malarias etc., especially at anti-tumor aspect, especially for melanoma, have prominent performance.The most also there is effect machine
Making the advantages such as special, molecular mass is little, hypotoxicity, be widely used DEVELOPMENT PROSPECT, is a very promising drug leads of class
Compound.
Currently, the source of betulic acid mainly has three kinds: first, extracts isolated from natural plants.Second, with in vain
Pine gum alcohol is precursor, obtains betulic acid by organic synthesis.3rd, with betulin as precursor, converted raw by microorganism
Become betulic acid.
At present, there are some researches show, Inonqqus obliquus can be separated to betulic acid.But, in experimentation, find from
In Inonqqus obliquus during extracting directly betulic acid, the amount obtained is the lowest, almost without, it is desirable that find a kind of highly efficient
Improve betulic acid yield method.
Summary of the invention
The invention provides a kind of method utilizing fatty acid-induced to extract betulic acid from Inonqqus obliquus, the method without
Need to introduce engineering strain, external enzyme or xenobiontics, post processing is simple, can be effectively improved betulic acid in Inonqqus obliquus
Yield.
A kind of method utilizing fatty acid-induced to extract betulic acid from Inonqqus obliquus, including: by the Pyropolyporus fomentarius (L.ex Fr.) Teng hole of activation
Bacterium (Inonotus obliquus) CFCC 83414 accesses fluid medium, in 24-26 DEG C of fermentation culture 8-14 days, in fermentation
Within the 5-8 days, add fatty acid, after having fermented, separating thallus and fermentation liquid, separated from thalline and fermentation liquid by post processing
Purification obtains betulic acid；
Described fatty acid is oleic acid, linoleic acid, stearic acid, palmitic acid, linolenic acid, palmitoleic acid, arachidonic acid, Petiolus Trachycarpi
At least one in acid.
Betulic acid belongs to a kind of secondary metabolite, mainly produces in the metabolic process of Inonqqus obliquus, Inonqqus obliquus
Fermentation period be generally 12 days.Fatty acid and derivant thereof are proved to be one can increase mycelial growth and cometabolism product
The derivant of produce amount.The present invention studies discovery, adds fatty acid at ferment middle and can be effectively improved Betula platyphylla Suk. fat in Inonqqus obliquus
The yield of acid.
In the present invention, described Inonqqus obliquus (Inonotus obliquus) CFCC 83414 bacterial strain is micro-purchased from China Forest
Biological inoculum preservation administrative center, strain number CFCC 83414.
The solid medium that described Inonqqus obliquus (Inonotus obliquus) CFCC 83414 activates is Rhizoma Solani tuber osi Portugal
Grape sugar agar synthetic medium and the mixture of bran water lixiviating solution.Potato dextrose agar synthetic medium can be commercially available
Product.
In described solid medium, raw material mass percent composition can be: Rhizoma Solani tuber osi leaches powder 1%, glucose 2%, fine jade
Fat 1.8%, chloromycetin 0.01%, surplus is bran water lixiviating solution；Wherein, bran water lixiviating solution is that wheat bran is according to 4-6% mass
Percent concentration is added to the water, and obtains through boiling, after remove impurity.
Solid medium can be prepared via a method which: is added to the water according to 4-6% mass percent concentration by wheat bran,
Bulky grain is removed by eight layers of filtered through gauze after boiling 0.4-0.6 hour；It is subsequently adding potato dextrose agar synthetic medium,
Mixing, boils, is divided in glass tubing, afterwards sterilizing 20min at 121 DEG C, be put into inclined-plane and treat that it solidifies after sterilizing.
Described fluid medium is potato glucose synthetic medium and the mixture of bran water lixiviating solution.Rhizoma Solani tuber osi Portugal
Grape sugar synthetic medium can be commercially available prod.
In described fluid medium, raw material mass percent composition can be: Rhizoma Solani tuber osi leaching powder 0.8%, glucose 2%,
Surplus is bran water lixiviating solution；Wherein, bran water lixiviating solution is that wheat bran is added to the water according to 4-6% mass percent concentration, warp
Boil, obtain after remove impurity.
Fluid medium can be prepared via a method which: is added to the water according to 4-6% mass percent concentration by wheat bran,
Bulky grain is removed by eight layers of filtered through gauze after boiling 0.4-0.6 hour；It is subsequently adding potato glucose synthetic medium, mixed
Even, subpackage, afterwards sterilizing 20min at 121 DEG C.Use this culture medium prescription, be more beneficial for growth and the metabolism of thalline.
Described fermentation temperature can be 25 DEG C, and fermentation time can be different according to fermentation mode difference.When using fermentation tank
During fermentation, incubation time can be 8-10 days；When using triangular flask fermentation, fermentation time can be 11-14 days.The present invention grinds
Studying carefully discovery fermentation tank treatment effect more preferable, fermentation tank, compared with triangular flask, is difficult to pollute, and ventilation is big, and dissolved oxygen is abundant, meanwhile,
Owing to Inonqqus obliquus is aerobic fungus, therefore with fermentor grown faster, fermentation period is shorter.
During triangular flask fermentation culture, within first 2-3 days, rotating speed is set to 0rpm/min, the most again with the rotating speed of 90-110rpm/min
Cultivate 2-3 days, cultivate with the rotating speed of 150-180r/min the most again.First stand 2～3 days, after being because just inoculating, in culture medium
Thalline is less, and required oxygen is the most less；Cultivate 2-3 days, when being because this with the rotating speed of 90-110rpm/min the most again
Phase, thalline is gradually increasing, so suitably to increase speed, to meet its demand to oxygen.The most again with 150-
The rotating speed of 180r/min is cultivated, and is because now its biomass and has reached its maximum, so suitably to strengthen rotating speed.So
Rotating speed is set, is more beneficial for thalli growth and metabolism.
Before addition, the aseptic organic system membrane filtration using 0.22 μm is degerming for described fatty acid.
Described fatty acid can be at least one in oleic acid, linoleic acid, stearic acid, palmitic acid；It is preferably oleic acid.Pre-reality
In testing, oleic acid, linoleic acid, stearic acid, these several acid of palmitic acid are carried out preliminary comparison, find that some acid can promote mycelia
The growth of body and the synthesis of promotion betulic acid, some acid can promote the synthesis of betulic acid but can suppress mycelial growth,
And oleic acid is the one that effect that mycelial growth can promote that betulic acid synthesizes both can have been promoted best.
The joining day of described oleic acid can be fermentation the 6-7 days.Because now adding, the betulic acid obtained after fermentation
Amount is at most.
In terms of every liter of fermentation liquid, the addition of described oleic acid can be 0.1-3.0g；It is preferably 1.0-2.0g；More preferably
1.0g.During because the amount adding oleic acid is very few, inducing effect is inconspicuous；When oleic acid addition is too high, it will suppression betulic acid
Generation.
Described post processing includes being extracted with ethyl acetate after bacterial cell disruption, is extracted with ethyl acetate by fermentation liquid simultaneously,
Combining extraction liquid afterwards, concentrates, obtains betulic acid.
Described bacterial cell disruption, extracting process be: is first washed twice with sterilized water by thalline, adds appropriate aseptic afterwards
Water, crushes 5 times with Ultrasonic Cell Disruptor, and each 1min (300W), the ethyl acetate being subsequently adding equivalent extracts, in 23-27
DEG C supersound extraction 0.8-1.2h, extracts 2-3 time, merges, is extracted liquid.
Described fermentation liquid extracting process is: the ethyl acetate of fermentation liquid equivalent extracted, in 23-27 DEG C of supersound extraction 0
.8-1.2h, extract 2-3 time, merge, be extracted liquid.
The present invention, in Inonqqus obliquus sweat, adds derivant fatty acid suitable opportunity, to improve Betula platyphylla Suk. fat
Acid yield, possesses following beneficial effect:
(1) by adding fatty acid at ferment middle, effectively facilitated betulic acid in Inonqqus obliquus and fermentation liquid thereof and produced
The raising of amount, when adding oleic acid, yield can improve 2-8 times.
(2) oleic acid, linoleic acid, stearic acid, palmitic acid, palmitoleic acid, arachidonic acid, Palmic acid etc. are Inonqqus obliquus
In intrinsic fatty acid.And oleic acid is as the one in fatty acid intrinsic in Inonqqus obliquus, there is multiple biological activity, it
Add without subsequent treatment.
Accompanying drawing explanation
Fig. 1 is the liquid chromatogram of betulic acid standard substance.
Fig. 2 is the liquid chromatogram of bacterial cell disruption liquid.
Fig. 3 is the liquid chromatogram of fermentation liquid
Fig. 4 is the process chart of the present invention.
Detailed description of the invention
For being best understood from the present invention, explain the present invention below in conjunction with embodiment.Material used by following embodiment, examination
Agent etc., if no special instructions, the most commercially obtain.
In following example: Inonqqus obliquus (Inonotus obliquus) CFCC 83414 bacterial strain is micro-purchased from China Forest
Biological inoculum preservation administrative center, strain number CFCC 83414.
Embodiment 1
1, the preparation of solid medium
Wheat bran is added to the water according to 5% mass percent concentration, removes by eight layers of filtered through gauze big after boiling 0.5 hour
Granule, obtains bran water lixiviating solution；It is subsequently adding PDA synthetic medium (commercially available), after mix homogeneously, is divided in after being boiled
In glass tubing, often pipe 10mL, afterwards sterilizing 20min at 121 DEG C.Go out and put the solidification of inclined-plane to inclined-plane after bacterium.
Wherein, raw materials quality percentage ratio consists of: Rhizoma Solani tuber osi leaches powder 1%, glucose 2%, agar 1.8%, chloromycetin
0.01%, surplus is bran water lixiviating solution.
2, the activation of strain
Inonqqus obliquus CFCC 83414 is inoculated on inclined-plane, cultivates about 15 days in the incubator of 25 DEG C, treat that it is long
Full whole inclined-plane.
3, the preparation of fluid medium
Wheat bran is added to the water according to the ratio of 5% (with the stereometer of water), and boils after half an hour with eight layers of yarn
Cloth is filtered to remove bulky grain, obtains bran water lixiviating solution；It is subsequently adding PDB synthetic medium (commercially available), after mix homogeneously, subpackage
In the conical flask of 500mL, every bottle of subpackage 100mL, afterwards sterilizing 20min at 121 DEG C.
Wherein, raw materials quality percentage ratio consists of: Rhizoma Solani tuber osi leaches powder 0.8%, and glucose 2%, surplus is wheat bran water logging
Extract.
4, inoculation
Each shaking flask 1 piece of 1cm of inoculation2Inonqqus obliquus CFCC 83414 strain.
5, fermentation
Cultivate at a temperature of 25 DEG C, first stand 2～3 days, cultivate 2～3 days with the rotating speed of 100rpm the most again, the most again with
The rotating speed of 180rpm is cultivated.
6, the addition of oleic acid
In normal sweat, (oleic acid need to be with the aseptic organic system filter membrane of 0.22 μm within the 6th day, to add oleic acid in fermentation
Filtration sterilization), every bottle adds 0.1g so that in fermentation liquid, the concentration of oleic acid is 1.0g/L.
7, thalline and the separation of fermentation liquid and extraction
Pour fermentation liquid and thalline into 50mL centrifuge tube, centrifugal 10min under the rotating speed of 3500r/min, by fermentation liquid and
Somatic cells separates.Thalline claims its weight in wet base after being washed with deionized water 2 times, the most often pipe adds the deionized water of about 15mL, uses
Ultrasonic Cell Disruptor crushes 5 times, each 1min, adds the ethyl acetate of equivalent, ultrasonic 1h at 25 DEG C, extracts 2 times；For each sample
Product, each sample of fermentation liquid takes 50mL, weighs its volume with graduated cylinder, is subsequently adding the 50mL ethyl acetate extraction of equivalent, 25 DEG C of temperature
Ultrasonic 1h under degree, afterwards with centrifuge, rotating speed is 3500r/min, and the time is 10min, each sample extraction 2 times.Afterwards
Steam instrument with rotation and carry out rotary evaporation concentration, until by its distilled-to-dryness, the parameter of rotary evaporation is as follows: rotating speed is 120r/min,
The temperature of water-bath is 50 DEG C.
Crystal 1-2mL methanol (chromatographically pure) that obtains dissolves, with the filtering with microporous membrane remove impurity of the organic system of 0.22 μm,
Wait sample introduction afterwards.
8, the mensuration of betulic acid concentration: reversed phase liquid chromatography (RP-HPLC).
Its chromatographic test strip part:
Chromatographic column DiamonsilC18 (250mm × 4.6mm, 5 μm)；
Flowing is acetonitrile/water (v/v=91/9) mutually；
Flow velocity 1.0mL/min；
Detection wavelength 210nm；
Column temperature 25 DEG C；
Sample size 20 μ L；
Run sample time 25min, gradient elution.
Draw standard curve: take 1ml standard substance sample introduction, under chromatographic test strip part, measure the peak area value (A) of correspondence, with
Peak area value A is vertical coordinate, and the concentration C of betulic acid is abscissa, draws standard curve, tries to achieve betulic acid through statistical disposition
Concentration with the equation of linear regression of peak area is: A=8.18575C+15.47603 (R2=0.99964)
Experimental group adds oleic acid when ferment middle, and matched group does not carries out any process.Measurement result shows, in matched group
In weight in wet base thalline, the content of betulic acid is 0.1004 μ g/g, adds the content of betulic acid in the experimental group wet thallus of oleic acid and is
0.6249μg/g.In matched group fermentation liquid, the content of betulic acid is 0.1180 μ g/mL, and adds the experimental group fermentation liquid of oleic acid
The content of middle betulic acid is 0.1873 μ g/mL.
From result, the yield of the experimental group betulic acid adding oleic acid is significantly higher than the matched group of natural fermentation.Can
See that adding oleic acid in good time, in appropriate amount can play the effect improving betulic acid yield.
Embodiment 2
With reference to the Inonqqus obliquus fermentation technology of embodiment 1, adding the oleic acid stage, every bottle adds 0.2g so that fermentation liquid
The concentration of middle oleic acid is 2.0g/L.Other conditions are with embodiment 1.
The detection method of betulic acid concentration is with embodiment 1.
According to the method for the present embodiment, after fermenting 12 days, detect that in wet thallus, the content of betulic acid is 0.6997 μ g/
G, in matched group, the content of betulic acid is 0.1004 μ g/g.
Experimental group adds oleic acid when ferment middle, and matched group does not carries out any process.Measurement result shows, matched group is sent out
In ferment liquid, the concentration of betulic acid is 0.1180 μ g/mL, adds the concentration of betulic acid in the experimental group fermentation liquid of oleic acid and is
0.2165μg/mL。
Show from the result of embodiment 1～2, add Inonqqus obliquus thalline or the fermentation of the oleic acid fermentation of 1.0～2.0g/L
In liquid, the content of betulic acid is above the comparison of natural fermentation；After illustrating to add oleic acid, betulic acid in thalline and fermentation liquid
Content be all significantly improved.
Embodiment 3
With reference to the Inonqqus obliquus fermentation technology of embodiment 1, sample being divided into five groups, wherein four groups is the not same order in fermentation
Section, is separately added into oleic acid for the 4th, 5,6,7 days, and each every bottle of fermentation liquid all adds oleic acid 0.1g so that in fermentation liquid, oleic acid is dense
Degree is 1.0g/L, and another set is blank.Other conditions are with embodiment 1.
The detection method of betulic acid concentration is with embodiment 1.
The present embodiment, in addition to betulic acid in survey fermentation liquid and thalline, has surveyed again the Biomass of each process group.Biological
The measurement of amount is carried out in accordance with the following methods: when, after thalline and separation of fermentative broth, being put into by thalline in 50mL centrifuge tube, and centrifuge tube is wanted
Weighing in advance, and finish label, prior to pre-freeze in the refrigerator of-80 DEG C a day, then on vacuum freeze drier, lyophilizing 48 was little
Time its moisture is all removed, weigh its quality afterwards.Deduct the quality of centrifuge tube, i.e. can get the dry weight of thalline.
According to the method for the present embodiment, the different oleic acid recorded after fermenting 12 days add the experimental result of time and are shown in Table 1.
As can be seen from the table, it is little on the impact of Inonqqus obliquus Biomass that different oleic acid adds the time, but right
The content impact of betulic acid is bigger.When within the 7th day, adding oleic acid, in fermentation liquid, the concentration of betulic acid is the highest, is 0.3251 μ g/
mL；When within the 4th day, adding oleic acid, in thalline, the content of betulic acid is the highest, is 8.85 μ g/g；And betulic acid total content is the highest
When being to add oleic acid on the 7th day.Consider above experimental result, it is known that the Best Times adding betulic acid is the 7th day.
The different oleic acid of table 1 adds the time impact on fermentation results
Embodiment 4
The present embodiment uses fermentation tank to ferment.The preparation of culture medium and the activation of strain are all with embodiment 1.
Compared with embodiment before, the following steps of the present embodiment are different:
1, the preparation of seed liquor
Inoculation 1cm2The strain of activation is in equipped with in the 500mL conical flask of 100mL culture medium, at 25 DEG C, the bar of 180rmp
Seed liquor is obtained after cultivating 48h in shaking table under part.
2, the pre-treatment of fermentation liquid
As described in Example 1, the fluid medium of 4L, and sterilizing are prepared.And the appropriate soybean oil that goes out is as froth breaking
Agent.After soda acid electrode calibration, fermentation tank is accessed, balance 4～more than 6 hours, make culture medium reach the temperature preset
Spend 25 DEG C.
3, inoculation
Around inoculation mouth, ethanol on point, then adds in fermentation tank by seed liquor rapidly, and seed liquor presses 10% (with fermentation
The stereometer of culture medium in tank) ratio add, i.e. 400mL.After inoculation, the temperature of fermentation tank is set to 25 DEG C, and connects and disappear
Infusion-soybean oil.Ferment 9 days.
4, fermentation liquid and the separation and Extraction of thalline
The separating step of fermentation liquid and thalline is with embodiment 1, but extraction step and some difference of embodiment 1.In order to find
A kind of more particularly suitable extracting mode, is therefore divided into different processing modes by bacterial cell disruption liquid and extracts.It is respectively as follows: 25 DEG C
Under ultrasonic 1h；Ultrasonic 1h at 60 DEG C；At 25 DEG C the most ultrasonic, directly extract；Liquid nitrogen grinding, the most ultrasonic.The present embodiment also has one
Dividing thalline to do frozen dried, freeze-drying process is with reference to embodiment 3, and the extracting mode of bacterial cell disruption liquid is supersound extraction at 25 DEG C
1h.The detection method of evaporation-concentration step and betulic acid is also with embodiment 1.
Different extraction processing modes the results are shown in Table 2.
Table 2 Different treatments is on the impact of betulic acid extracted amount in wet thallus
Test result indicate that, the temperature of extraction, and the most ultrasonic all result can be had an impact.By front two groups of Data Comparisons
Understand, effective than 60 DEG C of the extraction effect of 25 DEG C.First group and the 3rd group of Data Comparison are understood, same at a temperature of,
The effect of supersound extraction can be better.Being understood by last two groups of Data Comparisons, the effect of liquid nitrogen grinding is not very good.Comprehensively
Above four kinds of methods, it is known that at 25 DEG C, this method of supersound extraction 1h is best.
Above result is weight in wet base, and the content of the betulic acid that another set dry weight thalline detects is 612.56 μ g/
g。
For fermentation liquid, the present embodiment has only used a kind of this method of supersound extraction 1h at 25 DEG C to extract, and record is white
The content of pine gum acid is 3.2273 μ g/mL.
This example demonstrates that, with the amount of betulic acid all ratios in the thalline of ferment tank and in fermentation liquid with shake flask fermentation
Much higher, illustrate by the effect of fermentation tank more preferable.
Claims (10)
1. utilize the method that fatty acid-induced extracts betulic acid from Inonqqus obliquus, including: by the Inonqqus obliquus of activation
(Inonotus obliquus) CFCC 83414 accesses fluid medium, in 24-26 DEG C of fermentation culture 8-14 days, in fermentation the
Within 5-8 days, add fatty acid, after having fermented, separating thallus and fermentation liquid, separate pure from thalline and fermentation liquid by post processing
Change and obtain betulic acid；
Described fatty acid is in oleic acid, linoleic acid, stearic acid, palmitic acid, linolenic acid, palmitoleic acid, arachidonic acid, Palmic acid
At least one.
Method the most according to claim 1, it is characterised in that described fluid medium is that potato glucose synthesis is cultivated
Base and the mixture of bran water lixiviating solution.
Method the most according to claim 1, it is characterised in that described fermentation uses ferment tank, and fermentation time is 8-
10 days.
Method the most according to claim 1, it is characterised in that described fermentation uses triangular flask fermentation, and fermentation time is 11-
14 days；During cultivation, within first 2-3 days, rotating speed is set to 0rpm/min, cultivates 2-3 days with the rotating speed of 90-110rpm/min the most again, afterwards
Cultivate with the rotating speed of 150-180r/min again.
Method the most according to claim 1, it is characterised in that described fatty acid is oleic acid, linoleic acid, stearic acid, palmitin
At least one in acid.
Method the most according to claim 1, it is characterised in that described fatty acid is oleic acid.
Method the most according to claim 6, it is characterised in that the joining day of described oleic acid is fermentation the 6-7 days.
Method the most according to claim 6, it is characterised in that in terms of every liter of fermentation liquid, the addition of described oleic acid is
0.1-3.0g。
Method the most according to claim 1, it is characterised in that described post processing includes using ethyl acetate by after bacterial cell disruption
Extraction, is extracted with ethyl acetate fermentation liquid, afterwards combining extraction liquid simultaneously, concentrates, obtains betulic acid.
Method the most according to claim 9, it is characterised in that extracting process is: supersound extraction at being placed in 23-27 DEG C
0.8-1.2h。
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| CN201610525648.XA Pending CN106119333A (en) | 2016-06-30 | 2016-06-30 | A kind of method utilizing fatty acid-induced to extract betulic acid from Inonqqus obliquus |
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| CN107841522A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | The method for extracting betulic acid from Inonotus obliquus using jasmonate induction |
| 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 |
| CN113336865A (en) * | 2021-05-20 | 2021-09-03 | 杨培福 | Inonotus obliquus polysaccharide extraction element with edulcoration function |
| CN114767701A (en) * | 2022-05-09 | 2022-07-22 | 黑龙江中医药大学 | Medicine for treating acne and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107841522A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | The method for extracting betulic acid from Inonotus obliquus using jasmonate induction |
| 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 |
| CN113336865A (en) * | 2021-05-20 | 2021-09-03 | 杨培福 | Inonotus obliquus polysaccharide extraction element with edulcoration function |
| CN113336865B (en) * | 2021-05-20 | 2023-08-25 | 天津北洋百川生物技术有限公司 | Inonotus obliquus polysaccharide extraction element with edulcoration function |
| CN114767701A (en) * | 2022-05-09 | 2022-07-22 | 黑龙江中医药大学 | Medicine for treating acne and application thereof |
| CN114767701B (en) * | 2022-05-09 | 2023-10-20 | 黑龙江中医药大学 | Medicine for treating acne and application thereof |
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Application publication date: 20161116 |