CN104278070B - Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius - Google Patents
Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius Download PDFInfo
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- CN104278070B CN104278070B CN201410565891.5A CN201410565891A CN104278070B CN 104278070 B CN104278070 B CN 104278070B CN 201410565891 A CN201410565891 A CN 201410565891A CN 104278070 B CN104278070 B CN 104278070B
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Abstract
The invention discloses a method for improving the content of ergosterol in liquid fermentation products of phellinus igniarius. The method comprises the following steps: (1) preparing a seed solution, namely inoculating activated phellinus igniarius strains onto a liquid seed culture medium added with cyclocarya paliurus extracts and culturing for 3 days-7 days to obtain the seed solution; and (2) carrying out liquid fermentation culture on phellinus igniarius, namely inoculating the seed solution obtained in the step (1) which accounts for 5%-20% of the volume of a liquid fermentation culture medium onto the liquid fermentation culture medium, culturing for 1 days-4 days, adding ubiquitin protein and then continuously culturing for 2 days-4 days, and obtaining liquid fermentation products of phellinus igniarius after the fermentation is completed. According to the characteristic of the synthetic metabolic pathway of ergosterol in liquid fermentation products of medicinal fungi phellinus igniarius, by adding the cyclocarya paliurus extracts and ubiquitin protein at a specific fermentation stage, the content of phellinus igniarius ergosterol is substantially improved.
Description
Technical field
The invention belongs to bio-fermentation engineering field, and in particular to one kind improves Ergota in Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production
The method of sterol content.
Background technology
Phellinus igniarius (L. ex Fr.) Quel. (Phellinus igniarius) belongs to Basidiomycotina, shelf funguses guiding principle, Hymenochaetaceae, Phellinus.
Phellinus igniarius (L. ex Fr.) Quel. fungus extract has remarkable result after anticancer is shifted and prevents cancer operation in the clinical practice of recurrence etc., is
A kind of medicinal fungi of effective percentage highest in current internationally recognized biological anticancer preparation.
Because the particularity and complexity and external environment condition by physiological statuss is restricted, Phellinus igniarius (L. ex Fr.) Quel. shape in nature is caused
Rare into sporophore, particularly forming available sporophore needs for many years.And artificial culture is extremely difficult, condition of culture is harsh,
And growth cycle is up to 3-4, it is difficult to the market demand that satisfaction increasingly expands.Phellinus igniarius (L. ex Fr.) Quel. is produced using the method for Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation
Medicinal active ingredient because have the advantages that with short production cycle, labour force save and by external environment influence it is little be considered as one kind
Effective method.At present, both at home and abroad to phellinus igniarius mycelium culture production active pharmaceutical ingredient research and technique application mainly concentrate
Extract in the aspects such as separation in fermentation condition and product, overall fermentation level is not high.
Ergosterol, also known as ergosterol, is fatsoluble vitamin D2Precursor, can convert when being irradiated with
For vitamin D2.Ergosterol is the important component of fungal cell membrane, and it is in the integrity and membrane bound enzyme for guaranteeing membrane structure
The aspect such as activity, the mobility of film, cell viability and matter transportation plays an important role.Ergosterol is a kind of important medicine
Industrial chemicals, can be used for the production of the medicines such as cortisone, Progesterone, and in food, medicine and feed industry (Cao Long is widely used
Brightness, Li Xiao Jun, Zhao Wenhong etc. the progress [J] of ergosterol. China brewages, and 2014,33 (4)：9-11.).
Ubiquitin (Ubiquitin, Ub), i.e. ubiquitin protein, are a kind of polypeptides, and by 76 Amino acid profiles, its molecular weight is about
8.5kDa, separates first for 1975 from the pancreas of calf, is subsequently found in many different organisms and tissue, general
The aminoacid sequence of element has well-conserved.In recent years, with deepening continuously for studying ubiquitin, it is found that ubiquitin is not only
The labelling of protein degradation, or base of the cell maintenance to those protein levels produced by composing type regulation and environmental stimuluses
This regulative mode, intracellular protein ubiquitination participates in the various physiological processes of cell, and the labelling, DNA in protein degradation is repaiied
Again, play an important role in each vital movement such as gene transcription regulation and signal transduction, various diseases, the plant with the mankind
The physiological and biochemical procedure of thing exist and be closely connected (Huang Xinmin, Zhang Yanxia, Wan little Rong. the progress [J] of ubiquitin protein. it is wide
Eastern agricultural sciences, 2010,6:191-194.).
The liquid fermentation that ubiquitin protein is used for Phellinus igniarius (L. ex Fr.) Quel. is produced, the content of its ergosterol is improved, is not yet appeared in the newspapers both at home and abroad
Road.
The content of the invention
The present invention is directed to the deficiencies in the prior art, there is provided one kind is improved in Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production using ubiquitin protein
The method of Quantitative Determination of Ergosterol.
The technical solution used in the present invention is：
A kind of method for improving Quantitative Determination of Ergosterol in Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production, including step：
(1) prepared by seed liquor：Phellinus igniarius (L. ex Fr.) Quel. strain after activation is inoculated in into the liquid seeds culture of addition Cyclocarya paliurus Iljinskaja extract
Cultivate -7 days 3 days in base, obtain seed liquor；
(2) Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation and culture：By the seed liquor in step (1) by accounting for liquid fermentation medium volume 5%-20%
Amount be inoculated in liquid fermentation medium, after culture -4 days 1 day, add ubiquitin protein, then proceed to culture -4 days 2 days, eventually
After only fermenting, Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production is obtained.
The present invention according to the characteristics of Medicinal Fungus Phellinus igniarius liquid fermentation production ergosterol metabolic pathway of synthesizing, by addition
Cyclocarya paliurus Iljinskaja extract simultaneously adds ubiquitin protein in specific fermentation stage, and Ergota in Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production is greatly improved
The content of sterol.
Described Phellinus igniarius (L. ex Fr.) Quel. strain can adopt any one Phellinus igniarius (L. ex Fr.) Quel. strain, can adopt commercially available prod.Such as Phellinus igniarius (L. ex Fr.) Quel.
(Phellinus linteus) ACCC51181 strains, from Chinese agriculture Microbiological Culture Collection administrative center.
In order to reach more preferable invention effect, carry out following preferred：
In step (1), the addition of Cyclocarya paliurus Iljinskaja extract is 1g-10g in every liter of liquid seed culture medium.
Described Cyclocarya paliurus Iljinskaja extract can select commercially available prod, such as Xi'an Sai Ao Bioisystech Co., Ltd production
Cyclocarya paliurus Iljinskaja extract etc., it would however also be possible to employ prepared by existing method；It is preferred that the Cyclocarya paliurus Iljinskaja extract prepared as raw material with leaf of Cyclocarya paliurus Iljinskaja；
The Cyclocarya paliurus Iljinskaja extract being further preferably made up of leaf of Cyclocarya paliurus Iljinskaja ethanol extraction and leaf of Cyclocarya paliurus Iljinskaja water extract, can adopt following
Cyclocarya paliurus Iljinskaja extract prepared by preparation method, including：
Weigh a certain amount of leaf of Cyclocarya paliurus Iljinskaja, crush (mesh of 40 mesh of preferred mistake -100), first plus account for 10 times of amounts of leaf of Cyclocarya paliurus Iljinskaja weight -
The mass percentage concentration of 16 times of amounts extracts 1h-2h for the ethanol water of 60%-80% at 60 DEG C -80 DEG C, obtains after filtration
Clear liquid, vacuum lyophilization after supernatant concentration obtains extract I；Leaf of Cyclocarya paliurus Iljinskaja residue after above-mentioned alcohol extraction is added and accounts for leaf of Cyclocarya paliurus Iljinskaja
The water of weight -20 times of amounts of 10 times of amounts extracts 1h-2.5h at 85 DEG C -95 DEG C, and supernatant, supernatant concentration to 1/ are obtained after filtration
The dehydrated alcohol of concentrate volume -5 times of amounts of 2 times of amounts is added after 4 volumes, precipitate, precipitate vacuum lyophilization is collected by centrifugation
Extract II is obtained afterwards, merges said extracted thing I and extract II obtains Cyclocarya paliurus Iljinskaja extract.
In step (1), the Phellinus igniarius (L. ex Fr.) Quel. strain after described activation is in the liquid seed culture medium of addition Cyclocarya paliurus Iljinskaja extract
The temperature of culture be natural environment temperature, preferably 20 DEG C -30 DEG C.General 1L adds the liquid seeds culture of Cyclocarya paliurus Iljinskaja extract
2cm is accessed in base2-5cm2Phellinus igniarius (L. ex Fr.) Quel. strain truffle after the activation of size.
In step (1), the activation method of Phellinus igniarius (L. ex Fr.) Quel. strain is the conventional actication of culture method in this area, including：Inclined-plane is protected
The Phellinus igniarius (L. ex Fr.) Quel. strain of Tibetan is inoculated in PDA plate culture medium, carries out activation culture, 22 DEG C -30 DEG C of cultivation temperature, and incubation time 4 days -
10 days, the Phellinus igniarius (L. ex Fr.) Quel. strain after being activated.
The culture medium that described PDA plate culture medium and liquid seed culture medium is commonly used using this area seed culture,
Commercially available prod can be adopted.It is preferred that, described PDA plate culture medium：Rhizoma Solani tuber osi 200g, glucose 20g and agar 15g-20g, use
Water is settled to 1000mL.It is preferred that, described liquid seed culture medium：Glucose 5g-20g, yeast powder 4g, peptone 3g,
KH2PO41g and MgSO40.5g, with water 1000mL is settled to.
In step (2), described liquid fermentation medium is consisted of：In terms of 1L liquid fermentation mediums, glucose
20g, peptone 4g, Testa Tritici 5g, glutamic acid 0.5g-5g, KH2PO41.5g、MgSO4The water of 0.75g and surplus.
It is preferred that, add ubiquitin protein 0.2mg-3.0mg in every milliliters of liquid fermentation medium；Further preferably every milliliter of liquid
Add ubiquitin protein 0.8mg-2.4mg in body fermentation medium；Most preferably, ubiquitin is added in every milliliters of liquid fermentation medium
Albumen 1.6mg.
Described ubiquitin protein is using the ubiquitin protein extracted from the great majority food raw material such as medicine funguses or plant, Ke Yixuan
With commercially available prod, it would however also be possible to employ prepared by existing method, the ubiquitin protein extracted such as from Ganoderma raw material can adopt following preparation
It is prepared by method, including：The distilled water for accounting for Ganoderma superfine powder quality -30 times of amounts of 10 times of amounts is added in Ganoderma superfine powder, is added
The cellulase for accounting for Ganoderma superfine powder quality 0.5%-3% and the pectase for accounting for Ganoderma superfine powder quality 0.3%-2%, 35
DEG C of -55 DEG C stirring 1h-2h, are then homogenized under 70MPa-100MPa；10000rpm-30000rpm (rpm under the conditions of 3 DEG C -5 DEG C
Expression rev/min) centrifugation 10min-15min, collects supernatant and obtains crude protein liquid；Crude protein liquid is passed through into micro-filtration membrane (such as
0.22 μm of micro-filtration membrane) filtered solution is collected, to remove microgranule therein and antibacterial；The filtered solution is used at 3 DEG C -5 DEG C and is cut
Stay the ultrafilter membrane that molecular weight is 5000Da-10000Da to be concentrated, obtain ubiquitin protein.
Described Ganoderma superfine powder is obtained for Ganoderma Jing micronizing, and its preparation includes：By Ganoderma sporophore cleaning, drying
After carry out micronizing (micronizing is carried out such as in super micron mill), obtain Ganoderma superfine powder.Described micronizing is excellent
Temperature is selected to be 10 DEG C -40 DEG C, process time 3min-20min.
The enzyme activity of described pectase is preferably the U/g of 3.0 ten thousand U/g-5.0 ten thousand, the enzyme activity of cellulase and is preferably 2.0
The U/g of ten thousand U/g-3.0 ten thousand.
The temperature of described Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation and culture be natural environment temperature, generally 20 DEG C -30 DEG C, preferably 25
℃-30℃。
The present invention, described Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production can be carried through follow-up separating treatment from phellinus igniarius mycelium
Take, determine Quantitative Determination of Ergosterol.Described separating treatment is the conventional separation method in this area, for example, filter or be centrifuged；Can wrap
Include：By described Phellinus igniarius (L. ex Fr.) Quel. tunning filtered through gauze, extract in gained mycelium, determine Quantitative Determination of Ergosterol from filtering.Tool
The extraction step of body includes：Mycelium is rinsed well with water, in 55 DEG C of -65 DEG C of dryings to constant weight, by feed liquid mass ratio 1:10-
30 add CHCl3, ultrasound wave (preferred power 250W-300W, frequency 40kHz-50kHz) extraction 1h-1.5h, centrifuging and taking supernatant
Liquid, residue CHCl3Centrifuging and taking supernatant after washing, merges supernatant and obtains the extracting solution containing ergosterol.
Culture medium used by the present invention is both needed to sterilize, cool down after use, the condition of sterilizing using this area normal condition,
For example can sterilize 20min-30min at 120 DEG C -125 DEG C.
Cyclocarya paliurus Iljinskaja (Cyclocarya paliurus) also known as blue or green money Lee, money tree etc., are Juglandaceae cyclocarya plant,
It is Chinese distinctive single platymiscium.Cyclocarya paliurus Iljinskaja is a kind of tall and big fast-growing arbor, imparipinnate leafs, be distributed widely in Jiangxi,
The mountain area of the height above sea level 420m-2500m on the ground such as Zhejiang, Jiangsu, Anhui, Fujian, Taiwan, Hubei, Sichuan, Guizhou, trench or Calx
Rock mountain region.Its bark, leaveves have heat-clearing and toxic substances removing, pain-relieving functions.
Ganoderma (Ganoderma Lucidum (Leyss.ex Fr.) Karst.) profile is in umbrella, cap kidney shape, semicircle
Or subcircular, it is the sporophore of porous castor section fungi glossy ganoderma.With invigorating QI and tranquilization, effect of relieving cough and asthma, for dizziness sleeplessness,
Shortness of breath and palpitation, asthenia cough with asthma.
The present invention compared with the existing technology has the following advantages：
1. the present invention produces the liquid fermentation that ubiquitin protein is used for Phellinus igniarius (L. ex Fr.) Quel. ergosterol constituents, and optimizes fermentation side
Method, is greatly improved the content of ergosterol in Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production, can reach 2.15mg/g, the ergosterol of extraction
In can be used for food, medicine and feed industry.
2. phellinus igniarius mycelium liquid fermentation method of the present invention is simple, and reproducible, fermentation period is short, efficiency
Height, while by the use of natural product as raw materials for production, environment-protecting asepsis, low cost.Whole sweat is controllable, not by external environment condition
Condition is limited, and is especially suitable for industrialized production and application.The inventive method is equally applicable to fermentation tank large-scale production, tool
There is good industrial applications prospect.
3. the ubiquitin protein that the present invention is adopted can be extracted from the great majority food raw material such as medicine funguses and plant, wide material sources,
Cost is relatively low, and preparation method is also relatively easy, can scale extract production, the fermenting and producing of Phellinus igniarius (L. ex Fr.) Quel. ergosterol active substance is had
There is good facilitation effect.
Specific embodiment
Present invention is further elucidated with reference to some embodiments, but present disclosure and not only
It is limited to the following examples.
Phellinus igniarius (L. ex Fr.) Quel. (Phellinus linteus) ACCC51181 strains are purchased from the management of Chinese agriculture Microbiological Culture Collection
The heart.
Embodiment 1
First, material prepares
Pectase (enzyme activity is 5.0 ten thousand U/g), cellulase (enzyme activity is 3.0 ten thousand U/g), by Guangxi Pang Bo biology works
Journey company limited provides.
PDA plate culture medium：Rhizoma Solani tuber osi 200g, glucose 20g and agar 15g, with water 1000ml, natural pH are settled to,
Sterilize 20min at 121 DEG C.
The Phellinus igniarius (L. ex Fr.) Quel. strain of slant preservation is inoculated in PDA plate culture medium, activation culture is carried out, 25 DEG C of cultivation temperature,
Incubation time 7 days, the Phellinus igniarius (L. ex Fr.) Quel. strain truffle after being activated.
The preparation method of Cyclocarya paliurus Iljinskaja extract, including：A certain amount of leaf of Cyclocarya paliurus Iljinskaja is weighed, 100 mesh were crushed, is first added and is accounted for green grass or young crops
The mass percentage concentration of 13 times of amounts of money Folium Salicis Babylonicae weight is that 70% ethanol water extracts 1.0h at 60 DEG C, and supernatant is obtained after filtration
Liquid, vacuum lyophilization after supernatant concentration obtains extract I；Leaf of Cyclocarya paliurus Iljinskaja residue after above-mentioned alcohol extraction is added and accounts for leaf of Cyclocarya paliurus Iljinskaja weight
The distilled water of 10 times of amounts of amount extracts 1h at 85 DEG C, and supernatant is obtained after filtration, and concentration is added after supernatant concentration to 1/4 volume
Object accumulates the dehydrated alcohol of 2 times of amounts, and 5000rpm centrifugation 10min collect precipitate, extract is obtained after precipitate vacuum lyophilization
II, merge said extracted thing I and extract II obtains Cyclocarya paliurus Iljinskaja extract.
With the ubiquitin protein that Ganoderma prepares as raw material, preparation method includes：It is super by being put into after Ganoderma sporophore cleaning, drying
Ultra micro low-temperature grinding is carried out in atomizer, 25 DEG C of temperature, process time 10min obtains Ganoderma superfine powder.To Ganoderma superfine powder
Middle addition accounts for the distilled water of 20 times of amounts of Ganoderma superfine powder quality, adds and accounts for the cellulase of Ganoderma superfine powder quality 1% and account for
The pectase of Ganoderma superfine powder quality 0.5%, at 40 DEG C 1h is stirred, and is then placed in refiner being homogenized 3 times under 80MPa；4℃
Under the conditions of 10000rpm centrifugation 15min, collect supernatant obtain crude protein liquid；Crude protein liquid is collected by 0.22 μm of micro-filtration membrane
Filtered solution, to remove microgranule therein and antibacterial；By the filtered solution at 4 DEG C with the ultrafilter membrane that molecular cut off is 8000Da
Concentrated, obtained concentrated solution, as ubiquitin protein.
2nd, the preparation of Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production
(1) prepared by seed liquor：Take 2cm2Phellinus igniarius (L. ex Fr.) Quel. strain truffle after size activation is inoculated in 1L addition Cyclocarya paliurus Iljinskaja extracts
Liquid seed culture medium in, in every liter of liquid seed culture medium the addition of Cyclocarya paliurus Iljinskaja extract be 8g, 1L liquid seeds training
Support basis set becoming：Glucose 10g/L, yeast powder 4g/L, peptone 3g/L, KH2PO41g/L、MgSO4The water of 0.5g/L and surplus,
PH natures；25 DEG C are cultivated 4 days, obtain seed liquor.
(2) Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation and culture：Seed liquor is inoculated in into liquid by the amount for accounting for liquid fermentation medium volume 10%
In fermentation medium, after cultivating 2 days at 28 DEG C, ubiquitin protein is added, in every milliliters of liquid fermentation medium ubiquitin protein is added
1.6mg, then proceedes to culture 3 days, after terminating fermentation, obtains Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production.Wherein, the group of liquid fermentation medium
Become：Glucose 20g/L, peptone 4g/L, Testa Tritici 5g/L, glutamic acid 2.5g/L, KH2PO41.5g/L、MgSO40.75g/L and
The water of surplus.
3rd, determine
1. the measure of hypha biomass：8 layers of filtered through gauze of Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production Jing, filter gained mycelium distillation
Water is rinsed 3 times, is then put and dry in 60 DEG C of baking ovens to constant weight, as sample, is weighed.
2. ergosterol is extracted：Precision weighs 1g mycelium samples, is placed in 25mL volumetric flasks, adds 20mL CHCl3Liquid,
Ultrasonic-leaching 1h (power 250W, frequency 40kHz), then 4000r/min centrifugations 5min, takes supernatant, and residue is used
5mLCHCl34000r/min centrifugations 5min, takes supernatant after washing.Merge supernatant twice and obtain the extraction containing ergosterol
Liquid.
3. Quantitative Determination of Ergosterol is determined：By the extracting solution CHCl containing ergosterol3It is settled to 25mL and obtains extract, takes
2mL extracts, nitrogen is dissolved after drying up with 5mL methanol, and 0.45 μm of filtering with microporous membrane of Jing, filtrate is analyzed for HPLC.
High-efficient liquid phase chromatogram condition：Chromatographic column：Agilent C18Chromatographic column (200mm × 4.6mm, 5 μm)；Mobile phase：Methanol
(chromatographic grade), flow velocity 1.2mL/min；UV Detection wavelengths：Detection wavelength 282nm, 30 DEG C of column temperature, the μ L of sample size 10.
Testing result is shown in Table 1.
Embodiment 2
First, material prepares
Pectase (enzyme activity is 3.0 ten thousand U/g), cellulase (enzyme activity is 2.0 ten thousand U/g), by Guangxi Pang Bo biology works
Journey company limited provides.
PDA plate culture medium：Rhizoma Solani tuber osi 200g, glucose 20g and agar 20g, with water 1000ml, natural pH are settled to,
Sterilize 20min at 121 DEG C.
The Phellinus igniarius (L. ex Fr.) Quel. strain of slant preservation is inoculated in PDA plate culture medium, activation culture is carried out, 28 DEG C of cultivation temperature,
Incubation time 9 days, the Phellinus igniarius (L. ex Fr.) Quel. strain truffle after being activated.
The preparation method of Cyclocarya paliurus Iljinskaja extract, including：A certain amount of leaf of Cyclocarya paliurus Iljinskaja is weighed, 60 mesh were crushed, is first added and is accounted for green grass or young crops
The mass percentage concentration of 16 times of amounts of money Folium Salicis Babylonicae weight is that 80% ethanol water extracts 2h at 80 DEG C, and supernatant is obtained after filtration
Liquid, vacuum lyophilization after supernatant concentration obtains extract I；Leaf of Cyclocarya paliurus Iljinskaja residue after above-mentioned alcohol extraction is added and accounts for leaf of Cyclocarya paliurus Iljinskaja weight
The distilled water of 20 times of amounts of amount extracts 2.5h at 95 DEG C, and supernatant is obtained after filtration, adds after supernatant concentration to 1/4 volume dense
Contracting object accumulates the dehydrated alcohol of 5 times of amounts, and 5000rpm centrifugation 10min collect precipitate, must extract after precipitate vacuum lyophilization
Thing II, merges said extracted thing I and extract II obtains Cyclocarya paliurus Iljinskaja extract.
With the ubiquitin protein that Ganoderma prepares as raw material, preparation method includes：It is super by being put into after Ganoderma sporophore cleaning, drying
Ultra micro low-temperature grinding is carried out in atomizer, 40 DEG C of temperature, process time 3min obtains Ganoderma superfine powder.To Ganoderma superfine powder
Middle addition accounts for the distilled water of 30 times of amounts of Ganoderma superfine powder quality, adds and accounts for the cellulase of Ganoderma superfine powder quality 3% and account for
The pectase of Ganoderma superfine powder quality 0.3%, at 55 DEG C 1.5h is stirred, and is then placed in refiner being homogenized 3 times under 100MPa；
30000rpm centrifugations 10min under the conditions of 5 DEG C, collects supernatant and obtains crude protein liquid；Crude protein liquid is passed through into 0.22 μm of micro-filtration membrane
Filtered solution is collected, to remove microgranule therein and antibacterial；At 5 DEG C it is 10000Da's with molecular cut off by the filtered solution
Ultrafilter membrane is concentrated, and obtains concentrated solution, as ubiquitin protein.
2nd, the preparation of Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production
(1) prepared by seed liquor：Take 5cm2Phellinus igniarius (L. ex Fr.) Quel. strain truffle after size activation is inoculated in 1L addition Cyclocarya paliurus Iljinskaja extracts
Liquid seed culture medium in, in every liter of liquid seed culture medium the addition of Cyclocarya paliurus Iljinskaja extract be 10g, 1L liquid seeds training
Support basis set becoming：Glucose 15g/L, yeast powder 4g/L, peptone 3g/L, KH2PO41g/L、MgSO4The water of 0.5g/L and surplus,
PH natures；28 DEG C are cultivated 5 days, obtain seed liquor.
(2) Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation and culture：Seed liquor is inoculated in into liquid by the amount for accounting for liquid fermentation medium volume 15%
In fermentation medium, after cultivating 3 days at 28 DEG C, ubiquitin protein is added, in every milliliters of liquid fermentation medium ubiquitin protein is added
0.8mg, then proceedes to culture 4 days, after terminating fermentation, obtains Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production.Wherein, the group of liquid fermentation medium
Become：Glucose 20g/L, peptone 4g/L, Testa Tritici 5g/L, glutamic acid 5g/L, KH2PO41.5g/L、MgSO40.75g/L and remaining
The water of amount.
3rd, determine
1. the measure of hypha biomass：8 layers of filtered through gauze of Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production Jing, filter gained mycelium distillation
Water is rinsed 3 times, is then put and dry in 65 DEG C of baking ovens to constant weight, as sample, is weighed.
2. ergosterol is extracted：Precision weighs 1g samples, is placed in 25mL volumetric flasks, adds 6.8mLCHCl3Liquid, ultrasound wave
Extraction 1.5h (power 300W, frequency 50kHz), then 4000r/min centrifugations 5min, takes supernatant, residue 5mLCHCl3Wash
Rear 4000r/min centrifugations 5min is washed, supernatant is taken.Merge supernatant twice and obtain the extracting solution containing ergosterol.
3. Quantitative Determination of Ergosterol is determined：By the extracting solution CHCl containing ergosterol3It is settled to 25mL and obtains extract, takes
2mL extracts, nitrogen is dissolved after drying up with 5mL methanol, and 0.45 μm of filtering with microporous membrane of Jing, filtrate is analyzed for HPLC.
High-efficient liquid phase chromatogram condition, with embodiment 1.
Testing result is shown in Table 1.
Embodiment 3
First, material prepares
Pectase (enzyme activity is 4.0 ten thousand U/g), cellulase (enzyme activity is 2.0 ten thousand U/g), by Guangxi Pang Bo biology works
Journey company limited provides.
PDA plate culture medium：Rhizoma Solani tuber osi 200g, glucose 20g and agar 15g, with water 1000ml, natural pH are settled to,
Sterilize 20min at 121 DEG C.
The Phellinus igniarius (L. ex Fr.) Quel. strain of slant preservation is inoculated in PDA plate culture medium, activation culture is carried out, 30 DEG C of cultivation temperature,
Incubation time 4 days, the Phellinus igniarius (L. ex Fr.) Quel. strain truffle after being activated.
The preparation method of Cyclocarya paliurus Iljinskaja extract, including：A certain amount of leaf of Cyclocarya paliurus Iljinskaja is weighed, 40 mesh were crushed, is first added and is accounted for green grass or young crops
The mass percentage concentration of 10 times of amounts of money Folium Salicis Babylonicae weight is that 60% ethanol water extracts 1.5h at 70 DEG C, and supernatant is obtained after filtration
Liquid, vacuum lyophilization after supernatant concentration obtains extract I；Leaf of Cyclocarya paliurus Iljinskaja residue after above-mentioned alcohol extraction is added and accounts for leaf of Cyclocarya paliurus Iljinskaja weight
The distilled water of 15 times of amounts of amount extracts 1.5h at 90 DEG C, and supernatant is obtained after filtration, adds after supernatant concentration to 1/4 volume dense
Contracting object accumulates the dehydrated alcohol of 4 times of amounts, and 5000rpm centrifugation 10min collect precipitate, must extract after precipitate vacuum lyophilization
Thing II, merges said extracted thing I and extract II obtains Cyclocarya paliurus Iljinskaja extract.
With the ubiquitin protein that Ganoderma prepares as raw material, preparation method includes：It is super by being put into after Ganoderma sporophore cleaning, drying
Ultra micro low-temperature grinding is carried out in atomizer, 10 DEG C of temperature, process time 20min obtains Ganoderma superfine powder.To Ganoderma superfine powder
Middle addition accounts for the distilled water of 10 times of Ganoderma superfine powder quality amount, add the cellulase that accounts for Ganoderma superfine powder quality 0.5% and
The pectase of Ganoderma superfine powder quality 2% is accounted for, at 35 DEG C 2h is stirred, be then placed in refiner being homogenized 3 times under 70MPa；3℃
Under the conditions of 20000rpm centrifugation 10min, collect supernatant obtain crude protein liquid；Crude protein liquid is collected by 0.22 μm of micro-filtration membrane
Filtered solution, to remove microgranule therein and antibacterial；By the filtered solution at 3 DEG C with the ultrafilter membrane that molecular cut off is 5000Da
Concentrated, obtained concentrated solution, as ubiquitin protein.
2nd, the preparation of Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production
(1) prepared by seed liquor：Take 3cm2Phellinus igniarius (L. ex Fr.) Quel. strain truffle after size activation is inoculated in 1L addition Cyclocarya paliurus Iljinskaja extracts
Liquid seed culture medium in, in every liter of liquid seed culture medium the addition of Cyclocarya paliurus Iljinskaja extract be 5g, 1L liquid seeds training
Support basis set becoming：Glucose 20g/L, yeast powder 4g/L, peptone 3g/L, KH2PO41g/L、MgSO4The water of 0.5g/L and surplus,
PH natures；22 DEG C are cultivated 6 days, obtain seed liquor.
(2) Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation and culture：Seed liquor is inoculated in into liquid by the amount for accounting for liquid fermentation medium volume 20%
In fermentation medium, after cultivating 4 days at 30 DEG C, ubiquitin protein is added, in every milliliters of liquid fermentation medium ubiquitin protein is added
2.4mg, then proceedes to culture 2 days, after terminating fermentation, obtains Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production.Wherein, the group of liquid fermentation medium
Become：Glucose 20g/L, peptone 4g/L, Testa Tritici 5g/L, glutamic acid 4g/L, KH2PO41.5g/L、MgSO40.75g/L and remaining
The water of amount.
3rd, determine with embodiment 1.Testing result is shown in Table 1.
Embodiment 4
First, material prepares
Pectase (enzyme activity is 5.0 ten thousand U/g), cellulase (enzyme activity is 3.0 ten thousand U/g), by Guangxi Pang Bo biology works
Journey company limited provides.
PDA plate culture medium：Rhizoma Solani tuber osi 200g, glucose 20g and agar 15g, with water 1000ml, natural pH are settled to,
Sterilize 20min at 121 DEG C.
The Phellinus igniarius (L. ex Fr.) Quel. strain of slant preservation is inoculated in PDA plate culture medium, activation culture is carried out, 22 DEG C of cultivation temperature,
Incubation time 10 days, the Phellinus igniarius (L. ex Fr.) Quel. strain truffle after being activated.
Cyclocarya paliurus Iljinskaja extract and ubiquitin protein are with embodiment 1.
2nd, the preparation of Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production
(1) prepared by seed liquor：Take 3cm2Phellinus igniarius (L. ex Fr.) Quel. strain truffle after size activation is inoculated in 1L addition Cyclocarya paliurus Iljinskaja extracts
Liquid seed culture medium in, in every liter of liquid seed culture medium the addition of Cyclocarya paliurus Iljinskaja extract be 1g, 1L liquid seeds training
Support basis set becoming：Glucose 8g/L, yeast powder 4g/L, peptone 3g/L, KH2PO41g/L、MgSO4The water of 0.5g/L and surplus,
PH natures；30 DEG C are cultivated 3 days, obtain seed liquor.
(2) Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation and culture：Seed liquor is inoculated in into liquid by the amount for accounting for liquid fermentation medium volume 8% to send out
In ferment culture medium, after cultivating 1 day at 22 DEG C, ubiquitin protein is added, in every milliliters of liquid fermentation medium ubiquitin protein is added
0.2mg, then proceedes to culture 2 days, after terminating fermentation, obtains Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production.Wherein, the group of liquid fermentation medium
Become：Glucose 20g/L, peptone 4g/L, Testa Tritici 5g/L, glutamic acid 1.5g/L, KH2PO41.5g/L、MgSO40.75g/L and
The water of surplus.
3rd, determine with embodiment 1.Testing result is shown in Table 1.
Embodiment 5
First, material prepares
Pectase (enzyme activity is 5.0 ten thousand U/g), cellulase (enzyme activity is 3.0 ten thousand U/g), by Guangxi Pang Bo biology works
Journey company limited provides.
PDA plate culture medium：Rhizoma Solani tuber osi 200g, glucose 20g and agar 15g, with water 1000ml, natural pH are settled to,
Sterilize 20min at 121 DEG C.
The Phellinus igniarius (L. ex Fr.) Quel. strain of slant preservation is inoculated in PDA plate culture medium, activation culture is carried out, 22 DEG C of cultivation temperature,
Incubation time 10 days, the Phellinus igniarius (L. ex Fr.) Quel. strain truffle after being activated.
Cyclocarya paliurus Iljinskaja extract, is purchased from Xi'an Sai Ao Bioisystech Co., Ltd.
Ubiquitin protein, with embodiment 1.
2nd, the preparation of Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production
(1) prepared by seed liquor：Take 3cm2Phellinus igniarius (L. ex Fr.) Quel. strain truffle after size activation is inoculated in 1L addition Cyclocarya paliurus Iljinskaja extracts
Liquid seed culture medium in, in every liter of liquid seed culture medium the addition of Cyclocarya paliurus Iljinskaja extract be 6g, 1L liquid seeds training
Support basis set becoming：Glucose 5g/L, yeast powder 4g/L, peptone 3g/L, KH2PO41g/L、MgSO4The water of 0.5g/L and surplus,
PH natures；20 DEG C are cultivated 7 days, obtain seed liquor.
(2) Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation and culture：Seed liquor is inoculated in into liquid by the amount for accounting for liquid fermentation medium volume 5% to send out
In ferment culture medium, after cultivating 1 day at 20 DEG C, ubiquitin protein is added, in every milliliters of liquid fermentation medium ubiquitin protein is added
3mg, then proceedes to culture 2 days, after terminating fermentation, obtains Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production.Wherein, the composition of liquid fermentation medium
For：Glucose 20g/L, peptone 4g/L, Testa Tritici 5g/L, glutamic acid 0.5g/L, KH2PO41.5g/L、MgSO40.75g/L and remaining
The water of amount.
3rd, determine with embodiment 1.Testing result is shown in Table 1.
Comparative example 1
(1) shake-flask seed culture
1L liquid seed culture mediums：Glucose 10g/L, yeast powder 4g/L, peptone 3g/L, KH2PO41g/L、
MgSO4The water of 0.5g/L and surplus.PH natures, sterilize 20min at 121 DEG C.
Cultural method：Take 2cm2Phellinus igniarius (L. ex Fr.) Quel. strain truffle after activating in size embodiment 1 accesses 1L liquid after sterilizing cooling
In seed culture medium, 25 DEG C, shaking table culture 4 days under 120r/min obtain cultured seed liquor.
(2) shake flask fermentation culture
Liquid fermentation medium is with embodiment 1.PH natures, sterilize 20min at 121 DEG C.
Cultural method：Cultured seed liquor is accessed into liquid by the inoculum concentration for accounting for liquid fermentation medium volume 10% to send out
In ferment culture medium, at 25 DEG C, cultivate 12 days in 120r/min shaking tables.Each test set 3 it is parallel.
Testing result is shown in Table 1.
Comparative example 2 only adds Cyclocarya paliurus Iljinskaja extract, without ubiquitin protein
Except " two, it is prepared by preparation (1) seed liquor of Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production：Take 3cm2Phellinus igniarius (L. ex Fr.) Quel. bacterium after size activation
Plant truffle to be inoculated in the liquid seed culture medium of 1L addition Cyclocarya paliurus Iljinskaja extracts, Cyclocarya paliurus Iljinskaja is carried in every liter of liquid seed culture medium
The addition for taking thing is 6g, and 1L liquid seed culture mediums are consisted of：Glucose 5g/L, yeast powder 4g/L, peptone 3g/L,
KH2PO41g/L、MgSO4The water of 0.5g/L and surplus, pH natures；20 DEG C are cultivated 7 days, obtain seed liquor.(2) Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation
Culture：Seed liquor is inoculated in liquid fermentation medium by the amount for accounting for liquid fermentation medium volume 5%, at 20 DEG C 1 is cultivated
After it, continue to cultivate 2 days, after terminating fermentation, obtain Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production.Wherein, liquid fermentation medium is consisted of：
Glucose 20g/L, peptone 4g/L, Testa Tritici 5g/L, glutamic acid 0.5g/L, KH2PO41.5g/L、MgSO40.75g/L and surplus
Water." outside, remaining operation is with embodiment 5.Testing result is shown in Table 1.
Comparative example 3 only adds ubiquitin protein, without Cyclocarya paliurus Iljinskaja extract
Except " two, it is prepared by preparation (1) seed liquor of Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production：Take 3cm2Phellinus igniarius (L. ex Fr.) Quel. bacterium after size activation
Plant truffle and be inoculated in liquid seed culture medium, 1L liquid seed culture mediums consist of glucose 5g/L, yeast powder 4g/L, peptone
3g/L、KH2PO41g/L、MgSO4The water of 0.5g/L and surplus, pH natures；20 DEG C are cultivated 7 days, obtain seed liquor.(2) Phellinus igniarius (L. ex Fr.) Quel. liquid
Body fermentation culture：Seed liquor is inoculated in liquid fermentation medium by the amount for accounting for liquid fermentation medium volume 5%, at 20 DEG C
After culture 1 day, ubiquitin protein is added, in every milliliters of liquid fermentation medium ubiquitin protein 3mg is added, then proceed to culture 2 days,
After terminating fermentation, Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production is obtained.Wherein, liquid fermentation medium is consisted of：Glucose 20g/L, albumen
Peptone 4g/L, Testa Tritici 5g/L, glutamic acid 0.5g/L, KH2PO41.5g/L、MgSO4The water of 0.75g/L and surplus." outside, remaining behaviour
Make with embodiment 5.Testing result is shown in Table 1.
The Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation metabolite content testing result of table 1
Note：Compare with comparative example 1, Δ：P<0.05；ΔΔ：P<0.01；
Compare with comparative example 2, *：P<0.05；**：P<0.01；
Compare with comparative example 3, #：P<0.05；##：P<0.01.
By the data display of table 1, the present invention is according to Medicinal Fungus Phellinus igniarius liquid fermentation production ergosterol anabolism way
The characteristics of footpath, by adding Cyclocarya paliurus Iljinskaja extract and adding ubiquitin protein in specific fermentation stage, Phellinus igniarius (L. ex Fr.) Quel. is greatly improved
Quantitative Determination of Ergosterol.
Compared with comparative example 1, using Ergota steroid in the Phellinus igniarius (L. ex Fr.) Quel. metabolite mycelium of the inventive method liquid fermentation and culture
Alcohol content improves 44.4%-49.3%.
The change of each parameter in the range of preparation method of the present invention is limited has no effect on Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation metabolite
The combination of arbitrary parameter in the raising of middle Quantitative Determination of Ergosterol, therefore preparation method of the present invention is capable of achieving Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation generation
Thank to the raising of product Quantitative Determination of Ergosterol.Will not be described here.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvement can also be made, these improvement also should be regarded as the present invention's
Protection domain.
Claims (9)
1. it is a kind of improve Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production in Quantitative Determination of Ergosterol method, it is characterised in that including step：
(1) prepared by seed liquor：Phellinus igniarius (L. ex Fr.) Quel. strain after activation is inoculated in the liquid seed culture medium of addition Cyclocarya paliurus Iljinskaja extract
Culture -7 days 3 days, obtains seed liquor；
(2) Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation and culture：By the seed liquor in step (1) by the amount for accounting for liquid fermentation medium volume 5%-20%
In being inoculated in liquid fermentation medium, after cultivating -4 days 1 day, ubiquitin protein is added, then proceed to culture -4 days 2 days, terminate sending out
After ferment, Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production is obtained；
Described Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation production extracts through follow-up separating treatment from phellinus igniarius mycelium, determine ergosterol contains
Amount.
2. method according to claim 1, it is characterised in that in step (1), Cyclocarya paliurus Iljinskaja in every liter of liquid seed culture medium
The addition of extract is 1g-10g.
3. method according to claim 1, it is characterised in that described Cyclocarya paliurus Iljinskaja extract is with leaf of Cyclocarya paliurus Iljinskaja as raw material
The Cyclocarya paliurus Iljinskaja extract of preparation.
4. method according to claim 3, it is characterised in that described Cyclocarya paliurus Iljinskaja extract is by leaf of Cyclocarya paliurus Iljinskaja alcohol extracting thing
With leaf of Cyclocarya paliurus Iljinskaja water extract composition.
5. the method according to claim 1 or 3, it is characterised in that the preparation method of described Cyclocarya paliurus Iljinskaja extract, bag
Include：A certain amount of leaf of Cyclocarya paliurus Iljinskaja is weighed, is crushed, first plus accounted for the mass percentage concentration of 10 times of leaf of Cyclocarya paliurus Iljinskaja weight -16 times of amounts of amount and be
The ethanol water of 60%-80% obtains supernatant in 60 DEG C of -80 DEG C of extraction 1h-2h after filtration, vacuum is cold after supernatant concentration
Lyophilizing is dry to obtain extract I；Leaf of Cyclocarya paliurus Iljinskaja residue after above-mentioned alcohol extraction adds the water for accounting for leaf of Cyclocarya paliurus Iljinskaja weight -20 times of amounts of 10 times of amounts to exist
1h-2.5h is extracted at 85 DEG C -95 DEG C, supernatant is obtained after filtration, concentrate volume 2 is added after supernatant concentration to 1/4 volume
Again the dehydrated alcohol of -5 times of amounts of amount, are collected by centrifugation precipitate, and extract II is obtained after precipitate vacuum lyophilization, merge above-mentioned carrying
Take thing I and extract II obtains Cyclocarya paliurus Iljinskaja extract.
6. method according to claim 1, it is characterised in that in step (2), the temperature of described Phellinus igniarius (L. ex Fr.) Quel. liquid fermentation and culture
Spend for 20 DEG C -30 DEG C.
7. method according to claim 1, it is characterised in that described liquid fermentation medium is consisted of：With 1L liquid
Body fermentation medium meter, glucose 20g, peptone 4g, Testa Tritici 5g, glutamic acid 0.5g-5g, KH2PO41.5g、MgSO40.75g and
The water of surplus.
8. method according to claim 1, it is characterised in that add ubiquitin protein in per milliliters of liquid fermentation medium
0.2mg-3.0mg。
9. method according to claim 1, it is characterised in that the preparation method of described ubiquitin protein, including：To Ganoderma
The water for accounting for Ganoderma superfine powder quality -30 times of amounts of 10 times of amounts is added in superfine powder, is added and is accounted for Ganoderma superfine powder quality 0.5%-3%
Cellulase and account for the pectase of Ganoderma superfine powder quality 0.3%-2%, in 35 DEG C of -55 DEG C of stirrings 1h-2h, Ran Hou
It is homogenized under 70MPa-100MPa；10000rpm-30000rpm centrifugations 10min-15min under the conditions of 3 DEG C -5 DEG C, collects supernatant and obtains
To crude protein liquid；Crude protein liquid is collected into filtered solution by micro-filtration membrane, to remove microgranule therein and antibacterial；By the filtered solution
Concentrated with the ultrafilter membrane that molecular cut off is 5000Da-10000Da at 3 DEG C -5 DEG C, obtained ubiquitin protein.
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