CN104278070A - 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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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, be specifically related to a kind of method improving Quantitative Determination of Ergosterol in Phellinus liquid fermentation production.
Background technology
Phellinus (Phellinus igniarius) belongs to Basidiomycotina, shelf fungus guiding principle, Hymenochaetaceae, Phellinus.Phellinus fungus extract has unusual effect in anticancer transfer and the clinical application of to recur after preventing cancer operation etc., is the highest efficient a kind of medicinal fungi in current internationally recognized biological anticancer preparation.
Due to by the singularity of physiological status and the restriction of complicacy and outside atmosphere, cause Phellinus to form sporophore at occurring in nature rare, particularly forming available sporophore needs for many years.And artificial culture is extremely difficult, culture condition is harsh, and growth cycle reaches 3-4, is difficult to meet the market demand day by day expanded.Adopting the method production Phellinus active pharmaceutical ingredients of Phellinus liquid fermenting because have with short production cycle, labor force economizes and is considered to a kind of effective means by advantages such as external environment influence are little.At present, mainly concentrate in the aspect such as fermentation condition and product extraction and isolation phellinus igniarius mycelium cultivation production active pharmaceutical ingredient research and process application both at home and abroad, overall fermentation level is not high.
Ergosterol, also known as ergosterol, is liposoluble vitamin D
2precursor, can vitamins D be converted into when being subject to uviolizing
2.Ergosterol is the important component of fungal cell membrane, and it is guaranteeing the important role such as the integrity of membrane structure, mobility, cell viability and matter transportation with the activity of membrane bound enzyme, film.Ergosterol is a kind of important medicine chemical material, can be used for the production of the medicine such as cortisone, Progesterone, (Cao Long brightness; Li Xiao Jun is widely used in food, medicine and fodder industry, Zhao Wenhong etc. the progress [J] of ergosterol. China brewages, 2014,33 (4): 9-11.).
Ubiquitin (Ubiquitin, Ub), i.e. ubiquitin protein, it is a peptide species, by 76 Amino acid profiles, its molecular weight is about 8.5kDa, within 1975, separates first from the pancreas of calf, be found in many different organisms and tissue subsequently, the aminoacid sequence of ubiquitin has well-conserved.In recent years, along with deepening continuously of studying ubiquitin, find that ubiquitin is not only the mark of protein degradation, or cell maintains the basic regulative mode to the protein level that those produce by composing type adjustment and environmental stimulus, intracellular protein ubiquitination participates in the various physiological processes of cell, at the mark of protein degradation, DNA repairs, play an important role in each vital movement such as gene transcription regulation and signal transduction, with the various diseases of the mankind, the physiological and biochemical procedure of plant also exists the (Huang Xinmin that is closely connected, Zhang Yanxia, Wan little Rong. the progress [J] of ubiquitin protein. guangdong agricultural science, 2010, 6:191-194.).
Liquid fermenting ubiquitin protein being used for Phellinus is produced, and improve the content of its ergosterol, there is not been reported both at home and abroad.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of method utilizing ubiquitin protein to improve Quantitative Determination of Ergosterol in Phellinus liquid fermentation production.
The technical solution used in the present invention is:
Improve a method for Quantitative Determination of Ergosterol in Phellinus liquid fermentation production, comprise step:
(1) seed liquor preparation: the Phellinus strain inoculation after activation is cultivated 3 days-7 days in the liquid seed culture medium adding Cyclocarya paliurus extract, obtains seed liquor;
(2) Phellinus liquid fermentation and culture: the seed liquor in step (1) is inoculated in liquid fermentation medium by the amount accounting for liquid fermentation medium volume 5%-20%; cultivate after 1 day-4 days; add ubiquitin protein; then cultivation 2 days-4 days is continued; after stopping fermentation, obtain Phellinus liquid fermentation production.
The present invention, according to the feature of Medicinal Fungus Phellinus igniarius liquid fermentation production ergosterol metabolic pathway of synthesizing, by adding Cyclocarya paliurus extract and adding ubiquitin protein at specific fermentation stage, has increased substantially the content of ergosterol in Phellinus liquid fermentation production.
Described Phellinus bacterial classification can adopt any one Phellinus bacterial classification, can adopt commercially available prod.Such as Phellinus (Phellinus linteus) ACCC51181 bacterial classification, derives from Chinese agriculture Microbiological Culture Collection administrative center.
In order to reach better invention effect, carry out preferably following:
In step (1), in often liter of liquid seed culture medium, the addition of Cyclocarya paliurus extract is 1g-10g.
Described Cyclocarya paliurus extract can select commercially available prod, the Cyclocarya paliurus extract etc. that such as Xi'an Sai Ao Bioisystech Co., Ltd produces, and existing method also can be adopted to prepare; It is preferably Cyclocarya paliurus extract prepared by raw material with Cydocaryap paliurus (Baud.) Iljinsk.leaf; The preferred Cyclocarya paliurus extract be made up of Cydocaryap paliurus (Baud.) Iljinsk.leaf ethanol extraction and Cydocaryap paliurus (Baud.) Iljinsk.leaf water extract further, can adopt Cyclocarya paliurus extract prepared by following preparation method, comprise:
Take a certain amount of Cydocaryap paliurus (Baud.) Iljinsk.leaf, pulverize (preferred mistake 40 order-100 order), first adding the mass percentage concentration accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 10 times amount-16 times amount is that the aqueous ethanolic solution of 60%-80% is at 60 DEG C-80 DEG C lixiviate 1h-2h, obtain supernatant liquor after filtration, the lyophilize of supernatant concentration final vacuum obtains extract I; Cydocaryap paliurus (Baud.) Iljinsk.leaf residue after above-mentioned alcohol extracting adds the water lixiviate 1h-2.5h at 85 DEG C-95 DEG C accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 10 times amount-20 times amount, supernatant liquor is obtained after filtration, the dehydrated alcohol of enriched material volume 2 times amount-5 times amount is added after supernatant concentration to 1/4 volume, centrifugal collecting precipitate, obtain extract II after throw out vacuum lyophilization, merging said extracted thing I and extract II obtain Cyclocarya paliurus extract.
In step (1), the Phellinus bacterial classification after described activation is physical environment temperature adding the temperature of cultivating in the liquid seed culture medium of Cyclocarya paliurus extract, is preferably 20 DEG C-30 DEG C.2cm is accessed in the liquid seed culture medium of general 1L interpolation Cyclocarya paliurus extract
2-5cm
2phellinus bacterial classification bacterium block after the activation of size.
In step (1); the activation method of Phellinus bacterial classification is the actication of culture method of this area routine; comprise: by the Phellinus strain inoculation of slant preservation on PDA plate culture medium; carry out activation culture; culture temperature 22 DEG C-30 DEG C; incubation time 4 days-10 days, obtains the Phellinus bacterial classification after activating.
The substratum that described PDA plate culture medium and liquid seed culture medium all adopt this area seed culture conventional, can adopt commercially available prod.Preferably, described PDA plate culture medium: potato 200g, glucose 20g and agar 15g-20g, be settled to 1000mL with water.Preferably, described liquid seed culture medium: glucose 5g-20g, yeast powder 4g, peptone 3g, KH
2pO
41g and MgSO
40.5g, is settled to 1000mL with water.
In step (2), consisting of of described liquid fermentation medium: in 1L liquid fermentation medium, glucose 20g, peptone 4g, wheat bran 5g, L-glutamic acid 0.5g-5g, KH
2pO
41.5g, MgSO
4the water of 0.75g and surplus.
Preferably, ubiquitin protein 0.2mg-3.0mg is added in every milliliters of liquid fermention medium; Ubiquitin protein 0.8mg-2.4mg is added further in preferred every milliliters of liquid fermention medium; Most preferred, add ubiquitin protein 1.6mg in every milliliters of liquid fermention medium.
Described ubiquitin protein adopts the ubiquitin protein extracted from raw material such as great majority food medicine fungi or plant etc., commercially available prod can be selected, also existing method can be adopted to prepare, as the ubiquitin protein extracted from glossy ganoderma raw material, following preparation method can be adopted to prepare, comprise: in glossy ganoderma super-fine powder, add the distilled water accounting for glossy ganoderma super-fine powder quality 10 times amount-30 times amount, add the cellulase accounting for glossy ganoderma super-fine powder quality 0.5%-3% and the polygalacturonase accounting for glossy ganoderma super-fine powder quality 0.3%-2% again, 1h-2h, then homogenate under 70MPa-100MPa is stirred at 35 DEG C-55 DEG C; 10000rpm-30000rpm under 3 DEG C of-5 DEG C of conditions (rpm represent rev/min) centrifugal 10min-15min, collects supernatant liquor and obtains crude protein liquid; Crude protein liquid is collected filtered solution by microfiltration membrane (microfiltration membrane as 0.22 μm), to remove particulate wherein and bacterium; Described filtered solution is concentrated with the ultra-filtration membrane that molecular weight cut-off is 5000Da-10000Da at 3 DEG C-5 DEG C, obtains ubiquitin protein.
Described glossy ganoderma super-fine powder is that glossy ganoderma obtains through micronizing, and its preparation comprises: carrying out micronizing (as carried out micronizing in supper micron mill) by after Ganoderma sporophore cleaning, oven dry, obtaining glossy ganoderma super-fine powder.Described micronizing preferable temperature is 10 DEG C-40 DEG C, treatment time 3min-20min.
The enzyme activity of described polygalacturonase is preferably 3.0 ten thousand U/g-5.0 ten thousand U/g, the enzyme activity of cellulase is preferably 2.0 ten thousand U/g-3.0 ten thousand U/g.
The temperature of described Phellinus liquid fermentation and culture is physical environment temperature, is generally 20 DEG C-30 DEG C, is preferably 25 DEG C-30 DEG C.
The present invention, described Phellinus liquid fermentation production can extract through follow-up separating treatment, measure Quantitative Determination of Ergosterol from phellinus igniarius mycelium.Described separating treatment is the separation method of this area routine, such as filtration or centrifugal; Can comprise: by described Phellinus tunning filtered through gauze, extract from filtration gained mycelium, measure Quantitative Determination of Ergosterol.Concrete extraction step comprises: rinsed well by mycelium water, is dried to constant weight at 55 DEG C-65 DEG C, adds CHCl by feed liquid mass ratio 1:10-30
3, ultrasonic wave (preferred power 250W-300W, frequency 40kHz-50kHz) lixiviate 1h-1.5h, centrifuging and taking supernatant liquor, residue CHCl
3centrifuging and taking supernatant liquor after washing, merges the extracting solution that supernatant liquor obtains containing ergosterol.
The present invention's substratum used uses after all needing sterilizing, cooling, and the condition of sterilizing adopts the normal condition of this area, such as can at 120 DEG C-125 DEG C sterilizing 20min-30min.
Cyclocarya paliurus (Cyclocarya paliurus) has another name called blue or green money Lee, money tree etc., and being Juglandaceae cyclocarya plant, is Chinese distinctive autogenus plant.Cyclocarya paliurus is a kind of tall and big fast-growing arbor, and imparipinnate leaf, is distributed widely in the mountain area of the height above sea level 420m-2500m on the ground such as Jiangxi, Zhejiang, Jiangsu, Anhui, Fujian, Taiwan, Hubei, Sichuan, Guizhou, trench or Limestone Mountain.Its bark, leaf have clearing heat and detoxicating, pain-relieving functions.
Glossy ganoderma (Ganoderma Lucidum (Leyss.ex Fr.) Karst.) profile is umbrella, and cap kidney shape, semicircle or subcircular are the sporophore of porous castor section fungi glossy ganoderma.Have invigorating QI and tranquilization, relieving cough and asthma effect, for dizzy egersis, shortness of breath and palpitation, consumptive disease is coughed and is breathed heavily.
The present invention compared with the existing technology has the following advantages:
1. ubiquitin protein is used for the liquid fermenting production of Phellinus ergosterol constituents by the present invention; and optimize fermentation process; increased substantially the content of ergosterol in Phellinus liquid fermentation production, can reach 2.15mg/g, the ergosterol of extraction can be used in food, medicine and fodder industry.
2. phellinus igniarius mycelium liquid fermentation process of the present invention is simple, and reproducible, fermentation period is short, and efficiency is high, and utilize natural product as raw materials for production, environment-protecting asepsis, cost is low simultaneously.Whole fermenting process is controlled, does not limit by external environment condition, is very applicable to suitability for industrialized production and application.The inventive method is equally applicable to fermentor tank large-scale production, has good industrial applications prospect.
3. the ubiquitin protein that the present invention adopts can be eaten the raw materials such as medicine fungi and plant from great majority and extract; wide material sources, cost is lower, and preparation method is also relatively simple; scale can extract production, to the fermentative production of Phellinus ergosterol active substance, there is good facilitation effect.
Embodiment
Below in conjunction with some embodiments, content of the present invention is illustrated further, but content of the present invention is not limited in the following examples.
Phellinus (Phellinus linteus) ACCC51181 bacterial classification is purchased from Chinese agriculture Microbiological Culture Collection administrative center.
Embodiment 1
One, material prepares
Polygalacturonase (enzyme activity is 5.0 ten thousand U/g), cellulase (enzyme activity is 3.0 ten thousand U/g), by Guangxi, Pang Bo biotechnology company limited provides.
PDA plate culture medium: potato 200g, glucose 20g and agar 15g, be settled to 1000ml with water, natural pH, sterilizing 20min at 121 DEG C.
By the Phellinus strain inoculation of slant preservation on PDA plate culture medium, carry out activation culture, culture temperature 25 DEG C, incubation time 7 days, obtain the Phellinus bacterial classification bacterium block after activating.
The preparation method of Cyclocarya paliurus extract, comprise: take a certain amount of Cydocaryap paliurus (Baud.) Iljinsk.leaf, pulverized 100 orders, first adding the mass percentage concentration accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 13 times amount is that the aqueous ethanolic solution of 70% is at 60 DEG C of lixiviate 1.0h, obtain supernatant liquor after filtration, the lyophilize of supernatant concentration final vacuum obtains extract I; Cydocaryap paliurus (Baud.) Iljinsk.leaf residue after above-mentioned alcohol extracting adds the distilled water lixiviate 1h at 85 DEG C accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 10 times amount, supernatant liquor is obtained after filtration, the dehydrated alcohol of enriched material volume 2 times amount is added after supernatant concentration to 1/4 volume, 5000rpm centrifugal 10min collecting precipitation thing, obtain extract II after throw out vacuum lyophilization, merging said extracted thing I and extract II obtain Cyclocarya paliurus extract.
Be ubiquitin protein prepared by raw material with glossy ganoderma, preparation method comprises: Ganoderma sporophore is cleaned, dry after put into supper micron mill and carry out ultra micro pulverize at low temperature, temperature 25 DEG C, treatment time 10min, obtains glossy ganoderma super-fine powder.The distilled water accounting for glossy ganoderma super-fine powder quality 20 times amount is added in glossy ganoderma super-fine powder, add the cellulase accounting for glossy ganoderma super-fine powder quality 1% and the polygalacturonase accounting for glossy ganoderma super-fine powder quality 0.5% again, stir 1h at 40 DEG C, then put into refiner homogenate 3 times under 80MPa; The centrifugal 15min of 10000rpm under 4 DEG C of conditions, collects supernatant liquor and obtains crude protein liquid; Crude protein liquid is collected filtered solution by 0.22 μm of microfiltration membrane, to remove particulate wherein and bacterium; Described filtered solution is concentrated with the ultra-filtration membrane that molecular weight cut-off is 8000Da at 4 DEG C, obtains concentrated solution, be ubiquitin protein.
Two, the preparation of Phellinus liquid fermentation production
(1) seed liquor preparation: get 2cm
2phellinus bacterial classification bacterium block after size activation is inoculated in 1L and adds in the liquid seed culture medium of Cyclocarya paliurus extract; in often liter of liquid seed culture medium, the addition of Cyclocarya paliurus extract is that 8g, 1L liquid seed culture medium consists of: glucose 10g/L, yeast powder 4g/L, peptone 3g/L, KH
2pO
41g/L, MgSO
4the water of 0.5g/L and surplus, pH nature; Cultivate 4 days, obtain seed liquor for 25 DEG C.
(2) Phellinus liquid fermentation and culture: seed liquor is inoculated in liquid fermentation medium by the amount accounting for liquid fermentation medium volume 10%; after cultivating 2 days at 28 DEG C; add ubiquitin protein; ubiquitin protein 1.6mg is added in every milliliters of liquid fermention medium; then cultivation 3 days is continued; after stopping fermentation, obtain Phellinus liquid fermentation production.Wherein, the consisting of of liquid fermentation medium: glucose 20g/L, peptone 4g/L, wheat bran 5g/L, L-glutamic acid 2.5g/L, KH
2pO
41.5g/L, MgSO
4the water of 0.75g/L and surplus.
Three, measure
1. the mensuration of hypha biomass: Phellinus liquid fermentation production, through 8 layers of filtered through gauze, filters gained mycelium distilled water flushing 3 times, then puts and dry to constant weight in 60 DEG C of baking ovens, be sample, weigh.
2. ergosterol extracts: precision takes 1g mycelium sample, is placed in 25mL volumetric flask, adds 20mL CHCl
3liquid, ultrasonic-leaching 1h (power 250W, frequency 40kHz), then the centrifugal 5min of 4000r/min, gets supernatant liquor, residue 5mLCHCl
3the centrifugal 5min of 4000r/min after washing, gets supernatant liquor.Merge the extracting solution that twice supernatant liquor obtains containing ergosterol.
3. Quantitative Determination of Ergosterol measures: by the extracting solution CHCl containing ergosterol
3be settled to 25mL and obtain extraction liquid, get 2mL extraction liquid, nitrogen dries up rear 5mL dissolve with methanol, and through 0.45 μm of filtering with microporous membrane, filtrate is analyzed for HPLC.
High-efficient liquid phase chromatogram condition: chromatographic column: Agilent C
18chromatographic column (200mm × 4.6mm, 5 μm); Moving phase: methyl alcohol (chromatographic grade), flow velocity 1.2mL/min; UV determined wavelength: determined wavelength 282nm, column temperature 30 DEG C, sample size 10 μ L.
Detected result is in table 1.
Embodiment 2
One, material prepares
Polygalacturonase (enzyme activity is 3.0 ten thousand U/g), cellulase (enzyme activity is 2.0 ten thousand U/g), by Guangxi, Pang Bo biotechnology company limited provides.
PDA plate culture medium: potato 200g, glucose 20g and agar 20g, be settled to 1000ml with water, natural pH, sterilizing 20min at 121 DEG C.
By the Phellinus strain inoculation of slant preservation on PDA plate culture medium, carry out activation culture, culture temperature 28 DEG C, incubation time 9 days, obtain the Phellinus bacterial classification bacterium block after activating.
The preparation method of Cyclocarya paliurus extract, comprise: take a certain amount of Cydocaryap paliurus (Baud.) Iljinsk.leaf, pulverized 60 orders, first adding the mass percentage concentration accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 16 times amount is that the aqueous ethanolic solution of 80% is at 80 DEG C of lixiviate 2h, obtain supernatant liquor after filtration, the lyophilize of supernatant concentration final vacuum obtains extract I; Cydocaryap paliurus (Baud.) Iljinsk.leaf residue after above-mentioned alcohol extracting adds the distilled water lixiviate 2.5h at 95 DEG C accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 20 times amount, supernatant liquor is obtained after filtration, the dehydrated alcohol of enriched material volume 5 times amount is added after supernatant concentration to 1/4 volume, 5000rpm centrifugal 10min collecting precipitation thing, obtain extract II after throw out vacuum lyophilization, merging said extracted thing I and extract II obtain Cyclocarya paliurus extract.
Be ubiquitin protein prepared by raw material with glossy ganoderma, preparation method comprises: Ganoderma sporophore is cleaned, dry after put into supper micron mill and carry out ultra micro pulverize at low temperature, temperature 40 DEG C, treatment time 3min, obtains glossy ganoderma super-fine powder.The distilled water accounting for glossy ganoderma super-fine powder quality 30 times amount is added in glossy ganoderma super-fine powder, add the cellulase accounting for glossy ganoderma super-fine powder quality 3% and the polygalacturonase accounting for glossy ganoderma super-fine powder quality 0.3% again, stir 1.5h at 55 DEG C, then put into refiner homogenate 3 times under 100MPa; The centrifugal 10min of 30000rpm under 5 DEG C of conditions, collects supernatant liquor and obtains crude protein liquid; Crude protein liquid is collected filtered solution by 0.22 μm of microfiltration membrane, to remove particulate wherein and bacterium; Described filtered solution is concentrated with the ultra-filtration membrane that molecular weight cut-off is 10000Da at 5 DEG C, obtains concentrated solution, be ubiquitin protein.
Two, the preparation of Phellinus liquid fermentation production
(1) seed liquor preparation: get 5cm
2phellinus bacterial classification bacterium block after size activation is inoculated in 1L and adds in the liquid seed culture medium of Cyclocarya paliurus extract; in often liter of liquid seed culture medium, the addition of Cyclocarya paliurus extract is that 10g, 1L liquid seed culture medium consists of: glucose 15g/L, yeast powder 4g/L, peptone 3g/L, KH
2pO
41g/L, MgSO
4the water of 0.5g/L and surplus, pH nature; Cultivate 5 days, obtain seed liquor for 28 DEG C.
(2) Phellinus liquid fermentation and culture: seed liquor is inoculated in liquid fermentation medium by the amount accounting for liquid fermentation medium volume 15%; after cultivating 3 days at 28 DEG C; add ubiquitin protein; ubiquitin protein 0.8mg is added in every milliliters of liquid fermention medium; then cultivation 4 days is continued; after stopping fermentation, obtain Phellinus liquid fermentation production.Wherein, the consisting of of liquid fermentation medium: glucose 20g/L, peptone 4g/L, wheat bran 5g/L, L-glutamic acid 5g/L, KH
2pO
41.5g/L, MgSO
4the water of 0.75g/L and surplus.
Three, measure
1. the mensuration of hypha biomass: Phellinus liquid fermentation production, through 8 layers of filtered through gauze, filters gained mycelium distilled water flushing 3 times, then puts and dry to constant weight in 65 DEG C of baking ovens, be sample, weigh.
2. ergosterol extracts: precision takes 1g sample, is placed in 25mL volumetric flask, adds 6.8mLCHCl
3liquid, ultrasonic-leaching 1.5h (power 300W, frequency 50kHz), then the centrifugal 5min of 4000r/min, gets supernatant liquor, residue 5mLCHCl
3the centrifugal 5min of 4000r/min after washing, gets supernatant liquor.Merge the extracting solution that twice supernatant liquor obtains containing ergosterol.
3. Quantitative Determination of Ergosterol measures: by the extracting solution CHCl containing ergosterol
3be settled to 25mL and be extracted liquid, get 2mL extraction liquid, nitrogen dries up rear 5mL dissolve with methanol, and through 0.45 μm of filtering with microporous membrane, filtrate is analyzed for HPLC.
High-efficient liquid phase chromatogram condition, with embodiment 1.
Detected result is in table 1.
Embodiment 3
One, material prepares
Polygalacturonase (enzyme activity is 4.0 ten thousand U/g), cellulase (enzyme activity is 2.0 ten thousand U/g), by Guangxi, Pang Bo biotechnology company limited provides.
PDA plate culture medium: potato 200g, glucose 20g and agar 15g, be settled to 1000ml with water, natural pH, sterilizing 20min at 121 DEG C.
By the Phellinus strain inoculation of slant preservation on PDA plate culture medium, carry out activation culture, culture temperature 30 DEG C, incubation time 4 days, obtain the Phellinus bacterial classification bacterium block after activating.
The preparation method of Cyclocarya paliurus extract, comprise: take a certain amount of Cydocaryap paliurus (Baud.) Iljinsk.leaf, pulverized 40 orders, first adding the mass percentage concentration accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 10 times amount is that the aqueous ethanolic solution of 60% is at 70 DEG C of lixiviate 1.5h, obtain supernatant liquor after filtration, the lyophilize of supernatant concentration final vacuum obtains extract I; Cydocaryap paliurus (Baud.) Iljinsk.leaf residue after above-mentioned alcohol extracting adds the distilled water lixiviate 1.5h at 90 DEG C accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 15 times amount, supernatant liquor is obtained after filtration, the dehydrated alcohol of enriched material volume 4 times amount is added after supernatant concentration to 1/4 volume, 5000rpm centrifugal 10min collecting precipitation thing, obtain extract II after throw out vacuum lyophilization, merging said extracted thing I and extract II obtain Cyclocarya paliurus extract.
Be ubiquitin protein prepared by raw material with glossy ganoderma, preparation method comprises: Ganoderma sporophore is cleaned, dry after put into supper micron mill and carry out ultra micro pulverize at low temperature, temperature 10 DEG C, treatment time 20min, obtains glossy ganoderma super-fine powder.The distilled water accounting for glossy ganoderma super-fine powder quality 10 times amount is added in glossy ganoderma super-fine powder, add the cellulase accounting for glossy ganoderma super-fine powder quality 0.5% and the polygalacturonase accounting for glossy ganoderma super-fine powder quality 2% again, stir 2h at 35 DEG C, then put into refiner homogenate 3 times under 70MPa; The centrifugal 10min of 20000rpm under 3 DEG C of conditions, collects supernatant liquor and obtains crude protein liquid; Crude protein liquid is collected filtered solution by 0.22 μm of microfiltration membrane, to remove particulate wherein and bacterium; Described filtered solution is concentrated with the ultra-filtration membrane that molecular weight cut-off is 5000Da at 3 DEG C, obtains concentrated solution, be ubiquitin protein.
Two, the preparation of Phellinus liquid fermentation production
(1) seed liquor preparation: get 3cm
2phellinus bacterial classification bacterium block after size activation is inoculated in 1L and adds in the liquid seed culture medium of Cyclocarya paliurus extract; in often liter of liquid seed culture medium, the addition of Cyclocarya paliurus extract is that 5g, 1L liquid seed culture medium consists of: glucose 20g/L, yeast powder 4g/L, peptone 3g/L, KH
2pO
41g/L, MgSO
4the water of 0.5g/L and surplus, pH nature; Cultivate 6 days, obtain seed liquor for 22 DEG C.
(2) Phellinus liquid fermentation and culture: seed liquor is inoculated in liquid fermentation medium by the amount accounting for liquid fermentation medium volume 20%; after cultivating 4 days at 30 DEG C; add ubiquitin protein; ubiquitin protein 2.4mg is added in every milliliters of liquid fermention medium; then cultivation 2 days is continued; after stopping fermentation, obtain Phellinus liquid fermentation production.Wherein, the consisting of of liquid fermentation medium: glucose 20g/L, peptone 4g/L, wheat bran 5g/L, L-glutamic acid 4g/L, KH
2pO
41.5g/L, MgSO
4the water of 0.75g/L and surplus.
Three, same embodiment 1 is measured.Detected result is in table 1.
Embodiment 4
One, material prepares
Polygalacturonase (enzyme activity is 5.0 ten thousand U/g), cellulase (enzyme activity is 3.0 ten thousand U/g), by Guangxi, Pang Bo biotechnology company limited provides.
PDA plate culture medium: potato 200g, glucose 20g and agar 15g, be settled to 1000ml with water, natural pH, sterilizing 20min at 121 DEG C.
By the Phellinus strain inoculation of slant preservation on PDA plate culture medium, carry out activation culture, culture temperature 22 DEG C, incubation time 10 days, obtain the Phellinus bacterial classification bacterium block after activating.
Cyclocarya paliurus extract and ubiquitin protein are with embodiment 1.
Two, the preparation of Phellinus liquid fermentation production
(1) seed liquor preparation: get 3cm
2phellinus bacterial classification bacterium block after size activation is inoculated in 1L and adds in the liquid seed culture medium of Cyclocarya paliurus extract; in often liter of liquid seed culture medium, the addition of Cyclocarya paliurus extract is that 1g, 1L liquid seed culture medium consists of: glucose 8g/L, yeast powder 4g/L, peptone 3g/L, KH
2pO
41g/L, MgSO
4the water of 0.5g/L and surplus, pH nature; Cultivate 3 days, obtain seed liquor for 30 DEG C.
(2) Phellinus liquid fermentation and culture: seed liquor is inoculated in liquid fermentation medium by the amount accounting for liquid fermentation medium volume 8%; after cultivating 1 day at 22 DEG C; add ubiquitin protein; ubiquitin protein 0.2mg is added in every milliliters of liquid fermention medium; then cultivation 2 days is continued; after stopping fermentation, obtain Phellinus liquid fermentation production.Wherein, the consisting of of liquid fermentation medium: glucose 20g/L, peptone 4g/L, wheat bran 5g/L, L-glutamic acid 1.5g/L, KH
2pO
41.5g/L, MgSO
4the water of 0.75g/L and surplus.
Three, same embodiment 1 is measured.Detected result is in table 1.
Embodiment 5
One, material prepares
Polygalacturonase (enzyme activity is 5.0 ten thousand U/g), cellulase (enzyme activity is 3.0 ten thousand U/g), by Guangxi, Pang Bo biotechnology company limited provides.
PDA plate culture medium: potato 200g, glucose 20g and agar 15g, be settled to 1000ml with water, natural pH, sterilizing 20min at 121 DEG C.
By the Phellinus strain inoculation of slant preservation on PDA plate culture medium, carry out activation culture, culture temperature 22 DEG C, incubation time 10 days, obtain the Phellinus bacterial classification bacterium block after activating.
Cyclocarya paliurus extract, is purchased from Xi'an Sai Ao Bioisystech Co., Ltd.
Ubiquitin protein, with embodiment 1.
Two, the preparation of Phellinus liquid fermentation production
(1) seed liquor preparation: get 3cm
2phellinus bacterial classification bacterium block after size activation is inoculated in 1L and adds in the liquid seed culture medium of Cyclocarya paliurus extract; in often liter of liquid seed culture medium, the addition of Cyclocarya paliurus extract is that 6g, 1L liquid seed culture medium consists of: glucose 5g/L, yeast powder 4g/L, peptone 3g/L, KH
2pO
41g/L, MgSO
4the water of 0.5g/L and surplus, pH nature; Cultivate 7 days, obtain seed liquor for 20 DEG C.
(2) Phellinus liquid fermentation and culture: seed liquor is inoculated in liquid fermentation medium by the amount accounting for liquid fermentation medium volume 5%; after cultivating 1 day at 20 DEG C; add ubiquitin protein; ubiquitin protein 3mg is added in every milliliters of liquid fermention medium; then cultivation 2 days is continued; after stopping fermentation, obtain Phellinus liquid fermentation production.Wherein, the consisting of of liquid fermentation medium: glucose 20g/L, peptone 4g/L, wheat bran 5g/L, L-glutamic acid 0.5g/L, KH
2pO
41.5g/L, MgSO
4the water of 0.75g/L and surplus.
Three, same embodiment 1 is measured.Detected result is in table 1.
Comparative example 1
(1) shake-flask seed is cultivated
1L liquid seed culture medium: glucose 10g/L, yeast powder 4g/L, peptone 3g/L, KH
2pO
41g/L, MgSO
4the water of 0.5g/L and surplus.PH nature, sterilizing 20min at 121 DEG C.
Cultural method: get 2cm
2after Phellinus bacterial classification bacterium block access sterilizing cooling in size embodiment 1 after activation in 1L liquid seed culture medium, 25 DEG C, under 120r/min, shaking table cultivates 4 days, obtains cultured seed liquor.
(2) shake flask fermentation is cultivated
Liquid fermentation medium is with embodiment 1.PH nature, sterilizing 20min at 121 DEG C.
Cultural method: by cultured seed liquor by accounting in the inoculum size access liquid fermentation medium of liquid fermentation medium volume 10%, at 25 DEG C, cultivate 12 days in 120r/min shaking table.Each test establish 3 parallel.
Detected result is in table 1.
Comparative example 2 only adds Cyclocarya paliurus extract, does not add ubiquitin protein
Except " two, the preparation of preparation (1) seed liquor of Phellinus liquid fermentation production: get 3cm
2phellinus bacterial classification bacterium block after size activation is inoculated in 1L and adds in the liquid seed culture medium of Cyclocarya paliurus extract; in often liter of liquid seed culture medium, the addition of Cyclocarya paliurus extract is that 6g, 1L liquid seed culture medium consists of: glucose 5g/L, yeast powder 4g/L, peptone 3g/L, KH
2pO
41g/L, MgSO
4the water of 0.5g/L and surplus, pH nature; Cultivate 7 days, obtain seed liquor for 20 DEG C.(2) Phellinus liquid fermentation and culture: be inoculated in liquid fermentation medium by the amount accounting for liquid fermentation medium volume 5% by seed liquor, after cultivating 1 day, continues cultivation 2 days, after stopping fermentation, obtain Phellinus liquid fermentation production at 20 DEG C.Wherein, the consisting of of liquid fermentation medium: glucose 20g/L, peptone 4g/L, wheat bran 5g/L, L-glutamic acid 0.5g/L, KH
2pO
41.5g/L, MgSO
4the water of 0.75g/L and surplus." outside, all the other operations are with embodiment 5.Detected result is in table 1.
Comparative example 3 only adds ubiquitin protein, does not add Cyclocarya paliurus extract
Except " two, the preparation of preparation (1) seed liquor of Phellinus liquid fermentation production: get 3cm
2phellinus bacterial classification bacterium block after size activation is inoculated in liquid seed culture medium, and 1L liquid seed culture medium consists of glucose 5g/L, yeast powder 4g/L, peptone 3g/L, KH
2pO
41g/L, MgSO
4the water of 0.5g/L and surplus, pH nature; Cultivate 7 days, obtain seed liquor for 20 DEG C.(2) Phellinus liquid fermentation and culture: seed liquor is inoculated in liquid fermentation medium by the amount accounting for liquid fermentation medium volume 5%; after cultivating 1 day at 20 DEG C; add ubiquitin protein; ubiquitin protein 3mg is added in every milliliters of liquid fermention medium; then cultivation 2 days is continued; after stopping fermentation, obtain Phellinus liquid fermentation production.Wherein, the consisting of of liquid fermentation medium: glucose 20g/L, peptone 4g/L, wheat bran 5g/L, L-glutamic acid 0.5g/L, KH
2pO
41.5g/L, MgSO
4the water of 0.75g/L and surplus." outside, all the other operations are with embodiment 5.Detected result is in table 1.
Table 1 Phellinus liquid fermenting metabolite content detected result
Note: compare with comparative example 1, Δ: P<0.05; Δ Δ: 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 presentation of table 1, the present invention, according to the feature of Medicinal Fungus Phellinus igniarius liquid fermentation production ergosterol metabolic pathway of synthesizing, by adding Cyclocarya paliurus extract and adding ubiquitin protein at specific fermentation stage, has increased substantially Phellinus Quantitative Determination of Ergosterol.
Compared with comparative example 1, Quantitative Determination of Ergosterol in the Phellinus meta-bolites mycelium of the inventive method liquid fermentation and culture is adopted to improve 44.4%-49.3%.
In the scope that preparation method of the present invention limits, the change of each parameter does not affect the raising of Quantitative Determination of Ergosterol in Phellinus liquid fermenting meta-bolites, and therefore in preparation method of the present invention, the combination of arbitrary parameter all can realize the raising of Phellinus liquid fermenting meta-bolites Quantitative Determination of Ergosterol.Do not repeat them here.
The above is only the preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also make some improvement, and these improvement also should be considered as protection scope of the present invention.
Claims (9)
1. improve a method for Quantitative Determination of Ergosterol in Phellinus liquid fermentation production, it is characterized in that, comprise step:
(1) seed liquor preparation: the Phellinus strain inoculation after activation is cultivated 3 days-7 days in the liquid seed culture medium adding Cyclocarya paliurus extract, obtains seed liquor;
(2) Phellinus liquid fermentation and culture: the seed liquor in step (1) is inoculated in liquid fermentation medium by the amount accounting for liquid fermentation medium volume 5%-20%; cultivate after 1 day-4 days; add ubiquitin protein; then cultivation 2 days-4 days is continued; after stopping fermentation, obtain Phellinus liquid fermentation production.
2. method according to claim 1, is characterized in that, in step (1), in often liter of liquid seed culture medium, the addition of Cyclocarya paliurus extract is 1g-10g.
3. method according to claim 1, is characterized in that, described Cyclocarya paliurus extract is take Cydocaryap paliurus (Baud.) Iljinsk.leaf as Cyclocarya paliurus extract prepared by raw material.
4. method according to claim 3, is characterized in that, described Cyclocarya paliurus extract is made up of Cydocaryap paliurus (Baud.) Iljinsk.leaf alcohol extract and Cydocaryap paliurus (Baud.) Iljinsk.leaf water extract.
5. the method according to claim 1 or 3, it is characterized in that, the preparation method of described Cyclocarya paliurus extract, comprise: take a certain amount of Cydocaryap paliurus (Baud.) Iljinsk.leaf, pulverize, first add the mass percentage concentration accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 10 times amount-16 times amount be the aqueous ethanolic solution of 60%-80% at 60 DEG C-80 DEG C lixiviate 1h-2h, obtain supernatant liquor after filtration, the lyophilize of supernatant concentration final vacuum obtains extract I; Cydocaryap paliurus (Baud.) Iljinsk.leaf residue after above-mentioned alcohol extracting adds the water lixiviate 1h-2.5h at 85 DEG C-95 DEG C accounting for Cydocaryap paliurus (Baud.) Iljinsk.leaf weight 10 times amount-20 times amount, supernatant liquor is obtained after filtration, the dehydrated alcohol of enriched material volume 2 times amount-5 times amount is added after supernatant concentration to 1/4 volume, centrifugal collecting precipitate, obtain extract II after throw out vacuum lyophilization, merging said extracted thing I and extract II obtain Cyclocarya paliurus extract.
6. method according to claim 1, is characterized in that, in step (2), the temperature of described Phellinus liquid fermentation and culture is 20 DEG C-30 DEG C.
7. method according to claim 1, is characterized in that, consisting of of described liquid fermentation medium: in 1L liquid fermentation medium, glucose 20g, peptone 4g, wheat bran 5g, L-glutamic acid 0.5g-5g, KH
2pO
41.5g, MgSO
4the water of 0.75g and surplus.
8. method according to claim 1, is characterized in that, adds ubiquitin protein 0.2mg-3.0mg in every milliliters of liquid fermention medium.
9. method according to claim 1, it is characterized in that, the preparation method of described ubiquitin protein, comprise: in glossy ganoderma super-fine powder, add the water accounting for glossy ganoderma super-fine powder quality 10 times amount-30 times amount, add the cellulase accounting for glossy ganoderma super-fine powder quality 0.5%-3% and the polygalacturonase accounting for glossy ganoderma super-fine powder quality 0.3%-2% again, 1h-2h, then homogenate under 70MPa-100MPa is stirred at 35 DEG C-55 DEG C; The centrifugal 10min-15min of 10000rpm-30000rpm under 3 DEG C of-5 DEG C of conditions, collects supernatant liquor and obtains crude protein liquid; Crude protein liquid is collected filtered solution by microfiltration membrane, to remove particulate wherein and bacterium; Described filtered solution is concentrated with the ultra-filtration membrane that molecular weight cut-off is 5000Da-10000Da at 3 DEG C-5 DEG C, obtains ubiquitin protein.
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