CN105671100A - Method for synthesizing phellinus high-activity flavonoid compound - Google Patents
Method for synthesizing phellinus high-activity flavonoid compound Download PDFInfo
- Publication number
- CN105671100A CN105671100A CN201610075925.1A CN201610075925A CN105671100A CN 105671100 A CN105671100 A CN 105671100A CN 201610075925 A CN201610075925 A CN 201610075925A CN 105671100 A CN105671100 A CN 105671100A
- Authority
- CN
- China
- Prior art keywords
- phellinus
- seed
- culture medium
- flavone compound
- cultivation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biological fermentation engineering and particularly relates to a method for synthesizing a phellinus high-activity flavonoid compound. According to the method, a culture medium is mainly optimized, coumarin, phenylalanine and other matter are added into the culture medium, and by means of the first step of first-grade seed cultivation, the second step of second-grade seed cultivation, the third step of fermentation cultivation and the fourth step of extraction of the phellinus activity flavonoid compound, the yield of the phellinus flavonoid compound is greatly increased, and biological activity of the compound is also kept. By means of the method, the phellinus flavonoid compound both high in yield and high in anti-oxidation activity can be obtained, the content is high, activity is stable, the production period is short, pollution is little, cost is low, and the production method has huge industrial prospects.
Description
Technical field
The invention belongs to biological fermentation engineering field, relate to a kind of liquid fermentation method of efficient synthetic Phellinus active yellow ketone compounds.
Background technology
Phellinus (Phellinusigniarius); belong to Basidiomycotina (Basidiomycota); Hymenomycetes (Hymenomycetes); Aphyllophorales (Polyporales); Polyporaceae (Hymenochatetacae); the famous and precious medicinal fungi of Phellinus (Phellinus), claims again phelliuns igniarius (P.igniarius). Phellinus is a kind of traditional Chinese medicine, and mildly bitter flavor can sharp the five internal organs, soft heavily fortified point, toxin expelling, stop blooding, invigorate blood circulation and stomach antidiarrheal, among the people conventional with under treatment gonorrhoea, uterine bleeding band, sore cave gathers, weakness, splenasthenic diarrhea etc. The latest study proves, Phellinus has anti-oxidant, anti-inflammation, and a series of pharmacologically actives such as antitumor, and active component in Phellinus is mainly flavone compound and polysaccharide. Wherein flavone compound has antioxidation, antitumor, hypoglycemic, the effects such as immunological regulation. But owing to being subject to the restriction of various environmental conditions; the fructification rareness that Phellinus forms at occurring in nature; from its fructification, extract flavone compound; productive rate is low; activity is not high; and very difficult, therefore, extremely important with utilizing of the synthetic Flavonoid substances of Phellinus to utilization and exploitation by the flavone compound of the efficiently synthetic high activity of biological engineering method and high yield.
The people such as Liu Fan disclose a kind of liquid fermentation method (application publication number is CN103695316A) that improves phellinus igniarius mycelium yield of flavone, output can reach 0.6g/L, the people such as Zhu Hu disclose a kind of technique (application publication number is CN103627728A) of liquid-solid two-phase cultivation flavonoids from phellinus, and the maximum output that obtains flavones is 2.174g/L. the people such as glad happiness utilize fungal elicitor to improve yield of flavone in Phellinus liquid fermentation (application publication number is CN101702984A), the maximum output that obtains flavones is 0.34g/L, at present, also many documents that Phellinus fermentation medium is optimized have been reported at home, the output that the high people of Zhao Zi obtains flavones by the culture medium after optimizing is 0.128056g/L, the output that the people such as Xu Qian obtain flavones by the culture medium after optimizing is 0.21235g/L, these all provide method and fermentation medium for improving flavonoids from phellinus compounds output, but their output is all very low, all there is no to guarantee the biologically active of flavone compound in building-up process yet. use synthetic this type of reactive compound of formula in the present invention, can obtain the flavone compound up to 5.9-6.5g/L, and antioxidation activity is 5.1-6.7mmol/L, this output far away higher than above-mentioned patent or document is reported or the numerical value of publicity method, and the biologically active of having guaranteed for the first time to synthesize this compounds in building-up process, mainly because in the research of this formulation optimization, except considering that single factor is on the impact of the synthetic flavone compound productive rate of Phellinus, also from meeting the angle of the synthetic high yield of Phellinus and high activity flavone compound, add cumarin etc. to the medium component of guaranteeing that in building-up process, active group forms, and consider the reciprocation between medium component from affect the synthetic flavone compound productive rate of Phellinus and bioactive angle simultaneously, the productive rate and the bioactive culture medium condition that improve flavonoids from phellinus compounds have been formed simultaneously, this is unexistent in the current document of reporting or patent.
Although having now many technical systems of utilizing fungi or the synthetic flavone compound of plant cell, all there is following a few point defect in them:
1, in building-up process, only pay close attention to the output of flavone compound, and ignored its bioactive formation problem, this is unfavorable for obtaining highly active flavone compound.
2, the composition in the culture medium prescription of reporting is at present comparatively complicated, is Carbon and nitrogen sources greatly mainly with natural products, and this makes the quality of production process and activity have obvious batch of difference, is unfavorable for the stable synthetic of flavone compound.
3, the flavone compound output that these methods finally obtain is not high, and its biologically active does not ensure.
Therefore, we need to find a kind of method of efficient synthesizing activity flavone compound, and the present invention has has researched and solved the problems referred to above in Phellinus fermentation medium by the optimization of scientific statistics method-response surface. Not only building-up process is simple to operate, with low cost, can also in level of production scale, verify can the high yield synthetic highly active flavone compound that possesses; Verified this flavone compound in laboratory has hypoglycemic, anti-oxidant, anti-ageing and anti-trioxypurine effect clearly, is the important raw materials for production of this type of health-related drink and food of preparation, dietary supplements, food additives and related drugs.
Summary of the invention
The object of the invention is to overcome the existing defect of prior art, provide a kind of easy and simple to handle, with low cost, can be in the synthetic method that possesses highly active flavone compound of level of production high yield.
To achieve these goals, the technical solution used in the present invention is made up of following step:
(1) cultivation of first order seed
Phellinus the bacterial classification good activation of slant preservation is inoculated into and in seed culture medium, carries out first order seed cultivation, inoculum concentration is to access the bacterium piece that 4～5 block sizes are 5mm × 5mm in the seed culture medium of every 100ml, cultivation temperature is 27～30 DEG C, rotating speed is 150～180r/min, and incubation time is 7～9 days;
In above-mentioned 1L seed culture medium: glucose is that 15.0～22.0g, potato are 150.0～220.0g, KH2PO4Be 0.5～2.5g, MgSO4Be 0.2～0.5g, add water to 1L, the pH of this seed culture medium is 6.5;
(2) cultivation of secondary seed
Cultured first order seed in step (1) is received and in seed culture medium, carried out secondary seed cultivation, inoculum concentration is 5～15 (V/V) %, and cultivation temperature is 27～30 DEG C, and rotating speed is 150～180r/min, incubation time is 7～9 days, obtains the secondary seed of cultivating;
(3) fermented and cultured
The cultured secondary seed of step (2) is received in fermentation medium, inoculum concentration is 5～15 (V/V) %, cultivation temperature is 27～30 DEG C, rotating speed is 150～180r/min, cultivate and in fermentation medium, add D-101 macroreticular resin in 4～5 days, continue to cultivate 3～4 days;
In 1L fermentation medium: glucose is that 15.0～22.0g, peptone are that 2.0～7.0g, cumarin are that 0.02～0.06g, phenylalanine are that 0.2～0.6g, Tween-80 are 1.0～4.0g, cinnamic acid 0.05～0.5g, MgSO4Be 1.0～5.0g, KH2PO4Be 0.5～3.0g, add water to 1L, the pH of this fermentation medium is 6.5;
(4) extract Phellinus active yellow ketone compounds
After the fermented and cultured of step (3) completes, filter out D-101 macroreticular resin, utilize D-101 macroreticular resin that the flavone compound in fermentation medium is adsorbed, the methanol-eluted fractions with 80% out, obtains Phellinus active yellow ketone compounds.
The name of the Phellinus bacterial classification in above-mentioned steps (1) is called Phellinussp.P0988; preserving number is CCTCCNO:M2012080, and depositary institution is Chinese Typical Representative culture collection center, and address is Wuhan, China Wuhan University; postcode 430012, preservation date is on March 14th, 2012.
The optimization formula of above-mentioned steps (3) is: in 1L fermentation medium, glucose is that 18.0～20.0g, peptone are that 3.0～5.0g, cumarin are that 0.02～0.04g, phenylalanine are that 0.25～0.5g, cinnamic acid are 0.05～0.3g, MgSO4Be 1.0～3.0g, KH2PO4Be that 0.5～2.0g, Tween-80 are 1.0～3.0g, add water to 1L, the pH of this fermentation medium is 6.5.
The method of the synthetic Phellinus high activity flavone compound of the present invention, is to be mainly optimized at synthetic culture medium that Phellinus is fermented, and its technical advantage is as follows:
(1) by response surface design optimization method studied affect the synthetic flavone compound productive rate of Phellinus and bioactive medium component with and the reciprocation that exists each other. On statistical analysis basis, determine remarkable reciprocation and the primary influences affecting between the synthetic flavone compound productive rate of Phellinus and important medium component cumarin, phenylalanine and the Tween-80 of activity, thereby determined that these medium components are conducive to the synthetic flavone compound productive rate of Phellinus and the active concentration range forming most. This is never by publicity or reported on flavone compound synthetic.
(2) by having added cumarin, phenylalanine etc. to form the skeleton precursor substance of flavone compound in its fermentation medium; reduce the metabolic process of synthetic such material of Phellinus; shorten the production cycle, promoted the high activity forming process in Phellinus building-up process.
(3) the present invention has also added cinnamic acid, as the stimulus of secondary metabolite, promotes the synthetic and active forming process of flavones.
(4) this method is easy and simple to handle, with low cost, can obtain the highly active flavone compound of high yield, is widely used at food and medical technical field, and therefore, the present invention is for suitability for industrialized production flavone compound, significant
Detailed description of the invention
Further illustrate below with reference to experiment.
Embodiment 1
The method of the synthetic Phellinus high activity flavone compound of the present embodiment is made up of following steps:
(1) first order seed is cultivated
Phellinus the bacterial classification good activation of slant preservation is inoculated into and in seed culture medium, carries out first order seed cultivation, and inoculum concentration is to access the bacterium piece that 4 block sizes are 5mm × 5mm, 28 DEG C of cultivation temperature, rotating speed 160r/min, incubation time 9 days in the seed culture medium of every 100ml.
Phellinus bacterial classification is according to conventional method activation, specifically Phellinus bacterial classification is seeded on PDA culture medium, and 25～30 DEG C, aerobic lucifuge is cultured to Phellinus bacterial classification and covers with inclined-plane; The name of this Phellinus bacterial classification is called Phellinussp.P0988, and depositary institution is Chinese Typical Representative culture collection center, and address is Wuhan, China Wuhan University, postcode 430012, and deposit number is CCTCCNO:M2012080, preservation date is on March 14th, 2012.
In above-mentioned 1L seed culture medium: glucose 20.0g, potato 200.0g, KH2PO41g、MgSO40.5g, surplus is water, the pH of this seed culture medium is 6.5.
(2) secondary seed is cultivated
Cultured first order seed in step (1) is received and in seed culture medium, carried out secondary seed cultivation, inoculum concentration is 10 (V/V) %, it is the first order seed of inoculating 10mL in the seed culture medium of every 100mL, cultivation temperature is 28 DEG C, rotating speed is 160r/min, incubation time is 9 days, the secondary seed obtaining.
(3) fermented and cultured
Cultured secondary seed in step (2) is received in fermentation medium, inoculum concentration is 10 (V/V) %, it is the secondary seed of inoculating 10mL in the fermentation medium of every 100mL, cultivation temperature is 28 DEG C, rotating speed is 160r/min, cultivate 4 days, in fermentation medium, add D-101 macroreticular resin to adsorb, continue to cultivate 4 days;
In above-mentioned 1L fermentation medium: glucose 20.0g, peptone 4g, cumarin 0.03g, phenylalanine 0.35g, cinnamic acid 0.1g, MgSO41.5g、KH2PO41.0g, Tween-80 2.0g, add water to 1L, and the pH of this fermentation medium is 6.5.
(4) extract Phellinus active yellow ketone compounds
After completing, the fermented and cultured of step (3) filters out D-101 macroreticular resin; utilize D-101 macroreticular resin that the flavone compound generating in fermentation medium is adsorbed; methanol-eluted fractions with 80% out; obtain Phellinus active yellow ketone compounds; and the content that determines flavone compound is 5.76g/L, total antioxidant activity is 5.15mmol/L.
Embodiment 2
The method of the synthetic Phellinus high activity flavone compound of the present embodiment is made up of following steps:
(1) first order seed is cultivated
Phellinus the bacterial classification good activation of slant preservation is inoculated into and in seed culture medium, carries out first order seed cultivation, and inoculum concentration is to access the bacterium piece that 5 block sizes are 5mm × 5mm, 27 DEG C of cultivation temperature, rotating speed 180r/min, incubation time 8 days in the seed culture medium of every 100ml;
In above-mentioned 1L seed culture medium: glucose 18.0g, potato 180.0g, KH2PO41g、MgSO40.5g, adds water to 1L, and the pH of this seed culture medium is 6.5.
(2) secondary seed is cultivated
Cultured first order seed in step (1) is received and in seed culture medium, carried out secondary seed cultivation, inoculum concentration is 15 (V/V) %, it is the first order seed of inoculating 15mL in the seed culture medium of every 100mL, cultivation temperature is 27 DEG C, rotating speed is 180r/min, incubation time is 8 days, obtains secondary seed.
(3) fermented and cultured
Cultured secondary seed in step (2) is received in fermentation medium, inoculum concentration is 15 (V/V) %, it is the secondary seed of inoculating 15mL in the fermentation medium of every 100mL, cultivation temperature is 27 DEG C, rotating speed is 150r/min, cultivate 5 days, in fermentation medium, add D-101 macroreticular resin to adsorb, continue to cultivate 3 days;
In above-mentioned 1L fermentation medium: glucose 18.0g, peptone 3g, cumarin 0.04g, phenylalanine 0.4g, cinnamic acid 0.3g, MgSO44.0g、KH2PO40.5g, Tween-80 1.0g, add water to 1L, and the pH of this fermentation medium is 6.5.
(4) extract Phellinus active yellow ketone compounds
After completing, the fermented and cultured of step (3) filters out D-101 macroreticular resin; utilize D-101 macroreticular resin that the flavone compound generating in fermentation medium is adsorbed; methanol-eluted fractions with 80% out; obtain Phellinus active yellow ketone compounds; and the content that determines flavone compound is 4.054g/L, total antioxidant activity is 5.1964mmol/L.
Embodiment 3
The method of the synthetic Phellinus high activity flavone compound of the present embodiment is made up of following steps:
(1) first order seed is cultivated
Phellinus the bacterial classification good activation of slant preservation is inoculated into and in seed culture medium, carries out first order seed cultivation, and inoculum concentration is to access the bacterium piece that 5 block sizes are 5mm × 5mm, 30 DEG C of cultivation temperature, rotating speed 150r/min, incubation time 9 days in the seed culture medium of every 100ml;
In above-mentioned 1L seed culture medium: glucose 22.0g, potato 150.0g, KH2PO40.5g、MgSO40.5g, adds water to 1L, and the pH of this seed culture medium is 6.5.
(2) secondary seed is cultivated
Cultured first order seed in step (1) is received and in seed culture medium, carried out secondary seed cultivation, inoculum concentration is 5 (V/V) %, it is the first order seed of inoculating 5mL in the seed culture medium of every 100mL, cultivation temperature is 30 DEG C, rotating speed is 150r/min, incubation time is 9 days, obtains secondary seed.
(3) fermented and cultured
Cultured secondary seed in step (2) is received in fermentation medium, inoculum concentration is 5 (V/V) %, it is the secondary seed of inoculating 5mL in the fermentation medium of every 100mL, cultivation temperature is 30 DEG C, rotating speed is 180r/min, cultivate 4 days, in fermentation medium, add D-101 macroreticular resin to adsorb, continue to cultivate 3 days;
In above-mentioned 1L fermentation medium: glucose 20.0g, peptone 5g, cumarin 0.02g, phenylalanine 0.5g, cinnamic acid 0.05g, MgSO41.0g、KH2PO42.0g, Tween-80 3.0g, add water to 1L, and the pH of this fermentation medium is 6.5.
(4) extract Phellinus active yellow ketone compounds
After completing, the fermented and cultured of step (3) filters out D-101 macroreticular resin; utilize D-101 macroreticular resin that the flavone compound generating in fermentation medium is adsorbed; methanol-eluted fractions with 80% out; obtain Phellinus active yellow ketone compounds; and to determine its content be 4.73g/L, total antioxidant activity is 6.4mmol/L.
Embodiment 4
In the present embodiment, the seed culture based formulas of step (1) is: glucose 15.0g, potato 220.0g, KH2PO42.5g、MgSO40.2g, adds water to 1L, and the pH of this seed culture medium is 6.5.
In step (3), the formula of fermentation medium used is: glucose 15.0g, peptone 2g, cumarin 0.06g, phenylalanine 0.6g, cinnamic acid 0.5g, MgSO45.0g、KH2PO43.0g, Tween-80 4.0g, add water to 1L, and the pH of this fermentation medium is 6.5.
Other operation is all identical with embodiment 1, and productive rate and total antioxidant activity all approach with the result of embodiment 1.
Embodiment 5
In the present embodiment, the seed culture based formulas of step (1) is: glucose 22.0g, potato 150.0g, KH2PO42.0g、MgSO40.3g, adds water to 1L, and the pH of this seed culture medium is 6.5.
In step (3), the formula of fermentation medium used is: glucose 22.0g, peptone 7.0g, cumarin 0.02g, phenylalanine 0.2g, cinnamic acid 0.05g, MgSO41.0g、KH2PO40.5g, Tween-80 1.0g, add water to 1L, and the pH of this fermentation medium is 6.5.
Other operation is all identical with embodiment 1, and productive rate and total antioxidant activity all approach with the result of embodiment 1.
In order to verify technique effect of the present invention, applicant has done a large amount of experiments, now describes as an example of following experiment example.
(1) fermentation medium contrast
In the fermentation medium of comparative example 1: glucose 20.0g, peptone 4g, KH2PO41.0g、MgSO41.5g, cinnamic acid 0.1g, pH are 6.5.
In the fermentation medium of comparative example 2: glucose 20.0g, peptone 4g, KH2PO41.0g、MgSO41.5g, cinnamic acid 0.1g, cumarin 0.03g, pH are 6.5.
In the fermentation medium of comparative example 3: glucose 20.0g, peptone 4g, KH2PO41.0g、MgSO41.5g, cinnamic acid 0.1g, phenylalanine 0.35g, pH are 6.5.
In the fermentation medium of comparative example 4: glucose 20.0g, peptone 4g, KH2PO41.0g、MgSO41.5g, cinnamic acid 0.1g, Tween-80 2.0g, pH are 6.5.
Other step is all identical with embodiment 1, and experimental results is as following table 1:
Table 1 is the result contrast of each comparative example and embodiment 1
| Flavone compound | Embodiment 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 |
| Output | 5.76g/L | 1.90g/L | 2.63g/L | 2.95g/L | 3.35g/L |
| Total antioxidant activity | 5.15mmol/L | 0.74mmol/L | 1.44mmol/L | 3.64mmol/L | 1.45mmol/L |
As can be seen from Table 1, in the time adding respectively cumarin, phenylalanine and Tween-80, can improve flavone compound output and activity, but effect is also not obvious, and ought add three simultaneously, greatly improved output and the activity of flavone compound, this fully proves that cumarin, phenylalanine and Tween-80 have synergy, and this three has facilitation to flavone compound synthetic.
For verifying the relation of three in this experiment, analyze in conjunction with following experiment:
According to the design principle of the Central Composite experiment in response surface method, use respectively X1、X2、X3Represent cumarin, phenylalanine and Tween-80, then encode with-1,0,1 respectively by the basic, normal, high level of these 3 independents variable. Taking flavonoids from phellinus kind compound content and its total antioxidant activity as response contrived experiment, further select the allocation optimum concentration of significant factor and analyze the relation between three respectively, response surface experimental design with the results are shown in Table 2.
The experimental design of table 2 response surface and result
Taking flavonoid content as response, according to CCD contrived experiment result in upper table, use Minitab software to carry out Quadratic Regression Analysis to result wherein, obtain quadratic regression equation:
Y＝0.16458-0.1394X1+0.0406X2+0.1374X3+0.24055X1 2+0.40255X2 2+1.29355X3 2+0.26913X1X2+0.43137X1X3+1.58812X2X3
The variance analysis of regression equation is in table 3
The variance analysis of table 3 regression equation
Taking the total antioxidant activity of flavone compound as response, according to the experimental result of CCD design in table 2, use Minitab software to carry out Quadratic Regression Analysis to it, obtain quadratic regression equation and be
Y＝0.775-0.224X1+0.124X2-0.086X3+0.48X1 2+0.52X2 2+1.42X3 2+0.37X1X2+0.55X1X3+1.68X2X3
The variance analysis of regression equation is in table 4
The variance analysis of table 4 regression equation
Can be found out by table 3 and table 4, the reciprocation highly significant (P < 0.001) of equation, and the linearity of equation, quadratic term are also very remarkable, the impact that each experiment factor pair flavone compound is described is not simple linear relationship, their output and antioxidation activities on flavone compound have impact, further illustrate and between cumarin, phenylalanine and Tween-80, there is mutual coordination, interactional relation.
(2) impact of the addition of cumarin on flavone compound
In the fermentation medium of comparative example 4: glucose 20.0g, peptone 4g, cumarin 0.1g, phenylalanine 1.0g, MgSO41.5g、KH2PO41.0g, Tween-80 2.0g, pH are 6.5.
In the fermentation medium of comparative example 5: glucose 20.0g, peptone 4g, cumarin 0.005g, phenylalanine 0.05g, MgSO41.5g、KH2PO41.0g, Tween-80 2.0g, pH are 6.5.
Other step is all identical with embodiment 1, and experimental results is as following table 5:
Table 5 is the result contrast of each comparative example and embodiment 1
| Flavone compound | Embodiment 1 | Comparative example 4 | Comparative example 5 |
| Output | 5.76g/L | 1.13g/L | 1.99g/L |
| Total antioxidant activity | 5.15mmol/L | 1.11mmol/L | 1.91mmol/L |
The cumarin of different content has different impacts to flavone compound output and flavone compound activity as can be seen from Table 5, while exceeding the content of cumarin in this formula, the content of flavone compound and active can reduction, illustrate that cumarin too high levels can suppress the synthetic of flavone compound, and during lower than the content of cumarin in this formula, the content of flavone compound and activity also can reduce, this explanation cumarin content is too low, with the effect of other factor interactions a little less than, be unfavorable for the synthetic of flavone compound.
Claims (3)
1. a method for synthetic Phellinus high activity flavone compound, is characterized in that being made up of following step:
(1) cultivation of first order seed
Phellinus the bacterial classification good activation of slant preservation is inoculated into and in seed culture medium, carries out first order seed cultivation,Inoculum concentration is to access the bacterium piece that 4～5 block sizes are 5mm × 5mm in the seed culture medium of every 100ml, trainingFoster temperature is 27～30 DEG C, and rotating speed is 150～180r/min, and incubation time is 7～9 days;
In above-mentioned 1L seed culture medium: glucose be 15.0～22.0g, potato be 150.0～220.0g,KH2PO4Be 0.5～2.5g, MgSO4Be 0.2～0.5g, add water to 1L, the pH of this seed culture medium is 6.5;
(2) cultivation of secondary seed
Cultured first order seed in step (1) is received and in seed culture medium, is carried out secondary seed cultivation,Inoculum concentration is 5～15 (V/V) %, and cultivation temperature is 27～30 DEG C, and rotating speed is 150～180r/min, cultivatesTime is 7～9 days, obtains secondary seed;
(3) fermented and cultured
The cultured secondary seed of step (2) to be received in fermentation medium, inoculum concentration is 5～15 (V/V) %, cultivation temperature is 27～30 DEG C, rotating speed is 150～180r/min, cultivates 4～5 days to fermentationIn culture medium, add D-101 macroreticular resin, continue to cultivate 3～4 days;
In 1L fermentation medium: glucose is that 15.0～22.0g, peptone are 2.0～7.0g, cumarinBe 0.02～0.06g, phenylalanine be 0.2～0.6g, Tween-80 be 1.0～4.0g, cinnamic acid 0.05～0.5g、MgSO4Be 1.0～5.0g, KH2PO4Be 0.5～3.0g, add water to 1L, this fermentation mediumPH is 6.5;
(4) extract Phellinus active yellow ketone compounds
After the fermented and cultured of step (3) completes, filter out D-101 macroreticular resin, utilize D-101 macropore treeFat adsorbs the flavone compound generating in fermentation medium, and the methanol-eluted fractions with 80% out, obtainsTo Phellinus active yellow ketone compounds.
2. the method for synthetic Phellinus high activity flavone compound according to claim 1, is characterized in that instituteThe name of the Phellinus bacterial classification in the step (1) of stating is called Phellinussp.P0988, and preserving number is CCTCCNO:M2012080, depositary institution is Chinese Typical Representative culture collection center, address is Wuhan, China Wuhan University, protectsHiding the date is on March 14th, 2012.
3. the method for synthetic Phellinus high activity flavone compound according to claim 1, its feature existsIn the 1L fermentation medium of described step (3): glucose be 18.0～20.0g, peptone be 3.0～5.0g, cumarin be 0.02～0.04g, phenylalanine be 0.25～0.5g, cinnamic acid be 0.05～0.3g,MgSO4Be 1.0～3.0g, KH2PO4Be that 0.5～2.0g, Tween-80 are 1.0～3.0g, add water to 1L,The pH of this fermentation medium is 6.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610075925.1A CN105671100B (en) | 2016-02-03 | 2016-02-03 | A method of synthesis Phellinus high activity flavone compound |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610075925.1A CN105671100B (en) | 2016-02-03 | 2016-02-03 | A method of synthesis Phellinus high activity flavone compound |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105671100A true CN105671100A (en) | 2016-06-15 |
| CN105671100B CN105671100B (en) | 2019-04-12 |
Family
ID=56303948
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610075925.1A Active CN105671100B (en) | 2016-02-03 | 2016-02-03 | A method of synthesis Phellinus high activity flavone compound |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105671100B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112063663A (en) * | 2020-08-24 | 2020-12-11 | 浙江省农业科学院 | Fermentation method for high-yield phellinus igniarius extracellular flavonoids |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182550A (en) * | 2007-11-16 | 2008-05-21 | 上海市农业科学院 | Flavonoids from phellinus, method of producing the same and use |
| CN103695316A (en) * | 2013-12-04 | 2014-04-02 | 广东省农业科学院蚕业与农产品加工研究所 | Liquid fermentation method for increasing flavone yield of phellinus igniarius mycelium |
-
2016
- 2016-02-03 CN CN201610075925.1A patent/CN105671100B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182550A (en) * | 2007-11-16 | 2008-05-21 | 上海市农业科学院 | Flavonoids from phellinus, method of producing the same and use |
| CN103695316A (en) * | 2013-12-04 | 2014-04-02 | 广东省农业科学院蚕业与农产品加工研究所 | Liquid fermentation method for increasing flavone yield of phellinus igniarius mycelium |
Non-Patent Citations (2)
| Title |
|---|
| 刘伟: "药用菌桑黄代谢黄酮的调控研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 陈坚等: "微生物合成黄酮类化合物的研究进展", 《食品科学技术学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112063663A (en) * | 2020-08-24 | 2020-12-11 | 浙江省农业科学院 | Fermentation method for high-yield phellinus igniarius extracellular flavonoids |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105671100B (en) | 2019-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102351605B (en) | Culture method for liquid fermentation of rare edible-medicinal fungus Sparassis crispa and special medium thereof | |
| CN104285820B (en) | One strain selenium-enriched cordceps militaris strain and application thereof | |
| CN102199054B (en) | Production method and culture medium formula of zinc-rich edible mushroom | |
| CN101297821B (en) | Phellinus linteus mycelia active glucoprotein and use thereof and preparation | |
| CN103477994B (en) | Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran | |
| CN102816806B (en) | Production method of grifolan selenium compound | |
| CN1759760A (en) | Method for producing fruit juice of germinant unpolished rice and ganoderma lucidum, and production | |
| CN102210449A (en) | Liquid submerged fermentation culture of Thelephora ganbajun Zang and preparation methods of seasoning and selenium-enriched product of Thelephora ganbajun Zan | |
| CN1796539A (en) | Ferment for producing aweto in large scale and technique for processing power of fungus | |
| CN105969673A (en) | Fermentation method for increasing yield of haematochrome and uranidin in monascus purpureus | |
| CN103431368A (en) | Preparation method and comprehensive utilization of submerged fermentation product of cordyceps sinensis liquid | |
| CN103087893B (en) | Preparation method of composite coarse cereals monascus | |
| CN102286324B (en) | Method for preparing gingko golden fungus fruit wine | |
| CN104030843B (en) | A kind of Cordyccps-militaris-(L.)-link. Sporophore substratum adding hop residue | |
| CN103948023B (en) | The health food of a kind of develop immunitypty and improving water flood and two-step fermentation preparation method thereof | |
| CN110684672A (en) | Fermentation method of antioxidant cordyceps sobolifera mycelium | |
| CN102174414B (en) | Application of new morchella costata M8-13 liquid fermentation substance in development of health-care products and medicaments | |
| KR20100001375A (en) | New method for preparing the low molecular fucoidan from the brown algae fermentation | |
| CN105671100A (en) | Method for synthesizing phellinus high-activity flavonoid compound | |
| CN103555786A (en) | Method for producing polysaccharides through liquid state fermentation of rice bran and wheat bran complete material by using ganoderma lucidum mutant strain | |
| CN102584363B (en) | Method for producing medical mycelium or medical and edible dual-purpose mycelium by using yellow wine lees as liquid medium | |
| CN103876014B (en) | Compound phellinus oral liquid and preparation method thereof | |
| CN115039633A (en) | Artificial culture method for sporocarp of Isaria japonica | |
| CN102816807B (en) | Production method of grifolan manganese compound | |
| CN103468629B (en) | Production process for fermenting trichoderma spp by taking orange peels as raw materials |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |