CN109295120A - A method of flavones is obtained using Penicillium notatum bioconversion corn stigma - Google Patents
A method of flavones is obtained using Penicillium notatum bioconversion corn stigma Download PDFInfo
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Abstract
The present invention provides a kind of methods for obtaining flavones using Penicillium notatum bioconversion corn stigma, this method utilizes production flavones Penicillium notatum, it ferments to the culture solution containing corn stigma, flavones precursor substance in corn stigma is made full use of to carry out bioconversion in fermentation process, to largely accumulate flavonoid substance, then purifies and obtain high-purity flavones.Wherein, fermentation medium forms are as follows: corn stigma 30 ~ 50g of powder, 2 ~ 5g of yeast powder, 0.5 ~ 1g of dipotassium hydrogen phosphate, 0.5 ~ 1g of magnesium sulfate, 0.5 ~ 1g of potassium chloride, 0.01 ~ 0.1g of ferrous sulfate, vitamin B20.05 ~ 0.2g, vitamin B60.05 ~ 0.1g, 0.05 ~ 0.3g of vitamin C, 20 ~ 40g of maltose add water to 1L.The beneficial effect is that the method is at low cost, yield is high, and product purity is high, and activity is good, and step operation is simple, is suitble to industrial production.
Description
Technical field
The invention belongs to technical field of biological extraction, utilize Penicillium notatum bioconversion corn stigma more particularly, to a kind of
The method for obtaining flavones.
Background technique
Corn is important one of the grass family cereal crops in the world today, and it is me that corn stigma, which is that it dries filigree and column cap,
The traditional Chinese medicine of state, have expel the heat-evil it is treating stranguria, the effect of calming the liver and benefiting the lungs.Studies have shown that corn stigma contain multiple biological activities at
Point, such as volatile oil, saponin(e, flavones, there is extensive pharmacological effect, it is wherein many kinds of existing for flavones, and its chemistry
Structure very complicated, contains a keto-acid hydroxyl in its chemical structural formula, and most of its hydroxy derivatives present it is yellow
Color, so referred to as flavone compound.The pharmacological effect of flavone compound is significant, and having reduces myocardial oxygen consumption, increases hat
Shape artery and cerebrovascular flow, antibacterial, antitumor, diuresis, enhance immune, anti-oxidant, anti-aging, enhancing at hypoglycemic, reducing blood lipid
The functions such as immunity of organisms.China's corn planting is extensive, corn stigma resource very abundant, but development and utilization at present are seldom, except few
Amount be used as medicine it is outer be dropped, exploitation prospect is boundless.
Currently, Maize silk flavones extraction mostly uses extraction, enzyme process, microwave loss mechanisms and ultrasonic wave assisted extraction method etc..
The time that extraction generally expends is longer, and recovery rate is not high, it is easy to infiltrate impurity, make troubles for later separation purification.Enzyme
Although method can be such that flavones yield greatly improves, and avoid the use of a large amount of organic solvents, brought very for subsequent purifying technique
It is big convenient but at high cost, and take a long time.Microwave loss mechanisms have it is at low cost, extraction efficiency is high, selectivity is strong, extraction time
The advantages that short, solvent usage is lacked, but only rested at present to certain specific explorations extracted objects and carry out simple process conditions
On.Ultrasonic wave assisted extraction method is with recovery rate is relatively high, the used time is short, save solvent, reduces high temperature to the shadow for proposing ingredient
The advantages that ringing, but recovery rate is still to be improved.And the bis- enzymes of ultrasonic wave-cooperate with extraction method, while shortening extraction time, also greatly
The big recovery rate for improving flavones, but equally exist disadvantage at high cost.Big, flavones basis is expended and all there are raw material in above method
The low problem of content.
Summary of the invention
To solve the above problems, the present invention provides a kind of method for obtaining flavones using Penicillium notatum bioconversion corn stigma,
It using the Penicillium notatum for producing flavones, ferments, is made full use of in fermentation process yellow in corn stigma to the culture solution containing corn stigma
Ketone precursor substance carries out bioconversion, to largely accumulate flavonoid substance, then purifies the process for obtaining high-purity flavones.The side
Method comprises the following steps:
S1: by Penicillium strain after being activated on activation medium, by bacteria suspension concentration 104~106 CFU/ml, inoculation 1~5
Ml obtains fermentation liquid in 5 ~ 7d of shaking table culture in fermentation culture, and wherein shaking speed is 100 ~ 200rpm, cultivation temperature 25 ~ 35
DEG C, initial pH is 6 ~ 7, fermentation culture component are as follows: corn stigma 30 ~ 50g of powder, 2 ~ 5g of yeast powder, 0.5 ~ 1g of dipotassium hydrogen phosphate, sulphur
Sour 0.5 ~ 1g of magnesium, 0.5 ~ 1g of potassium chloride, 0.01 ~ 0.1g of ferrous sulfate, vitamin B20.05 ~ 0.2 g, vitamin B6 0.05
~ 0.1g, 0.05 ~ 0.3g of vitamin C, 20 ~ 40 g of maltose, add water to 1L；
S2: fermentation liquid in step S1 is taken to carry out 5~10 min of clasmatosis processing, 2000 ~ 4000 rpm are centrifuged 5 ~ 10 min, take
Supernatant, adjusting pH is 2~4, after 1~1.5 times of 40% ~ 60% ethanol solution of volume is added, 30~40 DEG C of 8~12h of standing；
S3: the supernatant of solution after taking step S2 to stand is concentrated into 200 mL, adjusts pH to 2~3,1~1.5 times of volume is added
Organic solvent extracts 2~3 times repeatedly, is concentrated to give flavones crude extract；
S4: will be added 1~1.5 times of 40 ~ 60% ethanol solution of volume extraction in step S3 flavones crude extract, 4000 ~ 6000 rpm turn
Speed centrifugation, collects 40 ~ 60 DEG C of solution and evaporates to obtain flavones concentrate；
S5: after flavones concentrate in step S4 by volume 1:100 ~ 500 plus distilled water dilution, adjusting pH is 2 ~ 3, by 10 ~
20mL/min flow velocity crosses D-101 macroreticular resin chromatography, finally elutes 3 ~ 10min with 40 ~ 60% ethanol solutions, wherein elution liquid stream
Speed is 50 ~ 100mL/min, collects eluent, 40 ~ 60 DEG C are concentrated to give Flavonoid substances.
Further, Penicillium notatum is the penicillium bacterial strain for producing flavones in the step S1.
Further, Penicillium notatum is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms in the step S1
Center, preservation date are on May 14th, 2012, deposit number are as follows: CGMCC NO.6113.
Further, activation medium is PDA culture medium in the step S1.
Further, fermentation medium component in the step S1 are as follows: corn stigma powder 50g, yeast powder 3g, dipotassium hydrogen phosphate
1g, magnesium sulfate 0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g, vitamin B2 0.1g, vitamin B60.1g, vitamin C
0.1g, maltose 30g, adds water to 1L.
Further, clasmatosis is Mechanical Crushing in the step S2, ultrasonication or the broken one kind of enzyme or it is a kind of with
On.
Further, organic solvent is ethyl acetate, one of butyl acetate or n-butanol in the step S3.
Further, organic solvent is ethyl acetate in the step S3.
Further, corn stigma powder is to crushed 40 ~ 60 mesh for after 40 ~ 60 DEG C of corn stigma dry 6 ~ 8h in the step S1
Sieve is made.
Compared with the existing technology, the present invention has the advantage that and effect:
1 production cost is low, and yield is high, and by-product is few, is convenient for separating-purifying, and purity is higher.
2 fermentation conditions are mildly easily controllable, are easy to be mass produced.
For 3 organic solvents using less, product is environmentally friendly convenient for recycling.
Specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.Unless stated otherwise, the present invention uses reagent, method and apparatus is the art conventional reagents, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are commercially available.
The preparation of culture medium in following embodiment and comparative example:
The preparation of activation medium: 200 g of fresh potato, by peeling potatoes, stripping and slicing is added suitable quantity of water and boils, 30 min；
Then by 6 layers of filtered through gauze of potato water cooking liquid, 1L is added water to；20 g of glucose is weighed, agar 20g is dissolved in potato water cooking liquid
In, pH is naturally, sterilizing.
Penicillium notatum used in the present embodiment and comparative example is commercially available acquisition, and the Penicillium is in penicillium communne
(Penicillium commune).Now be preserved in budapest treaty microorganism International Depository Authority: Chinese microorganism strain is protected
It hides administration committee's common micro-organisms center heart (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology of the academy of sciences, postcode: 100101；Preservation date is on May 14th, 2012, deposit number are as follows: CGMCC
NO.6113。
Embodiment 1:
The preparation of fermentation medium used in the embodiment: corn stigma powder 50g, yeast powder 3g, dipotassium hydrogen phosphate 1g, sulphur
Sour magnesium 0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g, vitamin B2 0.1g, vitamin B6 0.1g, vitamin C
0.1g, maltose 30g, adds water to 1L.
A method of flavones being obtained using Penicillium notatum bioconversion corn stigma, is included the following steps:
S1: by Penicillium notatum CGMCCNO.6113 after being activated on activation medium, by bacteria suspension concentration 104 CFU/ml, inoculation
1ml obtains fermentation liquid in shaking table culture 7d in fermentation culture, wherein shaking speed be 200rpm, 35 DEG C of cultivation temperature, initial pH
It is 7；
S2: take fermentation liquid in step S1 that driselase is added to carry out 37 DEG C of 5 min of water bath processing of clasmatosis, to discharge product in bacterium.
2000 rpm are centrifuged 5 min, take supernatant, and adjusting pH is 4, after 1 times of 40% ethanol solution of volume is added, 40 DEG C of standing 12h；
S3: the supernatant of solution after taking step S2 to stand is concentrated into 200 mL, adjusts pH to 2,1 times of volume of ethylacetate is added
It extracts 3 times repeatedly, is concentrated to give flavones crude extract 20mL；
S4: will be added the extraction of 40% ethanol solution of 20mL in flavones crude extract, 40 DEG C of solution steamings are collected in the centrifugation of 4000 rpm revolving speeds
Send out to obtain flavones concentrate 10mL；
S5: after adding 1000mL distilled water to dilute by 1:100 flavones concentrate in step S5, adjusting pH is 4, is flowed by 20mL/min
Speed crosses D-101 macroreticular resin chromatography, is finally eluted with 40% ethanol solution, collects eluent, 40 DEG C are concentrated to give Flavonoid substances
13.7g。
Embodiment 2:
The preparation of fermentation medium in the embodiment: corn stigma powder 50g, yeast powder 5g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate
0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g, vitamin B2 0.05g, vitamin B6 0.05g, vitamin C 0.05g,
Maltose 40g, adds water to 1L.
A method of flavones being obtained using Penicillium notatum bioconversion corn stigma, is included the following steps:
S1: by Penicillium notatum CGMCCNO.6113 after being activated on activation medium, by bacteria suspension concentration 106 CFU/ml, inoculation
1ml obtains fermentation liquid in shaking table culture 5d in fermentation culture, wherein shaking speed be 150rpm, 30 DEG C of cultivation temperature, initial pH
It is 6.6；
S2: take fermentation liquid in step S1 that driselase 2000u is added to carry out 37 DEG C of water bath processing 10min of clasmatosis, to discharge in bacterium
Product.4000 rpm are centrifuged 5 min, take supernatant, and adjusting pH is 4, after 1 times of 60% ethanol solution of volume is added, 40 DEG C of standings
12h；
S3: the supernatant of solution after taking step S2 to stand is concentrated into 200 mL, adjusts pH to 2,1 times of volumes of acetic acid butyl ester is added
It extracts 3 times repeatedly, is concentrated to give flavones crude extract 20mL；
S4: will be added the extraction of 60% ethanol solution of 20mL in flavones crude extract, the centrifugation of 6000 rpm revolving speeds collects 60 DEG C of solution
Evaporate to obtain flavones concentrate 10mL；
S5: after adding 1000mL distilled water to dilute by 1:100 flavones concentrate in step S4, adjusting pH is 4, is flowed by 30mL/min
Speed crosses D-101 macroreticular resin chromatography, is finally eluted with 40% ethanol solution, collects eluent, 60 DEG C are concentrated to give Flavonoid substances
9.8g。
Comparative example 1:
A method of flavones being obtained using Penicillium notatum bioconversion corn stigma, is included the following steps:
S1: taking fresh corn silk tap water to rinse, and the several seconds is first impregnated in 70% alcohol to remove air bubble, with 1.0% chlorine
Sour sodium sterilizes 10min, uses aseptic water washing 3 times later, and after 60 DEG C of dry 8h, crushing is sieved with 100 mesh sieve.
S2: 50g corn stigma powder 1000mL water is taken to dissolve；Adjusting pH is 4, after 1 times of 40% ethanol solution of volume is added, 40
DEG C stand 12h；
S3: the supernatant of solution after taking step S2 to stand is concentrated into 200 mL, adjusts pH to 2,1 times of volume of ethylacetate is added
It extracts 3 times repeatedly, is concentrated to give flavones crude extract 20mL；
S4: 40% ethanol solution will be added in flavones crude extract and dissolve centrifugation repeatedly, collect 40 DEG C of solution and evaporate to obtain flavones concentrate
10mL；
S5: after adding 1000mL distilled water to dilute by 1:100 flavones concentrate in step S5, adjusting pH is 4, is flowed by 20mL/min
Speed crosses D-101 macroreticular resin chromatography, is finally eluted with 40% ethanol solution, collects eluent, 40 DEG C are concentrated to give Flavonoid substances
1.1g。
Comparative example 1 the difference from embodiment 1 is that: by purification by liquid extraction directly from equivalent corn stigma powder, obtain flavones
Substance.
Comparative example 2
The preparation of fermentation medium in the comparative example: yeast powder 3g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, potassium chloride
0.5g, ferrous sulfate 0.01g, vitamin B20.1g, vitamin B6 0.1g, vitamin C 0.1g, maltose 30g are added water to
1L。
A method of flavones being obtained using Penicillium notatum bioconversion corn stigma, is included the following steps:
S1: by Penicillium notatum CGMCCNO.6113 after being activated on activation medium, by bacteria suspension concentration 104 CFU/ml, inoculation
1ml obtains fermentation liquid in shaking table culture 7d in fermentation culture, wherein shaking speed be 200rpm, 35 DEG C of cultivation temperature, initial pH
It is 7；
S2: take fermentation liquid in step S1 that driselase is added to carry out 37 DEG C of 5 min of water bath processing of clasmatosis, to discharge product in bacterium.
2000 rpm are centrifuged 5 min, take supernatant, and adjusting pH is 4, after 1 times of 40% ethanol solution of volume is added, 40 DEG C of standing 12h；
S3: the supernatant of solution after taking step S2 to stand is concentrated into 200 mL, adjusts pH to 2,1 times of volume of ethylacetate is added
It extracts 3 times repeatedly, is concentrated to give flavones crude extract 20mL；
S4: 40% ethanol solution will be added in flavones crude extract and dissolve centrifugation repeatedly, collect 40 DEG C of solution and evaporate to obtain flavones concentrate
10mL；
S5: after adding 1000mL distilled water to dilute by 1:100 flavones concentrate in step S4, adjusting pH is 4, is flowed by 20mL/min
Speed crosses D-101 macroreticular resin chromatography, is finally eluted with 40% ethanol solution, collects eluent, 40 DEG C are concentrated to give Flavonoid substances
4.1g。
Comparative example 2 the difference from embodiment 1 is that: corn stigma powder is not added in fermentation medium, directly utilize Penicillium notatum
Fermentation obtains flavonoid substance.
Claims (9)
1. a kind of method for obtaining flavones using Penicillium notatum bioconversion corn stigma, which is characterized in that the method includes as follows
Step:
S1: by Penicillium strain after being activated on activation medium, by bacteria suspension concentration 104~106 CFU/ml, inoculation 1~5
Ml obtains fermentation liquid in 5 ~ 7d of shaking table culture in fermentation culture, and wherein shaking speed is 100 ~ 200rpm, cultivation temperature 25 ~ 35
DEG C, initial pH is 6 ~ 7, fermentation culture component are as follows: corn stigma 30 ~ 50g of powder, 2 ~ 5g of yeast powder, 0.5 ~ 1g of dipotassium hydrogen phosphate, sulphur
Sour 0.5 ~ 1g of magnesium, 0.5 ~ 1g of potassium chloride, 0.01 ~ 0.1g of ferrous sulfate, vitamin B20.05 ~ 0.2 g, vitamin B6 0.05
~ 0.1g, 0.05 ~ 0.3g of vitamin C, 20 ~ 40 g of maltose, add water to 1L；
S2: fermentation liquid in step S1 is taken to carry out clasmatosis 5~10 min of processing, 2000 ~ 4000 rpm revolving speeds centrifugation 5 ~ 10
Min takes supernatant, and adjusting pH is 2~4, after 1~1.5 times of 40% ~ 60% ethanol solution of volume is added, 30~40 DEG C of standings 8~
12h；
S3: the supernatant of solution after taking step S2 to stand is concentrated into 200 mL, adjusts pH to 2~3,1~1.5 times of volume is added
Organic solvent extracts 2~3 times repeatedly, is concentrated to give flavones crude extract；
S4: will be added 1~1.5 times of 40 ~ 60% ethanol solution of volume extraction in step S3 flavones crude extract, 4000 ~ 6000 rpm turn
Speed centrifugation, collects 40 ~ 60 DEG C of solution and evaporates to obtain flavones concentrate；
S5: after flavones concentrate in step S4 by volume 1:100 ~ 500 plus distilled water dilution, adjusting pH is 2 ~ 3, by 10 ~
20mL/min flow velocity crosses D-101 macroreticular resin chromatography, finally elutes 3 ~ 10min with 40 ~ 60% ethanol solutions, wherein elution liquid stream
Speed is 50 ~ 100mL/min, collects eluent, 40 ~ 60 DEG C are concentrated to give Flavonoid substances.
2. the method for obtaining flavones using Penicillium notatum bioconversion corn stigma according to claim 1, which is characterized in that described
Penicillium notatum is the penicillium bacterial strain for producing flavones in step S1.
3. the method for obtaining flavones using Penicillium notatum bioconversion corn stigma according to claim 1, which is characterized in that described
Penicillium notatum is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in step S1, and preservation date is 2012
On May 14, in, deposit number are as follows: CGMCC NO.6113.
4. the method for obtaining flavones using Penicillium notatum bioconversion corn stigma according to claim 1, which is characterized in that described
Activation medium is PDA culture medium in step S1.
5. the method for obtaining flavones using Penicillium notatum bioconversion corn stigma according to claim 1, which is characterized in that described
Fermentation culture component in step S1 are as follows: corn stigma powder 50g, yeast powder 3g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, chlorination
Potassium 0.5g, ferrous sulfate 0.01g, vitamin B20.1g, vitamin B6 0.1g, vitamin C 0.1g, maltose 30g add
Water is to 1L.
6. the method for obtaining flavones using Penicillium notatum bioconversion corn stigma according to claim 1, which is characterized in that described
Clasmatosis is the broken one or more of Mechanical Crushing, ultrasonication or enzyme in step S2.
7. the method for obtaining flavones using Penicillium notatum bioconversion corn stigma according to claim 1, which is characterized in that described
Organic solvent is one of ethyl acetate, butyl acetate or n-butanol in step S3.
8. the method for obtaining flavones using Penicillium notatum bioconversion corn stigma according to claim 1, which is characterized in that described
Organic solvent is ethyl acetate in step S3.
9. the method for obtaining flavones using Penicillium notatum bioconversion corn stigma according to claim 1, which is characterized in that described
Corn stigma powder is after 40 ~ 60 DEG C of corn stigma dry 6 ~ 8h, will to crushed 40 ~ 60 meshes to be made in step S1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107050099A (en) * | 2017-04-21 | 2017-08-18 | 贵阳中医学院 | The high efficiency extraction equipment and its extracting method of a kind of caragana general flavone |
| CN113564055A (en) * | 2021-07-26 | 2021-10-29 | 浙江树人学院（浙江树人大学） | Penicillium lanuginosum DZ-9-67 and application thereof in total flavone extraction of eucommia ulmoides leaves |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107050099A (en) * | 2017-04-21 | 2017-08-18 | 贵阳中医学院 | The high efficiency extraction equipment and its extracting method of a kind of caragana general flavone |
| CN113564055A (en) * | 2021-07-26 | 2021-10-29 | 浙江树人学院（浙江树人大学） | Penicillium lanuginosum DZ-9-67 and application thereof in total flavone extraction of eucommia ulmoides leaves |
| CN113564055B (en) * | 2021-07-26 | 2023-09-01 | 浙江树人学院（浙江树人大学） | Felt Mao Qingmei DZ-9-67 and application thereof in extraction of total flavonoids of eucommia ulmoides leaves |
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