CN108977475A - A kind of production method of Guava Leaf flavone aglycone - Google Patents
A kind of production method of Guava Leaf flavone aglycone Download PDFInfo
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Abstract
The invention discloses a kind of production methods of Guava Leaf flavone aglycone, comprising the following steps: (1) flavone compound is extracted from Guava Leaf；(2) flavone compound obtained in step (1) is added in the fluid nutrient medium containing streptomyces griseus (Streptomyces griseus) and is cultivated, oscillation and fermentation cultivation obtains fermentation liquid；(3) the oscillation enzymatic hydrolysis of fermentation liquid obtained in step (2) is obtained into enzymatic hydrolysis solution；(4) enzymatic hydrolysis solution left standstill, centrifugation, precipitating obtained in step (3) are dissolved with methanol, the methanol solution containing flavone aglycone is obtained after filtering.The production method of Guava Leaf flavone aglycone of the present invention is easy, easily operated, and the impurity content of finally obtained flavone aglycone is small, subsequent purification simple process.
Description
Technical field
The invention belongs to guava field more particularly to a kind of production methods of guava leaf extract.
Background technique
Guava (Psidium guaijava L.) be Myrtaceae Psidium plant, be tropical evergreen dungarunga or
Shrub originates in tropical America, China is passed to before several centuries, the ground such as present China Fujian, Guangdong, Taiwan have extensive kind
It plants.Chinese medicine thinks that its mild-natured taste is dry and astringent, has the function of astringing to arrest diarrhea etc., volatile oil has antibacterial, antivirus action, to digestion
System also plays the role of spasmolysis, enhancing gastrointestinal peristalsis.Modern research shows that containing there are many important aromatics second levels in guava
Metabolite such as polyphenol chromocor class compound has scholar to think that exactly these phenolic compounds assign a variety of medicinal function of guava
Effect.Thus, there are preferable prospect and necessity for the flavone aglycone resource progress developmental research of Guava Leaf.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the shortcomings of to mention in background above technology and defect, one kind is provided
The production method of Guava Leaf flavone aglycone at low cost, quality is high.In order to solve the above technical problems, technology proposed by the present invention
Scheme are as follows:
A kind of production method of Guava Leaf flavone aglycone, comprising the following steps:
(1) flavone compound is extracted from Guava Leaf；
(2) flavone compound obtained in step (1) is added to containing streptomyces griseus (Streptomyces
Griseus ATCC 13273) fluid nutrient medium in cultivate, under the conditions of 25-40 DEG C, sent out within oscillation and fermentation cultivation 6-8 days
Ferment solution；
(3) by fermentation liquid obtained in step (2) under the conditions of 50-55 DEG C, oscillation enzymatic hydrolysis 3-4h obtains enzymatic hydrolysis solution；
(4) enzymatic hydrolysis solution left standstill, centrifugation, precipitating obtained in step (3) are dissolved with methanol, is obtained after filtering containing Huang
The methanol solution of ketoside member.
In the production method of above-mentioned Guava Leaf flavone aglycone, streptomyces griseus firstly the need of elder generation with potato culture into
Row expands culture as seed, inoculates to new potato culture.
In the production method of above-mentioned Guava Leaf flavone aglycone, it is preferred that the fluid nutrient medium containing streptomyces griseus
For standard potato culture, formula are as follows: peeling fresh potato 200g, glucose 20g, MgSO4·7H2O 1.5g、
KH2PO43g, 1L distilled water, the initial pH value of the standard potato culture is without adjusting.It is metabolized and is produced by streptomyces griseus
Raw rhamnosidase and β-D-Glucose glycosides enzyme etc. can be such that the flavone glycoside ingredient in extract acts on by microbial fermentation
It is converted into flavone aglycone (Quercetin, Kaempferol and Isorhamnetin etc.), is converted with microorganism, product amount can be increased,
The shortcomings that overcoming chemical synthesis, and because microbial fermentation is acted in neutrality, under room temperature mostly, reduce product isomery, disappear
The unfavorable factors such as rotation and rearrangement reaction.
In the production method of above-mentioned Guava Leaf flavone aglycone, it is preferred that the fluid nutrient medium containing streptomyces griseus
The inoculum concentration of middle streptomyces griseus is 10000-60000 spore of every milliliter of inoculation of medium.It is furthermore preferred that being 10000-
60000 spores.
In the production method of above-mentioned Guava Leaf flavone aglycone, it is preferred that in the step (2), the flavonoid
The additional amount of object and the mass ratio control of fluid nutrient medium are (0.5-1.5): 5.
In the production method of above-mentioned Guava Leaf flavone aglycone, it is preferred that extracted from Guava Leaf in the step (1)
The method of flavone compound is buck or diluted alkaline alcohol extraction method, organic solvent extraction.Since flavones ingredient has mostly
There is phenolic hydroxyl group, therefore available alkaline water such as sodium carbonate, sodium hydroxide, calcium hydroxide aqueous solution or diluted alkaline alcohol such as ethyl alcohol leach,
Flavone compound is precipitated after leachate is acidified.Organic solvent extraction is current most popular method both at home and abroad, is had
Solvent extracts, product yield height simple to equipment requirement, industrialized production easy to accomplish, but higher cost, impurity content
Higher, the organic solvent for being usually used in extracting flavonoids has methanol, ethyl alcohol, acetone, acetic acid ethyl ester, ether etc., it is contemplated that methanol, acetone
Deng be more toxic, preferably take ethyl alcohol be Extraction solvent.
In the production method of above-mentioned Guava Leaf flavone aglycone, it is preferred that extracted from Guava Leaf in the step (1)
The method of flavone compound is microwave-assisted eutectic extractive technique, process conditions are as follows: with the processing kind of 200W microwave intensity
After pomegranate leaf 2min, mixed for 4:1 ratio as eutectic solvent, control using butanediol and choline chloride according to molal volume ratio
Solid-liquid ratio is 1:22, extracts 40min at 65 DEG C.When microwave radiation exaraction, the polar molecule being extracted is quick in microwave electric field
It turns to and aligns, to generate tearing and phase mutual friction causes to generate heat, it is ensured that the quick transmitting of energy and sufficiently benefit
With, be easy to dissolve out and discharge.Meanwhile can protect environment using low eutectic solutions, reduce waste liquid and the pollution of the mankind is generated.
In the production method of above-mentioned Guava Leaf flavone aglycone, it is preferred that extracted from Guava Leaf in the step (1)
The method of flavone compound is ultrasonic wave extraction, process conditions are as follows: using 75% ethyl alcohol as solution, control solid-liquid ratio is
1:28 extracts 30min at 42 DEG C.Ultrasonic wave extraction is to reinforce guava effective component using the cavitation of ultrasonic wave
Leaching extract, in addition, such as mechanical oscillation of the second order effect of ultrasonic wave, emulsification, spread, smash, chemical effect etc. can also accelerate
The dispersal events of target component are simultaneously sufficiently mixed with solvent, are conducive to extract.
In the production method of above-mentioned Guava Leaf flavone aglycone, it is preferred that flavone compound also passes through in the step (1)
It crosses high-speed countercurrent chromatography and isolates and purifies process, process conditions are as follows: using petroleum ether-ethyl acetate-alcohol-water as two-phase
Dicyandiamide solution, each component are prepared according to the ratio that volume ratio is 2: 2: 3: 1, are stood 6-10h and are obtained respectively as upper and lower phase
Solution component is injected upper phase in HSCCC separating pipe with maximum flow rate, and adjustment engine speed is after upper phase is full of entire pipeline
720r/min, then lower phase is injected with 1.5mL/min flow velocity；It is flowed out to mobile phase from column outlet, two-phase reaches dynamic in separating pipe
After state balance, the solution obtained through ultrasonic wave extraction is injected by sampling valve.After ultrasonic wave extraction, using efficient
Adverse current chromatogram (HSCCC) technology can obtain the flavone extractive of higher purity, be conducive to separating and purifying flavone class compound
Improve the quality of final products.
In the production method of above-mentioned Guava Leaf flavone aglycone, it is preferred that the flavone compound warp in the step (1)
It adds in the fluid nutrient medium containing streptomyces griseus and cultivates after freeze-drying.
In the production method of above-mentioned Guava Leaf flavone aglycone, it is preferred that using fibre when oscillation enzymatic hydrolysis in the step (3)
Tie up the mixed solvent of plain enzyme and pectase.The mixed solvent of cellulase and pectase can convert oil-soluble flavonoids to
Glycoside soluble easily in water is dissolved in enzymatic hydrolysis solution and is conducive to extract, and improves flavones content, and passes through enzyme reaction for plant tissue point
Solution makes to extract resistance to mass tranfer reduction, accelerates reaction process.In addition, the impurity in extracting solution can be made to decompose removal, thus after simplifying
It is continuous to isolate and purify process.
Compared with the prior art, the advantages of the present invention are as follows:
1, the present invention uses the fluid nutrient medium culture flavone compound containing streptomyces griseus, and streptomyces griseus catalysis is yellow
For hydroxyl on ketone parent nucleus at methoxyl group, this process is that one of mode of biologic detoxication and Structures of Natural Products modification generate surely
One of the important reaction for determining compound, reduces the unfavorable factors such as product isomery, racemization and rearrangement reaction.In addition, microorganism sends out
Ferment effect is acted in neutrality, under room temperature mostly, can more conveniently control reaction environment, is reduced in large-scale production,
To the big cost input of the control conditions such as workshop temperature, humidity, economy.
2, the production method of Guava Leaf flavone aglycone of the present invention is easy, easily operated, finally obtained flavone aglycone
Impurity content is small, subsequent purification simple process.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is the structural schematic diagram of extracting flavonoids equipment in embodiment 5.
Fig. 2 is the Section View in the face A-A in Fig. 1.
Marginal data:
1, Multifunction tank；2, desorption column；3, rotary harrow；4, circulating tank is leached；5, guide shell；6, inverted conical shape gas is kept off
Plate；7, the snorkel of provided with electromagnetic valve；8, fixed link；9, collet heating device；101, first pipe；102, second pipe；103,
Third pipeline；104 the 4th pipelines；105, the second ventilation forcing pipe；107, the first ventilation forcing pipe；108, exhaust pipe.
Specific embodiment
To facilitate the understanding of the present invention, the present invention is made below in conjunction with Figure of description and preferred embodiment more complete
Face meticulously describes, but the protection scope of the present invention is not limited to the following specific embodiments.
Unless otherwise defined, all technical terms used hereinafter and the normally understood meaning of those skilled in the art
It is identical.Technical term used herein is intended merely to the purpose of description specific embodiment, is not intended to the limitation present invention
Protection scope.
Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention can pass through city
Field is commercially available or can be prepared by existing method.
Embodiment 1:
A kind of production method of Guava Leaf flavone aglycone, comprising the following steps:
(1) it under the conditions of 45 DEG C, to Guava Leaf by 80% methanol aqueous solution mechanical shaking extraction 8h, filters, in triplicate,
Combined extract, then extracting solution is obtained into extract powder by being freeze-dried；
(2) flavone compound obtained in step (1) is added to containing streptomyces griseus (Streptomyces
Griseus ATCC 13273) fluid nutrient medium in cultivate (additional amount of flavone compound and the quality of fluid nutrient medium
It is 1:5 than control), every milliliter of culture medium inoculated amount is 3 × 105A spore, under the conditions of 25-40 DEG C, oscillation and fermentation cultivation 6-8
It obtains fermentation liquid；Wherein, the fluid nutrient medium containing streptomyces griseus is standard potato culture, formula are as follows: peeling
Fresh potato 200g, glucose 20g, MgSO4·7H2O 1.5g、KH2PO43g, 1L distilled water, the standard potato culture
The initial pH value of base is without adjusting；
(3) by fermentation liquid obtained in step (2) under the conditions of 50-55 DEG C, oscillation enzymatic hydrolysis 3-4h is digested (10U/
The cellulase and pectase of ml) solution；
(4) enzymatic hydrolysis solution left standstill, centrifugation, precipitating obtained in step (3) are dissolved with methanol, is obtained after filtering containing Huang
The methanol solution of ketoside member.
Comparative example 1:
This comparative example compared with Example 1, the difference is that only and handle without step (2).But it is used in step (3)
Enzyme include a variety of enzyme α-rhamnosidase, beta-glucosidase, zytase, cellulase, pectase, alpha-amylase etc. connection
It closes and uses, usage amount usually requires the enzyme amount of 20U/ml or so.
Through detecting, the obtained flavone aglycone of final production is handled by streptomyces griseus, and is not passed through at streptomyces griseus
Reason, but directly adopt the flavone aglycone that progress bioenzymatic conversion produces after organic solvent extracts and compare, as a result such as the following table 1
It is shown.
Table 1: flavone aglycone yield and flavones total conversion in embodiment 1 and comparative example 1
Embodiment 1 is compared with comparative example 1, although the total conversion of the interest rate of each flavone aglycone in comparative example 1 and flavones
Compared to the height of embodiment 1, but the type of enzyme used in comparative example 1 and dosage are significantly more than embodiment 1, this is in cost
It is worthless.
Embodiment 2:
A kind of production method of Guava Leaf flavone aglycone, comprising the following steps:
(1) using 75% ethyl alcohol as solution, the solid-liquid ratio for controlling Guava Leaf and ethyl alcohol is 1:28, ultrasonic wave extraction at 42 DEG C
Time 30min, then extracting solution is obtained into extract powder by being freeze-dried；
(2)-(4) are identical as (2)-(4) the step of embodiment 1.
Embodiment 3:
A kind of production method of Guava Leaf flavone aglycone, comprising the following steps:
(1) after handling Guava Leaf 2min with 200W microwave intensity, with butanediol and choline chloride according to molal volume ratio
It is used as eutectic solvent for the mixing of 4:1 ratio, control solid-liquid ratio is 1:22, extracts 40min at 65 DEG C, then extracting solution is passed through
Freeze-drying, obtains extract powder；
(2)-(4) are identical as (2)-(4) the step of embodiment 1.
Embodiment 4:
A kind of production method of Guava Leaf flavone aglycone, comprising the following steps:
(1) identical as step (1) in embodiment 3；
(2) extract powder obtained in step (1) is isolated and purified into process, high-speed counter-current through high-speed countercurrent chromatography
Chromatographic technique isolates and purifies the process conditions of process are as follows: using petroleum ether-ethyl acetate-alcohol-water as two phase solvent system,
Each component is prepared according to the ratio that volume ratio is 2: 2: 3: 1, is stood 6-10h and is obtained the solution component respectively as upper and lower phase, uses
Maximum flow rate injects upper phase in HSCCC separating pipe, and adjustment engine speed is 720r/min after upper phase is full of entire pipeline, then
Lower phase is injected with 1.5mL/min flow velocity；It is flowed out to mobile phase from column outlet, after two-phase reaches dynamic equilibrium in separating pipe, by
Sampling valve injects the solution obtained through ultrasonic wave extraction；
(3)-(5) are identical as (2)-(4) the step of embodiment 1.
Flavone aglycone obtained in embodiment 2-4 and total conversion result are as shown in table 2 below.
Table 2: flavone aglycone obtained in embodiment 2-4 and total conversion
As shown in Table 2, the flavone aglycone obtained by high performance countercurrent chromatography separation and purification treatment final production, and does not pass through
The flavone aglycone that high performance countercurrent chromatography separation and purification treatment final production obtains is compared, and is further purified due to passing through, flavonoids
Compound purity is higher, Quercetin, Kaempferol, Isorhamnetin yield increase.
Embodiment 5:
A kind of production method of Guava Leaf flavone aglycone, comprising the following steps:
(1) flavone compound is extracted from Guava Leaf using the extracting flavonoids equipment in the present embodiment；
(2)-(4) are identical as (2)-(4) the step of embodiment 1.
As depicted in figs. 1 and 2, the extracting flavonoids equipment in the present embodiment, including Multifunction tank 1 and desorption column 2, it is multi-functional
The middle and upper part of tank 1 is equipped with the slag-drip opening that opens or closes of function according to Multifunction tank 1, be equipped in Multifunction tank 1 for row
Cinder notch cooperates rotary harrow 3 that flotation dreg be discharged from slag-drip opening, the bottom of Multifunction tank 1 equipped with it is being connect with desorption column 2, be used for
Infusion solution after flotation is sent with absorbent particles to the second pipe 102 of desorption column 2, Multifunction tank 1 is interior to be equipped with stirring
Device；Function according to Multifunction tank 1, which opens or closes, to be referred to: when Multifunction tank 1 is for when leaching, slag-drip opening closing to rotate
Rake 3 does not work；When Multifunction tank 1 is used for flotation, slag-drip opening is opened, and rotary harrow 3 works.
In the present embodiment, extracting flavonoids equipment further includes leaching circulating tank 4, leaches and passes through between circulating tank 4 and slag-drip opening
First pipe 101 is connected, and is equipped between leaching circulating tank 4 and desorption column 2 and enters leaching for regenerated sorbent granules, adsorption tail liquid
(desorption column 2 carries solid-liquid separation function to the third pipeline 103 of circulating tank 4, when the infusion solution and adsorbent after flotation out
When grain is sent to desorption column 2, desorption column 2 can retain absorbent particles, and infusion solution can then flow out desorption column 2 and obtain absorption tail
Liquid, therefore, third pipeline 103 are connected with leaching circulating tank 4 again after being converged by two branch lines, this two branch line is respectively
Enter the pipeline for leaching circulating tank 4 for adsorption tail liquid and regenerated sorbent granules), leach circulating tank 4 bottom be equipped with it is more
That functional pot 1 connects, flotation dreg, regenerated sorbent granules, adsorption tail liquid for leaching in circulating tank 4 enter Multifunction tank 1
The 4th pipeline 104.In addition, regenerated sorbent granules can also directly pass through the second pipe under the effect of the pressure in the present embodiment
Road 102 enters Multifunction tank 1, and regenerated sorbent granules leach in circulating tank 4 without being stored at this time.
In the present embodiment, the 4th pipeline 104 is connected by second pipe 102 with Multifunction tank 1.4th pipeline 104 is logical
It crosses second pipe 102 and connects the usage amount that can reduce pipeline, can also reduce the quantity in 1 upper opening of Multifunction tank.
In the present embodiment, agitating device is the gas-lifting type agitating device in Multifunction tank 1, gas-lifting type agitating device packet
Include guide shell 5, inverted conical shape gas baffle 6 and liquid flowing side in the function control Multifunction tank 1 for foundation Multifunction tank 1
The snorkel 7 of the provided with electromagnetic valve of formula, guide shell 5 are fixedly arranged in Multifunction tank 1 by fixed link 8, inverted conical shape gas baffle 6
It is fixedly arranged on 5 lower section of guide shell, the snorkel 7 of provided with electromagnetic valve is equipped with exhaust outlet with lower section above inverted conical shape gas baffle 6；
Refer to according to liquid flow patterns in the function control Multifunction tank 1 of Multifunction tank 1: when Multifunction tank 1 is used to leach, only
Exhaust open positioned at 6 lower section of inverted conical shape gas baffle, it is in interior for making the outside in guide shell 5 of the liquid in Multifunction tank 1
It circulates to reach and leach function (liquid flow direction as shown in figure 11 inside solid arrow direction of Multifunction tank)；Work as Multifunction tank
1 be used for flotation when, opened above inverted conical shape gas baffle 6 with the exhaust outlet of lower section, make the liquid in Multifunction tank 1
Outside can not form internal circulation flow to reach flotation function (liquid flow direction Multifunction tank 1 as shown in figure 1 in guide shell 5
Interior dotted line arrow direction).
In the present embodiment, the top of Multifunction tank 1 is equipped with exhaust pipe 108, and the bottom of desorption column 2 is equipped with the first ventilation pressurization
Pipe 107 is communicated with the second ventilation forcing pipe 105 in first pipe 101.In the present embodiment, the first ventilation forcing pipe 107 and the
Two ventilation forcing pipes 105 can directly be connected to (situation as shown in fig. 1) with the air inlet of the snorkel of provided with electromagnetic valve 7, only
It wants to achieve the purpose that ventilation pressurization.
In the present embodiment, the outer wall of Multifunction tank 1 is equipped with collet heating device 9, and the top of Multifunction tank 1 is equipped with charging
Mouth and liquid-feeding tube (feed opening and liquid-feeding tube are not shown in Fig. 1) leach 4 bottom of circulating tank and are equipped with slag notch.Slag notch is preferable
It is the branch line outputed on the 4th pipeline 104, can reduce the open amount leached on circulating tank 4 in this way.
The Multifunction tank 1 of the present embodiment can realize the multi-functionals such as leaching-absorption-flotation, i.e. reachable in same equipment
To multiple effect, without by tank infusion solution and leached mud export be separated by solid-liquid separation again, it is easy to operate, can also reduce solid
Temperature losses in liquid separation process, energy utilization rate are high；The effect for leaching circulating tank 4 is temporarily to store flotation dreg, regeneration
It is multi-functional only need to will to leach the indentation of the substance in circulating tank 4 when needing multiple leaching operation for absorbent particles and adsorption tail liquid
Tank 1, whole process, which can be realized, continuously or semi-continuously to be operated.
The extracting flavonoids equipment of the present embodiment is former for when leaching, belonging to the leaching absorption mixing leached and adsorbed while carrying out
Reason mode.When for leaching, Guava Leaf, absorbent particles and leaching agent are under the action of gas-lifting type agitating device multi-functional
It is in internal circulation flow in tank 1, achievees the effect that adsorb when leaching.When for flotation, the control of gas-lifting type agitating device is multi-functional
Liquid in tank 1 can not form internal circulation flow, and there is the air-flow of rising in outside in guide shell 5, recycle 80-100 mesh or so
Guava Leaf and the heat-resisting polystyrene type macroporous absorbent resin absorbent particles specific gravity of 20-40 mesh difference, 80-100 mesh is left
Right Guava Leaf can float on the upper layer of Multifunction tank 1, under the action of rotary harrow 3, upper layer Guava Leaf (i.e. flotation dreg) base
This whole can be entered by flotation leaches circulating tank 4, to realize the separation of Guava Leaf and absorbent particles, and infusion solution with
The separation of absorbent particles then passes through force feed and realizes into desorption column.
Step (1) in the present embodiment is specific as follows:
A, by the Guava Leaf of 100g 80-100 mesh or so and the heat-resisting polystyrene type macroporous absorption tree of 200g 20-40 mesh
Rouge absorbent particles HD-P is added by the feed opening at 1 top of Multifunction tank, and close the lid closing, then passes through the Multifunction tank 1
The weak alkaline aqueous solution leaching agent that the pH value that solid-liquid ratio is 1:15 is 8.5 is added in the liquid-feeding tube at top；
B, it is heated by the collet heating device 9 outside Multifunction tank 1, controlling temperature in Multifunction tank 1 is 85 DEG C, opens bottom
The snorkel 7 of portion's provided with electromagnetic valve, control draft speed are 1-20vvm, and air agitation makes the liquid in Multifunction tank 1 carry out gas lift
Circulation in formula leaches the absorption mode (first pipe 101 and second pipe 102 being connected at this time with Multifunction tank 1 into mixing
On valve all close), control leach and absorption 0.5-1.5 hours；
C, after absorption is leached in mixing, flotation rotary harrow 3 is opened, (is connected at this time with Multifunction tank 1 into flotation mode
The valve opening in first pipe 101 connect, the valve on second pipe 102 are closed, and the 4th are managed with leach that circulating tank 4 is connected
Valve on road 104 is closed), the snorkel 7 of provided with electromagnetic valve is opened, adjusting Ventilation Rate is 5-25vvm, utilizes 80-100 mesh
The difference of the heat-resisting polystyrene type macroporous absorbent resin absorbent particles specific gravity of Guava Leaf and 20-40 mesh of left and right, makes 80-
The whole flotation of the Guava Leaf (flotation dreg) of 100 mesh or so, which enter, leaches circulating tank 4, and the suction of infusion solution and load flavones
Attached dose of particle is then retained in 1 in Multifunction tank；
D, after flotation, the valve in first pipe 101 is closed, opens the valve on second pipe 102, opens third
Valve on pipeline 103, then the absorbent particles for loading flavones and infusion solution are pressed into along second pipe 102 into desorption column 2
Top, the absorbent particles for loading flavones are trapped in desorption column 2, and solution (i.e. adsorption tail liquid) then from 2 bottom of desorption column from
Dynamic return is leached in circulating tank 4；After cleaning solution being passed through into desorption column 2 again to remove the impurity in absorbent particles, then use
The flavones that loads is in the ethanol solution desorption absorbent particles of 30%-95% to get to flavonoids solution；
E, the slag-drip opening slag dumping for leaching circulating tank 4, plate compression are opened.
When extracting flavone compound using the extracting flavonoids equipment in the present embodiment, uses flotation and be classified, flotation dreg
Can repeatedly circulating leaching is without separation of solid and liquid repeatedly, cooling and the warming temperature of general technology, leaching rate is high, and energy consumption
It is lower.In addition, the leaching of flavones and being adsorbed in a reactor carries out, it is good not only to mix mass transfer effect, but also because of product quilt
Balance when adsorbing and having broken Leach reaction is greatly improved leaching rate and leaching effect, improves adsorption efficiency.
Using the extracting flavonoids method in the present embodiment, the extracted amount of flavone compound is higher.
Embodiment 6:
A kind of production method of Guava Leaf flavone aglycone, comprising the following steps:
(1) identical as step (1) in embodiment 5；
(2) extract powder obtained in step (1) is isolated and purified into process, high-speed counter-current through high-speed countercurrent chromatography
Chromatographic technique isolates and purifies the process conditions of process are as follows: using petroleum ether-ethyl acetate-alcohol-water as two phase solvent system,
Each component is prepared according to the ratio that volume ratio is 2: 2: 3: 1, is stood 6-10h and is obtained the solution component respectively as upper and lower phase, uses
Maximum flow rate injects upper phase in HSCCC separating pipe, and adjustment engine speed is 720r/min after upper phase is full of entire pipeline, then
Lower phase is injected with 1.5mL/min flow velocity；It is flowed out to mobile phase from column outlet, after two-phase reaches dynamic equilibrium in separating pipe, by
Sampling valve injects the solution obtained through ultrasonic wave extraction；
(3)-(5) are identical as (2)-(4) the step of embodiment 1.
Flavone aglycone obtained in embodiment 5-6 and total conversion result are as shown in table 3 below.
Table 3: flavone aglycone yield and flavones total conversion in embodiment 5-6
Claims (10)
1. a kind of production method of Guava Leaf flavone aglycone, which comprises the following steps:
(1) flavone compound is extracted from Guava Leaf；
(2) flavone compound obtained in step (1) is added to containing streptomyces griseus (Streptomyces griseus)
Fluid nutrient medium in cultivate, oscillation and fermentation cultivation obtains fermentation liquid；
(3) the oscillation enzymatic hydrolysis of fermentation liquid obtained in step (2) is obtained into enzymatic hydrolysis solution；
(4) enzymatic hydrolysis solution left standstill, centrifugation, precipitating obtained in step (3) are dissolved with methanol, is obtained after filtering containing flavonoid glycoside
The methanol solution of member.
2. the production method of Guava Leaf flavone aglycone according to claim 1, which is characterized in that the strepto- containing grey
The fluid nutrient medium of bacterium be standard potato culture, formula are as follows: peeling fresh potato 200g, glucose 20g,
MgSO4·7H2O 1.5g、KH2PO43g, 1L distilled water.
3. the production method of Guava Leaf flavone aglycone according to claim 1, which is characterized in that the strepto- containing grey
The inoculum concentration of streptomyces griseus is 500-200000 spore of every milliliter of inoculation of medium in the fluid nutrient medium of bacterium.
4. the production method of Guava Leaf flavone aglycone according to claim 3, which is characterized in that the strepto- containing grey
The inoculum concentration of streptomyces griseus is 10000-60000 spore of every milliliter of inoculation of medium in the fluid nutrient medium of bacterium.
5. the production method of Guava Leaf flavone aglycone according to claim 1, which is characterized in that in the step (2),
The additional amount of the flavone compound and the mass ratio control of fluid nutrient medium are (0.5-1.5): 5.
6. the production method of Guava Leaf flavone aglycone according to any one of claims 1-5, which is characterized in that described
The method for extracting flavone compound in step (1) from Guava Leaf is microwave-assisted eutectic extractive technique, technique item
Part are as follows: after 200W microwave intensity processing Guava Leaf 2min, with butanediol and choline chloride according to molal volume ratio for 4:1 ratio
Example mixing is used as eutectic solvent, and control solid-liquid ratio is 1:22, extracts 40min at 65 DEG C.
7. the production method of Guava Leaf flavone aglycone according to any one of claims 1-5, which is characterized in that described
The method for extracting flavone compound in step (1) from Guava Leaf is ultrasonic wave extraction, process conditions are as follows: with
75% ethyl alcohol is solution, and control solid-liquid ratio is 1:28, extracts 30min at 42 DEG C.
8. the production method of Guava Leaf flavone aglycone according to any one of claims 1-5, which is characterized in that described
Flavone compound also passes through high-speed countercurrent chromatography and isolates and purifies process, process conditions in step (1) are as follows: by petroleum
Ether-ethyl acetate, alcohol and water as two phase solvent system, prepare according to the ratio that volume ratio is 2: 2: 3: 1, quiet by each component
It sets 6-10h and obtains the solution component respectively as upper and lower phase, injected upper phase in HSCCC separating pipe with maximum flow rate, to upper phase
It is 720r/min full of engine speed is adjusted after entire pipeline, then lower phase is injected with 1.5mL/min flow velocity；Go out to mobile phase from column
Mouth outflow after two-phase reaches dynamic equilibrium in separating pipe, injects the solution obtained through ultrasonic wave extraction by sampling valve.
9. the production method of Guava Leaf flavone aglycone according to any one of claims 1-5, which is characterized in that described
It adds in the fluid nutrient medium containing streptomyces griseus and cultivates after flavone compound in step (1) is freeze-dried.
10. the production method of Guava Leaf flavone aglycone according to any one of claims 1-5, which is characterized in that institute
State mixed solvent when oscillation digests in step (3) using cellulase and pectase.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112226479A (en) * | 2020-11-06 | 2021-01-15 | 江西邦泰绿色生物合成生态产业园发展有限公司 | Method for improving lutein extraction rate of marigold by using multiple enzymes |
| CN115119935A (en) * | 2022-06-21 | 2022-09-30 | 广东轻工职业技术学院 | Guava fruit powder with intestinal conditioning function and preparation method and application thereof |
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| EP0378921A2 (en) * | 1988-12-21 | 1990-07-25 | Idaho Research Foundation, Inc. | Enhancement of plant metabolite production by timed elicitation |
| CN101768613A (en) * | 2010-03-09 | 2010-07-07 | 中国农业大学 | Preparation method of flavone genin of seabuckthorn leaves |
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| EP0378921A2 (en) * | 1988-12-21 | 1990-07-25 | Idaho Research Foundation, Inc. | Enhancement of plant metabolite production by timed elicitation |
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| CN112226479A (en) * | 2020-11-06 | 2021-01-15 | 江西邦泰绿色生物合成生态产业园发展有限公司 | Method for improving lutein extraction rate of marigold by using multiple enzymes |
| CN115119935A (en) * | 2022-06-21 | 2022-09-30 | 广东轻工职业技术学院 | Guava fruit powder with intestinal conditioning function and preparation method and application thereof |
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