CN108977475B - Production method of guava leaf flavonoid aglycone - Google Patents
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Abstract
The invention discloses a production method of guava leaf flavonoid aglycone, which comprises the following steps: (1) extracting flavonoids from folium Psidii Guajavae; (2) adding the flavonoid compound obtained in the step (1) into a liquid culture medium containing Streptomyces griseus to culture, and performing shaking fermentation culture to obtain a fermentation solution; (3) oscillating the fermentation solution obtained in the step (2) for enzymolysis to obtain an enzymolysis solution; (4) and (4) standing and centrifuging the enzymolysis solution obtained in the step (3), dissolving the precipitate with methanol, and filtering to obtain a methanol solution containing flavonoid aglycone. The production method of the guava leaf flavonoid aglycone is simple and convenient, is easy to operate, and the finally obtained flavonoid aglycone has small impurity content and simple subsequent purification process.
Description
Technical Field
The invention belongs to the field of guavas, and particularly relates to a production method of a guava leaf extract.
Background
Guava (Psidium guajajava L.) is a plant of genus Psidium of family Myrtaceae, is a tropical evergreen small tree or shrub, is native to tropical America, is introduced into China for centuries, and is widely planted in Fujian, Guangdong, Taiwan and the like in China. The traditional Chinese medicine considers that the volatile oil has the effects of smoothing nature, relieving taste, astringing, stopping diarrhea and the like, and the volatile oil has the effects of resisting bacteria and viruses, relieving spasm of digestive systems and enhancing gastrointestinal motility. Modern researches show that guava contains a plurality of important aromatic secondary metabolites such as polyphenol flavonoids, and people of the modern researches think that the phenolic compounds endow the guava with various medicinal effects. Therefore, the development and research of the flavonoid aglycone resource of the guava leaf have better prospect and need.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings in the background technology and provide a production method of guava leaf flavonoid aglycone with low cost and high quality. In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for producing guava leaf flavonoid aglycone comprises the following steps:
(1) extracting flavonoids from folium Psidii Guajavae;
(2) adding the flavonoid compound obtained in the step (1) into a liquid culture medium containing Streptomyces griseus ATCC13273 for culture, and performing shaking fermentation culture at the temperature of 25-40 ℃ for 6-8 days to obtain a fermentation solution;
(3) carrying out oscillation enzymolysis on the fermentation solution obtained in the step (2) for 3-4h at the temperature of 50-55 ℃ to obtain an enzymolysis solution;
(4) and (4) standing and centrifuging the enzymolysis solution obtained in the step (3), dissolving the precipitate with methanol, and filtering to obtain a methanol solution containing flavonoid aglycone.
In the production method of the guava leaf flavonoid aglycone, the streptomyces griseus firstly needs to be subjected to amplification culture by using a potato culture medium as a seed and then is inoculated to a new potato culture medium.
In the above method for producing guava leaf flavonoid aglycone, preferably, the liquid culture medium containing streptomyces griseochromogenes is a standard potato culture medium, and the formula thereof is as follows: peeled fresh potato 200g, glucose 20g, MgSO4·7H2O 1.5g、KH2PO43g, 1L of distilled water, and the initial pH value of the standard potato culture medium does not need to be adjusted. Through rhamnosidase, beta-D-glucosidase and the like generated by metabolism of streptomyces griseus, flavonoid glycoside components in the extract are converted into flavonoid aglycone (quercetin, kaempferol, isorhamnetin and the like) through microbial fermentation, and the flavonoid aglycone is converted by microorganisms, so that the product amount can be increased, the defect of chemical synthesis is overcome, and adverse factors such as product isomerization, racemization, rearrangement reaction and the like are reduced because most microbial fermentation is performed under neutral and room temperature conditions.
In the above method for producing guava leaf flavonoid aglycone, preferably, the inoculation amount of the streptomyces griseus in the liquid culture medium containing the streptomyces griseus is 10000-60000 spores per ml of the culture medium. More preferably, 10000-.
In the above method for producing guava leaf flavonoid aglycone, preferably, in the step (2), the mass ratio of the added amount of the flavonoid compound to the liquid culture medium is controlled to (0.5-1.5): 5.
in the above method for producing guava leaf flavonoid aglycone, preferably, the method for extracting flavonoid compounds from guava leaves in the step (1) is extraction with alkaline water or alkaline diluted alcohol, or extraction with an organic solvent. Since the flavonoids have phenolic hydroxyl group, alkaline water such as sodium carbonate, sodium hydroxide, calcium hydroxide aqueous solution or alkaline diluted alcohol such as ethanol can be used for leaching, and the leachate is acidified to separate out flavonoids. The organic solvent extraction method is the most widely used method at home and abroad at present, the organic solvent extraction has simple requirements on equipment, high product yield and high impurity content, industrial production is easy to realize, but the cost is high, the organic solvent commonly used for flavone extraction is methanol, ethanol, acetone, acetic ether, diethyl ether and the like, and the ethanol is preferably adopted as the extraction solvent in consideration of the high toxicity of the methanol, the acetone and the like.
In the above method for producing guava leaf flavonoid aglycone, preferably, the method for extracting flavonoid compounds from guava leaves in the step (1) is a microwave-assisted eutectic extraction technology, and the process conditions are as follows: treating guava leaves with 200W microwave intensity for 2min, and then adding butanediol and choline chloride according to a molar volume ratio of 4: 1 proportion is mixed as eutectic solvent, and the ratio of material to liquid is controlled to be 1: extracting at 22 and 65 deg.C for 40 min. When the microwave-assisted extraction is carried out, the extracted polar molecules are quickly turned and directionally arranged in a microwave electric field, so that the tearing and the mutual friction are generated to cause heating, the quick transmission and the full utilization of energy can be ensured, and the dissolution and the release are easy. Meanwhile, the eutectic solution is adopted, so that the environment can be protected, and the pollution of waste liquid to human is reduced.
In the above method for producing guava leaf flavonoid aglycone, preferably, the method for extracting flavonoid compounds from guava leaves in the step (1) is an ultrasonic extraction technique, and the process conditions are as follows: taking 75% ethanol as a solution, and controlling the ratio of materials to liquid to be 1: extracting at 28 and 42 deg.C for 30 min. The ultrasonic extraction technology is to utilize the cavitation of ultrasonic waves to enhance the leaching and extraction of the effective components of the guava, and in addition, the secondary effects of the ultrasonic waves, such as mechanical vibration, emulsification, diffusion, crushing, chemical effects and the like, can also accelerate the diffusion and release of the target components and can be fully mixed with a solvent, thereby being beneficial to extraction.
In the above method for producing guava leaf flavonoid aglycone, preferably, the flavonoid compound in the step (1) is further subjected to a separation and purification process by a high-speed counter-current chromatography technology, and the process conditions are as follows: taking petroleum ether-ethyl acetate-ethanol-water as a two-phase solvent system, preparing the components according to the volume ratio of 2: 3: 1, standing for 6-10h to obtain solution components respectively serving as an upper phase and a lower phase, injecting the upper phase into an HSCCC separation tube at the maximum flow rate, adjusting the rotating speed of a main engine to 720r/min after the upper phase is filled in a whole pipeline, and then injecting the lower phase at the flow rate of 1.5 mL/min; after the mobile phase flows out from the column outlet and the two phases reach dynamic balance in the separating tube, the solution obtained by the ultrasonic extraction technology is injected by the sample injection valve. After the ultrasonic extraction technology, the high-efficiency countercurrent chromatography (HSCCC) technology is used for separating and purifying the flavonoid compounds, so that the flavone extract with higher purity can be obtained, and the quality of the final product can be improved.
In the above method for producing guava leaf flavonoid aglycone, preferably, the flavonoid compound in the step (1) is freeze-dried and then added into a liquid culture medium containing streptomyces griseus for culture.
In the above method for producing guava leaf flavonoid aglycone, preferably, a mixed solvent of cellulase and pectinase is used in the step (3) during shaking and enzymolysis. The mixed solvent of cellulase and pectinase can convert oil-soluble flavonoid into water-soluble glycoside, and dissolve in enzymolysis solution to facilitate extraction, improve flavone content, decompose plant tissue by enzyme reaction, reduce extraction mass transfer resistance, and accelerate reaction process. In addition, impurities in the extracting solution can be decomposed and removed, so that the subsequent separation and purification process is simplified.
Compared with the prior art, the invention has the advantages that:
1. the invention adopts a liquid culture medium containing streptomyces griseus to culture the flavonoid compound, the streptomyces griseus catalyzes the hydroxyl on the flavonoid mother nucleus to form methoxyl, the process is one of biological detoxification modes and is also one of important reactions for producing stable compounds by modifying the structure of natural products, and adverse factors such as product isomerism, racemization, rearrangement reaction and the like are reduced. In addition, the microbial fermentation mostly acts under the conditions of neutrality and room temperature, the reaction environment can be controlled more conveniently, the large cost investment on the control conditions of the temperature, the humidity and the like of a factory building is reduced during large-scale production, and the method is economical and economical.
2. The production method of the guava leaf flavonoid aglycone is simple and convenient, is easy to operate, and the finally obtained flavonoid aglycone has small impurity content and simple subsequent purification process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
Fig. 1 is a schematic structural diagram of a flavone extracting apparatus in embodiment 5.
Fig. 2 is a sectional view taken along the plane a-a in fig. 1.
Illustration of the drawings:
1. a multifunctional tank; 2. a desorption column; 3. rotating the harrow; 4. a leaching circulation tank; 5. a draft tube; 6. an inverted conical gas baffle; 7. a vent pipe with an electromagnetic valve; 8. fixing the rod; 9. a jacket heating device; 101. a first conduit; 102. a second conduit; 103. a third pipeline; 104 a fourth conduit; 105. a second venting pressurization pipe; 107. a first ventilation pressurization pipe; 108. and (4) exhausting the gas.
Detailed Description
In order to facilitate an understanding of the invention, the invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example 1:
a method for producing guava leaf flavonoid aglycone comprises the following steps:
(1) performing oscillation extraction on guava leaves for 8 hours by using 80% methanol water solution at the temperature of 45 ℃, performing suction filtration, repeating for three times, combining extracting solutions, and performing freeze drying on the extracting solutions to obtain extract powder;
(2) flavonoid compound obtained in the step (1)Adding into liquid culture medium containing Streptomyces griseus ATCC13273, and culturing (mass ratio of flavonoids to liquid culture medium is 1: 5), wherein the inoculation amount per ml culture medium is 3 × 105Culturing spores at 25-40 deg.C for 6-8 days to obtain fermentation solution; wherein, the liquid culture medium containing the streptomyces griseochromogenes is a standard potato culture medium, and the formula of the liquid culture medium is as follows: peeled fresh potato 200g, glucose 20g, MgSO4·7H2O 1.5g、KH2PO43g of 1L of distilled water, and the initial pH value of the standard potato culture medium does not need to be adjusted;
(3) carrying out oscillatory enzymolysis on the fermentation solution obtained in the step (2) for 3-4h at 50-55 ℃ to obtain an enzymolysis (10U/ml cellulase and pectinase) solution;
(4) and (4) standing and centrifuging the enzymolysis solution obtained in the step (3), dissolving the precipitate with methanol, and filtering to obtain a methanol solution containing flavonoid aglycone.
Comparative example 1:
this comparative example differs from example 1 only in that it has not been subjected to the treatment of step (2). However, the enzymes used in step (3) include a combination of a plurality of enzymes, such as α -rhamnosidase, β -glucosidase, xylanase, cellulase, pectinase, and α -amylase, and the amount of the enzyme used is usually about 20U/ml.
Through detection, compared with the flavonoid aglycone which is not processed by the streptomyces griseus but directly extracted by an organic solvent and then converted by a biological enzyme, the flavonoid aglycone finally produced by processing the streptomyces griseus is shown in the following table 1.
Table 1: flavone aglycone yield and total flavone conversion in example 1 and comparative example 1
Example 1 compared with comparative example 1, although the interest rate of each flavonoid aglycone and the total conversion rate of the flavonoid in comparative example 1 were high compared with example 1, the kind and amount of the enzyme used in comparative example 1 were significantly more than those of example 1, which was not preferable in terms of cost.
Example 2:
a method for producing guava leaf flavonoid aglycone comprises the following steps:
(1) taking 75% ethanol as solution, controlling the ratio of guava leaf to ethanol at 1:28, performing ultrasonic extraction at 42 deg.C for 30min, and freeze drying the extractive solution to obtain extract powder;
(2) - (4) is the same as steps (2) to (4) of example 1.
Example 3:
a method for producing guava leaf flavonoid aglycone comprises the following steps:
(1) treating guava leaves with 200W microwave intensity for 2min, and then adding butanediol and choline chloride according to a molar volume ratio of 4: 1 proportion is mixed as eutectic solvent, and the ratio of material to liquid is controlled to be 1: extracting at 22, 65 deg.C for 40min, and freeze drying the extractive solution to obtain extract powder;
(2) - (4) is the same as steps (2) to (4) of example 1.
Example 4:
a method for producing guava leaf flavonoid aglycone comprises the following steps:
(1) same as in step (1) in example 3;
(2) separating and purifying the extract powder obtained in the step (1) by using a high-speed counter-current chromatography technology, wherein the process conditions of the separation and purification process by using the high-speed counter-current chromatography technology are as follows: taking petroleum ether-ethyl acetate-ethanol-water as a two-phase solvent system, preparing the components according to the volume ratio of 2: 3: 1, standing for 6-10h to obtain solution components respectively serving as an upper phase and a lower phase, injecting the upper phase into an HSCCC separation tube at the maximum flow rate, adjusting the rotating speed of a main engine to 720r/min after the upper phase is filled in a whole pipeline, and then injecting the lower phase at the flow rate of 1.5 mL/min; after the mobile phase flows out from the column outlet and the two phases reach dynamic balance in the separation tube, injecting the solution obtained by the ultrasonic extraction technology through a sample injection valve;
(3) - (5) is the same as in steps (2) - (4) of example 1.
The flavonoid aglycones obtained in examples 2 to 4 and the results of the total conversion are shown in Table 2 below.
Table 2: flavonoid aglycones obtained in examples 2-4 and Total conversion
As can be seen from Table 2, the flavonoid aglycone finally produced by the separation and purification treatment of the high performance counter current chromatography was higher in purity of the flavonoid compounds and higher in the yields of quercetin, kaempferol and isorhamnetin than the flavonoid aglycone finally produced without the separation and purification treatment of the high performance counter current chromatography due to further purification.
Example 5:
a method for producing guava leaf flavonoid aglycone comprises the following steps:
(1) flavone extraction equipment in the embodiment is adopted to extract flavonoid compounds from guava leaves;
(2) - (4) is the same as steps (2) to (4) of example 1.
As shown in fig. 1 and fig. 2, the flavone extracting apparatus in this embodiment includes a multifunctional tank 1 and a desorption column 2, a slag discharge port opened or closed according to the function of the multifunctional tank 1 is disposed at the middle upper portion of the multifunctional tank 1, a rotary rake 3 for discharging the flotation slag from the slag discharge port in cooperation with the slag discharge port is disposed in the multifunctional tank 1, a second pipeline 102 connected to the desorption column 2 and used for sending the leaching solution and the adsorbent particles subjected to flotation to the desorption column 2 is disposed at the bottom of the multifunctional tank 1, and a stirring device is disposed in the multifunctional tank 1; the functional opening or closing according to the multi-functional can 1 means: when the multifunctional tank 1 is used for leaching, the slag discharge port is closed, and the rotary harrow 3 does not work; when the multifunctional tank 1 is used for flotation, the slag discharge port is opened, and the rotary rake 3 works.
In this embodiment, the flavone extracting apparatus further includes a leaching circulation tank 4, the leaching circulation tank 4 is connected to the slag discharge port through a first pipeline 101, a third pipeline 103 for regenerating adsorbent particles and adsorbing tail liquid to enter the leaching circulation tank 4 is provided between the leaching circulation tank 4 and the desorption column 2 (the desorption column 2 has a solid-liquid separation function, when the leached solution and the adsorbent particles after flotation are sent to the desorption column 2, the desorption column 2 intercepts the adsorbent particles, and the leached solution flows out of the desorption column 2 to obtain the adsorbing tail liquid, so that the third pipeline 103 is connected to the leaching circulation tank 4 after being converged by two branch pipelines, which are respectively pipelines for adsorbing the tail liquid and the regenerated adsorbent particles to enter the leaching circulation tank 4), and the bottom of the leaching circulation tank 4 is provided with a floating slag, which is connected to the leaching circulation tank 4 and is connected to the multifunctional tank 1, and is used for leaching the flotation slag in the leaching circulation tank 4, The regenerated adsorbent particles and the adsorption tail liquid enter a fourth pipeline 104 of the multifunctional tank 1. In addition, in this embodiment, the regenerated sorbent particles can also be directly fed into the multifunctional tank 1 through the second pipeline 102 under the pressure, and the regenerated sorbent particles do not need to be stored in the leaching circulation tank 4.
In this embodiment, the fourth pipe 104 is connected to the multipurpose tank 1 through the second pipe 102. The connection of the fourth pipe 104 through the second pipe 102 can reduce the amount of use of the pipe and also reduce the number of openings on the multipurpose tank 1.
In this embodiment, the stirring device is an air-lift stirring device disposed in the multifunctional tank 1, the air-lift stirring device includes a draft tube 5, an inverted cone-shaped gas baffle 6, and a vent pipe 7 with an electromagnetic valve for controlling the liquid flow mode in the multifunctional tank 1 according to the function of the multifunctional tank 1, the draft tube 5 is fixedly disposed in the multifunctional tank 1 through a fixing rod 8, the inverted cone-shaped gas baffle 6 is fixedly disposed below the draft tube 5, and the vent pipe 7 with the electromagnetic valve is provided with exhaust ports above and below the inverted cone-shaped gas baffle 6; the method for controlling the liquid flowing mode in the multifunctional tank 1 according to the functions of the multifunctional tank 1 is as follows: when the multifunctional tank 1 is used for leaching, only the exhaust port below the inverted conical gas baffle 6 is opened, so that liquid in the multifunctional tank 1 flows in an internal circulation manner inside and outside the guide cylinder 5 to achieve a leaching function (the liquid flowing direction is as shown by the solid arrow in the multifunctional tank 1 in fig. 1); when the multifunctional tank 1 is used for flotation, the exhaust ports above and below the inverted cone-shaped gas baffle 6 are opened, so that the liquid in the multifunctional tank 1 cannot form internal circulation flow inside and outside the guide cylinder 5 to achieve the flotation function (the liquid flow direction is as shown by the dotted line arrow direction inside the multifunctional tank 1 in fig. 1).
In this embodiment, the top of the multifunctional tank 1 is provided with an exhaust pipe 108, the bottom of the desorption column 2 is provided with a first aeration pressure pipe 107, and the first pipeline 101 is communicated with a second aeration pressure pipe 105. In this embodiment, the first ventilation pressurizing pipe 107 and the second ventilation pressurizing pipe 105 can be directly communicated with the air inlet of the ventilation pipe 7 with the electromagnetic valve (as in the case shown in fig. 1), as long as the purpose of ventilation pressurizing can be achieved.
In this embodiment, the outer wall of the multifunctional tank 1 is provided with a jacket heating device 9, the top of the multifunctional tank 1 is provided with a feed inlet and a liquid feeding pipe (the feed inlet and the liquid feeding pipe are not shown in fig. 1), and the bottom of the leaching circulation tank 4 is provided with a slag outlet. The tap hole may preferably be a branch line from the fourth pipe 104, which reduces the number of openings in the leaching circulation tank 4.
The multifunctional tank 1 of the embodiment can realize multiple functions of leaching, adsorption, flotation and the like, can achieve multiple effects in the same equipment, does not need to lead out leaching solution and leaching slag in the tank and then carry out solid-liquid separation, is simple to operate, can reduce temperature loss in the solid-liquid separation process, and has high energy utilization rate; the leaching circulation tank 4 is used for temporarily storing flotation slag, regenerating adsorbent particles and adsorbing tail liquid, when multiple leaching operations are needed, only the substances in the leaching circulation tank 4 are needed to be pressed into the multifunctional tank 1, and the whole process can realize continuous or semi-continuous operation.
When the flavone extraction equipment is used for leaching, the extraction and adsorption principle mode belongs to the mode of simultaneous leaching and adsorption. When the multifunctional pot is used for leaching, the guava leaves, the adsorbent particles and the leaching agent flow in an internal circulation manner in the multifunctional pot 1 under the action of the air-lift stirring device, so that the effect of leaching while adsorbing is achieved. When the pneumatic-lifting type stirring device is used for flotation, the liquid in the multifunctional tank 1 cannot form internal circulation flow under the control of the pneumatic-lifting type stirring device, rising air flow exists on the inner side and the outer side of the guide cylinder 5, the guava leaves of about 80-100 meshes and the heat-resistant polystyrene macroporous adsorption resin adsorbent particles of 20-40 meshes are utilized to be different in specific gravity, the guava leaves of about 80-100 meshes can float on the upper layer of the multifunctional tank 1, and basically all the guava leaves (namely flotation slag) on the upper layer can enter the leaching circulation tank 4 through flotation under the action of the rotary rake 3, so that the guava leaves and the adsorbent particles are separated, and the leaching solution and the adsorbent particles are separated through being pressed into a desorption column.
The step (1) in this example is specifically as follows:
A. adding 100g of guava leaves of about 80-100 meshes and 200g of heat-resistant polystyrene macroporous adsorbent resin particles HD-P of 20-40 meshes into the multifunctional tank 1 through a feed inlet at the top, covering the multifunctional tank with a cover for sealing, and adding the mixture into the multifunctional tank 1 through a liquid adding pipe at the top, wherein the mixture ratio of the mixture to the liquid is 1: 15 a weakly alkaline aqueous solution leachant having a pH of 8.5;
B. heating by a jacket heating device 9 outside the multifunctional tank 1, controlling the temperature in the multifunctional tank 1 to be 85 ℃, opening a vent pipe 7 with a solenoid valve at the bottom, controlling the venting speed to be 1-20vvm, conducting air-lift type internal circulation on liquid in the multifunctional tank 1 by venting and stirring, entering a mixed leaching and adsorption mode (at the moment, valves on a first pipeline 101 and a second pipeline 102 connected with the multifunctional tank 1 are all closed), and controlling leaching and adsorption for 0.5-1.5 hours;
C. after the mixed leaching adsorption is finished, starting a flotation rotary rake 3, entering a flotation mode (at the moment, a valve on a first pipeline 101 connected with the multifunctional tank 1 is opened, a valve on a second pipeline 102 is closed, and a valve on a fourth pipeline 104 connected with the leaching circulation tank 4 is closed), opening a vent pipe 7 with a solenoid valve, adjusting the ventilation rate to be 5-25vvm, and making the guava leaves (flotation slag) of about 80-100 meshes enter the leaching circulation tank 4 in a flotation mode by utilizing the difference of specific gravity of the guava leaves of about 80-100 meshes and adsorbent particles of heat-resistant polystyrene type macroporous adsorbent resin of 20-40 meshes, and keeping the leaching solution and the adsorbent particles loaded with flavone in the multifunctional tank 1;
D. after the flotation is finished, closing a valve on the first pipeline 101, opening a valve on the second pipeline 102, opening a valve on the third pipeline 103, pressing the flavone-loaded adsorbent particles and the leaching solution into the top of the desorption column 2 along the second pipeline 102, intercepting the flavone-loaded adsorbent particles in the desorption column 2, and automatically returning the solution (i.e. the adsorption tail solution) into the leaching circulation tank 4 from the bottom of the desorption column 2; then introducing a washing solution into the desorption column 2 to remove impurities in the adsorbent particles, and desorbing the flavone loaded in the adsorbent particles by using a 30-95% ethanol solution to obtain a flavone solution;
E. opening a slag discharge port of the leaching circulation tank 4 to discharge slag, and performing plate-frame filter pressing.
When the flavone extraction equipment in the embodiment is used for extracting the flavonoid compounds, flotation classification is adopted, flotation slag can be circularly leached for many times without repeated solid-liquid separation, temperature reduction and temperature rise operations of a common process, the leaching rate is high, and the energy consumption is lower. In addition, the leaching and the adsorption of the flavone are carried out in one reactor, so that the mixing mass transfer effect is good, the balance of the leaching reaction is broken due to the adsorption of the product, the leaching rate and the leaching effect can be greatly improved, and the adsorption efficiency is improved.
By adopting the flavone extraction method in the embodiment, the extraction amount of the flavonoid compound is higher.
Example 6:
a method for producing guava leaf flavonoid aglycone comprises the following steps:
(1) same as in step (1) in example 5;
(2) separating and purifying the extract powder obtained in the step (1) by using a high-speed counter-current chromatography technology, wherein the process conditions of the separation and purification process by using the high-speed counter-current chromatography technology are as follows: taking petroleum ether-ethyl acetate-ethanol-water as a two-phase solvent system, preparing the components according to the volume ratio of 2: 3: 1, standing for 6-10h to obtain solution components respectively serving as an upper phase and a lower phase, injecting the upper phase into an HSCCC separation tube at the maximum flow rate, adjusting the rotating speed of a main engine to 720r/min after the upper phase is filled in a whole pipeline, and then injecting the lower phase at the flow rate of 1.5 mL/min; after the mobile phase flows out from the column outlet and the two phases reach dynamic balance in the separation tube, injecting the solution obtained by the ultrasonic extraction technology through a sample injection valve;
(3) - (5) is the same as in steps (2) - (4) of example 1.
The flavonoid aglycones obtained in examples 5 to 6 and the results of the total conversion are shown in Table 3 below.
Table 3: flavonoid aglycone yields and Total flavonoid conversions in examples 5-6
Claims (6)
1. The production method of the guava leaf flavonoid aglycone is characterized by comprising the following steps:
(1) extracting flavonoids from folium Psidii Guajavae;
(2) adding the flavonoid compound obtained in the step (1) into streptomyces griseus (A)Streptomyces griseus) Culturing in the liquid culture medium, and performing shaking fermentation culture to obtain a fermentation solution;
(3) oscillating the fermentation solution obtained in the step (2) for enzymolysis to obtain an enzymolysis solution;
(4) standing and centrifuging the enzymolysis solution obtained in the step (3), dissolving the precipitate with methanol, and filtering to obtain a methanol solution containing flavonoid aglycone;
the method for extracting the flavonoid compounds from the guava leaves in the step (1) is a microwave-assisted eutectic extraction technology, and the process conditions are as follows: treating guava leaves with 200W microwave intensity for 2min, and then adding butanediol and choline chloride according to a molar volume ratio of 4: 1 proportion is mixed as eutectic solvent, and the ratio of material to liquid is controlled to be 1: extracting at 22, 65 deg.C for 40 min;
the flavonoid compound in the step (1) is further subjected to a high-speed counter-current chromatography separation and purification process, and the process conditions are as follows: taking petroleum ether-ethyl acetate-ethanol-water as a two-phase solvent system, preparing the components according to the volume ratio of 2: 3: 1, standing for 6-10h to obtain solution components respectively serving as an upper phase and a lower phase, injecting the upper phase into an HSCCC separation tube at the maximum flow rate, adjusting the rotating speed of a main engine to 720r/min after the upper phase is filled in a whole pipeline, and then injecting the lower phase at the flow rate of 1.5 mL/min; after the mobile phase flows out from the column outlet and the two phases reach dynamic balance in the separation tube, injecting the solution obtained by the ultrasonic extraction technology through a sample injection valve;
and (3) adopting a mixed solvent of cellulase and pectinase during the oscillating enzymolysis.
2. The method for producing guava leaf flavonoid aglycone according to claim 1, wherein the liquid medium containing streptomyces griseus is a standard potato medium and has a formula of: peeled fresh potato 200g, glucose 20g, MgSO4·7H2O 1.5g、KH2PO4 3g, 1L of distilled water.
3. The method for producing guava leaf flavonoid aglycone according to claim 1, wherein the amount of Streptomyces griseus inoculated per ml of the culture medium is 500-200000 spores per ml of the culture medium.
4. The method for producing guava leaf flavonoid aglycone according to claim 3, wherein the amount of Streptomyces griseus inoculated in the Streptomyces griseus-containing liquid medium is 10000-60000 spores per ml of the medium.
5. The method for producing guava leaf flavonoid aglycone according to claim 1, wherein the mass ratio of the flavonoid compound to the liquid medium in the step (2) is controlled to be (0.5-1.5): 5.
6. the method for producing guava leaf flavonoid aglycone according to any one of claims 1 to 5, wherein the flavonoid compound obtained in the step (1) is freeze-dried and then cultured in a liquid medium containing Streptomyces griseus.
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