CN108912184A - A method of steviol glycoside RA being obtained from chrysanthemum using STEVIA REBAUDIANA extracting solution - Google Patents
A method of steviol glycoside RA being obtained from chrysanthemum using STEVIA REBAUDIANA extracting solution Download PDFInfo
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- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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Abstract
The present invention relates to the methods that STEVIA REBAUDIANA in vitro culture improves RA content in STEVIA REBAUDIANA blade, belong to cell biology and biological field.STEVIA REBAUDIANA leaf extract is sprayed application in chrysanthemum " Mount Taishan jewel " plant, to make to contain steviol glycoside RA ingredient in " Mount Taishan jewel " blade, and utilize chrysanthemum resistance, the characteristics such as biomass is big, make in " Mount Taishan jewel " entire plant containing steviol glycoside RA ingredient, and then chrysanthemum for tea use is made to become more sweet.Solution route：Obtain steviol glycoside crude extract, steviol glycoside crude extract sprays chrysanthemum plant, steviol glycoside, macroreticular resin absorbing method purifying steviol glycoside crude extract, Rotary Evaporators method are evaporated stevioside extracting solution in chrysanthemum, high performance liquid chromatograph analysis chrysanthemum leaf steviol glycoside content in cellulose Enzymatic Extraction chrysanthemum leaf.
Description
Technical field
The present invention relates to STEVIA REBAUDIANA (Stevia rebaudiana Bertoni) in vitro cultures to improve RA in STEVIA REBAUDIANA blade
The method of content, belongs to cell biology and biological field.
Background technique
STEVIA REBAUDIANA is composite family (Compositae) sweetleaf Chrysanthemum (Stevia) herbaceous plant, originates in South America Paraguay area,
Contain steviol glycoside (stevioside, ST) in its blade, it is that natural high sugariness is low that sugariness, which is 200-300 times of sucrose,
Energy type sweet substance.It is proved through modern science, STEVIA REBAUDIANA has preferably hypertension, diabetes, artery sclerosis, patients with coronary heart disease
Control efficiency.Stevia rebaudiana tea is drunk, can help to release mental fatigue, boost metabolism, be beneficial to health.Currently, existing
Research shows that steviol glycoside substance has, a variety of chemical combination such as rebaudioside A (Rebaudioside A, RA), RB, RC, RD, RA
Object.Wherein stevioside (ST) and Rebaudioside A (RA) proportion are maximum (generally 90% or more), rebaudioside C (RC),
Dole gram glycosides A (DA), the content of steviolbioside (RA) are less.Recent study proves, the steviolbioside RA in STEVIA REBAUDIANA
Sugariness be higher than steviolbioside ST, and bitter taste is weaker than steviolbioside ST, thus the steviol glycoside RA ingredient in STEVIA REBAUDIANA will
With wider array of application prospect.But steviol glycoside RA content is less in STEVIA REBAUDIANA blade at present, if steviol glycoside RA ingredient
It can be enriched in the chrysanthemum body of root of Roundfruit Licorice, using the chrysanthemum plant feature bigger than STEVIA REBAUDIANA biomass, then stevioside
The yield of glycosides RA will greatly increase.
Chrysanthemum (Chrysanthemum morifolium RAat.) is composite family Chrysanthemum herbaceos perennial.Chrysanthemum originates in
China, with more than 1600 years cultivation histories.China's Dendranthema morifolium Varieties are more than more than 3000 at present, and cultivated area reaches 8900 public affairs
Hectare, chrysanthemum be mainly used for afforestation, quilt, interior decoration, tea use, it is medicinal and make wine etc..The tea that this testing sieve is selected is used
Chrysanthemum " Mount Taishan jewel " flower is in foresythia, flower size about 13cm, and the soup look bubbled out is golden yellow, is a kind of to be very suitable to do tea
Chrysanthemum, and its cultivated area constantly expands.But current " Mount Taishan jewel " though the sweet slightly bitter taste of tea flavour that flower bubbles out, such as
Fruit can make the tea flavour of chrysanthemum for tea use become more sweet using the steviol glycoside extracting solution in STEVIA REBAUDIANA, then its market prospects will
It can be more wide.
Therefore, it is contemplated that STEVIA REBAUDIANA leaf extract can be sprayed application in chrysanthemum " Mount Taishan jewel " plant, to make
Contain steviol glycoside RA ingredient in " Mount Taishan jewel " blade, and utilize chrysanthemum resistance, the characteristics such as biomass is big make that " Mount Taishan is bright
Contain steviol glycoside RA ingredient in the entire plant of pearl ", and then chrysanthemum for tea use is made to become more sweet.
Summary of the invention
It is an object of the present invention to steviol glycoside RA be obtained from chrysanthemum using STEVIA REBAUDIANA extracting solution, to solve above-mentioned ask
Topic.
Solution of the invention：
1, steviol glycoside crude extract is obtained
The blade on STEVIA REBAUDIANA kind " profit agriculture " plant is picked, blade is put into drying, drying box temperature in drying box and is set
It is 55 DEG C, it is spare after dry 48h.
After dry STEVIA REBAUDIANA blade 500g is crushed, pour into 80 meshes and filter, the powder that will filter out weigh 50g in
In beaker, it is added 230ml pure water and 2% cellulase of 5ml, PH control is 5 or so, the water-bath in 50 DEG C of water-baths
90min is filtered with Buchner funnel, after centrifugation, takes supernatant liquid filtering.In the residue of taking-up, further be added 230ml pure water and
After 2% cellulase of 5ml, 45min is heated in water-bath, is filtered, is taken filtrate.Then, in filtrate be added 5g calcium oxide and
After 4g aluminum aluminum sulfate, impurity 40min, agitation and filtration constant volume into 1L volumetric flask, to obtain STEVIA REBAUDIANA are removed in 50 DEG C of water-baths
Steviol glycoside crude extract 1L in blade.
2, steviol glycoside crude extract sprays chrysanthemum plant
Steviol glycoside crude extract 1L in the STEVIA REBAUDIANA blade obtained by above-mentioned 1 process is diluted to 10 times.Work as chrysanthemum
When flower plant height reaches 30cm, the morning 9：00 utilizes sprayer, and it is primary that dilution is sprayed chrysanthemum plant, sprays steviol glycoside every time
The method of crude extract is complete stool sprays, and drop of water is degree to be sprayed on chrysanthemum leaf, and spray is primary weekly, continuous spraying
4 times.The 6th week morning 9 every one week after spraying 4 times, after starting experiment：It 00 point, when observing on chrysanthemum plant without dewdrop, takes
Chrysanthemum overground part, removal have the blade of scab, filter out a certain amount of healthy and strong, disease-free spot blade, put it into drying box
It is dry.Drying box temperature is set as 55 DEG C, a length of 48h when dry, spare after dry.
3, steviol glycoside in cellulose Enzymatic Extraction chrysanthemum leaf
Using method identical with above-mentioned 1 process, steviol glycoside crude extract in Dendranthema morifolium Varieties " Mount Taishan jewel " blade is extracted.
After dry chrysanthemum leaf 500g is crushed, pours into 80 meshes and filter, the powder that will filter out weighs 50g in beaker, adds
Entering 2% cellulase of 230ml pure water and 5ml, the water-bath 90min in 50 DEG C of water-baths is filtered with Buchner funnel, after centrifugation,
Take supernatant liquid filtering.In the residue of taking-up, after 230ml pure water and 2% cellulase of 5ml further is added, in water-bath
45min is heated, filters, takes filtrate.Then, it after 5g calcium oxide and 4g aluminum aluminum sulfate being added in filtrate, is removed in 50 DEG C of water-baths
Impurity 40min, agitation and filtration constant volume into 1L volumetric flask, to obtain the steviol glycoside crude extract 1L in chrysanthemum leaf.
4, macroreticular resin absorbing method purifies steviol glycoside crude extract
By by the chrysanthemum leaf steviol glycoside crude extract of above-mentioned 3 procedure extraction, resin is adsorbed by being equipped with macroporous type
Chromatographic column, at this point, steviol glycoside almost all discards filtrate by resin adsorption.It is adsorbed with 95% ethanol elution resin
Steviol glycoside, the color until resin bleaches collect eluent.By eluent through 0.45 μm of filtering with microporous membrane.
5, Rotary Evaporators method is evaporated stevioside extracting solution in chrysanthemum
It will be evaporated on the Rotary Evaporators at 45 DEG C by the above-mentioned obtained filtrate of 4 process, chrysanthemum leaf stevioside
Glycosides ingredient is evaporated in round-bottomed flask, is dissolved with 95% ethyl alcohol of mobile phase of 3ml.
6, high performance liquid chromatograph analyzes chrysanthemum leaf steviol glycoside content
99%RA, 85%RC, 98.5%STV standard items 5mg are accurately weighed, is placed in 10mL volumetric flask, uses mobile phase
The dissolution of 95% ethyl alcohol, and constant volume shakes up rear spare to 5ml.Precision draws each 20 μ L of sample solution of contrast solution and processing, point
Not Zhu Ru high performance liquid chromatograph, the content of each steviol glycoside ingredient is calculated with external standard method.High performance liquid chromatograph detector bar
Part is set as：Instrument model, 515 type of U.S. Waters；Detector, Waters2487 dual wavelength ultraviolet detector；Chromatographic column,
250 × 4.6 nh 2 column of Kromasil；Mobile phase, acetonitrile: water=80: 20；Wavelength, 210nm；Sensitivity, 0.5AUFS；Flow velocity,
0.9ml/min；Sample volume, 10ul.
Chrysanthemum leaf steviol glycoside content is analyzed by high performance liquid chromatograph, it is found that total steviol glycoside content reaches
0.81%, RA content are 0.50%.
Beneficial effect：
Due to using the above method, the chrysanthemum plant in blade containing steviol glycoside RA is obtained.
Present invention obtains the methods that steviol glycoside RA ingredient is obtained from chrysanthemum leaf.Difference is detected in chrysanthemum leaf
Steviol glycoside component content is as follows.
Different steviol glycoside component contents in 1 chrysanthemum leaf of table
Note：Control：Show the control for not spraying steviol glycoside extracting solution；Treatment1,2,3：Show to spray stevia rebaudianum
The processing of glucosides extracting solution.
Detailed description of the invention
Fig. 1：" Mount Taishan jewel " cuttage field planting.
Fig. 2：Pick STEVIA REBAUDIANA blade.
Fig. 3：The dry blade of STEVIA REBAUDIANA for extracting steviol glycoside crude extract.
Fig. 4：When the plant height of chrysanthemum plant reaches about 30cm, start to spray steviol glycoside crude extract.
Fig. 5：When the plant height of chrysanthemum plant reaches about 80cm, sprays steviol glycoside crude extract 4 times, terminated to spray.
Fig. 6 detects different steviol glycoside component contents.
Specific embodiment
Solution of the invention：
1, steviol glycoside crude extract is obtained
STEVIA REBAUDIANA kind ' the blade on profit agriculture ' plant is picked, blade is put into drying, drying box temperature in drying box and is set
It is 55 DEG C, it is spare after dry 48h.
After dry STEVIA REBAUDIANA blade 500g is crushed, pour into 80 meshes and filter, the powder that will filter out weigh 50g in
In beaker, it is added 230ml pure water and 2% cellulase of 5ml, PH control is 5 or so, the water-bath in 50 DEG C of water-baths
90min is filtered with Buchner funnel, after centrifugation, takes supernatant liquid filtering.In the residue of taking-up, further be added 230ml pure water and
After 2% cellulase of 5ml, 45min is heated in water-bath, is filtered, is taken filtrate.Then, in filtrate be added 5g calcium oxide and
After 4g aluminum aluminum sulfate, impurity 40min, agitation and filtration constant volume into 1L volumetric flask, to obtain STEVIA REBAUDIANA are removed in 50 DEG C of water-baths
Steviol glycoside crude extract 1L in blade.
2, steviol glycoside crude extract sprays chrysanthemum plant
Steviol glycoside crude extract 1L in the STEVIA REBAUDIANA blade obtained by above-mentioned 1 process is diluted to 10 times.Work as chrysanthemum
When flower plant height reaches 30cm, the morning 9：00 utilizes sprayer, and it is primary that dilution is sprayed chrysanthemum plant, sprays steviol glycoside every time
The method of crude extract is complete stool sprays, and drop of water is degree to be sprayed on chrysanthemum leaf, and spray is primary weekly, continuous spraying
4 times.The 6th week morning 9 every one week after spraying 4 times, after starting experiment：It 00 point, when observing on chrysanthemum plant without dewdrop, takes
Chrysanthemum overground part, removal have the blade of scab, filter out a certain amount of healthy and strong, disease-free spot blade, put it into drying box
It is dry.Drying box temperature is set as 55 DEG C, a length of 48h when dry, spare after dry.
3, steviol glycoside in cellulose Enzymatic Extraction chrysanthemum leaf
Using method identical with above-mentioned 1 process, steviol glycoside crude extract in Dendranthema morifolium Varieties " Mount Taishan jewel " blade is extracted.
After dry chrysanthemum leaf 500g is crushed, pours into 80 meshes and filter, the powder that will filter out weighs 50g in beaker, adds
Entering 2% cellulase of 230ml pure water and 5ml, the water-bath 90min in 50 DEG C of water-baths is filtered with Buchner funnel, after centrifugation,
Take supernatant liquid filtering.In the residue of taking-up, after 230ml pure water and 2% cellulase of 5ml further is added, in water-bath
45min is heated, filters, takes filtrate.Then, it after 5g calcium oxide and 4g aluminum aluminum sulfate being added in filtrate, is removed in 50 DEG C of water-baths
Impurity 40min, agitation and filtration constant volume into 1L volumetric flask, to obtain the steviol glycoside crude extract 1L in chrysanthemum leaf.
4, macroreticular resin absorbing method purifies steviol glycoside crude extract
By by the chrysanthemum leaf steviol glycoside crude extract of above-mentioned 3 procedure extraction, resin is adsorbed by being equipped with macroporous type
Chromatographic column, at this point, steviol glycoside almost all discards filtrate by resin adsorption.It is adsorbed with 95% ethanol elution resin
Steviol glycoside, the color until resin bleaches collect eluent.By eluent through 0.45 μm of filtering with microporous membrane.
5, Rotary Evaporators method is evaporated stevioside extracting solution in chrysanthemum
It will be evaporated on the Rotary Evaporators at 45 DEG C by the above-mentioned obtained filtrate of 4 process, chrysanthemum leaf stevioside
Glycosides ingredient is evaporated in round-bottomed flask, is dissolved with 95% ethyl alcohol of mobile phase of 3ml.
6, high performance liquid chromatograph analyzes chrysanthemum leaf steviol glycoside content
99%RA, 85%RC, 98.5%STV standard items 5mg are accurately weighed, is placed in 10mL volumetric flask, uses mobile phase
The dissolution of 95% ethyl alcohol, and constant volume shakes up rear spare to 5ml.Precision draws each 20 μ L of sample solution of contrast solution and processing, point
Not Zhu Ru high performance liquid chromatograph, the content of each steviol glycoside ingredient is calculated with external standard method.High performance liquid chromatograph detector bar
Part is set as：Instrument model, 515 type of U.S. Waters；Detector, Waters2487 dual wavelength ultraviolet detector；Chromatographic column,
250 × 4.6 nh 2 column of Kromasil；Mobile phase, acetonitrile: water=80: 20；Wavelength, 210nm；Sensitivity, 0.5AUFS；Flow velocity,
0.9ml/min；Sample volume, 10ul.
Chrysanthemum leaf steviol glycoside content is analyzed by high performance liquid chromatograph, it is found that total steviol glycoside content reaches
0.81%, RA content are 0.50%.
Claims (6)
1. a kind of method for obtaining steviol glycoside RA from chrysanthemum using STEVIA REBAUDIANA extracting solution, steviol glycoside crude extract is sprayed
On chrysanthemum plant, after being handled one month by experimental program, with steviol glycoside in cellulose Enzymatic Extraction chrysanthemum leaf, with macropore tree
Rouge purification via adsorption-based process steviol glycoside crude extract, is evaporated stevioside extracting solution in chrysanthemum with Rotary Evaporators method, finally utilizes height
Effect liquid phase chromatogram instrument analyzes chrysanthemum leaf steviol glycoside content.
2. the method for obtaining steviol glycoside crude extract using cellulase such as the acquisition in right 1, which is characterized in that picking sweet tea
Blade is put into drying in drying box by the leaf chrysanthemum kind ' blade on profit agriculture ' plant, and drying box temperature is set as 55 DEG C, dry 48h
It is spare afterwards, after dry STEVIA REBAUDIANA blade 500g is crushed, pour into 80 meshes and filter, the powder that will filter out weigh 50g in
In beaker, it is added 230ml pure water and 2% cellulase of 5ml, PH control is 5 or so, the water-bath in 50 DEG C of water-baths
90min is filtered with Buchner funnel, after centrifugation, takes supernatant liquid filtering, in the residue of taking-up, be further added 230ml pure water and
After 2% cellulase of 5ml, 45min is heated in water-bath, filter, take filtrate, then in filtrate be added 5g calcium oxide and
After 4g aluminum aluminum sulfate, impurity 40min, agitation and filtration constant volume into 1L volumetric flask, to obtain STEVIA REBAUDIANA are removed in 50 DEG C of water-baths
Steviol glycoside crude extract 1L in blade.
3. such as the method sprayed steviol glycoside crude extract on chrysanthemum plant in right 1, which is characterized in that will pass through above-mentioned
The steviol glycoside crude extract 1L in STEVIA REBAUDIANA blade that 1 process obtains is diluted to 10 times, when chrysanthemum plant height reaches 30cm, on
Noon 9：00 utilizes sprayer, dilution sprays to chrysanthemum plant is primary, the method for spraying steviol glycoside crude extract every time is complete
Strain sprays, and drop of water is degree to be sprayed on chrysanthemum leaf, and spray is primary weekly, and continuous spraying 4 times, every one after spraying 4 times
Week, the 6th week morning 9 after starting experiment：00 point, when observing on chrysanthemum plant without dewdrop, chrysanthemum overground part is taken, removal has
The blade of scab filters out a certain amount of healthy and strong, disease-free spot blade, puts it into dry in drying box.
4. as purified the method for steviol glycoside crude extract with macroreticular resin absorbing method in right 1, which is characterized in that use macropore
Resin adsorption method purifies steviol glycoside crude extract, by by the chrysanthemum leaf steviol glycoside crude extract of above-mentioned 3 procedure extraction, leads to
The chromatographic column equipped with macroporous type absorption resin is crossed, at this point, steviol glycoside almost all discards filtrate by resin adsorption, with 95%
The adsorbed steviol glycoside of ethanol elution resin eluent is collected, by eluent through 0.45 μm of micropore until resin bleaches color
Membrane filtration.
5. such as the method for being evaporated stevioside extracting solution in chrysanthemum with Rotary Evaporators method in right 1, which is characterized in that with rotation
When evaporimeter method is evaporated stevioside extracting solution in chrysanthemum, it will be steamed by rotation of the above-mentioned obtained filtrate of 4 process at 45 DEG C
It is evaporated on hair instrument, chrysanthemum leaf steviol glycoside ingredient is evaporated in round-bottomed flask, is dissolved with 95% ethyl alcohol of mobile phase of 3ml.
6. such as the method using high performance liquid chromatograph analysis chrysanthemum leaf steviol glycoside content in right 1, which is characterized in that
When analyzing chrysanthemum leaf steviol glycoside content using high performance liquid chromatograph, 99%RA, 85%RC, 98.5%STV are accurately weighed
Standard items 5mg is placed in 10mL volumetric flask, is dissolved with 95% ethyl alcohol of mobile phase, and constant volume shakes up rear spare, precision suction to 5ml
Contrast solution and each 20 μ L of the sample solution of processing are taken, high performance liquid chromatograph is injected separately into, each stevia rebaudianum is calculated with external standard method
The content of glycoside component, high performance liquid chromatograph testing conditions are set as：Instrument model, 515 type of U.S. Waters；Detector,
Waters2487 dual wavelength ultraviolet detector；Chromatographic column, 250 × 4.6 nh 2 column of Kromasil；Mobile phase, acetonitrile: water=80:
20；Wavelength, 210nm；Sensitivity, 0.5AUFS；Flow velocity, 0.9ml/min；Sample volume, 10ul pass through high performance liquid chromatograph point
Chrysanthemum leaf steviol glycoside content is analysed, it is found that total steviol glycoside content reaches 0.81%, RA content is 0.50%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108918731A (en) * | 2018-06-28 | 2018-11-30 | 安徽蚌埠惠农甜叶菊高科技发展有限公司 | A kind of method of a large amount of acquisition STEVIA REBAUDIANA blade product steviol glycoside RM |
| CN111662347A (en) * | 2019-03-06 | 2020-09-15 | 山东农业大学 | Method for quickly decoloring stevia extract |
| CN114261127A (en) * | 2021-12-10 | 2022-04-01 | 湖南绿蔓生物科技股份有限公司 | Sweet glycoside stoste's intelligence squeezes extraction element in sweetleaf chrysanthemum |
-
2018
- 2018-05-29 CN CN201810612576.1A patent/CN108912184A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108918731A (en) * | 2018-06-28 | 2018-11-30 | 安徽蚌埠惠农甜叶菊高科技发展有限公司 | A kind of method of a large amount of acquisition STEVIA REBAUDIANA blade product steviol glycoside RM |
| CN111662347A (en) * | 2019-03-06 | 2020-09-15 | 山东农业大学 | Method for quickly decoloring stevia extract |
| CN114261127A (en) * | 2021-12-10 | 2022-04-01 | 湖南绿蔓生物科技股份有限公司 | Sweet glycoside stoste's intelligence squeezes extraction element in sweetleaf chrysanthemum |
| CN114261127B (en) * | 2021-12-10 | 2023-12-26 | 湖南绿蔓生物科技股份有限公司 | Intelligent squeezing and extracting device for stevioside stock solution in stevia rebaudiana |
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Application publication date: 20181130 |
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| WD01 | Invention patent application deemed withdrawn after publication |