CN112110962A - Method for separating and purifying stevioside from stevioside-containing source - Google Patents
Method for separating and purifying stevioside from stevioside-containing source Download PDFInfo
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- CN112110962A CN112110962A CN202010949185.6A CN202010949185A CN112110962A CN 112110962 A CN112110962 A CN 112110962A CN 202010949185 A CN202010949185 A CN 202010949185A CN 112110962 A CN112110962 A CN 112110962A
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- stevioside
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/256—Polyterpene radicals
Abstract
The invention discloses a method for separating and purifying stevioside from stevioside-containing raw materials, which comprises the following steps: extracting a stevioside raw material from the stevioside-containing raw material by using an organic extracting agent; performing primary separation in a column containing an adsorbent; wherein the preliminary separation comprises: the first step is as follows: eluting with an organic solvent mixture at a pressure of 50psi to 100 psi to produce a crystallized stevioside fraction to provide a preliminarily purified stevioside compound and a mother liquor, and concentrating said mother liquor. The separation and purification process provided by the embodiment of the invention is simple in steps, easy to operate, suitable for industrial large-scale preparative separation and purification, high in purity of the obtained product and high in application value.
Description
Technical Field
The invention relates to a separation and purification process of compounds, in particular to a method for separating and purifying stevioside from a stevioside-containing source.
Background
Steviolin is a novel diterpene glucoside active substance extracted from the leaves of stevia rebaudiana Bertoni of a sweet wild Compositae originally produced in yerba mate and the amantadine Brazil boundary, and the diterpene glucoside substances comprise stevioside, rebaudioside A, B, C, D, E, F, M and other sweet substances. Because of their super strong sweetness (about 300-450 times of sucrose sweetness), and their comfortable and good taste, rebaudioside a, D, and M compounds are widely used in the food industry as high-potency sweeteners.
However, stevioside limits its use as a sweetener in the food industry due to its characteristic unpleasant post-bitter taste and licorice flavor. Stevioside has almost no other use than in kimchi processing in korea and seasoning in soy sauce processing in japan, and its use is rejected in western countries such as europe and america.
For hundreds of years, the original residents in south America, paraguay, Brazil and the like have used stevia rebaudiana leaves to drink tea for controlling and treating diabetes. With the intensive research on stevia in recent years, it has been found that: besides being used as a sweetener, the stevioside separated from the stevia rebaudiana can effectively reduce the postprandial blood sugar concentration of experimental animals and does not generate the risk of hypoglycemia. Further research finds that the stevioside can effectively stimulate islet beta-cells to secrete insulin and inhibit alpha-cells to secrete glucagon, thereby achieving the purpose of reducing postprandial blood sugar of type II diabetes patients.
The indigestibility of the stevioside ensures that the ingestion of the stevioside does not cause the increase of the blood sugar concentration, and the stevioside can stimulate the islet cells of a type II diabetes patient to secrete insulin, so that the postprandial blood sugar concentration of the type II diabetes patient can be effectively reduced, the stevioside is suitable for the diabetes patient, and clinical researches find that the stevioside has the function of improving the insulin, and particularly has the function of resisting hyperglycemia on type II diabetes. Can not cause the rise of blood sugar value, eliminates the 'three more' symptoms (polyphagia, polydipsia and polyuria) of the diabetics after taking the product, and is a nutritional sugar substitute which is most suitable for the diabetics to eat. The stevioside has the effect of reducing the contents of cholesterol and triglyceride in blood, and can also reduce the concentration of low-density lipoprotein (LDL) in a human body without influencing the concentration of high-density lipoprotein (HDL), so that the ratio of HDL to LDL is increased.
Recent studies have demonstrated that stevioside has a unique hypoglycemic mechanism of action in experimental animal models of diabetes, and this mechanism is unique among all compounds found in humans to date, and is expected to be a new therapeutic agent for diabetes. Through research on a series of diterpene glucosides of stevia sugars, scientists find that only stevioside in the series of stevioside substances has the function of reducing the blood sugar of experimental animals with diabetes, but other stevioside substances do not have the function, so that the separation and purification of the stevioside are very necessary.
Disclosure of Invention
In order to solve the above technical problems, embodiments of the present invention provide a method for separating and purifying stevioside from a steviol glycoside-containing source.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
the embodiment of the invention provides a method for separating and purifying stevioside from stevioside-containing raw materials, which comprises the following steps:
extracting a stevioside raw material from the stevioside-containing raw material by using an organic extracting agent; performing primary separation in a column containing an adsorbent; wherein the preliminary separation comprises:
the first step is as follows: eluting with an organic solvent mixture at a pressure of 50psi to 100 psi to produce a crystallized stevioside fraction to provide a preliminarily purified stevioside compound and a mother liquor, and concentrating said mother liquor.
Further, the first step further includes: removing lignan and flavonoid impurities.
Further, steviol glycoside-containing materials include various stevia extracts and concentrates of mother liquors remaining after crystallization of rebaudioside a.
Further, the steviol glycoside-containing material comprises a botanical source that includes individual cultivar members of stevia rebaudiana.
Further, the steviol glycoside comprises stevioside, rebaudioside a, rebaudioside B, rebaudioside C, or rebaudioside D.
Further, the organic extractant is a mixture of methanol, ethanol, propanol or a combination thereof and water.
Further, the adsorbent comprises macroporous polystyrene-divinylbenzene polymer resin.
Embodiments of the present invention also provide a method for separating and purifying stevioside from a steviol glycoside-containing source, comprising:
providing an intermediate pressure column comprising an adsorbent; a first step of separating and purifying a stevioside moiety by elution with an organic solvent; crystallizing the stevioside portion to provide a semi-purified stevioside and a mother liquor; a second step of crystallizing said semi-purified stevioside by heating with methanol to obtain at least a substantially purified stevioside.
Further, the first step also includes removing lignan and flavonoid impurities.
Further, the organic solvent comprises a mixture of water and ethanol.
Further, the organic solvent is passed through the column at a pressure of 50psi to 100 psi.
Embodiments of the present invention also provide a method for separating and purifying stevioside from a steviol glycoside-containing source, comprising: providing a source of steviol glycosides; providing a medium-pressure column comprising a polystyrene-divinylbenzene polymeric resin; eluting with an organic solvent at a pressure between 50 and 100 psi to elute the purified stevioside component and remove the flavone and lignan impurities; crystallizing the stevioside component to provide a semi-purified stevioside and a mother liquor; the semi-purified stevioside was thermally crystallized by 90-95% methanol at a temperature of 55-58 c to obtain a primary purified stevioside.
Further, the purified stevioside is at least 97% pure.
The embodiment of the invention provides a method for separating and purifying stevioside from a stevioside-containing source, which comprises the steps of firstly carrying out primary separation on raw materials by using a chromatographic column, then carrying out primary purification through recrystallization, and finally carrying out further purification through secondary recrystallization to obtain a stevioside finished product with the content of more than 97%. The separation and purification process provided by the embodiment of the invention is simple in steps, easy to operate, suitable for industrial large-scale preparative separation and purification, high in purity of the obtained product and high in application value.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention, belong to the scope of protection of the invention.
In some embodiments of the invention, a method of isolating and purifying stevioside from a steviol glycoside-containing source comprises:
extracting a stevioside raw material from the stevioside-containing raw material by using an organic extracting agent; performing primary separation in a column containing an adsorbent; wherein the preliminary separation comprises:
the first step is as follows: eluting with an organic solvent mixture at a pressure of 50psi to 100 psi to produce a crystallized stevioside fraction to provide a preliminarily purified stevioside compound and a mother liquor, and concentrating said mother liquor.
Further, the first step further includes: removing lignan and flavonoid impurities.
Further, steviol glycoside-containing materials include various stevia extracts and concentrates of mother liquors remaining after crystallization of rebaudioside a.
Further, the steviol glycoside-containing material comprises a botanical source that includes individual cultivar members of stevia rebaudiana.
Further, the steviol glycoside comprises stevioside, rebaudioside a, rebaudioside B, rebaudioside C, or rebaudioside D.
Further, the organic extractant is a mixture of methanol, ethanol, propanol or a combination thereof and water.
Further, the adsorbent comprises macroporous polystyrene-divinylbenzene polymer resin.
Embodiments of the present invention also provide a method for separating and purifying stevioside from a steviol glycoside-containing source, comprising:
providing an intermediate pressure column comprising an adsorbent; a first step of separating and purifying a stevioside moiety by elution with an organic solvent; crystallizing the stevioside portion to provide a semi-purified stevioside and a mother liquor; a second step of crystallizing said semi-purified stevioside by heating with methanol to obtain at least a substantially purified stevioside.
Further, the first step also includes removing lignan and flavonoid impurities.
Further, the organic solvent comprises a mixture of water and ethanol.
Further, the organic solvent is passed through the column at a pressure of 50psi to 100 psi.
Embodiments of the present invention also provide a method for separating and purifying stevioside from a steviol glycoside-containing source, comprising: providing a source of steviol glycosides; providing a medium-pressure column comprising a polystyrene-divinylbenzene polymeric resin; eluting with an organic solvent at a pressure between 50 and 100 psi to elute the purified stevioside component and remove the flavone and lignan impurities; crystallizing the stevioside component to provide a semi-purified stevioside and a mother liquor; the semi-purified stevioside was thermally crystallized by 90-95% methanol at a temperature of 55-58 c to obtain a primary purified stevioside.
Further, the purified stevioside is at least 97% pure.
In other embodiments of the present invention, a process for separating and purifying stevioside from a steviol glycoside-containing material comprises the steps of:
step S10, providing a raw material containing stevioside;
step S20, dissolving the raw materials in an intermediate solvent, and adding the raw materials into a chromatographic column, wherein the chromatographic column comprises an adsorption filler;
step S30, adding eluent into the chromatographic column for elution, and collecting the fraction containing stevioside;
step S40, recrystallizing the stevioside-containing fraction for the first time to obtain a solid crude product containing the stevioside and a mother liquor;
step S50, recrystallizing the solid crude product for the second time, and collecting a filter cake containing the stevioside;
and step S60, washing the filter cake by using a washing solvent.
Firstly, the raw materials are subjected to primary separation by using a chromatographic column, then primary purification is carried out by recrystallization, and finally further purification is carried out by secondary recrystallization, so that a stevioside finished product with the content of more than 97 percent can be obtained. The separation and purification process provided by the embodiment of the invention is simple in steps, easy to operate, suitable for industrial large-scale preparative separation and purification, high in purity of the obtained product and high in application value.
Further, the raw materials of the embodiment of the present invention include, but are not limited to: an aqueous solution of a mother liquor sugar for rebaudioside a crystallization process. Wherein, the mother liquor sugar comprises the following components in percentage by weight: rebaudioside D3-4.5%, rebaudioside A25-30%, stevioside 25-30%, rebaudioside C9-12.5%, and the balance other glycosides.
In addition, in the embodiment of the invention, the content determination method of the stevioside in the crude product of the stevioside, the crude product of the stevioside and the finished product of the stevioside can be used for detecting by using a High Performance Liquid Chromatography (HPLC).
In the examples of the present invention, "content" means a mass percentage unless otherwise specified.
Further, the stevioside raw material contains 43% stevioside and 20% rebaudioside A in percentage by weight, and the balance of other stevioside and impurities.
Further, step S40: carrying out primary recrystallization on the stevioside raw material to obtain a stevioside crude product, which comprises the following steps:
step S41, mixing the first solvent and the stevioside raw material, and stirring;
and step S42, separating for the first time to obtain the crude product of the stevioside.
Further, in step S41: mixing a first solvent and a stevioside raw material, and stirring; further comprising: after heating to a first preset temperature, maintaining the first preset temperature and continuing stirring for a first preset time;
and after the temperature is reduced to a second preset temperature, continuously stirring.
Further, the first separation may be performed by centrifugal separation. Furthermore, the mother liquor after separation is concentrated to recover the first solvent, so that the cost is saved.
Further, the first solvent is a 90% concentration by volume aqueous solution of methanol.
Further, the first preset temperature is 58 to 62 ℃, for example, the first preset temperature is 58 ℃, 59 ℃, 60 ℃, 61 ℃, or 62 ℃; the second preset temperature is 20-30 ℃, for example: the second preset temperature is 23 ℃, 25 ℃, 28 ℃ and the like; and/or the first preset time is 3-5 h, for example, the first preset time is 3h, 3.5h, 4h, 4.2h, 4.5h, 5h, and the like.
Further, step S50: recrystallizing the crude solid product for a second time and collecting a filter cake containing the stevioside, comprising:
s51, mixing a second solvent with the crude solid product;
s52, dripping water until the crude solid product is completely dissolved to obtain a dissolved solution;
s53, crystallizing, separating and collecting a filter cake containing the stevioside.
Specifically, mixing a second solvent with the crude stevioside, heating to a third preset temperature, and continuously stirring; dripping water until the crude product of the stevioside is completely dissolved to obtain a dissolved solution; raising the temperature to a fourth preset temperature again, and continuing to stir for a second preset time; cooling and crystallizing; separating and washing to obtain the stevioside finished product.
In the embodiment of the present invention, "water" is preferably water that does not affect the product, such as distilled water or pure water for experiments.
The dropwise addition is favorable for controlling the content of the solvent, reducing the precipitation of impurities and improving the purity of subsequent crystallization.
Further, before raising the temperature again to the fourth preset temperature, the process further includes: the solution was filtered hot.
Further, the second solvent is a 95% volume concentration methanol aqueous solution.
Further, the fourth preset temperature is 55 to 60 ℃, for example, the fourth preset temperature is 55 ℃, 58 ℃, 60 ℃ and the like; and/or the second preset time is 3-5 h, for example, the second preset time is 3h, 4h, 4.5h, 5h, and the like.
Further, after washing the filter cake with a washing solvent, the method further comprises the following steps:
milling the filter cake.
Further, the adsorptive filler comprises a reversed-phase polystyrene/divinylbenzene polymeric adsorptive filler.
Further, the eluent is an aqueous solution of ethanol.
Further, the intermediate solvent is water.
In some other embodiments of the present invention, a method for isolating and purifying stevioside from a steviol glycoside-containing source comprises the steps of:
providing a raw material containing stevioside;
dissolving the raw materials in an intermediate solvent, and adding the raw materials into a chromatographic column filled with an inverse polystyrene/divinylbenzene polymer adsorption filler;
adding an eluent into the chromatographic column for elution, and collecting a fraction containing the stevioside;
carrying out primary recrystallization on the fraction containing the stevioside by using a first solvent to obtain a solid crude product containing the stevioside and a mother liquor;
carrying out secondary recrystallization on the crude solid product by using a second solvent, and collecting a filter cake containing the stevioside;
washing the filter cake with a washing solvent to obtain the stevioside finished product.
Further, the first solvent and the second solvent are both methanol aqueous solutions, and the content of methanol in the first solvent is lower than that in the second solvent.
Further, in the solid crude product, the weight percentage of the stevioside is 85-95%;
and/or
In the stevioside finished product, the weight percentage of the stevioside is more than 97 percent.
The technical solution of the present invention is further described below with reference to specific examples, and it should be understood that the present invention is not limited to the following examples.
Example one
40 kg of mother liquor sugar (the substance component: rebaudioside D3-4.5%, rebaudioside A25-30%, stevioside 25-30%, rebaudioside C9-12.5% and other glycosides) from the rebaudioside A crystallization process was dissolved in 100l of water under heating to make a 40% aqueous solution. Then the aqueous solution is pumped into a medium-pressure stainless steel industrial preparative chromatographic column (column size 800 mm x 3500 mm), the chromatographic column is pre-filled with reversed-phase polystyrene/divinylbenzene polymer adsorption filler, and the particle size of the filler is about 100-150 microns. Then, the column was eluted with 20% ethanol in 4 bed volumes at a flow rate of 1.5 bed volumes per hour, and the eluate was collected in 500L per fraction. Then, 30% ethanol was flowed through the column at a flow rate of 1.5 bed volumes per hour, 6 bed volumes. The eluates were collected for each 500L and subjected to HPLC trace detection, the column was then eluted with 35% and 40% ethanol solutions (each 5 bed volumes), and the eluates were collected for each 500L, and the fractions were combined according to the HPLC detection results to obtain fractions containing 7-15% rebaudioside D, 40-50% rebaudioside A and 40-50% stevioside, and 20-30 rebaudioside C, i.e., a stevioside-containing fraction, respectively. And evaporating and concentrating the elution solution of the fraction, and then performing spray drying to obtain high-content rebaudioside D, rebaudioside A, rebaudioside C and stevioside raw material crude products.
Example two
1600 liters of 90% strength methanol was pumped into a 3000 liter stainless steel reaction tank, then 400 kg of crude stevioside feedstock from the preparation of example one (containing 43% stevioside, 20% rebaudioside a, and other minor amounts of steviol glycosides and impurities) was added, stirring was started and the jacket of the reaction tank was heated by passing heat transfer oil through it, the temperature in the reaction tank was raised to 60 + 2 ℃, maintained at that temperature and kept stirring for 4 hours, during which time white crystals formed, then gradually lowered to 25 ℃, and crystallization continued for 8-10 hours with stirring. The crystals were then separated by centrifugation. The mother liquor was concentrated to recover most of the ethanol solvent, and then the concentrated solution was pumped into a container for spray drying to obtain yellow powder. The cake is a crude solid product containing stevioside, and the content of the crude product is 85-95%.
400 kg (dry weight about 240-250 kg) of the wet crude stevioside-containing solid was suspended in 2000L of methanol (95%), heated to 45 deg.C, and stirred continuously for 30 minutes. Water (-50L) was added in a dropwise fashion until the material was completely dissolved and the solution was clear. The clean solution was filtered while hot and then warmed to 55-60 ℃ with continued stirring for 4 hours, followed by cooling to 25-30 ℃ overnight. The precipitated solid was filtered by centrifugation, the filter cake was washed with methanol (2 × 50L, 90%), and the washed filter cake was dried by a microwave moving bed dryer and milled to 100 mesh white powder. The final product was analyzed by high performance liquid chromatography as a white crystalline powder containing >97% stevioside.
EXAMPLE III
3200 liters of 90% strength methanol was pumped into a 5600 liter stainless steel reaction tank, then 800 kg of crude stevioside raw material (containing 43% stevioside, 20% rebaudioside a, and other small amounts of stevioside and impurities) prepared by the method of example was added, stirring was started and the jacket of the reaction tank was heated by introducing heat transfer oil, the temperature in the reaction tank was raised to 60 ℃, the temperature was maintained and stirring was maintained for 4.3 hours, during which time white crystals were formed, then the temperature was gradually lowered to 25 ℃, and the stirring crystallization was continued for 10 hours. The crystals were then separated by centrifugation. The mother liquor was concentrated to recover most of the ethanol solvent, and then the concentrated solution was pumped into a container for spray drying to obtain yellow powder. The centrifuged cake is the crude stevioside product, and the content of the crude stevioside product is usually 89%.
800 kg (dry weight: about 480 kg) of wet crude stevioside-containing solid was suspended in 4000L of methanol (95%), heated to 45 deg.C, and continuously stirred for 35 minutes. Water (approximately 100L) was added dropwise until the starting material was completely dissolved and the solution was clear. The clean solution was filtered while hot and then warmed to 58 ℃ for 4 hours with continued stirring, followed by cooling to 25 ℃ overnight. The precipitated solid was filtered by centrifugation, the filter cake was washed with methanol (2 × 100L, 90%), and the washed filter cake was dried by a microwave moving bed dryer and milled into 100 mesh white powder. The final product was analyzed by high performance liquid chromatography to be a white crystalline powder of stevioside with a content of 99%.
Example four
1500 liters of 90% strength methanol was pumped into a 2800 liter stainless steel reaction tank, then 400 kg of crude stevioside feedstock from the preparation of example one (42% stevioside, 22% rebaudioside a, and other minor amounts of steviol glycosides and impurities) was added, stirring was started and the jacket of the reaction tank was heated by passing heat transfer oil through it, the temperature in the reaction tank was raised to 61 ℃, maintained at that temperature and kept stirring for 4 hours during which time white crystals formed, then the temperature was gradually lowered to 25 ℃ and crystallization was continued with stirring for 9 hours. The crystals were then separated by centrifugation. The mother liquor was concentrated to recover most of the ethanol solvent, and then the concentrated solution was pumped into a container for spray drying to obtain yellow powder. The centrifugal cake is a crude solid product containing stevioside, and the content of the crude product containing stevioside is 91 percent.
380 kg (about 245kg dry weight) of wet crude stevioside-containing solid was suspended in 1900L methanol (95%), heated to 48 deg.C, and stirred continuously for 38 minutes. Distilled water (-50L) was added in drops until the material was completely dissolved and the solution was clear. The clean solution was filtered while hot and then warmed to 55-60 ℃ with continued stirring for 3.8 hours, followed by cooling to 26 ℃ overnight. The precipitated solid was filtered by centrifugation, the filter cake was washed with methanol (2 × 50L, 90%), and the washed filter cake was dried by a microwave moving bed dryer and milled to 100 mesh white powder. Analyzing the final product by high performance liquid chromatography to obtain the final product of stevioside, wherein the final product is stevioside white crystalline powder with the content of 98 percent.
Other operations of the separation and purification process according to the embodiments of the present invention are understood and easily performed by those skilled in the art, and thus will not be described in detail.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention.
Claims (13)
1. A process for the separation and purification of stevioside from a steviol glycoside-containing material, comprising:
extracting a stevioside raw material from the stevioside-containing raw material by using an organic extracting agent; performing primary separation in a column containing an adsorbent; wherein the preliminary separation comprises:
the first step is as follows: eluting with an organic solvent mixture at a pressure of 50psi to 100 psi to produce a crystallized stevioside fraction to provide a preliminarily purified stevioside compound and a mother liquor, and concentrating said mother liquor.
2. The method of claim 1, wherein the first step further comprises: removing lignan and flavonoid impurities.
3. The method of claim 1, wherein the steviol glycoside-containing material comprises extracts of various stevia rebaudiana and concentrates of mother liquors remaining after crystallization of rebaudioside a.
4. The method of claim 3, wherein the steviol glycoside-containing material comprises a plant source comprising members of each cultivar of stevia rebaudiana.
5. The method of claim 3, wherein the steviol glycoside comprises stevioside, rebaudioside A, rebaudioside B, rebaudioside C or rebaudioside D.
6. The method of claim 1, wherein the organic extractant is a mixture of methanol, ethanol, propanol, or a combination thereof and water.
7. The method of claim 1, wherein the adsorbent comprises a macroporous polystyrene-divinylbenzene polymeric resin.
8. A process for the isolation and purification of stevioside from a steviol glycoside-containing source which comprises:
providing an intermediate pressure column comprising an adsorbent; a first step of separating and purifying a stevioside moiety by elution with an organic solvent; crystallizing the stevioside portion to provide a semi-purified stevioside and a mother liquor; a second step of crystallizing said semi-purified stevioside by heating with methanol to obtain at least a substantially purified stevioside.
9. The method of claim 8, wherein the first step further comprises removing lignan and flavonoid impurities.
10. The method of claim 8, wherein the organic solvent comprises a mixture of water and ethanol.
11. The method of claim 10, wherein the organic solvent is passed through the column at a pressure of 50psi to 100 psi.
12. A process for the isolation and purification of stevioside from a steviol glycoside-containing source which comprises: providing a source of steviol glycosides; providing a medium-pressure column comprising a polystyrene-divinylbenzene polymeric resin; eluting with an organic solvent at a pressure between 50 and 100 psi to elute the purified stevioside component and remove the flavone and lignan impurities; crystallizing the stevioside component to provide a semi-purified stevioside and a mother liquor; the semi-purified stevioside was thermally crystallized by 90-95% methanol at a temperature of 55-58 c to obtain a primary purified stevioside.
13. The method of claim 12, wherein the purified stevioside is at least 97% pure.
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| CN113201034A (en) * | 2021-04-25 | 2021-08-03 | 四川大学 | Obtaining high-purity stevioside from primary crystallization mother liquor of stevioside through secondary crystallization and enriching rebaudioside C |
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