CN109535209B - Process method for extracting and purifying stevioside from stevia rebaudiana - Google Patents
Process method for extracting and purifying stevioside from stevia rebaudiana Download PDFInfo
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Abstract
The invention relates to a process method for extracting and purifying stevioside from stevia rebaudiana. Soaking dry stevia rebaudiana leaves in ethanol, press-filtering, collecting filter residues, and recovering ethanol from the filtrate; atomizing the stevia leaf slurry by a high-pressure sprayer, extracting a target component of stevioside by using ethanol and/or water as a solvent, and filtering to obtain a stevia leaf alcohol water extracting solution; the obtained stevia rebaudiana leaf alcohol water extract passes through a ceramic ultrafiltration membrane, and a permeate is collected; the permeate passes through a ceramic nanofiltration membrane to obtain an extract concentrated solution; purifying the concentrated solution by a reversed-phase filler chromatographic column to obtain crude stevioside; or further purifying the crude sugar to obtain crude stevioside; and (3) purifying to obtain stevioside crude sugar, decoloring by using gel chromatography to obtain RA crude sugar, concentrating the RA crude sugar, and then recrystallizing to obtain high-purity RA with the purity of more than 97%. The method has the advantages of simple process, controllable cost, strong operability, high product purity and yield, and obvious advantages compared with the traditional process, wherein the final total yield of the stevioside is more than 95%.
Description
Technical Field
The invention relates to a method for extracting and purifying stevioside from stevia rebaudiana Bertoni.
Background
Stevia sugar is a mixture of diterpene glycosides containing 8 components extracted from leaves of stevia rebaudiana Bertoni, the leaves of stevia rebaudiana Bertoni contain 8% -16% of stevioside, and belongs to tetracyclic diterpene glycosides according to the division of natural phytochemistry, the sweetness of the stevioside is 200-300 times of that of cane sugar, the calorie of the stevioside is only 1/300 of cane sugar, the stevioside is gradually favored by people due to the characteristics of high sweetness, low calorie, safety, no toxicity, pure nature and the like, and the stevioside is widely applied to industries such as food, beverage, medicine, daily chemical industry and the like. Among the known 9 glycosides of stevioside, the content, taste and sweetness of each ingredient are different, and among them, stevioside (stevioside), rebaudioside a (ra), and rebaudioside c (rc) are higher, accounting for more than 90%. RA has the highest sweetness, the sweetness characteristic of RA is similar to that of cane sugar, and the novel natural sweetener has the characteristics of high sweetness, low calorie, easy dissolution, heat resistance, stability and the like. It can be used for treating obesity, hypertension, and hyperglycemia.
In the conventional process, Liyuxiu et al report a method for extracting steviol glycosides with different alcohol solutions (methanol, ethanol-water). However, the traditional heating and refluxing method has high energy consumption, acetone is adopted for crystallization subsequently, the volatility is high, the alumina column chromatography separation and purification separation effect is low, and the final purity is only 90%.
Shitongsen and the like disclose the separation and purification of stevioside by solvent extraction, silica gel column chromatography, reversed phase column chromatography or Sephadex LH-20 and recrystallization. However, the traditional organic solvent extraction method takes long time at room temperature and the energy consumption is large, so the amount of ultrasonic and microwave extract materials is small, and the method is not suitable for industrial production. Due to the lack of extraction and purification means before column chromatography, the highest purity of final RA is only 90.80% because of more impurities in the sample during column chromatography.
Chenfei et al (CN 106046075 a) and pristinamin (CN 105418703A) disclose the process routes of ethanol soaking, ether extraction, and separation of stevioside by macroporous adsorption resin adsorption. Among them, the main technical defect is that the introduction of the organic solvent ether with high toxicity causes great harm to personnel and environment. Low resin utilization, large consumption of resin, solvent and water, difficult treatment of acid-base wastewater and low product yield. The process route of compound enzyme wall breaking and supercritical extraction is further introduced by the step of the chen flight and the like. Wherein, the complex enzyme and the supercritical extraction have higher cost and are not beneficial to industrial production.
Therefore, the existing water extraction process of stevioside has more impurities in the extracting solution and difficult separation and purification; long extraction time, high water consumption and high organic waste water yield. The traditional stevioside extraction needs to add a certain amount of expensive preservative TG, which causes difficult separation and increases the cost. The process for removing impurities by flocculating the extracting solution adopts chemical flocculation, and a flocculating agent needs to be added, so that a flocculating solution contains a large amount of non-ferrous metal ions and needs to be removed by ion exchange resin, and the whole production process is complicated, long in production period and high in production cost. The current mainstream process for separating stevioside by resin adsorption has the disadvantages of low resin utilization degree, high resin, solvent and water consumption, difficult treatment of acid-base wastewater and low product yield, and becomes an industry pain point.
Disclosure of Invention
The invention aims to provide a process method for extracting and purifying stevioside from stevia rebaudiana.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for extracting and purifying stevioside from stevia rebaudiana Bertoni comprises soaking dry leaves of stevia rebaudiana Bertoni in ethanol, press filtering, collecting filter residue, and recovering ethanol from the filtrate; atomizing the stevia leaf slurry obtained after pulping into fine fog drops by a high-pressure sprayer, leaching out stevioside target components by a countercurrent contact mode by taking ethanol and/or water as a solvent, and filtering to obtain a stevia leaf alcohol-water extracting solution; passing the obtained stevia leaf alcohol water extract through a ceramic ultrafiltration membrane, and collecting a permeate; the permeate passes through a ceramic nanofiltration membrane, and the extract concentrated solution is obtained by collection and interception; purifying the concentrated solution by a reversed-phase filler chromatographic column to obtain crude stevioside; and further carrying out hydrophilic chromatography purification and gel chromatography decoloration on the crude sugar to obtain RA crude sugar, and concentrating and then recrystallizing the RA crude sugar to obtain the high-purity RA with the purity of more than 97%.
The stevia rebaudiana leaf slurry is prepared by removing sandy soil impurities, airing, crushing stevia rebaudiana leaves, repeatedly soaking crushed stevia rebaudiana leaves in absolute ethyl alcohol, filtering, pulping the filtered crushed stevia rebaudiana leaves by using ethanol and/or water, and standing by.
Repeatedly soaking the stevia rebaudiana crushed leaves in absolute ethyl alcohol, namely washing and crushing the dried stevia rebaudiana leaves, repeatedly soaking in absolute ethyl alcohol, performing filter pressing after soaking, combining soaking filter residues collected by filter pressing after repeated soaking, and recycling ethanol from filtrate for reuse; wherein, the ratio of the broken stevia leaves to the absolute ethyl alcohol in the soaking process is 1: 1.5-20 (mass ratio), and the soaking time is 1-3 h.
And leaching the stevia leaf slurry obtained after pulping for 20-60 min at room temperature by using ethanol and/or water as a solvent through a high-pressure sprayer to realize atomization into fine fog drops, further leaching out target components of stevioside, and filtering to obtain a leaching solution.
And mixing and pulping the soaked filter residue and ethanol and/or water according to the material-liquid ratio of 1:6-12 (mass ratio). Wherein, the concentration of the solvent ethanol/water mixed solvent is 30-70% (volume percentage); the extraction temperature was normal temperature (25 ℃).
The leaching liquor is sequentially subjected to two-stage membrane treatment by an ultrafiltration membrane and a nanofiltration membrane; wherein, the molecular weight cut-off of the ultrafiltration membrane passing through the leaching solution is more than 10kD (the diameter aperture is 10-50 nm); the ceramic sodium filter membrane has a molecular weight cut-off of 0.2-1 kD (pore diameter of 1-2 nm).
Gradient elution is carried out on the concentrated solution through a reversed-phase filler chromatographic column, an eluent is an alcohol-water system, elution components with the stevioside content of 30-100wt% are collected through HPLC test, wherein the elution components with the stevioside content of 30-80wt% (namely, the stevioside content is less than 80%) are concentrated through a nanofiltration membrane, the concentrated elution components are dissolved in an ethanol-water system after concentration, gradient elution is carried out through a hydrophilic chromatographic column after dissolution, the elution components with the stevioside content of 80-100wt% are collected through HPLC test, then the elution components with the stevioside content of 80-100wt% collected through the reversed-phase filler chromatographic column are combined and subjected to nanofiltration concentration, and the concentrated solution is decolorized through a gel chromatographic column to obtain RA crude sugar; the volume of alcohol and water in the eluent of the reversed-phase filler chromatographic column is 0-10: 10-0; the volume ratio of ethanol to water in an ethanol-water system of the eluent of the hydrophilic chromatographic is 0-10: 10-0; the volume ratio of ethanol to water in the ethanol-water system for dissolving the elution component is 0-10: 10-0.
The mass ratio of solute in the concentrated solution to the reversed-phase filler in the purification process is 1: 5-1: 80, the solute is mainly total glycosides of stevioside.
The filler in the reversed-phase filler chromatographic column is a medium-low pressure chromatographic column filler with the particle size of 40-60 mu m; and in the purification process, the collected components in each step are concentrated by a nanofiltration membrane to obtain ethanol, and the ethanol is recycled and reused.
The medium and low pressure chromatographic column filler is Silica gel bonded C18 filler, SKR series Silica gel, HP-Silica, Bio-C18, UniPMM 50-Carb taking polyacrylate as a framework, Jiazhong special filler (35-100 mu m), PS-DVB filler bonded with acetic acid and the like,
the hydrophilic chromatographic packing is Polar-Silica, Polar-100, Polar-Diol and the like, preferably Polar-Silica (the pore diameter is 40-60 nm);
the gel decolorizing filler is G-type sephadex, sepharose, sephadex, etc., preferably Octyl sepharose.
Mixing the RA crude sugar and a solvent according to a volume ratio of 1:8-1:10, and recrystallizing by adopting a circulating crystallization method, wherein the solvent is a mixture of two or more of chloroform, methanol, ethanol, ethyl acetate and water.
The above recrystallization solvent is preferably 90-95% ethanol solution, RA content is increased to 97% or more, refining yield is improved, and impurities are removed completely and solubilized substances are removed sufficiently in crude sugar extraction.
The invention has the advantages that:
according to the method for extracting and purifying stevioside, firstly, ethanol is adopted for pretreatment, impurities with lower polarity are removed, the viscosity of subsequent extracting solution is reduced, and the subsequent membrane filtration and chromatographic separation and purification are facilitated; secondly, continuous countercurrent normal-temperature high-pressure atomization extraction is adopted, the extraction time is shortened from the traditional 20-60 hours to 3-8 hours, and the extraction solvent is saved by more than 60%; in the process, high pressure and alcohol and/or water extraction are adopted, the addition of harmful preservatives in the traditional water extraction process is eliminated, the guarantee is provided for the natural green safety of the product, the dosage of the extraction water is greatly reduced, the leaching of other impurity components is reduced, the energy consumption and the environmental protection pressure are reduced, and the separation and filtration efficiency is improved; and then the product is purified by a chromatographic technique, the selectivity and the separation efficiency of the preparative chromatographic technique are high, the production period and the product quality are superior to those of macroporous adsorption resin, the regeneration treatment of resin and acid-base solution is not needed, a large amount of waste water is reduced, the using amount of gel filler is small, the sample loading amount is large, the decoloring effect is good, most of pigments can be removed, and the cost has obvious advantages compared with that of the decoloring macroporous resin.
The method has the advantages of simple process, controllable cost, strong operability, high product purity and yield, and suitability for factory production, and combines continuous countercurrent extraction and preparative chromatographic separation in the process, so that the yield and purity of the target are greatly improved; high-purity RA (the content is more than 97 percent) can be directly obtained, the obtained product has light color, good taste and water solubility, the final total yield of stevioside is more than 95 percent, and the method has obvious advantages compared with the prior art.
Drawings
FIG. 1 is a flow chart of the extraction and purification process provided in the embodiment of the present invention.
FIG. 2 is an HPLC chart of the extraction sample solution provided by the embodiment of the invention.
FIG. 3 is a water + 10% methanol eluent HPLC (GP-C18 reverse phase packing) plot as provided by an example of the present invention.
FIG. 4 is a 30% methanol eluent HPLC (GP-C18 reverse phase packing) chart as provided by the examples of the present invention.
FIG. 5 is a 70% methanol eluent HPLC (GP-C18 reverse phase packing) plot as provided by an example of the present invention.
FIG. 6 is a 100% methanol eluent HPLC (GP-C18 reverse phase packing) plot provided by an example of the present invention.
FIG. 7 is a 70% ethanol eluate HPLC (Polar-Silica hydrophilic chromatography packing) chart provided by an example of the present invention.
FIG. 8 is a graph of RA HPLC after recrystallization provided by an example of the present invention.
FIG. 9 is an HPLC chart of RA standard provided in the examples of the present invention, wherein the time to peak is 7.5 min.
Detailed Description
The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention. In this embodiment, 1 part by mass of the crushed stevia rebaudiana leaves obtained by drying in the sun to remove impurities such as sandy soil and the like and then crushing is taken as a reference.
Example 1
A process for extracting purified steviol glycoside from stevia rebaudiana Bertoni comprises the following steps:
pretreatment of stevia rebaudian leaves: air-drying stevia rebaudiana leaves, removing impurities such as sandy soil, crushing, and weighing 1 part by mass of the crushed stevia rebaudiana leaves;
soaking pretreatment: placing the crushed stevia rebaudiana leaves into an extraction tank, adding 1 part by mass of ethanol to soak for two times, each time for 1.5 hours, combining and filtering the secondary leaching liquor, and distilling the filtrate to recover the ethanol.
High-pressure atomization continuous countercurrent extraction: continuous countercurrent extraction equipment was used for the experiments. Adding appropriate amount of water into the filter residue, pulping to obtain suspension, dispersing the liquid into fine droplets by high pressure atomizer, spraying into countercurrent extraction tank, allowing 50% ethanol water solution to flow into the extraction tank from the bottom under high pressure, and allowing the ethanol water solution and the ethanol water solution to contact sufficiently to allow extraction to be complete, wherein the tested extraction rate of stevioside is 99%; wherein the volume ratio of the stevia rebaudiana leaf slurry to the 50% ethanol aqueous solution is 1:8, the pressure of a high-pressure spraying machine is 2-4MPa, the flow rate is 200-250L/h, the extraction temperature in the extraction tank is room temperature, and the extraction time is 30 min.
And (3) two-stage membrane filtration and concentration: centrifugally separating leaching solution and sweet stevia residue, wherein the leaching solution passes through a first-stage ceramic ultrafiltration membrane (50nm), the inlet pressure of the first-stage ceramic membrane is 0.20MPa, the outlet pressure is 0.15MPa, and the average process flux is 100 (L.m)-2·h-1) Left and right; the permeate passes through a ceramic nanofiltration membrane, the operating pressure is 0.4Mpa, and the process average flux is 180 (L.m)-2·h-1) And the permeate is used as an extraction solvent for recycling, and the trapped liquid is further concentrated to obtain the stevioside concentrated solution.
And (3) reversed phase chromatography purification: loading the concentrated solution to a glass chromatographic column which is soaked and balanced by methanol in advance and is filled with 15g of GP-C18 reversed phase chromatographic packing, carrying out gradient elution by sequentially adopting pure water, 10 percent methanol aqueous solution (v/v), 30 percent methanol aqueous solution, 70 percent methanol aqueous solution and 100 percent methanol, wherein the dosage of each eluent is 20 column volumes, and collecting to obtain an impurity effluent liquid and a crude sugar effluent liquid. By HPLC test, pure water and 10% methanol eluent are mainly impurity effluent (figure 3), 30% methanol aqueous solution-100% methanol eluent is crude sugar effluent, and the components are collected, as shown in figure 4-6, wherein the total content of stevioside STV, RA and RC in 70% methanol aqueous solution eluent reaches 89.2%, the content of three glycosides in 30% methanol aqueous solution eluent reaches 68.7%, the content of three glycosides in 100% methanol eluent reaches 80.4%, 70% methanol aqueous solution and 100% methanol eluent are reserved as target 1, 30% methanol eluent is reserved as target 2, and concentrate of target 1 concentrated by nanofiltration is reserved.
Hydrophilic chromatographic purification and gel decoloration: concentrating the elution component (namely the target 2) with low glucoside content in the crude product by using a nanofiltration membrane, continuously loading the concentrated elution component into a hydrophilic chromatographic filler Polar-Silica (with the particle size of 60 mu m), eluting and eluting by using pure water and ethanol/water in sequence, performing gradient elution by using pure water, 10% ethanol aqueous solution, 30% ethanol aqueous solution, 70% ethanol aqueous solution and 100% methanol in sequence, wherein the using amount of each eluent is 12 column volumes, collecting 70% ethanol aqueous solution eluent, performing HPLC (high performance liquid chromatography) test spectrogram as shown in figure 7, and performing nanofiltration concentration on the eluent, wherein the normalized content of STV, RA and RC is 82.5%.
Subjecting the concentrate of target 1 and hydrophilic chromatographic 70% ethanol water concentrate to Sephadex G-75, adsorbing and decolorizing, eluting with water, and spray drying to obtain RA crude sugar.
And (3) recrystallization: recrystallizing the RA crude sugar with 90% ethanol solution, wherein the ratio of sample to solvent is 1:8(v/v), the HPLC detection chromatogram is shown in FIG. 8, the RA purity is 97.3%, and the total yield is 78.9%. (percentage by mass)
Example 2
A process for extracting purified steviol glycoside from stevia rebaudiana Bertoni comprises the following steps:
pretreatment of stevia rebaudiana: air drying stevia rebaudiana leaf, removing impurities such as sand and the like, and crushing.
Soaking pretreatment: weighing 1 part by mass of the broken stevia leaves, placing the broken stevia leaves in an extraction tank, adding 2 parts by mass of edible ethanol according to the weight of the stevia leaves, soaking for 2 hours at normal temperature, carrying out centrifugal filtration, distilling the filtrate to recover the ethanol, testing the solid content of the filtrate to be 1.6%, testing the ethanol extract by HPLC (high performance liquid chromatography) to ensure that the total stevioside accounts for 0.18% (accounting for the weight of the stevia leaves), soaking and extracting the stevioside with little loss (about 1.5%) by the ethanol, and leaching to remove impurities such as tannin, pectin, pigment and the like to facilitate membrane filtration.
High-pressure atomization continuous countercurrent extraction: adding a proper amount of 50% ethanol solution into filter residues, dispersing and pulping to prepare suspension, dispersing feed liquid into fine liquid drops through a high-pressure atomizer, spraying the fine liquid drops into a countercurrent extraction tank, enabling 50% ethanol solvent to flow into the extraction tank from the bottom under high-level pressure, enabling the ethanol solvent and the solvent to be fully contacted, enabling the extraction to be completely carried out, enabling the ratio of solute to solvent to be 1:6, enabling the pressure of the high-pressure atomizer to be 2-4MPa, enabling the flow rate to be 200 plus materials at 250L/h, enabling the extraction temperature in the extraction tank to be room temperature, and testing the extraction rate of stevioside to be 98.8%.
And (3) two-stage membrane filtration and concentration: centrifugally separating leaching solution and sweet stevia residue, wherein the leaching solution passes through a first-stage ceramic ultrafiltration membrane (50nm), the inlet pressure of the first-stage ceramic membrane is 0.20MPa, the outlet pressure is 0.15MPa, and the average process flux is 100 (L.m)-2·h-1) Left and right; the permeate passes through a ceramic nanofiltration membrane, the operating pressure is 0.4Mpa, and the process average flux is 180 (L.m)-2·h-1) And the permeate is used as an extraction solvent for recycling, and the trapped liquid is further concentrated to obtain the stevioside concentrated solution.
And (3) reversed phase chromatography purification: loading the concentrated solution to a glass chromatographic column which is soaked and balanced by methanol in advance and is filled with 30g of Uni PMM50-Carb polyacrylate skeleton microsphere reversed-phase chromatographic packing (the loading capacity is about 120 mg/g), carrying out gradient elution by sequentially adopting pure water, 10% ethanol aqueous solution (v/v), 20% ethanol aqueous solution, 40% ethanol aqueous solution, 70% ethanol aqueous solution and 100% ethanol, wherein the dosage of each eluent is 15 column volumes, and collecting to obtain impurity effluent and crude sugar effluent. By HPLC test, the pure water, 10% ethanol water solution and 20% ethanol water solution eluent mainly contain no stevioside, the 40% ethanol water solution, 70% ethanol water solution and 100% ethanol eluent have fewer impurities and are mainly target glycoside components, wherein the total normalized content of stevioside STV, RA and RC in the 70% ethanol water solution eluent reaches 90.4%, the normalized content of the 40% ethanol water solution and the 100% ethanol water solution reaches 68.9%, the 70% ethanol water solution eluent is reserved as a target 1, the 40% ethanol water solution and the 100% ethanol eluent are reserved as a target 2, and the concentrate of the target 1 after nanofiltration concentration is reserved.
Hydrophilic chromatographic purification and gel decoloration: and concentrating the target 2 by using a nanofiltration membrane, continuously loading the concentrated target into a hydrophilic chromatographic filler Polar-Silica (with the particle size of 60 mu m), eluting and eluting by using pure water and ethanol/water, performing gradient elution by using pure water, 10% ethanol aqueous solution, 30% ethanol aqueous solution, 50% ethanol aqueous solution, 70% ethanol aqueous solution and 100% ethanol in sequence, using 15 column volumes of each eluent, collecting 70% ethanol aqueous solution eluent, testing the RA normalization content by using HPLC (high performance liquid chromatography), and performing nanofiltration concentration on the eluent.
Respectively passing the concentrate of the target 1 and the concentrate of hydrophilic chromatogram 70% ethanol water solution through Sephadex G-15, adsorbing and decolorizing, eluting with water, and spray drying the decolorized receiving solution to obtain RA crude sugar.
And (3) recrystallization: and recrystallizing the RA crude sugar with 93% ethanol solution, wherein the volume ratio of the sample to the solvent is 1:10, the RA purity is 97.2% by HPLC detection, and the total yield is 88.9%.
Example 3
A process for extracting purified steviol glycoside from stevia rebaudiana Bertoni comprises the following steps:
pretreatment of stevia rebaudiana: air drying stevia rebaudiana leaf, removing impurities such as sand and the like, and crushing.
Soaking pretreatment: weighing 1 part by mass of the stevia rebaudiana leaves, placing the stevia rebaudiana leaves into an extraction tank, adding 5 parts by mass of edible ethanol according to the weight of the stevia rebaudiana leaves, soaking for 2.5 hours at normal temperature, centrifuging, filtering, and distilling the filtrate to recover the ethanol.
High-pressure atomization continuous countercurrent extraction: adding a proper amount of 60% ethanol solution into stevia rebaudiana residue, dispersing and pulping to prepare a suspension, dispersing feed liquid into fine liquid drops through a high-pressure atomizer, spraying the fine liquid drops into a countercurrent extraction tank, allowing 60% ethanol solvent to flow into the extraction tank from the bottom under high pressure, fully contacting the two to ensure complete extraction, wherein the ratio of solute to total solvent is 1:10, the pressure of the high-pressure atomizer is 2-4MPa, the flow rate is 200 plus materials and 250L/h, the extraction temperature in the extraction tank is room temperature, and the extraction rate of stevioside is tested to be 99.2%.
And (3) two-stage membrane filtration and concentration: centrifugally separating leaching solution and sweet stevia residue, wherein the leaching solution passes through a first-stage ceramic ultrafiltration membrane (50nm), the inlet pressure of the first-stage ceramic membrane is 0.20MPa, the outlet pressure is 0.15MPa, and the average process flux is 100 (L.m)-2·h-1) Left and right; the permeate passes through a ceramic nanofiltration membrane, the operating pressure is 0.4Mpa, and the process average flux is 180 (L.m)-2·h-1) And the permeate is used as an extraction solvent for recycling, and the trapped liquid is further concentrated to obtain the stevioside concentrated solution.
And (3) reversed phase chromatography purification: and (3) loading the concentrated solution to a glass chromatographic column which is soaked and balanced by ethanol in advance and is filled with 30g of Jiazhong special reverse phase chromatographic packing (50 mu m), carrying out gradient elution by sequentially adopting pure water, 10% ethanol aqueous solution, 70% ethanol aqueous solution and 100% ethanol according to 12% of the resin amount, wherein the dosage of each eluent is 15 column volumes, and collecting to obtain impurity effluent liquid and crude sugar effluent liquid. By HPLC test, the pure water and 10% ethanol water solution eluent is mainly impurities, the 70% ethanol water solution and 100% ethanol eluent have fewer impurities and are mainly target glycoside components, wherein the total glycoside content in the 70% ethanol water solution eluent reaches 87.5%, the total glycoside content in the 100% ethanol water solution eluent reaches 92.5%, and the 70% ethanol water solution eluent and the 100% ethanol eluent are reserved as target objects.
Hydrophilic chromatographic purification and gel decoloration: nano-filtering and concentrating the target eluent, passing through agarose gel, adsorbing and decoloring, and carrying out spray drying on the water eluent, decoloring and receiving liquid to obtain RA crude sugar.
And (3) recrystallization: recrystallizing the RA crude sugar with 95% ethanol solution, wherein the ratio of the sample to the solvent is 1:9, the RA purity is 97.0% by HPLC detection, and the total yield is 91.2%.
Example 4
A process for extracting purified steviol glycoside from stevia rebaudiana Bertoni comprises the following steps:
pretreatment of stevia rebaudiana: air drying stevia rebaudiana leaf, removing impurities such as sand and the like, and crushing.
Soaking pretreatment: weighing 1 part by mass of stevia rebaudiana leaf fragments, placing the stevia rebaudiana leaf fragments into an extraction tank, and soaking the stevia rebaudiana leaf fragments in ethanol at a material-liquid ratio of 1:1.5 for 1 h.
High-pressure atomization continuous countercurrent extraction: adding appropriate amount of 30% ethanol solution into the filter residue, dispersing and pulping to obtain suspension, dispersing the material liquid into fine liquid drops by high pressure atomizer, spraying into countercurrent extraction tank, allowing 30% ethanol solvent to flow into the extraction tank from the bottom under high pressure, and allowing the two to contact sufficiently to perform extraction completely, wherein the material-liquid ratio is 1:6, and the extraction temperature is normal temperature (about 25 deg.C); the leaching time is 20 min.
And (3) two-stage membrane filtration and concentration: centrifugally separating leaching solution and sweet stevia residue, wherein the leaching solution passes through a first-stage ceramic ultrafiltration membrane (50nm), the inlet pressure of the first-stage ceramic membrane is 0.20MPa, the outlet pressure is 0.15MPa, and the average process flux is 100 (L.m)-2·h-1) Left and right; the permeate passes through a ceramic nanofiltration membrane, the operating pressure is 0.4Mpa, and the process average flux is 180 (L.m)-2·h-1) And the permeate is used as an extraction solvent for recycling, and the trapped liquid is further concentrated to obtain the stevioside concentrated solution.
And (3) reversed phase chromatography purification: loading the concentrated solution to a glass chromatographic column which is soaked and balanced by methanol in advance and is filled with SKR series silica gel reverse phase chromatographic packing, carrying out gradient elution by sequentially adopting pure water, 10 percent, 20 percent, 40 percent, 70 percent methanol aqueous solution and 100 percent methanol aqueous solution, using 15 column volumes of eluent for each part, and collecting to obtain impurity effluent liquid and crude sugar effluent liquid. By HPLC test, pure water, 10% methanol water solution and 20% methanol water solution eluent mainly contain stevioside as impurities, 40% methanol water solution, 70% methanol water solution and 100% methanol eluent have fewer impurities and are mainly target glycoside components, wherein the total normalized content of stevioside STV, RA and RC in the 70% methanol water solution eluent reaches 93.6%, the normalized content of the 40% methanol water solution and the 100% methanol water solution is 75.9%, the 70% methanol water solution eluent is reserved as a target 1, the 40% methanol water solution and the 100% methanol eluent are reserved as a target 2, and the concentrate of the target 1 after nanofiltration concentration is reserved.
Hydrophilic chromatographic purification and gel decoloration: concentrating the target 2 by using a nanofiltration membrane, continuously loading the concentrated target into a hydrophilic chromatographic filler Polar-100 chromatographic column, performing gradient elution by sequentially adopting pure water, 20% ethanol aqueous solution, 40% ethanol aqueous solution, 60% ethanol aqueous solution, 80% ethanol aqueous solution and 100% ethanol, using 15 column volumes of eluent in each part, collecting 80% ethanol eluent, testing the RA normalization content by using HPLC (high performance liquid chromatography) to obtain 98.7%, and performing nanofiltration concentration on the eluent. Respectively passing the concentrate of the target 1 and the 80% ethanol concentrate through Sephadex G-15, adsorbing and decolorizing, eluting with water, and spray drying to obtain RA crude sugar.
And (3) recrystallization: the RA crude sugar is recrystallized by using a 90% ethanol solution, the ratio of the sample to the solvent is 1:8, the RA purity is 99.3% by HPLC detection, and the total yield is 95.6%.
Example 5
A process for extracting purified steviol glycoside from stevia rebaudiana Bertoni comprises the following steps:
pretreatment of stevia rebaudiana: air drying stevia rebaudiana leaf, removing impurities such as sand and the like, and crushing.
Soaking pretreatment: weighing 1 part by mass of the broken stevia rebaudiana leaves, placing the broken stevia rebaudiana leaves into an extraction tank, and soaking the stevia rebaudiana leaves in ethanol at a material-liquid ratio of 1:20 for 3 hours.
High-pressure atomization continuous countercurrent extraction: adding appropriate amount of 70% ethanol solution into the filter residue, dispersing and pulping to obtain suspension, dispersing the material liquid into fine liquid drops by a high-pressure atomizer, spraying into a countercurrent extraction tank, allowing 70% ethanol solvent to flow into the extraction tank from the bottom under high pressure, and allowing the two to contact sufficiently to allow complete extraction, wherein the material-liquid ratio is 1: 12; the extraction temperature is normal temperature (about 25 ℃); the leaching time is 60 min.
And (3) two-stage membrane filtration and concentration: centrifugally separating leaching solution and stevia rebaudiana residue, wherein the leaching solution passes through a first-stage ceramic ultrafiltration membrane (50nm), the inlet pressure of the first-stage ceramic membrane is 0.20MPa, the outlet pressure is 0.15MPa, and the average process flux is 100 (L.m)-2·h-1) Left and right; the permeate passes through a ceramic nanofiltration membrane, and the operating pressure is 04MPa, process mean flux 180 (L.m)-2·h-1) And the permeate is used as an extraction solvent for recycling, and the trapped liquid is further concentrated to obtain the stevioside concentrated solution.
And (3) reversed phase chromatography purification: and (2) loading the concentrated solution to a glass chromatographic column which is soaked and balanced by methanol in advance and is filled with HP-Silica reversed phase chromatographic packing, performing gradient elution by sequentially adopting pure water, 10 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent methanol aqueous solution and 100 percent methanol, wherein the dosage of each eluent is 15 column volumes, and collecting to obtain impurity effluent liquid and crude sugar effluent liquid. Through HPLC test, pure water and methanol eluent with 10% -30% mainly contain no stevioside as impurity, 40% -70% methanol and methanol eluent have less impurity and mainly contain target glucoside component, wherein the total normalized content of stevioside STV, RA and RC in the methanol eluent with 70% reaches 91.4%, and the methanol eluent with 70% is reserved as a target 1. Combining 40%, 50%, 60% and 100% methanol aqueous solutions, the total content of stevioside STV, RA and RC reached 71.4%, and the final product was designated as target 2.
Hydrophilic chromatographic purification and gel decoloration: concentrating the target 2 by using a nanofiltration membrane, continuously loading the concentrated target into a hydrophilic chromatographic filler Polar-100 chromatographic column, performing gradient elution by sequentially adopting pure water, 20% ethanol aqueous solution, 40% ethanol aqueous solution, 60% ethanol aqueous solution, 80% ethanol aqueous solution and 100% ethanol, using 15 column volumes of eluent in each part, collecting 80% ethanol aqueous solution eluent, testing the RA normalized content by using HPLC (high performance liquid chromatography) to be 88.5%, and performing nanofiltration concentration on the eluent. Respectively passing the concentrate of the target substance 1 and the concentrate of 70% ethanol water solution through sephadex G-15, adsorbing and decoloring, carrying out water eluent, and carrying out spray drying on a receiving solution after decoloring to obtain RA crude sugar.
And (3) recrystallization: recrystallizing the RA crude sugar with 95% ethanol solution, wherein the ratio of the sample to the solvent is 1:10, the RA purity is 97.4% by HPLC detection, and the total yield is 87.6%.
In conclusion, the extraction and purification method adopts ethanol pretreatment to remove impurities with lower polarity, so that the viscosity of subsequent extracting solution is reduced, and the subsequent membrane filtration and chromatographic separation and purification are facilitated; continuous countercurrent normal-temperature high-pressure atomization extraction is adopted, the extraction time is shortened from the traditional 20-60 hours to 3-8 hours, and the extraction solvent is saved by more than 60%; high pressure and alcohol-water mixed extraction, the addition of harmful preservative in the traditional water extraction process is cancelled, the natural green safety of the product is ensured, the using amount of leaching water is greatly reduced, the leaching of other impurity components is reduced, and the energy consumption and the environmental protection pressure are reduced. The preparation chromatographic technique has high selectivity and separation efficiency, the production period and the product quality are superior to those of macroporous adsorption resin, resin and acid-base solution regeneration treatment are not needed, a large amount of waste water is reduced, the using amount of gel filler is small, the sample loading amount is large, the decoloring effect is good, most of pigments can be removed, and the cost has obvious advantage compared with that of the decoloring macroporous resin. The method has the advantages of simple process, strong operability, high product purity and yield, and suitability for factory production. The method creatively combines continuous countercurrent extraction, prepares chromatographic separation, determines final process parameters through a large number of experiments, and greatly improves the yield and the purity of the target product. From examples 1-5, it can be seen that example 4 works best, fully illustrating the invention requiring the best process parameters to be obtained after numerous experiments.
The above-mentioned embodiments only express some embodiments of the present invention, and the description is specific, but not construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the following claims.
Claims (3)
1. A process method for extracting and purifying stevioside from stevia rebaudiana is characterized by comprising the following steps: soaking dry stevia rebaudiana leaves in ethanol, press-filtering, collecting filter residues, and recovering ethanol from the filtrate; atomizing the stevia leaf slurry obtained after pulping into fine fog drops by a high-pressure sprayer, leaching out stevioside target components by a countercurrent contact mode by taking ethanol and/or water as a solvent, and filtering to obtain a stevia leaf alcohol-water extracting solution; the obtained stevia rebaudiana leaf alcohol water extract passes through a ceramic ultrafiltration membrane, and a permeate is collected; the permeate passes through a ceramic nanofiltration membrane, and the extract concentrated solution is obtained by collection and interception; purifying the concentrated solution by a reversed-phase filler chromatographic column to obtain crude stevioside; or further purifying the crude sugar by hydrophilic chromatography to obtain crude stevioside; the crude stevioside obtained by the purification is decolored by gel chromatography to obtain RA crude sugar, and RA crude sugar is concentrated and then recrystallized to obtain RA with the purity of more than 97 percent;
the stevia rebaudiana leaf slurry is prepared by removing sandy soil impurities, airing, crushing stevia rebaudiana leaves, repeatedly soaking crushed stevia rebaudiana leaves in absolute ethyl alcohol, filtering, pulping the filtered crushed stevia rebaudiana leaves by using ethanol and/or water, and preparing the slurry for later use;
repeatedly soaking the stevia rebaudiana crushed leaves in absolute ethyl alcohol, namely washing and crushing the dried stevia rebaudiana leaves, repeatedly soaking in absolute ethyl alcohol, performing filter pressing after soaking, combining soaking filter residues collected by filter pressing after repeated soaking, and recycling ethanol from filtrate for reuse; wherein the mass ratio of the stevia rebaudiana crushed leaves to the absolute ethyl alcohol liquid in each soaking process is 1: 1.5-20, and the soaking time is 1-3 h;
leaching the stevia leaf slurry obtained after pulping for 20-60 min at room temperature by using ethanol and/or water as a solvent through a high-pressure sprayer to realize atomization into fine fog drops, further leaching out stevioside target components, and filtering to obtain a leaching solution;
the leaching liquor is sequentially subjected to two-stage membrane treatment by an ultrafiltration membrane and a nanofiltration membrane; wherein the ultrafiltration membrane used for leaching solution has the molecular weight cutoff of more than 10kD and the diameter aperture of 10-50 nm; the ceramic nanofiltration membrane has a molecular weight cut-off of 0.2-1 kD and a pore diameter of 1-2 nm;
gradient elution is carried out on the concentrated solution through a reversed-phase filler chromatographic column, an eluent is an alcohol-water system, elution components with the stevioside content of 30-100wt% are collected through HPLC test, wherein the elution components with the stevioside content of 30-80wt% are concentrated through a nanofiltration membrane, dissolved in an ethanol-water system after being concentrated, gradient elution is carried out through a hydrophilic chromatographic column after being dissolved, the elution components with the stevioside content of 80-100wt% are collected through HPLC test, then the elution components and the elution components with the stevioside content of 80-100wt% collected through the reversed-phase filler chromatographic column are combined and subjected to nanofiltration concentration, and RA crude sugar is obtained after concentrated solution is decolored through a gel chromatographic column; the volume ratio of alcohol to water in the eluent of the reversed-phase filler chromatographic column is 0-10: 10-0; the volume ratio of ethanol to water in an ethanol-water system of the eluent of the hydrophilic chromatographic is 0-10: 10-0; the volume ratio of ethanol to water in an ethanol-water system for dissolving the elution component is 0-10: 10-0;
mixing the RA crude sugar and a solvent according to a volume ratio of 1:8-1:10, and recrystallizing by adopting a circulating crystallization method, wherein the solvent is a mixture of two or more of chloroform, methanol, ethanol, ethyl acetate and water.
2. The process of claim 1, wherein the step of extracting purified stevia glycosides from stevia rebaudiana Bertoni comprises the steps of: mixing and pulping the soaked filter residue and ethanol and/or water according to the material liquid mass ratio of 1: 6-12;
wherein the volume percentage of the solvent ethanol/water mixed solvent is 30-70%.
3. The process of claim 1, wherein the step of extracting purified stevia glycosides from stevia rebaudiana Bertoni comprises the steps of: the filler in the reversed-phase filler chromatographic column is a medium-low pressure chromatographic column filler with the particle size of 40-60 mu m; and in the purification process, the collected components in each step are concentrated by a nanofiltration membrane to obtain ethanol, and the ethanol is recycled and reused.
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