阅读说明：本技术 一种从甜叶菊中提取莱鲍迪苷a、甜菊苷和甜菊多酚的方法及其应用 (Method for extracting rebaudioside A, stevioside and stevia rebaudiana polyphenol from stevia rebaudiana and application of method ) 是由 范斌 张宝堂 谭家忠 廖娜 雷桂芳 于 2021-10-26 设计创作，主要内容包括：本发明公开了一种从甜叶菊中提取莱鲍迪苷A、甜菊苷和甜菊多酚的方法及其应用,提取方法包括,向热水浸提甜叶菊粉末后得到的浸提液中加入饱和石灰水调节pH,静置沉淀后,离心分离；将离心后的上清液进行后处理得到甜菊糖苷粗品,随后加入正丙醇后加热并趁热过滤,分别纯化滤液和滤饼得到甜菊苷和莱鲍迪苷A；将离心后的沉淀加稀盐酸溶解并调pH后离心,取上清液用聚酰胺树脂纯化得到甜菊多酚。本发明通过利用莱鲍迪苷A和甜菊苷在正丙醇里面具有不同溶解性的特点,能高效率分离甜菊糖苷里面的两个主要成分；采用80％以上纯度的莱鲍迪苷A粗品进行结晶能缩短纯化时间,避免直接结晶时莱鲍迪苷A部分溶解于母液而未能使其充分析出,且能得到含量在95％以上的甜菊苷。(The invention discloses a method for extracting rebaudioside A, stevioside and stevia rebaudiana polyphenol from stevia rebaudiana and application thereof, wherein the extraction method comprises the steps of adding saturated limewater into a leaching solution obtained after leaching stevia rebaudiana powder by hot water to adjust the pH value, standing for precipitation, and performing centrifugal separation; carrying out post-treatment on the centrifuged supernatant to obtain a stevioside crude product, adding n-propanol, heating, filtering while hot, and respectively purifying the filtrate and the filter cake to obtain stevioside and rebaudioside A; and adding dilute hydrochloric acid into the centrifuged precipitate to dissolve, adjusting the pH value, centrifuging, taking the supernatant, and purifying with polyamide resin to obtain the stevia polyphenol. The method has the advantages that the characteristics of different solubilities of rebaudioside-A and stevioside in n-propanol are utilized, so that two main components in the stevioside can be separated efficiently; the rebaudioside A crude product with the purity of more than 80% is adopted for crystallization, so that the purification time can be shortened, the rebaudioside A is prevented from being partially dissolved in mother liquor during direct crystallization and being not fully separated out, and stevioside with the content of more than 95% can be obtained.)

1. A method for extracting rebaudioside A, stevioside and stevia polyphenol from stevia rebaudiana is characterized in that,

comprises the steps of (a) preparing a mixture of a plurality of raw materials,

adding saturated limewater into leaching solution obtained after leaching stevia rebaudiana powder with hot water to adjust pH, standing for precipitation, and centrifuging;

carrying out post-treatment on the supernatant obtained after centrifugal separation to obtain a stevioside crude product, then adding n-propanol, heating, filtering while hot, and respectively purifying the filtrate and the filter cake to obtain stevioside and rebaudioside A;

dissolving the precipitate obtained after centrifugal separation with dilute hydrochloric acid, adjusting pH to 4.0-6.5, centrifuging, collecting supernatant, and purifying with polyamide resin to obtain stevia polyphenol.

2. The method of claim 1, wherein the rebaudioside A, stevioside, and stevia polyphenol are extracted from stevia rebaudiana Bertoni,

the adding amount of the saturated lime water is that the pH value of the leaching liquor is adjusted to 8.5-11.0;

and/or the standing time is 0.5-2.0 h.

3. The method of claim 1, wherein the rebaudioside A, stevioside, and stevia polyphenol are extracted from stevia rebaudiana Bertoni,

the n-propanol is added in an amount of 0.5-0.8ml corresponding to 1g of stevia powder;

and/or the heating temperature and the heat preservation time after the n-propanol is added are respectively 50-65 ℃ and 3-5 min.

4. The method of extracting rebaudioside A, stevioside and stevia polyphenol from stevia rebaudiana Bertoni according to claim 1 or 3,

and the post-treatment comprises the steps of putting the supernate on a nonpolar macroporous resin column, washing with deionized water until the effluent liquid is neutral, then washing with an ethanol water solution, collecting the eluate, carrying out reduced pressure concentration, recovering ethanol, and then further concentrating until a pasty stevioside crude product is obtained.

5. The method of claim 4, wherein the rebaudioside A, stevioside, and stevia polyphenol are extracted from stevia rebaudiana Bertoni,

the nonpolar macroporous resin column is one of D101, AB-8 and HP20 macroporous resin columns;

and/or controlling the flow rate of the supernatant liquid on the macroporous resin column to be 0.5-1.5 BV/h;

and/or the concentration of the ethanol aqueous solution is 60-75 wt%, and the dosage and the flushing flow rate of the ethanol aqueous solution are respectively 3-5BV and 1-2 BV/h.

6. The method of extracting rebaudioside A, stevioside, and stevia polyphenol from stevia rebaudiana as claimed in any of claims 1 to 5,

the process of purifying to obtain stevioside comprises washing the filter cake with deionized water at normal temperature, mixing the filtrate with the obtained washing liquid, concentrating and drying;

and/or the process of purifying to obtain rebaudioside A comprises the steps of heating and dissolving a filter cake by using 50-80 wt% of ethanol water solution, crystallizing at low temperature, and sequentially filtering, washing and drying.

7. The method of claim 6, wherein the rebaudioside A, stevioside, and stevia polyphenol are extracted from stevia rebaudiana Bertoni,

the amount of the ethanol aqueous solution for dissolving the filter cake by heating is 0.05-0.5ml corresponding to 1g of stevia powder;

and/or the temperature and the time of the crystallization are respectively 4-10 ℃ and 6-12 h.

8. The method of extracting rebaudioside A, stevioside, and stevia polyphenol from stevia rebaudiana as claimed in any of claims 1 to 7,

the concentration of the dilute hydrochloric acid for dissolving the precipitate is 0.5-1.5 mol/L;

and/or purifying the supernatant by polyamide resin, wherein the process comprises subjecting the centrifuged supernatant to polyamide resin column, eluting with deionized water and ethanol aqueous solution, discarding the effluent and water eluate of the column, collecting the alcohol eluate, concentrating and drying the alcohol eluate;

preferably, the dosage and the elution flow rate of the deionized water are respectively 4-6BV and 1-3BV/h, and/or the concentration of the ethanol aqueous solution is 50-70 wt%, and the dosage and the elution flow rate of the ethanol aqueous solution are respectively 3-5BV and 1-3 BV/h.

9. The method of extracting rebaudioside A, stevioside, and stevia polyphenol from stevia rebaudiana as claimed in any of claims 1 to 8,

the process for extracting stevia rebaudiana powder with hot water to obtain the leaching liquor comprises adding stevia rebaudiana powder into deionized water according to the ratio of 1g to 5-20ml, extracting at 70-90 deg.C for 1-3h, extracting under the same conditions for 2-3 times, and mixing the leaching liquors.

10. Use of the method of any one of claims 1-9 for extracting rebaudioside a, stevioside, and stevia polyphenols from stevia rebaudiana Bertoni.

Technical Field

The invention relates to the technical field of extraction of effective components of plants, and particularly relates to a method for extracting rebaudioside A, stevioside and stevia polyphenol from stevia rebaudiana and application of the method.

Background

With the serious health problems caused by the increased consumption of sucrose, low calorie sweeteners are increasingly appreciated by consumers.

Steviol glycosides are a group of sweet substances rich in the stems and leaves of Stevia rebaudiana (Stevia rebaudiana Bertoni), and their main components are rebaudioside a (ra) and Stevioside (STV), accounting for about 90 wt% of steviol glycosides. The stevioside is safe and non-toxic, has stable physicochemical properties, is a sweetener with high sweetness and low calorific value, can reduce the incidence rate of diabetes and obesity while meeting the requirement of sweetness, and plays roles in protecting the kidney and promoting the oral health. Besides the obvious advantages of being used as a natural sweetener, the STV and RA also have the effects of reducing blood sugar, promoting the secretion of insulin and glucagon, reducing blood pressure, enhancing immunity and the like, and are widely applied to the industries of wine brewing, beverages, chemical industry, medicines and the like.

Because the STV and the RA have the same aglycone, the oxygen-glycoside bond of the RA at the C13 position has one more glucosyl group than the STV, and the structure and the polarity of the RA are very close, which brings certain difficulty to the separation work.

In the prior art, the separation and purification method generally adopted by stevioside mainly comprises the following steps: recrystallization, column chromatography, preparative chromatography, molecular imprinting and high-speed counter-current chromatography.

The Chinese patent application with the publication number of CN111777652A discloses a method for extracting stevioside and rebaudioside A from stevia rebaudiana Bertoni, which uses a natural eutectic solvent consisting of 1, 2-propylene glycol, glycerol and water to extract the stevia rebaudiana Bertoni, and compared with water extraction and alcohol extraction, the method can greatly improve the extraction rate of the stevioside and the rebaudioside A; however, the patent application does not disclose how to further separate the extract.

Chinese patent application with publication number CN103483402A discloses a method for purifying and preparing stevioside and rebaudioside-A, which comprises the steps of sequentially carrying out alcohol extraction, macroporous resin column chromatography, silica gel column chromatography, recrystallization and the like on stevia rebaudiana to obtain the stevioside and the rebaudioside-A with the content of more than 98 percent; however, the method claimed in the patent application uses a large amount of toxic and harmful solvents, and silica gel column chromatography is only suitable for small-scale preparation in a laboratory.

The Chinese patent application with the publication number of CN104098734A discloses a preparation method of a phenylboronic acid group modified macroporous resin and application of the phenylboronic acid group modified macroporous resin in separation of rebaudioside A and stevioside, the resin can selectively adsorb rebaudioside A in stevioside, and the purity and recovery rate of the rebaudioside A and stevioside are high; however, the method of the invention is complex in process and has no mass production to meet the requirement of industrial production.

In summary, most of the methods disclosed in the prior art have the disadvantages of large solvent consumption, long time consumption, complex process, expensive equipment, too few samples to be processed, unsuitability for industrial production, and the like. Therefore, research and development of a method for separating rebaudioside a and stevioside, main components of stevioside, are urgently needed.

In addition, in the stevia extract, steviol glycosides are the main products, and stevia polyphenols are the by-products in the steviol glycoside extraction process. When stevioside is extracted, iron salt and calcium salt are usually used for complexing and impurity removal, so that stevia polyphenol is mixed into waste residues, and even worse, more organic and inorganic mixed solid waste can be generated.

Stevia rebaudiana polyphenol comprises chlorogenic acid, flavonoid, flavonol, flavanol, isoflavone, procyanidine and the like, and at least 30 stevia rebaudiana polyphenol with definite structures are reported at present. Stevia rebaudiana polyphenol is a water-soluble polyphenol compound, has good and stable free radical scavenging capacity, also has the performances of resisting inflammation, promoting urination, resisting hypertension, reducing blood sugar, resisting tumor and oxidation, and also has the effects of reducing blood pressure and blood fat. If high-purity stevia rebaudiana Bertoni polyphenol can be obtained while high-purity main products rebaudioside A and stevioside are obtained, the comprehensive utilization value of stevia rebaudiana is greatly improved.

In view of this, the invention is particularly proposed.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for extracting rebaudioside A, stevioside and stevia rebaudiana polyphenol from stevia rebaudiana and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for extracting rebaudioside A, stevioside and stevia polyphenol from stevia rebaudiana Bertoni comprises,

adding saturated limewater into leaching solution obtained after leaching stevia rebaudiana powder with hot water to adjust pH, standing for precipitation, and centrifuging;

carrying out post-treatment on the supernatant obtained after centrifugal separation to obtain a stevioside crude product, then adding n-propanol, heating, filtering while hot, and respectively purifying the filtrate and the filter cake to obtain stevioside and rebaudioside A;

dissolving the precipitate obtained after centrifugal separation with dilute hydrochloric acid, adjusting pH to 4.0-6.5, centrifuging, collecting supernatant, and purifying with polyamide resin to obtain stevia polyphenol.

In the technical scheme, the adding amount of the saturated lime water is that the pH of the leaching liquor is adjusted to 8.5-11.0.

In the technical scheme, after the saturated lime water is added and the pH is adjusted, the standing time is 0.5-2.0 h.

In the above technical scheme, the addition amount of n-propanol is 0.5-0.8ml corresponding to 1g of stevia powder.

In the technical scheme, the heating temperature and the heat preservation time after the n-propanol is added are respectively 50-65 ℃ and 3-5 min.

Further, in the above technical scheme, the post-treatment comprises that the supernatant is put on a nonpolar macroporous resin column, washed by deionized water until the effluent liquid is neutral, then washed by ethanol water solution and the eluate is collected, and further concentrated after the ethanol is recovered by reduced pressure concentration until a paste stevioside crude product is obtained.

In a specific embodiment of the invention, the non-polar macroporous resin column is one of a D101, AB-8 and HP20 macroporous resin column.

In the specific embodiment of the invention, the flow rate of the supernatant liquid on the macroporous resin column is controlled to be 0.5-1.5 BV/h.

In a specific embodiment of the present invention, the concentration of the ethanol aqueous solution is 60 to 75 wt%, and the amount of the ethanol aqueous solution and the rinsing flow rate are 3 to 5BV and 1 to 2BV/h, respectively.

Further, in the above technical scheme, the purifying process to obtain stevioside comprises washing the filter cake with deionized water at normal temperature, combining the filtrate with the obtained washing solution, concentrating, and drying.

Still further, in the above technical scheme, the process of purifying to obtain rebaudioside a comprises dissolving the filter cake in 50-80 wt% aqueous ethanol solution under heating, crystallizing at low temperature, and sequentially filtering, washing with water, and drying.

In a specific embodiment of the present invention, the amount of the ethanol aqueous solution for heat dissolving the filter cake is 0.05-0.5ml corresponding to 1g of stevia powder.

In a particular embodiment of the invention, the crystallization temperature and time are 4-10 ℃ and 6-12h, respectively.

Still further, in the above technical solution, the concentration of the dilute hydrochloric acid used for dissolving the precipitate is 0.5-1.5 mol/L.

Still further, in the above technical solution, the process of purifying the supernatant with polyamide resin comprises subjecting the centrifugally separated supernatant to a polyamide resin column, eluting with deionized water and an ethanol aqueous solution in sequence, discarding the effluent and the water wash of the column, collecting the alcohol wash, and concentrating and drying the alcohol wash.

In a preferred embodiment of the present invention, the amount of the deionized water and the elution flow rate are 4 to 6BV and 1 to 3BV/h, respectively.

In another preferred embodiment of the present invention, the concentration of the aqueous ethanol solution is 50 to 70 wt%, and the amount of the aqueous ethanol solution and the elution flow rate are 3 to 5BV and 1 to 3BV/h, respectively.

Still further, in the above technical solution, the process of leaching stevia rebaudiana powder with hot water to obtain the leaching solution includes that the stevia rebaudiana powder is mixed with a water-oil mixture in a ratio of 1 g: adding 5-20ml of the extract into deionized water, extracting at 70-90 deg.C for 1-3h, extracting for 2-3 times under the same conditions, and mixing the extractive solutions.

The invention also provides application of the method in extracting rebaudioside A, stevioside and stevia polyphenol in stevia rebaudiana.

Compared with the prior art, the invention has the following advantages:

(1) according to the method, the characteristics that rebaudioside A and stevioside have different solubilities in n-propanol are utilized, two main components in the stevioside are separated, and the method has high efficiency;

(2) compared with the method that stevioside is directly crystallized by using a solvent, the method provided by the invention adopts the rebaudioside A crude product with the purity of more than 80% for crystallization, so that the time required for purification can be greatly shortened, the yield of target components in unit time is improved, the rebaudioside A can be prevented from being partially dissolved in mother liquor and being not fully separated out during direct crystallization, and the stevioside with the content of more than 95% can be obtained;

(3) according to the method provided by the invention, the saturated limewater is added into the stevia leaching liquor for precipitation, so that part of impurities in the leaching liquor can be effectively removed, the rebaudioside A and stevioside content in the supernatant after centrifugal separation of the leaching liquor is improved, and the acidic substances are not required to be removed by alkali after water washing in macroporous resin chromatography, so that the rebaudioside A and stevioside content in the obtained stevioside is higher, and the subsequent purification is facilitated;

(4) the method provided by the invention has the advantages of coherent and simple process, strong operability, low production cost, high recovery rate and purity of the obtained product, no use of toxic and harmful chemical solvents, safety, environmental protection and suitability for industrial production, can achieve a comprehensive utilization effect on stevia rebaudiana, obviously shortens the time required by the process compared with the extraction of one kind of components from a batch of raw materials, and reduces the loss of plant raw materials, thereby greatly reducing the production cost and having wide practical application prospect.

Drawings

FIG. 1 is an HPLC chromatogram of stevioside extracted in example 1 of the present invention;

FIG. 2 is an HPLC chromatogram of rebaudioside A extracted in example 1 of the present invention;

FIG. 3 is an HPLC chromatogram of stevioside extracted in example 2 of the present invention;

FIG. 4 is an HPLC chromatogram of rebaudioside A extracted in example 2 of the present invention;

FIG. 5 is an HPLC chromatogram of stevioside extracted in example 3 of the present invention;

FIG. 6 is an HPLC chromatogram of rebaudioside A extracted in example 3 of this invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.

It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the examples, the means used are conventional in the art unless otherwise specified.

The terms "comprises," "comprising," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion.

For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The reagents and equipment used in the examples and comparative examples of the present invention were all commercially available products.

The contents of rebaudioside a, stevioside and stevia polyphenol in the stevia leaf raw materials used in the examples and comparative examples of the present invention were 6.24 wt%, 4.56 wt% and 11.06 wt%, respectively.

Example 1

The embodiment of the invention provides a method for extracting rebaudioside A, stevioside and stevia rebaudiana polyphenol from stevia rebaudiana, which specifically comprises the following steps:

s1, water extraction

Weighing 100g of dried and pulverized stevia rebaudiana leaf raw material, performing reflux extraction with 1000ml of pure water at the temperature of 80 ℃ for 90min, separating to obtain filter residue and filtrate, performing reflux extraction on the filter residue for 60min twice respectively with 700ml of pure water under the same condition, combining 3 times of obtained water extract, and concentrating to 300 ml;

s2, precipitation

Adding saturated limewater into the concentrated solution obtained in S1 to adjust pH to 10, standing for 1h, and centrifuging to obtain supernatant and precipitate;

s3, stevioside purification

Taking the supernatant obtained in S2 to pass through a D101 macroporous resin column, controlling the flow rate of sample application to be 1BV/h, after sample application is completed, firstly washing the effluent to be neutral by water and removing water-soluble impurities, wherein the washing volume is 5BV and the flow rate is 2BV/h, then washing the column by 68 wt% of ethanol water solution, the elution volume is 3.5BV and the flow rate is 2BV/h, collecting eluent, decompressing, concentrating and recovering ethanol, then concentrating to be dry to obtain stevioside, adding 50ml of n-propanol into stevioside, heating to 60 ℃, keeping the temperature, stirring to fully dissolve out stevioside, filtering while hot to obtain filtrate and filter cake, washing the filter cake by 50ml of cold deionized water, combining the filtrate and the water washing solution, concentrating and drying to obtain stevioside, wherein the content is 95.63% by HPLC (the result is shown in figure 1), and the recovery rate is 93.58%; heating and dissolving the filter cake with 20ml of 70 wt% ethanol water solution, standing and crystallizing at 5 ℃ for 10h, filtering, washing with water and drying to obtain a rebaudioside A refined product, wherein the rebaudioside A content is 96.17% and the recovery rate is 95.17% by HPLC (HPLC determination result shown in figure 2);

s4, purifying stevia rebaudiana polyphenol

Dissolving the precipitate obtained in S2 with 0.8mol/L dilute hydrochloric acid solution, adjusting pH to 4, centrifuging, discarding the precipitate, purifying the supernatant polyamide resin with a sample loading flow rate of 1BV/h, washing with water for 4BV, eluting with 60 wt% ethanol water solution for 3.5BV, discarding the effluent liquid on the column and the water washing liquid, collecting the ethanol washing liquid, concentrating and drying the ethanol washing liquid to obtain stevia rebaudiana polyphenol with polyphenol content of 95.37% and recovery rate of 93.94% by UV detection.

Example 2

The embodiment of the invention provides a method for extracting rebaudioside A, stevioside and stevia rebaudiana polyphenol from stevia rebaudiana, which specifically comprises the following steps:

s1, water extraction

Weighing 1000g of dried and pulverized folium Stevlae Rebaudianae raw material, reflux-extracting with 12000ml of pure water at 78 deg.C for 100min, separating to obtain residue and filtrate, reflux-extracting the residue with 9000ml of pure water under the same conditions for 90min twice, mixing 3 times to obtain water extractive solutions, and concentrating to 3500 ml;

s2, precipitation

Adding saturated limewater into the concentrated solution obtained in S1 to adjust pH to 9.5, standing for 80min, and centrifuging to obtain supernatant and precipitate;

s3, stevioside purification

Taking the supernatant obtained in S2 to pass through an AB-8 macroporous resin column, controlling the flow rate of sample application to be 1BV/h, after sample application is finished, firstly washing the effluent to be neutral by water and removing water-soluble impurities, wherein the washing volume is 5BV and the flow rate is 2.5BV/h, then washing the column by 65 wt% of ethanol water solution, the elution volume is 4BV and the flow rate is 2BV/h, collecting eluent, decompressing, concentrating and recovering ethanol, then concentrating to be dry to obtain stevioside, adding 600ml of n-propanol into stevioside, heating to 60 ℃, keeping the temperature, stirring to fully dissolve the stevioside, filtering while hot to obtain filtrate and filter cake, washing the filter cake by 200ml of cold deionized water, combining the filtrate and the water washing solution, concentrating and drying to obtain the stevioside, wherein the content is 95.02% by HPLC (the result is shown in figure 3), and the recovery rate is 94.97%; heating and dissolving the filter cake with 250ml of 72 wt% ethanol water solution, standing and crystallizing at 5 ℃ for 10h, filtering, washing with water and drying to obtain a rebaudioside A refined product, wherein the rebaudioside A content is 96.84% and the recovery rate is 96.27% by HPLC (shown in figure 4);

s4, purifying stevia rebaudiana polyphenol

Dissolving the precipitate obtained in S2 with 1mol/L dilute hydrochloric acid solution, adjusting pH to 4.5, centrifuging, discarding the precipitate, purifying the supernatant polyamide resin with a sample loading flow rate of 0.8BV/h, washing with water for 5BV, eluting with 60 wt% ethanol water solution for 3.5BV, discarding the effluent liquid on the column and the water washing solution, collecting the ethanol washing solution, concentrating and drying the ethanol washing solution to obtain stevia rebaudiana polyphenol with polyphenol content of 96.62% and recovery rate of 93.75% through UV detection.

Example 3

The embodiment of the invention provides a method for extracting rebaudioside A, stevioside and stevia rebaudiana polyphenol from stevia rebaudiana, which specifically comprises the following steps:

s1, water extraction

Weighing 100kg of dried and pulverized folium Stevlae Rebaudianae raw material, reflux-extracting with 1000L of pure water at 80 deg.C for 90min, separating to obtain filter residue and filtrate, reflux-extracting the filter residue with 800L of pure water under the same conditions for 60min twice, mixing 3 times to obtain water extractive solutions, and concentrating to 250L;

s2, precipitation

Adding saturated limewater into the concentrated solution obtained in S1 to adjust pH to 10.0, standing for 1h, and centrifuging to obtain supernatant and precipitate;

s3, stevioside purification

Taking the supernatant obtained in S2 to pass through an HP-20 macroporous resin column, controlling the sample flow rate to be 1BV/h, after the sample is finished, firstly washing the effluent to be neutral by water and removing water-soluble impurities, wherein the washing volume is 5BV and the flow rate is 2BV/h, then washing the column by 62 wt% of ethanol water solution, the elution volume is 3BV and the flow rate is 2BV/h, collecting eluent, decompressing, concentrating and recovering ethanol, concentrating to be dry to obtain stevioside, adding 50L of n-propanol into stevioside, heating to 60 ℃, keeping the temperature, stirring to fully dissolve out stevioside, filtering while hot to obtain filtrate and filter cake, washing the filter cake by 50L of cold deionized water, combining the filtrate and the water washing solution, concentrating and drying to obtain the stevioside, and determining by HPLC (the result is shown in figure 5), the content is 96.01%, and the recovery rate is 92.91%; heating and dissolving the filter cake with 180L of 74 wt% ethanol water solution, standing at 5 ℃ for crystallization for 12h, filtering, washing with water and drying to obtain rebaudioside A refined product, wherein the rebaudioside A content is 95.97% and the recovery rate is 93.86% by HPLC (shown in figure 6);

s4, purifying stevia rebaudiana polyphenol

Dissolving the precipitate obtained in S2 with 0.8mol/L dilute hydrochloric acid solution, adjusting pH to 4.5, centrifuging, discarding the precipitate, purifying the supernatant with polyamide resin at a sample loading flow rate of 0.8BV/h, washing with water for 5BV, eluting with 60 wt% ethanol water solution for 4BV, discarding the effluent of the column and the water washing solution, collecting the ethanol washing solution, concentrating and drying the ethanol washing solution to obtain stevia rebaudiana polyphenol with polyphenol content of 96.51% and recovery rate of 94.73% by UV detection.

Comparative example 1

This comparative example provides a method for extracting rebaudioside a, stevioside and stevia polyphenol from stevia rebaudiana, which has the same specific steps as example 1, except that in step S3, the step of adding n-propanol into stevioside and heating for dissolution is omitted, and the stevioside obtained by macroporous resin column chromatography is directly subjected to twice crystallization, crystals and mother liquor are separated, the crystals are directly dried, and the mother liquor is dried after concentration.

Through detection, the rebaudioside A content obtained in the comparative example 1 after extraction and purification is 92.91%, and the recovery rate is 74.32%; the content of stevioside is 60.14 percent, and the recovery rate is 89.79 percent; in addition, the time required for the whole process was extended by 48 hours as compared with example 1.

Comparative example 2

This comparative example provides a method for extracting rebaudioside a, stevioside and stevia rebaudiana polyphenol from stevia rebaudiana, the specific steps are the same as those in example 1, and the difference is that in step S2, the steps of adding saturated lime water to the concentrated solution, adjusting pH and performing centrifugal separation are omitted, the concentrated solution is directly subjected to macroporous resin column chromatography, the effluent liquid on the column is collected, the concentrated and dried solution is stevia rebaudiana polyphenol, the eluate of the 60 wt% ethanol water solution is eluted after water washing and impurity removal, the eluate of the 60 wt% ethanol water solution is stevioside, the stevioside is directly subjected to twice crystallization, crystals and mother liquor are separated, the crystals are directly dried, and the mother liquor is dried after concentration.

Through detection, the rebaudioside A content obtained by extraction and purification in the comparative example 2 is 85.78%, and the recovery rate is 69.39%; the content of stevioside is 54.89%, and the recovery rate is 82.31%; the content of stevia polyphenol is 35.72 percent, and the recovery rate is 40.19 percent; in addition, the time required for the entire process was extended by 60 hours as compared with example 1.

By comparing the results of examples 1 to 3 and comparative examples 1 to 2, it can be seen that:

(1) according to the embodiment of the invention, the characteristics that rebaudioside-A and stevioside have different solubilities in n-propanol are utilized, so that two main components in the stevioside are separated, and the efficiency is higher;

(2) compared with the method that stevioside is directly crystallized by using a solvent, the method provided by the embodiment of the invention adopts the rebaudioside A crude product with the purity of more than 80% for crystallization, so that the time required for purification can be greatly shortened, the yield of target components in unit time is improved, the rebaudioside A can be prevented from being partially dissolved in mother liquor and being not fully separated out during direct crystallization, and the stevioside with the content of more than 95% can be obtained;

(3) according to the method provided by the embodiment of the invention, the saturated limewater is added into the stevia leaching liquor for precipitation, so that part of impurities in the leaching liquor can be effectively removed, the rebaudioside A and stevioside content in the supernatant after centrifugal separation of the leaching liquor is improved, and the acidic substances are not required to be removed by alkali after water washing during macroporous resin chromatography, so that the rebaudioside A and stevioside content in the obtained stevioside is higher, and the subsequent purification is facilitated;

(4) the method provided by the embodiment of the invention has the advantages of coherent and simple process, strong operability and low production cost, the prepared product has high recovery rate and purity, no toxic and harmful chemical solvents are used in the whole process, the method is safe and environment-friendly, is suitable for industrial production, can achieve a comprehensive utilization effect on stevia rebaudiana, obviously shortens the time required by the process compared with the extraction of one kind of components from a batch of raw materials, and reduces the loss of plant raw materials, thereby greatly reducing the production cost and having wide practical application prospect.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention.

It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.