CN115710295A - Method for extracting rebaudioside-A from reaction liquid obtained by catalyzing stevioside through biological enzyme method - Google Patents
Method for extracting rebaudioside-A from reaction liquid obtained by catalyzing stevioside through biological enzyme method Download PDFInfo
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Abstract
The invention relates to a method for extracting rebaudioside A from reaction liquid of stevioside catalyzed by a biological enzyme method, which is characterized by comprising the following steps: (1) Carrying out heat treatment and solid-liquid separation on reaction liquid prepared by a biological enzyme method, and refining the solution by adopting macroporous adsorption resin; (2) Washing the separation column with distilled water or deionized water and 20-70% ethanol after adsorption, collecting ethanol desorption solution, and concentrating the ethanol desorption solution by evaporation to obtain concentrated solution; (3) according to the concentrated solution: mixing the organic reagent and the mixture at a volume ratio of 1-10, adding rebaudioside-A with a purity of 95% -99% of 0.1-1 per mill of the total mass as seed crystal, and crystallizing at 20-50 ℃ for 10-60h at 50-200 rpm/min; solid-liquid separation, solid washing and drying. The invention has the advantages that: the process is simple, the crystallization temperature is low, the stirring speed is low, and the energy consumption is low; the rebaudioside A content can be improved from 10% -30% to 60-95.5%, and after repeated recrystallization, the rebaudioside A content can reach 98-99%.
Description
Technical Field
The invention belongs to the field of sweetener production and preparation, relates to stevioside preparation, and particularly relates to a method for extracting rebaudioside A from reaction liquid of stevioside catalyzed by a biological enzyme method.
Background
The natural sweeteners steviol glycosides are a class of extracts with a common aglycone (steviol) and differ in the number and type of carbohydrate residues at the C13 and C19 positions, the major glycosides found in stevia leaves being stevioside ST (2-10%) and rebaudioside C (1-2%). Other glycosides, such as rebaudiosides B, E and F, steviolbioside and rubusoside, are found at much lower levels (approximately 0-1%).
Stevioside ST, the main ingredient of the natural sweetener stevioside described above, has the least astringent, least bitter and least lingering aftertaste, and therefore has the most desirable organoleptic attributes, however, even in a highly purified state, stevioside still has undesirable taste attributes such as bitter, sweet aftertaste, licorice, and the like. These undesirable taste attributes in stevia sweeteners have been one of the major obstacles in commercialization, affecting the taste quality of stevia, limiting its broader use.
Rebaudioside A (RA for short) is one of the components of stevioside, has the advantages of low calorie and health care function of stevioside, and has good taste and high sweetness which is 200-300 times of that of cane sugar.
At present, the research on the purification method of various single components in stevioside is focused internationally. Because the taste of a single component is relatively single, the single component can be directly added into food or medicines, and a series of researches on properties and the like can be carried out by taking a single-component pure product as a research object. And RA is one of single components in the stevioside mixture. Because the content of RA in stevioside is very low, the current mainstream method is to directly extract RA by using stevia rebaudiana, and the RA obtained by the method has insufficient purity and still contains a lot of STV to influence the sweet taste. If the reaction liquid for catalyzing stevioside by using the biological enzyme method is used as a raw material, the purification of a single component RA is a necessary step in the RA production technology, and a reliable method can be provided for realizing the production of a high-purity RA product.
Disclosure of Invention
The invention aims to solve the problem that the purity of the purified rebaudioside-A is not high (10-30%) in the prior art, and provides a method for extracting rebaudioside-A from a reaction liquid obtained by catalyzing stevioside with a biological enzyme method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for extracting rebaudioside A from reaction liquid of stevioside catalyzed by a biological enzyme method is characterized by comprising the following steps:
(1) Carrying out heat treatment (treatment at 100-150 ℃ for 30-60 min) on reaction liquid (containing 10-30% rebaudioside A) prepared by a biological enzyme method, then carrying out solid-liquid separation (centrifugation, filtration or flocculation), and removing precipitates to obtain solution; refining the solution with macroporous adsorbent resin to remove non-diterpene compounds;
(2) Washing the separation column with distilled water or deionized water after adsorption, washing the separation column with 20-70% ethanol by volume fraction, collecting ethanol desorption solution (only containing diterpene compounds and stevioside), evaporating and concentrating the ethanol desorption solution to obtain 60-70% concentrated solution;
(3) According to the concentrated solution: mixing the concentrated solution and the organic reagent according to the volume ratio of the organic reagent of 1 to 3-10, adding high-purity rebaudioside A (the purity is 95% -99%) with the mass of 0.1-1 per mill of the mixture as seed crystals, controlling the stirring speed to be 50-200rpm/min, and crystallizing at 20-50 ℃ for 10-60 hours; and then carrying out solid-liquid separation, washing the solid (removing mother liquor impurities), and drying (drying at 35-55 ℃ for 4-12 h) to obtain the rebaudioside A with the purity of 60-95.5%.
Further, the rebaudioside A with the purity of 60-95.5% is continuously recrystallized for 1-3 times, so that the rebaudioside A with the purity of 98-99% can be obtained.
Further, the reaction liquid prepared by the biological enzyme method in the step (1) is obtained by converting components such as stevioside ST in a stevioside raw material into rebaudioside A by using a biological enzyme method, wherein a commercialized stevia extract containing stevioside ST with at least 90% of the total glycoside content is used as a starting material.
Further, the macroporous adsorption resin is a resin product with high selectivity only for adsorbing diterpene compounds (stevioside); such as: d series, LX, XDA series, HB-8, and the like.
Further, the evaporative concentration is vacuum evaporative concentration.
Further, the organic solvent is a methanol solution with the volume fraction of 95-99.9% and/or an ethanol solution with the volume fraction of 95-99.9%.
According to the invention, the rebaudioside A with purity of more than 60% is obtained by carrying out heat treatment, macroporous resin adsorption, analysis, concentration, crystallization, separation, washing and drying on reaction liquid prepared by a biological enzyme method and containing 10% -30% of rebaudioside A; multiple recrystallizations can be performed on this basis in order to obtain rebaudioside a in a higher purity.
Compared with the prior art, the invention has the following advantages:
1. the invention has simple process, lower crystallization temperature, low stirring speed and low energy consumption;
2. the solvent used in the invention can be concentrated, recovered and recycled, so that lower environmental pollution and higher economic benefit are realized;
3. by the treatment of the invention, the rebaudioside A content can be improved from 10-30% to 60-95.5%, and after repeated recrystallization, the rebaudioside A content can reach 98-99%.
Detailed description of the preferred embodiment
A method for extracting rebaudioside A from reaction liquid of stevioside catalyzed by a biological enzyme method comprises the following specific implementation steps:
example 1
(1) Carrying out heat treatment (treatment at 100 ℃ for 30 min) on 1L of reaction liquid (containing rebaudioside-A12%) prepared by a biological enzyme method, and then carrying out centrifugation to remove precipitates to obtain a solution; refining the solution with macroporous adsorbent resin (D series) to remove non-diterpene compounds;
(2) After adsorption, washing the separation column with deionized water, then washing the separation column with ethanol with volume fraction of 40%, collecting ethanol desorption solution (only containing diterpene compounds and stevioside), and carrying out vacuum evaporation concentration on the ethanol desorption solution (vacuum degree is-0.075 MPa) to obtain 200ml of concentrated solution;
(3) Adding 500ml of 99.9 volume percent methanol aqueous solution into the concentrated solution, uniformly mixing, placing the mixture into a three-neck flask, controlling the water bath temperature to be 35 ℃, stirring at the speed of 100rpm/min, adding 0.7g of rebaudioside-A with the purity of 99 percent as seed crystals, and continuously crystallizing for 24 hours; and (3) carrying out suction filtration on the crystallization mixed solution by using a Buchner funnel, washing the obtained crystal by using ethanol water solution with the volume fraction of 99%, and placing the crystal in a vacuum drying oven at 70 ℃ for drying overnight to obtain 41.6g of rebaudioside-A.
Table 1 shows the results of the liquid phase assay of the rebaudioside a product of example 1:
| name of sample | RA | RE | RF | ST |
| Ethanol desorption concentrate (%) | 12.2 | 1.7 | Not detected out | 86.1 |
| First crystalline product (%) | 94.5 | Undetected output | Not detected out | 5.5 |
The purity of RA in the product can be improved to 94.5% by adopting the method of example 1.
Example 2
(1) Carrying out heat treatment (treatment at 130 ℃ for 35 min) on 1L of reaction liquid (containing rebaudioside-A12%) prepared by a biological enzyme method, and then carrying out centrifugation to remove precipitates to obtain a solution; refining the solution with macroporous adsorbent resin (LX, XDA series) to remove non-diterpene compounds;
(2) After adsorption, washing the separation column with deionized water, then washing the separation column with ethanol with volume fraction of 40%, collecting ethanol desorption solution (only containing diterpene compounds and stevioside), and carrying out vacuum evaporation concentration on the ethanol desorption solution (vacuum degree is-0.075 MPa) to obtain 200ml of concentrated solution;
(3) Adding 500ml of 99.9 volume percent methanol aqueous solution into the concentrated solution, uniformly mixing, placing the mixture into a three-neck flask, controlling the water bath temperature to be 35 ℃, stirring at the speed of 100rpm/min, adding 0.7g of 99 percent rebaudioside-A serving as a seed crystal, and continuously crystallizing for 24 hours; carrying out suction filtration on the crystallized mixed solution by using a Buchner funnel, and washing the obtained crystal by using an ethanol water solution with the volume fraction of 99%;
(4) Performing recrystallization, adding 400ml of deionized water, heating to 100 ℃, concentrating to 200ml, adding 500ml of 99.9 volume percent methanol aqueous solution, uniformly mixing, placing in a three-neck flask, controlling the temperature of water bath to be 35 ℃, stirring at 100rpm/min, adding 0.7g of rebaudioside-A with the purity of 99 percent as seed crystals, and continuously crystallizing for 24 hours; and placing the mixture in a vacuum drying oven at 70 ℃ for drying overnight to obtain 35.5g of rebaudioside A.
Table 2 is a liquid phase assay of the rebaudioside a product of example 2:
| name of sample | RA | RE | RF | ST |
| Ethanol desorption concentrate (%) | 12.2 | 1.7 | Undetected | 86.1 |
| Second crystalline product (%) | 98.5 | Undetected output | Undetected | 1.5 |
The RA content of the product can be increased to 98.5% by adopting the method of example 2.
Example 3
(1) Carrying out heat treatment (treatment at 150 ℃ for 30 min) on 1L of reaction liquid (containing rebaudioside-A27%) prepared by a biological enzyme method, and then carrying out centrifugation to remove precipitates to obtain a solution; refining the solution with macroporous adsorbent resin (HB-8) to remove non-diterpene compounds;
(2) Washing the separation column with distilled water after adsorption, washing the separation column with 60% ethanol by volume fraction, collecting ethanol desorption solution (only containing diterpene compounds and stevioside), and vacuum evaporating and concentrating the ethanol desorption solution (vacuum degree of-0.08 MPa) to obtain 200ml concentrated solution;
(3) Adding 500ml of 99.9 volume percent methanol aqueous solution into the concentrated solution, uniformly mixing, placing the mixture into a three-neck flask, controlling the water bath temperature to be 35 ℃, stirring at the speed of 100rpm/min, adding 0.7g of 99 percent rebaudioside-A serving as a seed crystal, and continuously crystallizing for 24 hours; and (3) carrying out suction filtration on the crystallization mixed solution by using a Buchner funnel, washing the obtained crystal by using an ethanol water solution with the volume fraction of 99%, and placing the crystal in a vacuum drying oven at 70 ℃ for overnight drying to obtain 62.4g of rebaudioside-A.
Table 3 is a liquid phase assay of the rebaudioside a product of example 3:
| sample name | RA | RE | RF | ST |
| Ethanol desorption concentrate (%) | 16.2 | 1.1 | Not detected out | 82.7 |
| First crystalline product (%) | 95.4 | Undetected output | Not detected out | 4.6 |
The purity of RA in the product can be improved to 95.4% by adopting the method of example 3.
Example 4
(1) Carrying out heat treatment (treatment at 100 ℃ for 30 min) on 2L of reaction liquid (containing rebaudioside-A12%) prepared by a biological enzyme method, and then carrying out centrifugation to remove precipitates to obtain a solution; refining the solution with macroporous adsorbent resin (HB-8) to remove non-diterpene compounds;
(2) After adsorption, washing the separation column by using deionized water, then washing the separation column by using ethanol with the volume fraction of 40%, collecting ethanol desorption solution (only containing diterpene compounds and stevioside), and carrying out vacuum evaporation concentration on the ethanol desorption solution (the vacuum degree is-0.088 MPa) to obtain 200ml of concentrated solution;
(3) Adding 500ml of 99.9 volume percent methanol aqueous solution into the concentrated solution, uniformly mixing, placing the mixture into a three-neck flask, controlling the water bath temperature to be 35 ℃, stirring at the speed of 100rpm/min, adding 0.7g of rebaudioside-A with the purity of 99 percent as seed crystals, and continuously crystallizing for 24 hours; and (3) carrying out suction filtration on the crystallization mixed solution by using a Buchner funnel, washing the obtained crystal by using ethanol water solution with the volume fraction of 99%, and placing the crystal in a vacuum drying oven at 70 ℃ for drying overnight to obtain 58.4g of rebaudioside-A.
Table 4 is a liquid phase assay of the rebaudioside a product of example 4:
| sample name | RA | RE | RF | ST |
| Ethanol desorption concentrate (%) | 16.2 | 1.2 | Not detected out | 82.6 |
| First crystalline product (%) | 94.7 | Undetected output | Not detected out | 5.3 |
The RA content of the product can be increased to 94.7 percent by adopting the method of example 4.
Example 5
(1) Carrying out heat treatment (150 ℃ for 40 min) on 1L of reaction liquid (containing rebaudioside-A12%) prepared by a biological enzyme method, and then carrying out centrifugation to remove precipitates to obtain a solution; refining the solution with macroporous adsorbent resin (D series) to remove non-diterpene compounds;
(2) Washing the separation column with distilled water after adsorption, washing the separation column with 65% ethanol by volume fraction, collecting ethanol desorption solution (only containing diterpene compounds and stevioside), and vacuum evaporating and concentrating the ethanol desorption solution (vacuum degree of-0.088 MPa) to obtain 200ml concentrated solution;
(3) Adding 500ml of 99.9 volume percent methanol aqueous solution into the concentrated solution, uniformly mixing, placing the mixture into a three-neck flask, controlling the water bath temperature to be 35 ℃, stirring at the speed of 100rpm/min, adding 0.7g of rebaudioside-A with the purity of 99 percent as seed crystals, and continuously crystallizing for 48 hours; and (3) carrying out suction filtration on the crystallization mixed solution by using a Buchner funnel, washing the obtained crystal by using ethanol water solution with the volume fraction of 99%, and placing the crystal in a vacuum drying oven at 70 ℃ for drying overnight to obtain 41.6g of rebaudioside-A.
Table 5 is a liquid phase assay of the rebaudioside a product of example 5:
| sample name | RA | RE | RF | ST |
| Ethanol desorption concentrate (%) | 12.4 | 1.8 | Not detected out | 85.8 |
| First crystalline product (%) | 97.5 | Undetected output | Not detected out | 2.5 |
The RA content of the product can be increased to 95.5 percent by adopting the method of example 5.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; those skilled in the art can make many possible variations and modifications to the disclosed solution, or to modify equivalent embodiments, without departing from the scope of the solution, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Claims (7)
1. A method for extracting rebaudioside A from reaction liquid obtained by catalyzing stevioside through biological enzyme method is characterized by comprising the following steps:
(1) Carrying out heat treatment on reaction liquid prepared by a biological enzyme method, then carrying out solid-liquid separation, and removing precipitates to obtain a solution; refining the solution by using macroporous adsorption resin to remove non-diterpene compounds;
(2) After adsorption, washing the separation column with distilled water or deionized water, then washing the separation column with 20-70% by volume of ethanol, collecting ethanol desorption solution, carrying out evaporation concentration on the ethanol desorption solution, and concentrating to obtain a concentrated solution with the mass concentration of 60-70%;
(3) According to the concentrated solution: mixing the concentrated solution and the organic reagent according to a volume ratio of the organic reagent of 1 to 3-10, adding rebaudioside A with purity of 95-99 percent of 0.1-1 per mill of the mass of the mixture as seed crystals, controlling the stirring speed to be 50-200rpm/min, and crystallizing at 20-50 ℃ for 10-60 hours; and then carrying out solid-liquid separation, washing the solid, and drying to obtain the rebaudioside A with the purity of 60-95.5%.
2. The method for extracting rebaudioside A from reaction liquid obtained by catalyzing stevioside through a biological enzyme method, according to claim 1, is characterized in that: the reaction liquid prepared by the biological enzyme method is obtained by using a commercialized stevia extract containing at least 90 percent of total glycoside content of stevioside ST as a starting material and converting components such as stevioside ST in a stevioside raw material into rebaudioside A by using the biological enzyme method.
3. The method for extracting rebaudioside A from reaction liquid obtained by catalyzing stevioside through a biological enzyme method, according to claim 1, is characterized in that: the reaction liquid prepared by the biological enzyme method contains 10 to 30 percent of rebaudioside-A.
4. The method for extracting rebaudioside A from reaction liquid obtained by catalyzing stevioside through a biological enzyme method, according to claim 1, is characterized in that: the heat treatment temperature is 100-150 deg.C, and the heat treatment time is 30-60min.
5. The method for extracting rebaudioside A from reaction liquid obtained by catalyzing stevioside through biological enzyme method according to claim 1, wherein the rebaudioside A is extracted from the reaction liquid obtained by catalyzing stevioside through biological enzyme method through the following steps: the drying temperature is 35-55 ℃, and the drying time is 4-12h.
6. The method for extracting rebaudioside A from reaction liquid obtained by catalyzing stevioside through biological enzyme method according to claim 1, wherein the rebaudioside A is extracted from the reaction liquid obtained by catalyzing stevioside through biological enzyme method through the following steps: the macroporous adsorption resin is a resin product only having high selectivity on adsorbing diterpene compounds; such as: d series, LX, XDA series, HB-8.
7. The method for extracting rebaudioside A from reaction liquid obtained by catalyzing stevioside through a biological enzyme method, according to claim 1, is characterized in that: the evaporative concentration is vacuum evaporative concentration.
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