CN115710295A - Method for extracting rebaudioside-A from reaction liquid obtained by catalyzing stevioside through biological enzyme method - Google Patents

Abstract

The invention relates to a method for extracting rebaudioside A from a stevioside reaction solution catalyzed by a biological enzyme method, which is characterized in that: (1) the reaction solution prepared by a biological enzyme method is subjected to heat treatment and solid-liquid separation, and the solution is (2) Wash the separation column with distilled water or deionized water and 20‑70% ethanol after the adsorption is completed, collect the ethanol desorption liquid, and evaporate and concentrate the ethanol analysis liquid to obtain a concentrated liquid; (3) press the concentration Solution: the volume ratio of organic reagent is 1:3‑10, mix the two, add rebaudioside A with a purity of 95%‑99% of the total mass of 0.1‰‑1‰ as a seed crystal, and run at 50‑200rpm/min , Crystallization at 20-50°C for 10-60h; solid-liquid separation, washing the solid, and drying. The present invention has the advantages of simple process, low crystallization temperature, low stirring speed and low energy consumption; the content of rebaudioside A can be increased from 10%-30% to 60-95.5%, and after multiple recrystallizations, rebaudioside A The content of glycoside A can reach 98‑99%.

Description

A method for extracting rebaudioside A from stevioside reaction liquid catalyzed by biological enzyme method

technical field

The invention belongs to the field of production and preparation of sweeteners, and relates to the preparation of steviol glycosides, in particular to a method for extracting rebaudioside A from a stevioside reaction liquid catalyzed by a biological enzymatic method.

Background technique

The natural sweetener steviol glycosides is a class of extracts with a common aglycone (steviol) and differs by the number and type of carbohydrate residues at C13 and C19 positions, the main glycoside found in stevia leaves is stevioside ST (2-10%) and Rebaudioside C (1-2%). Other glycosides, such as rebaudiosides B, E and F, steviolbioside and rubusoside, were found at much lower levels (approximately 0-1%).

Stevioside ST has the least astringency, the least bitterness and the least persistent aftertaste among the main components of the above-mentioned natural sweetener steviol glycosides, so it has the most popular sensory attributes, however, even in a highly purified state, Steviosides still have undesirable taste attributes such as bitterness, sweet aftertaste, licorice, etc. These undesirable taste attributes in stevia sweeteners have become one of the major obstacles to commercialization, affecting the taste quality of stevia and limiting its wider application.

Rebaudioside A (RA for short) is one of the components of stevioside. In addition to the advantages of low calorie and health benefits of stevioside, it also has a good taste and high sweetness. It is sweetened by sucrose 200-300 times the degree.

At present, the international focus is on the research on the purification methods of various single components in stevioside. Because the taste of a single component is relatively simple, it can be directly added to food or medicine, and a series of properties and other researches can be carried out with a single-component pure product as the research object. RA is one of the single components in the stevia mixture. Because the content of RA in stevia is very small, the current mainstream method is to use stevia to extract directly. The purity of RA obtained by this method is not enough, and it still contains a lot of STV to affect the sweetness. If it is possible to use the biological enzymatic method to catalyze the stevioside reaction liquid as a raw material to purify single-component RA, it will be a necessary step in the RA production technology, and it will also provide a reliable method for the production of high-purity RA products.

Contents of the invention

The purpose of the present invention is to solve the problem that the purity of rebaudioside A purified in the prior art is not high (10-30%), and to provide a method for extracting rebaudioside A from the stevioside reaction liquid catalyzed by biological enzymatic method method.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A method for extracting rebaudioside A from a bioenzyme-catalyzed stevioside reaction liquid, characterized in that it comprises the following steps:

(1) Heat-treat the reaction solution (containing 10%-30% rebaudioside A) prepared by the biological enzyme method (100-150°C for 30-60min), followed by solid-liquid separation (centrifugation, filtration or flocculation), Remove the precipitated solution; the solution is refined with a macroporous adsorption resin to remove non-diterpene compounds;

(2) After the adsorption is completed, wash the separation column with distilled water or deionized water, then wash the separation column with ethanol with a volume fraction of 20-70%, and collect the ethanol desorption solution (only containing diterpene compounds and stevioside), and analyze the ethanol The liquid is evaporated and concentrated to obtain a concentrated solution with a mass concentration of 60-70%;

(3) According to the volume ratio of concentrated liquid: organic reagent is 1:3-10, mix the concentrated liquid and organic reagent, and then add high-purity rebaudioside A (purity 95% -99%) A as a seed crystal, control the stirring speed at 50-200rpm/min, and crystallize at 20-50°C for 10-60h; then perform solid-liquid separation, and wash the solid (remove mother liquor impurities), and dry ( Dry at 35-55°C for 4-12h) to obtain rebaudioside A with a purity of 60-95.5%.

Further, continue to recrystallize rebaudioside A with a purity of 60-95.5% for 1-3 times to obtain rebaudioside A with a purity of 98-99%.

Further, the reaction solution prepared by the bio-enzyme method in the step (1) is a commercial stevia extract containing stevioside ST with a total glycoside content of at least 90% as a starting material, and the stevioside is made by using a bio-enzyme method The reaction solution obtained by converting stevioside ST and other components in the raw material into rebaudioside A.

Further, the macroporous adsorption resin is a resin product with high selectivity only for the adsorption of diterpene compounds (stevioside); such as: D series, LX, XDA series, HB-8, etc.

Further, the evaporative concentration is vacuum evaporative concentration.

Further, the organic solvent is a methanol solution with a volume fraction of 95-99.9% and/or an ethanol solution with a volume fraction of 95-99.9%.

In the present invention, the reaction solution prepared by a biological enzyme method with a rebaudioside A content of 10%-30% is subjected to heat treatment, macroporous resin adsorption, analysis, concentration, crystallization, separation, washing, and drying to obtain rebaudioside A with a purity of more than 60%. Baudioside A; in order to obtain rebaudioside A with higher purity, multiple recrystallizations can be carried out on this basis.

Compared with prior art, the present invention has following advantage:

1. The process of the present invention is simple, the crystallization temperature is lower, the stirring speed is low, and the energy consumption is low;

2. The solvent used in the present invention can be concentrated, recycled and reused, which realizes lower environmental pollution and higher economic benefits;

3. Through the treatment of the present invention, the content of rebaudioside A can be increased from 10%-30% to 60-95.5%, and after multiple recrystallizations, the content of rebaudioside A can reach 98-99%.

Specific implementation content

A method for extracting rebaudioside A from a bioenzymatically catalyzed stevioside reaction liquid, the specific implementation steps are as follows:

Example 1

(1) Heat-treat 1L of the reaction solution (containing 12% rebaudioside A) prepared by the bio-enzyme method (100°C for 30 minutes), and then centrifuge to remove the precipitated solution; the solution is made of macroporous adsorption resin (D series) refined to remove non-diterpenoids;

(2) After the adsorption is completed, wash the separation column with deionized water first, then wash the separation column with ethanol with a volume fraction of 40%, and collect the ethanol desorption solution (only containing diterpene compounds and stevioside), and vacuum evaporate the ethanol analysis solution Concentrate (vacuum degree is -0.075MPa) to obtain 200ml concentrate;

(3) Add 500ml of methanol aqueous solution with a volume fraction of 99.9% to the concentrated solution, mix well and place in a three-necked flask, control the temperature in a water bath at 35°C, and stir at a speed of 100rpm/min, add 0.7g of rebaudioside with a purity of 99% A was used as the seed crystal, and the crystallization continued for 24 hours; the crystallization mixture was suction-filtered with a Buchner funnel, the obtained crystals were washed with an aqueous ethanol solution with a volume fraction of 99%, and dried overnight in a vacuum oven at 70°C to obtain 41.6g of Rebaudi Glycoside A.

Table 1 is the liquid phase determination result of rebaudioside A product in embodiment 1:

Sample name RA RE RF ST Ethanol desorption concentrate (%) 12.2 1.7 not detected 86.1 Primary crystallization product (%) 94.5 not detected not detected 5.5

The RA purity in the product can be improved to 94.5% by adopting the method of Example 1.

Example 2

(1) Heat-treat 1L of the reaction solution (containing 12% rebaudioside A) prepared by the bio-enzyme method (130°C for 35 minutes), and then centrifuge to remove the precipitated solution; use macroporous adsorption resin (LX, XDA series) are refined to remove non-diterpenoids;

(2) After the adsorption is completed, wash the separation column with deionized water first, then wash the separation column with ethanol with a volume fraction of 40%, and collect the ethanol desorption solution (only containing diterpene compounds and stevioside), and vacuum evaporate the ethanol analysis solution Concentrate (vacuum degree is -0.075MPa) to obtain 200ml concentrate;

(3) Add 500ml of methanol aqueous solution with a volume fraction of 99.9% to the concentrated solution, mix well and place in a three-necked flask, control the temperature in a water bath at 35°C, and stir at a speed of 100rpm/min, add 0.7g of rebaudioside with a purity of 99% A is used as a seed crystal, and the crystallization is continued for 24 hours; the crystallization mixture is suction-filtered with a Buchner funnel, and the obtained crystals are washed with an aqueous ethanol solution with a volume fraction of 99%;

(4) For recrystallization, add 400ml of deionized water, heat to 100°C, concentrate to 200ml, add 500ml of methanol aqueous solution with a volume fraction of 99.9%, mix well and place in a three-necked flask, control the temperature of the water bath at 35°C, and the stirring speed is 100 rpm/min, add 0.7 g of rebaudioside A with a purity of 99% as a seed crystal, and continue to crystallize for 24 hours; place it in a vacuum oven at 70°C and dry overnight to obtain 35.5 g of rebaudioside A.

Table 2 is the liquid phase determination result of rebaudioside A product in embodiment 2:

Sample name RA RE RF ST Ethanol desorption concentrate (%) 12.2 1.7 not detected 86.1 Secondary crystallization products (%) 98.5 not detected not detected 1.5

The RA content in the product can be raised to 98.5% by adopting the method of Example 2.

Example 3

(1) Heat-treat 1L of the reaction solution (containing 27% rebaudioside A) prepared by the biological enzyme method (150°C for 30 minutes), and then centrifuge to remove the precipitated solution; the solution is made of macroporous adsorption resin (HB-8 ) are refined to remove non-diterpenoids;

(2) After the adsorption is completed, wash the separation column with distilled water first, then wash the separation column with ethanol with a volume fraction of 60%, and collect the ethanol desorption liquid (containing only diterpene compounds and stevioside), and vacuum evaporate the ethanol desorption liquid ( The vacuum degree is -0.08MPa), and 200ml of concentrated solution is obtained;

(3) Add 500ml of methanol aqueous solution with a volume fraction of 99.9% to the concentrated solution, mix well and place in a three-necked flask, control the temperature in a water bath at 35°C, and stir at a speed of 100rpm/min, add 0.7g of rebaudioside with a purity of 99% A was used as the seed crystal, and the crystallization continued for 24 hours; the crystallization mixture was filtered with a Buchner funnel, and the obtained crystals were washed with an aqueous ethanol solution with a volume fraction of 99%, and dried overnight in a vacuum oven at 70°C to obtain 62.4g of Rebaudi Glycoside A.

Table 3 is the liquid phase determination result of rebaudioside A product in embodiment 3:

Sample name RA RE RF ST Ethanol desorption concentrate (%) 16.2 1.1 not detected 82.7 Primary crystallization product (%) 95.4 not detected not detected 4.6

The RA purity in the product can be improved to 95.4% by adopting the method of Example 3.

Example 4

(1) Heat-treat 2L of the reaction solution (containing 12% rebaudioside A) prepared by the bio-enzyme method (100°C for 30 minutes), and then centrifuge to remove the precipitated solution; use macroporous adsorption resin (HB-8 ) are refined to remove non-diterpenoids;

(2) After the adsorption is completed, wash the separation column with deionized water first, then wash the separation column with ethanol with a volume fraction of 40%, and collect the ethanol desorption solution (only containing diterpene compounds and stevioside), and vacuum evaporate the ethanol analysis solution Concentrate (vacuum degree is -0.088MPa) to obtain 200ml concentrate;

(3) Add 500ml of methanol aqueous solution with a volume fraction of 99.9% to the concentrated solution, mix well and place in a three-necked flask, control the temperature in a water bath at 35°C, and stir at a speed of 100rpm/min, add 0.7g of rebaudioside with a purity of 99% A was used as the seed crystal, and the crystallization was continued for 24 hours; the crystallization mixture was suction-filtered with a Buchner funnel, and the obtained crystals were washed with an aqueous ethanol solution with a volume fraction of 99%, and dried overnight in a vacuum oven at 70°C to obtain 58.4g of Rebaudi Glycoside A.

Table 4 is the liquid phase determination result of rebaudioside A product in embodiment 4:

Sample name RA RE RF ST Ethanol desorption concentrate (%) 16.2 1.2 not detected 82.6 Primary crystallization product (%) 94.7 not detected not detected 5.3

The RA content in the product can be raised to 94.7% by adopting the method of Example 4.

Example 5

(1) Heat-treat 1L of the reaction solution (containing 12% rebaudioside A) prepared by the biological enzyme method (150°C for 40 minutes), and then centrifuge to remove the precipitated solution; the solution is made of macroporous adsorption resin (D series) refined to remove non-diterpenoids;

(2) After the adsorption is completed, wash the separation column with distilled water first, then wash the separation column with ethanol with a volume fraction of 65%, and collect the ethanol desorption liquid (only containing diterpene compounds and stevioside), and vacuum evaporate the ethanol desorption liquid ( The vacuum degree is -0.088 MPa), and 200ml of concentrated solution is obtained;

(3) Add 500ml of methanol aqueous solution with a volume fraction of 99.9% to the concentrated solution, mix well and place in a three-necked flask, control the temperature in a water bath at 35°C, and stir at a speed of 100rpm/min, add 0.7g of rebaudioside with a purity of 99% A was used as the seed crystal, and the crystallization was continued for 48 hours; the crystallization mixture was suction-filtered with a Buchner funnel, and the obtained crystals were washed with an aqueous ethanol solution with a volume fraction of 99%, and dried overnight in a vacuum oven at 70°C to obtain 41.6g of Rebaudi Glycoside A.

Table 5 is the liquid phase assay result of rebaudioside A product in embodiment 5:

Sample name RA RE RF ST Ethanol desorption concentrate (%) 12.4 1.8 not detected 85.8 Primary crystallization product (%) 97.5 not detected not detected 2.5

The RA content in the product can be raised to 95.5% by adopting the method of Example 5.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any form; any person skilled in the art can use the above disclosure without departing from the scope of the technical solution of the present invention. Methods and Technical Contents Many possible changes and modifications are made to the technical solution of the present invention, or modified into equivalent embodiments with equivalent changes. Therefore, any simple modifications, equivalent replacements, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention, which do not deviate from the technical solutions of the present invention, still fall within the protection scope of the technical solutions of the present invention.

Claims (7)

1. a method for extracting rebaudioside A from bioenzyme catalyzed stevioside reaction liquid, is characterized in that comprising the steps: (1) heat-treat the reaction solution prepared by the biological enzyme method, and then conduct solid-liquid separation to remove the precipitated solution; refine the solution with macroporous adsorption resin to remove non-diterpene compounds; (2) After the adsorption is completed, wash the separation column with distilled water or deionized water, then wash the separation column with ethanol with a volume fraction of 20-70%, and collect the ethanol desorption solution, evaporate and concentrate the ethanol analysis solution, and concentrate to obtain a mass concentration of 60-70% concentrate; (3) According to the volume ratio of concentrated solution: organic reagent is 1:3-10, mix the concentrated solution and organic reagent, and then add rebaudioside A with a purity of 95%-99% and a mass of 0.1‰-1‰ of the mixture As a seed crystal, control the stirring speed at 50-200rpm/min, and crystallize at 20-50°C for 10-60h; then carry out solid-liquid separation, wash the solid, and dry to obtain Reiba with a purity of 60-95.5%. Diglycoside A. 2. according to claim 1, a kind of method for extracting rebaudioside A from the bio-enzyme method catalyzed stevioside reaction liquid, is characterized in that: the reaction liquid that described bio-enzyme method makes is with commercialization containing stevioside The stevia extract with a total ST content of at least 90% of the total glycosides is used as a starting material, and the reaction liquid obtained by converting stevioside ST and other components in the stevioside raw material into rebaudioside A by using a biological enzyme method. 3. according to claim 1, a kind of method extracting rebaudioside A from the bioenzyme method catalyzed stevioside reaction liquid, is characterized in that: the reaction liquid that bioenzyme method makes contains rebaudioside A 10%- 30%. 4. A method for extracting rebaudioside A from a stevioside reaction solution catalyzed by biological enzymatic method according to claim 1, characterized in that: the heat treatment temperature is 100-150°C, and the heat treatment time is 30-60min. 5. A method for extracting rebaudioside A from the bioenzyme-catalyzed stevioside reaction liquid according to claim 1, characterized in that: the drying temperature is 35-55°C, and the drying time is 4-5°C. 12h. 6. according to claim 1, a kind of method extracting rebaudioside A from biological enzymatic method catalyzed stevioside reaction liquid, is characterized in that: described macroporous adsorption resin has higher selectivity only to adsorption diterpene compound Sexual resin products; such as: D series, LX, XDA series, HB-8. 7. A method for extracting rebaudioside A from a stevioside reaction solution catalyzed by a bio-enzyme method according to claim 1, characterized in that: said evaporative concentration is vacuum evaporative concentration.