CN106967133B - A method of extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor - Google Patents

Abstract

A method for extracting high-purity rebaudioside D from a refined stevioside mother liquor, comprising the following steps: (1) crystallization and impurity removal: adding the steviol glycoside refined mother liquor to an alcohol solution, dissolving, crystallizing, and filtering to obtain a filtrate; (2) Reverse-phase silica gel sample mixing: Concentrate the filtrate to no alcohol, add reverse-phase silica gel, mix evenly, and dry to obtain mixed sample silica gel; (3) Column packing: In the empty chromatography column, first load reverse-phase silica gel , and then load the mixed sample silica gel; (4) Elution: elute the chromatographic column with eluent, collect the effluent, concentrate, and dry to obtain RD crude product; (5) Refining: use organic solvent or organic solvent for RD crude product Dissolve in water, cool, crystallize, filter, and dry to obtain RD fine product. The RD obtained by the method of the present invention is white crystalline powder, the purity can be as high as 99.0wt%, and the yield can be as high as 92.4%. The method of the present invention is environmentally friendly, low in cost, pure and pollution-free, and has high added value, and is suitable for industrial production.

Description

A method for extracting high-purity rebaudioside D from stevioside refined mother liquor

technical field

The invention relates to a method for extracting rebaudioside D, in particular to a method for extracting high-purity rebaudioside D from stevioside refined mother liquor.

Background technique

The main glycosides in steviol glycosides are stevioside (ST) and rebaudioside A (RA), other known glycosides include rebaudioside B (RB), rebaudioside C (RC), rebaudioside D (RD), rebaudioside F (RF), dulcoside A, rubusoside and steviolbioside.

In recent years, studies have found that the best-tasting component of steviol glycosides is not RA, which has the highest sweetness, but RD. The sweetness of RD is about 150 times that of sucrose, and it has a good taste and no bitterness. However, the content of RD in stevia The content is very low, only about 1%. Therefore, it is difficult to extract high-purity RD with high recovery rate.

CN102060892A discloses a method for purifying stevioside RD, which is to flow the mother liquid sugar material liquid through a macroporous adsorption resin, then elute with ethanol, collect the eluate in sections, concentrate, dry and refine to obtain RD. However, due to the poor adsorption selectivity of macroporous resins, the operability of collecting RD by segmental elution and determining the critical point is low, and it is difficult to enrich RD. Therefore, the yield of RD is not high.

CN102406113A discloses a preparation method of RA and RD compound stevioside, which uses Stevia rebaudiana as raw material, through water extraction, macroporous resin adsorption, elution, decolorization, crystallization, recrystallization and other steps to obtain the mixed RA and RD product. However, this method cannot obtain a high content of RD.

CN103709215A discloses a method for extracting rebaudioside D from stevioside crystallization mother liquor sugar, which is to heat the stevioside mother liquor sugar with water and methanol, stir, and repeatedly crystallize to obtain RD products. However, this method has a low yield due to too many times of crystallization, and the purity of RD is only about 50%.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the above-mentioned defects in the prior art, to provide a product obtained from stevioside refined mother liquor with high purity, high yield, high added value, environmental protection and low cost, which is suitable for industrial production A method for extracting high-purity rebaudioside D from

The technical scheme adopted by the present invention to solve its technical problems is as follows: a method for extracting high-purity rebaudioside D from stevioside refined mother liquor, comprising the following steps:

(1) Crystallization and impurity removal: add the refined mother liquor of steviol glycosides to the alcohol solution, heat to dissolve, cool and crystallize, filter to obtain the filtrate;

(2) Reverse-phase silica gel sample mixing: Concentrate the filtrate obtained in step (1) under reduced pressure to no alcohol, add reverse-phase silica gel to the concentrated solution, mix well, and dry to constant weight to obtain sample mixing silica gel;

(3) Column packing: In the empty chromatographic column, first load the reversed-phase silica gel, and then load the mixed sample silica gel obtained in step (2) on the reversed-phase silica gel;

(4) Elution: use the eluent to elute the chromatographic column packed in step (3), collect the target elution effluent, concentrate, dry, and obtain the crude product of rebaudioside D;

(5) Refining: heating and dissolving the crude rebaudioside D obtained in step (4) with an organic solvent or water, cooling, crystallizing, filtering, and drying to obtain the refined rebaudioside D.

Preferably, in step (1), the mass content of rebaudioside D in the stevioside refined mother liquor is 5-30% (more preferably 6-10%). The stevioside refined mother liquor of the present invention is derived from the mother liquor produced when high-purity rebaudioside A (RA) and stevioside (ST) are prepared by crystallization with an organic solvent using stevia rebaudiana extract as a raw material.

Preferably, in step (1), the amount of the alcohol solution used is 4-20 times (more preferably 5-10 times) the mass of the stevioside refining mother liquor.

Preferably, in step (1), the alcohol in the alcohol solution is one or more of methanol, ethanol, isopropanol or n-butanol, and the volume concentration of the alcohol solution is 50-99%.

Preferably, in step (1), the temperature of the heating and dissolving is 40-60°C.

Preferably, in step (1), the cooling crystallization temperature is 10-25° C., and the time is 12-24 hours.

In step (1), the filter residue after crystallization and filtration contains high content of RA and ST, which can be extracted by existing technology.

Preferably, in step (2), the temperature of the vacuum concentration is 60-90° C., and the vacuum degree is -0.06-0.09 MPa.

Preferably, in step (2), the drying temperature is 60-90°C.

Preferably, in step (2), the amount of the reversed-phase silica gel is 1-2 times the mass of the concentrate. The purpose of sample mixing is to make the material fully adsorb with the reversed-phase silica gel; if the amount of reversed-phase silica gel is too small, the material cannot be completely adsorbed; if the amount of reversed-phase silica gel is too much, it will easily cause waste and increase the difficulty of elution.

Preferably, in step (2), the type of the reversed-phase silica gel is one or more of C4, C8 or C18.

Preferably, in step (3), the amount of the reversed-phase silica gel is 1-20 times (more preferably 5-15 times) the mass of the mixed sample silica gel. The purpose of adding the reversed-phase silica gel first is to provide a buffer area so that various components can be retained in the reversed-phase silica gel for different times during the elution process. If the amount of reversed-phase silica gel is too small, the relative time of retention of various components is too close to achieve the purpose of separation; if the amount of reversed-phase silica gel is too much, it will easily cause waste and increase the amount of eluent.

Preferably, in step (3), the aspect ratio of the chromatographic column is 5-20:1. If the aspect ratio is too small, the separation effect will be poor; if the aspect ratio is too large, the amount of eluent and the elution time will be increased.

Preferably, in step (4), the amount of the eluent used is 2-10 BV, and the elution flow rate is 0.5-2.0 BV/h. The 1BV=1 column bed volume. The target elution effluent is the elution effluent segment containing rebaudioside D detected by thin layer chromatography.

Preferably, in step (4), the eluent is methanol, ethanol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate with a volume concentration of 40-95% (more preferably 50-80%) One or more of ester solution or water.

Preferably, in step (4), concentrate until there is no solvent, and dry to constant weight.

Preferably, in step (5), the amount of the organic solvent or water used is 4-20 times (more preferably 4.5-10 times) the mass of the crude rebaudioside D.

Preferably, in step (5), the organic solvent is methanol, ethanol, isopropanol, n-butanol, acetone or ethyl acetate solution with a volume concentration of 40-95% (more preferably 60-90%) one or several.

Preferably, in step (5), the heating and dissolving temperature is 40-60°C.

Preferably, in step (5), the crystallization temperature is 10-25° C., and the time is 12-24 hours.

The principle of the method of the present invention is: since the content of RD in the stevioside refined mother liquor is generally not high, it is difficult to reach the concentration where RD can crystallize after dissolving with alcohol, and RD will not crystallize out, while other impurity components, such as RA and ST, have high content , then a large amount of crystallization can be precipitated in the alcohol solution. After the precipitated impurities are filtered first, most of the RD is left in the crystallization mother liquor, thereby increasing the content of RD and reducing the difficulty of subsequent reversed-phase silica gel column chromatography. . One of the characteristics of reversed-phase silica gel is that during the elution process, the highly polar components flow out first, and the less polar components flow out later. RD is the most polar among all steviol glycosides. Therefore, the elution of reversed-phase silica gel column The neutralization is the first to flow out, so that RD can be efficiently separated from other glycosides, and finally the high-purity RD product can be obtained through recrystallization.

The beneficial effects of the inventive method are as follows:

(1) The method of the present invention can extract high-purity RD from the cheap steviol glycoside refined mother liquor, and the obtained RD fine product is white crystalline powder, and the purity of RD can be as high as 99.0wt% through high-performance liquid chromatography detection, and the RD's The final yield can be as high as 92.4%, which improves the utilization rate of stevia resources;

(2) The raw material of the method of the present invention is natural, the physical extraction method adopted, the production process is green and environmentally friendly, the cost is low, the product is pure and pollution-free, the price of RD is more than 10 times that of the raw material, the added value of the product is high, and it is suitable for industrial production .

Detailed ways

The present invention will be further described below in conjunction with embodiment.

The steviol glycoside refining mother liquor used in the embodiment of the present invention comes from Hunan Huacheng Biological Resources Co., Ltd., which uses stevia extract as raw material and uses organic solvents to crystallize the mother liquor produced when high-purity RA and ST are prepared. Detected by external standard method, the mass content of RD is 6.5%; C4, C8 and C18 reversed-phase silica gel used in the examples of the present invention are all purchased from Dalian Yilite Analytical Instrument Co., Ltd.; raw materials or chemical reagents used in the examples of the present invention , unless otherwise specified, were obtained through conventional commercial channels.

Example 1

(1) Crystallization and impurity removal: Add 100g of stevioside refined mother liquor to 500g of ethanol solution with a volume concentration of 80%, heat to dissolve at 40°C, then cool and crystallize at 25°C for 16h, filter to obtain the filtrate;

(2) Reverse-phase silica gel sample mixing: Concentrate the filtrate obtained in step (1) at 65°C and a vacuum of -0.06MPa to no alcohol under reduced pressure to obtain 60g of concentrated solution, and add C4 reverse-phase silica gel to 60g of concentrated solution 60g, mix evenly, and dry at 70°C until constant weight to obtain 100g mixed sample silica gel;

(3) Column packing: first load 1000g of C4 reverse-phase silica gel into an empty chromatography column with a height-to-diameter ratio of 15:1, and then load 100g of sample-mixing silica gel obtained in step (2) on the C4 reverse-phase silica gel;

(4) Elution: Use 3BV of acetonitrile solution with a volume concentration of 70% to elute the column after packing in step (3) at a flow rate of 0.5BV/h, and detect it by thin layer chromatography. Collect the target eluted effluent containing rebaudioside D, concentrate until free of acetonitrile, and dry to constant weight to obtain 7.6g of crude rebaudioside D;

(5) Refining: 7.6g of crude rebaudioside D obtained in step (4) was dissolved in 38g of 85% methanol solution at 50°C by heating, cooled to room temperature, and crystallized at 10°C for 24 hours , filtered, and dried to obtain 6.1g rebaudioside D fine product.

The rebaudioside D refined product obtained in this example is a white crystalline powder, which is detected by the high-performance liquid chromatography external standard method. The purity of the obtained rebaudioside D refined product is 98.5wt%, and the final yield of rebaudioside D is 92.4%.

Example 2

(1) Crystallization and impurity removal: Add 50g of stevioside refined mother liquor to 300g of isopropanol solution with a volume concentration of 55%, heat to dissolve at 60°C, then cool and crystallize at 15°C for 12h, filter to obtain filtrate;

(2) Reverse-phase silica gel sample mixing: Concentrate the filtrate obtained in step (1) at 85°C and a vacuum of -0.08MPa to no alcohol under reduced pressure to obtain 35g of concentrated liquid, and add C8 reverse-phase silica gel to the 35g concentrated liquid 70g, mixed evenly, and dried to constant weight at 90°C to obtain 82g mixed sample silica gel;

(3) Column packing: first load 440g of C8 reverse-phase silica gel into an empty chromatography column with a height-to-diameter ratio of 10:1, and then load 82g of mixed sample silica gel obtained in step (2) on the C8 reverse-phase silica gel;

(4) Elution: Use 5BV of methanol solution with a volume concentration of 60% to elute the column after packing in step (3) at a flow rate of 1BV/h, detect by thin layer chromatography, and collect The target eluted effluent containing rebaudioside D was concentrated to no methanol, and dried to constant weight to obtain 3.9 g of crude rebaudioside D;

(5) Refining: 3.9 g of crude rebaudioside D obtained in step (4) was dissolved in 30 g of ethanol solution with a volume concentration of 90% at 55°C, cooled to room temperature, and crystallized at 10°C for 18 hours , filtered, and dried to obtain 2.95g rebaudioside D fine product.

The rebaudioside D refined product obtained in this example is a white crystalline powder, which is detected by the high-performance liquid chromatography external standard method. The purity of the obtained rebaudioside D refined product is 99.0wt%, and the final yield of rebaudioside D is 89.9%.

Example 3

(1) Crystallization and impurity removal: add 1 kg of stevioside refined mother liquor to 8 kg of methanol solution with a volume concentration of 95%, heat to dissolve at 50°C, then cool and crystallize at 20°C for 24 hours, filter to obtain the filtrate;

(2) Reverse-phase silica gel sample mixing: Concentrate the filtrate obtained in step (1) at 60°C and a vacuum of -0.09MPa to no alcohol under reduced pressure to obtain 650g of concentrated liquid, and add C18 reverse-phase silica gel to 650g of concentrated liquid 650g, mixed evenly, dried to constant weight at 75°C to obtain 1.1kg mixed sample silica gel;

(3) Column packing: first load 16.5kg of C18 reversed-phase silica gel into an empty chromatography column with a height-to-diameter ratio of 20:1, and then load 1.1kg of mixed sample silica gel obtained in step (2) on the C18 reversed-phase silica gel ;

(4) Elution: Use 10BV of ethanol solution with a volume concentration of 50% to elute the chromatographic column after packing in step (3) at a flow rate of 1.5BV/h, and detect it by thin layer chromatography. Collect the target eluted effluent containing rebaudioside D, concentrate to no ethanol, and dry to constant weight to obtain 77g of crude rebaudioside D;

(5) Refining: 77g of crude rebaudioside D obtained in step (4) was dissolved in 380g of n-butanol solution with a volume concentration of 60% at 55°C, cooled to room temperature, and crystallized at 25°C After 24 hours, filter and dry to obtain 60g of rebaudioside D fine product.

The rebaudioside D refined product obtained in this example is a white crystalline powder, which is detected by the high-performance liquid chromatography external standard method. The purity of the obtained rebaudioside D refined product is 98.6wt%, and the final yield of rebaudioside D is 91.0%.

Claims (23)

1. a kind of method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, which is characterized in that including following step It is rapid:

(1) crystallization removal of impurities: steviol glycoside refinement mother liquor is added in alcoholic solution, is dissolved by heating, cooling crystallization, and filtering obtains filtrate； The mass content of rebaudioside D is 5~30% in the steviol glycoside refinement mother liquor；

(2) reverse phase silica gel mixes sample: filtrate decompression obtained by step (1) being concentrated into no alcohol, reverse phase silica gel is added in concentrate, is mixed It closes uniformly, it is dry to constant weight, sample silica gel must be mixed；

(3) it fills column: in empty chromatographic column, being initially charged with reverse phase silica gel, then be packed on reverse phase silica gel and mix sample silicon obtained by step (2) Glue；

(4) elute: the chromatographic column after with eluant, eluent step (3) are filled with column elutes, and collects target and elutes efflux, is concentrated, It is dry, obtain rebaudioside D crude product；

(5) it refines: rebaudioside D crude product organic solvent obtained by step (4) or water is dissolved by heating, cooling, crystallization, filtering, It is dry, obtain rebaudioside D fine work；The organic solvent is methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution One or more of.

2. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 1, feature exist In in step (1), the dosage of the alcoholic solution is 4~20 times of steviol glycoside refinement mother liquor quality；Alcohol in the alcoholic solution For one or more of methanol, ethyl alcohol, isopropanol or n-butanol, the volumetric concentration of alcoholic solution is 50~99%.

3. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special Sign is, in step (1), the temperature of the heating for dissolving is 40~60 DEG C；The temperature of the cooling crystallization is 10~25 DEG C, when Between for 12~for 24 hours.

4. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special Sign is, in step (2), the temperature of the reduced pressure is 60~90 DEG C, and vacuum degree is -0.06~-0.09MPa；It is described dry Dry temperature is 60~90 DEG C.

5. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature exist In in step (2), the temperature of the reduced pressure is 60~90 DEG C, and vacuum degree is -0.06~-0.09MPa；The drying Temperature is 60~90 DEG C.

6. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special Sign is, in step (2), the dosage of the reverse phase silica gel is 1~2 times of concentrate quality；The type of the reverse phase silica gel is One or more of C4, C8 or C18.

7. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature exist In in step (2), the dosage of the reverse phase silica gel is 1~2 times of concentrate quality；The type of the reverse phase silica gel is C4, C8 Or one or more of C18.

8. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 4, feature exist In in step (2), the dosage of the reverse phase silica gel is 1~2 times of concentrate quality；The type of the reverse phase silica gel is C4, C8 Or one or more of C18.

9. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special Sign is, in step (3), the dosage of the reverse phase silica gel is 1~20 times for mixing sample silica gel quality.

10. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature It is, in step (3), the dosage of the reverse phase silica gel is 1~20 times for mixing sample silica gel quality.

11. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 4, feature It is, in step (3), the dosage of the reverse phase silica gel is 1~20 times for mixing sample silica gel quality.

12. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 6, feature It is, in step (3), the dosage of the reverse phase silica gel is 1~20 times for mixing sample silica gel quality.

13. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special Sign is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The elution Agent is methanol, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or the water of volumetric concentration 40~95% One or more of.

14. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature It is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The eluant, eluent For in the methanol of volumetric concentration 40~95%, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water One or more.

15. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 4, feature It is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The eluant, eluent For in the methanol of volumetric concentration 40~95%, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water One or more.

16. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 6, feature It is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The eluant, eluent For in the methanol of volumetric concentration 40~95%, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water One or more.

17. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 9, feature It is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The eluant, eluent For in the methanol of volumetric concentration 40~95%, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water One or more.

18. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special Sign is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic Solvent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.

19. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.

20. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 4, feature It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.

21. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 6, feature It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.

22. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 9, feature It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.

23. 3 method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 1, feature It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.