Method for separating and purifying rebaudioside F and rebaudioside C from stevia mother liquor sugar
The separation of rebaudioside F and rebaudioside C in stevia mother sugars through gradient analysis and liquid chromatography has solved the problems of high separation difficulty and low purity in the prior art, and achieved efficient and pure separation effect.
Patent Information
- Application Number
- CN202311285037.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-28
- Publication Date
- 2025-03-28
- Estimated Expiration
- 2043-09-28
AI Technical Summary
The prior art is difficult to effectively isolate rebaudioside F and rebaudioside C in stevia mother liquor sugar, resulting in high difficulty in isolation and purification, low separation efficiency and low purity of the isolated product.
Rebaudioside F and Rebaudioside C were gradually separated and purified by diluting the stevia mother liquor sugar and adsorbing through macroporous adsorption resin, using gradient analytical method and regular phase medium and low pressure liquid chromatography technology. The specific steps include multiple gradient analysis and concentration treatments, combined with methanol dilution and crystallization steps, and finally further purification by reverse phase high-pressure liquid chromatography.
The efficient separation of rebaudioside F and rebaudioside C in stevia mother sugar was achieved, and the separation efficiency and product purity were improved. The content of rebaudioside C reached more than 85 wt%, and the content of rebaudioside F reached more than 90 wt%.
Smart Images
Figure CN119708095A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of glycoside separation, and in particular to a method for separating and purifying rebaudioside F and rebaudioside C from stevia mother liquor sugar. Background Art
[0002] Stevioside is a mixture of various terpene glycosides, which have the same stevioside aglycone core and various sugar groups connected to C13 and C19. At present, the structures of more than ten terpene glycosides have been identified. Most of the glycosides in natural stevia leaves are rebaudioside A (2% to 10%), stevioside (2% to 10%) and rebaudioside C (1% to 2%). Other glycosides such as rebaudioside B, rebaudioside D, rebaudioside E and rebaudioside F, stevioside and rubusoside exist in stevia leaves at low levels (less than 1%). Among them, rebaudioside F and rebaudioside C have very similar structures and properties. The only difference is that rebaudioside C is connected to a glucose group at the C13 position, while rebaudioside F is connected to a xylose group at the C13 position.
[0003] Since the structures of rebaudioside F and rebaudioside C are very similar, the separation of the two is more difficult. The existing technology cannot effectively separate rebaudioside F and rebaudioside C. At present, the separation research of glycosides in stevia mother liquor sugar mainly focuses on the separation of stevioside STV, rebaudioside A and rebaudioside B. Therefore, a new separation and purification method is urgently needed to solve the above technical problems. Summary of the invention
[0004] In order to solve the technical problems of high difficulty in separation and purification of rebaudioside F and rebaudioside C in the existing stevia mother liquor sugar, low separation efficiency, and low purity of the separated product, the present invention provides a method for separating and purifying rebaudioside F and rebaudioside C in stevia mother liquor sugar. In view of this, the present invention is implemented by the following technical scheme.
[0005] In a first aspect, the present invention provides a method for separating and purifying rebaudioside C from stevia mother liquor sugar, comprising:
[0006] Diluting stevia mother liquid sugar with water to prepare a mother liquid sugar dilution solution;
[0007] The mother sugar dilution liquid is adsorbed by a macroporous adsorption resin, and a first gradient analysis is performed using an ethanol aqueous solution with a mass concentration of 30 to 70 wt % to obtain a first analysis solution;
[0008] The first analytical solution is concentrated and dried, the dried mixture is dissolved in high-concentration methanol, stirred, crystallized, and filtered to obtain a first filter cake and a first filtrate, and the first filtrate is concentrated to obtain a concentrated solution with a solid content of 10 to 50 wt%;
[0009] The concentrated solution is diluted with 80-95 wt % methanol, the diluted solution is passed through a normal phase medium and low pressure liquid chromatography column, and a second gradient analysis is performed using an ethanol aqueous solution with a mass concentration of 70-95 wt % to obtain a second analysis solution;
[0010] The second analytical solution is concentrated and dried, and the dried mixture is dissolved in 10-50 wt % methanol, stirred, crystallized, and filtered to obtain a second filter cake and a second filtrate, wherein the content of rebaudioside C in the second filter cake is higher than 85 wt %.
[0011] Compared with the prior art, in the method for separating and purifying rebaudioside C from stevia mother liquor sugar of the present invention, the stevia mother liquor sugar contains rebaudioside F, rebaudioside C, rebaudioside A and stevioside, and after the mother liquor sugar dilution is passed through a macroporous adsorption resin column, the rebaudioside F, rebaudioside C, rebaudioside A and stevioside in the mother liquor sugar dilution are adsorbed by the macroporous adsorption resin, and the rebaudioside F, rebaudioside C, rebaudioside A and stevioside adsorbed by the macroporous adsorption resin can be separated and purified by using an ethanol aqueous solution with a mass concentration of 30 to 70 wt%. Stevioside is analyzed to obtain a first analysis solution; the first analysis solution is concentrated and dried to remove the ethanol aqueous solution in the first analysis solution, and the dried mixture is dissolved in high-concentration methanol, and the high-concentration methanol is used as a crystallization solvent for rebaudioside A and stevioside. During the stirring process, rebaudioside A and stevioside are crystallized and precipitated. After filtering, a first filter cake containing rebaudioside A and stevioside and a first filtrate containing rebaudioside F and rebaudioside C can be obtained. This step can remove most of the rebaudioside A and stevioside in the stevia mother liquor sugar. After the above-mentioned first filtrate is concentrated to a solid content of 10 to 50wt%, it is diluted with 80 to 95wt% of methanol, and the dilution is passed through a normal phase medium and low pressure liquid chromatography column. Rebaudioside F and rebaudioside C in the dilution are adsorbed by the stationary phase, and a second gradient analysis is performed using an ethanol aqueous solution with a mass concentration of 70 to 95wt% to obtain a second analysis solution containing rebaudioside F and rebaudioside C. In this step, impurities other than rebaudioside F and rebaudioside C can be further removed by passing the dilution through a normal phase medium and low pressure liquid chromatography column and then analyzing. The second analytical solution is concentrated and dried to remove the ethanol aqueous solution, and the dried mixture is dissolved in 10-50wt% methanol. Rebaudioside C is crystallized during the stirring process, and a second filter cake containing rebaudioside C and a second filtrate containing rebaudioside F are obtained after filtration. The content of rebaudioside C in the second filter cake is higher than 85wt%, and can be directly used as a rebaudioside C product. Through the above technical scheme of the present invention, efficient separation of rebaudioside C and rebaudioside F in stevia mother liquid sugar is achieved, and the technical problems of high difficulty in separation and purification of rebaudioside F and rebaudioside C in stevia mother liquid sugar, low separation efficiency, and low purity of the separated product are solved.
[0012] Furthermore, in the method for separating and purifying rebaudioside C from stevia mother liquor sugar of the present invention, the mass ratio of mother liquor sugar to water in the mother liquor sugar dilution is 1:(5-10); and / or,
[0013] In the process of diluting the concentrated solution with 80-95 wt % of methanol, the volume ratio of the concentrated solution to the methanol is 1:(5-10).
[0014] Furthermore, in the method for separating and purifying rebaudioside C from stevia mother liquor sugar of the present invention, the concentration of the high concentration methanol is not less than 90wt%; and / or,
[0015] The dried mixture is dissolved in high-concentration methanol, and the ratio of the mass of the mixture to the volume of the high-concentration methanol is 1:(1.5-5).
[0016] Furthermore, in the method for separating and purifying rebaudioside C from stevia mother liquor sugar of the present invention, the dried mixture is dissolved in 10-50 wt % methanol, and the ratio of the mass of the mixture to the volume of methanol is 1:(1-4).
[0017] In a second aspect, the present invention provides a method for separating and purifying rebaudioside F from stevia mother liquor sugar, using the second filtrate, comprising:
[0018] The second filtrate is concentrated and dried, the dried mixture is dissolved in a 60-70 wt % methanol solution and stirred evenly, after being passed through a reverse phase high pressure liquid chromatography column, a third gradient analysis is performed using a 50-70 wt % methanol solution to obtain a third analysis solution;
[0019] After the third analytical solution is concentrated and dried, a rebaudioside F product is obtained, wherein the rebaudioside F content in the rebaudioside F product is higher than 85 wt %.
[0020] Compared with the prior art, the beneficial effects of the method for separating and purifying rebaudioside F from stevia mother liquor sugar provided by the present invention are the same as the beneficial effects of the method for separating and purifying rebaudioside C from stevia mother liquor sugar described in the above technical scheme, and are not described in detail here.
[0021] Furthermore, in the method for separating and purifying rebaudioside F from stevia mother liquor sugar of the present invention, the third gradient analysis using 50-70wt% methanol solution to obtain a third analysis solution comprises:
[0022] A third gradient analysis is performed using a 50-70wt% methanol solution to obtain a plurality of third analysis solutions of different concentration segments, and the content of rebaudioside F in each segment of the third analysis solution is detected. The third analysis solutions with a rebaudioside F content higher than 90wt% are combined, concentrated, and dried to obtain a rebaudioside F product with a rebaudioside F content higher than 90wt%.
[0023] Furthermore, in the method for separating and purifying rebaudioside F from stevia mother liquor sugar of the present invention, after the third gradient analysis using 50-70wt% methanol solution to obtain a third analysis solution of multiple different concentration ranges, the method further comprises:
[0024] The third analytical solutions with a rebaudioside F content lower than 90 wt % are combined, concentrated and dried;
[0025] The dried mixture is dissolved in 10-50wt% methanol, stirred, crystallized, and filtered to obtain a third filter cake and a third filtrate, wherein the rebaudioside C content in the third filter cake is higher than 90wt%; the ratio of the volume of the 10-50wt% methanol to the mass of the dried mixture is (1-4):1.
[0026] In a third aspect, the present invention provides a rebaudioside C product, which is separated and obtained by using the above-mentioned method for separating and purifying rebaudioside C from stevia mother liquor sugar, and the content of rebaudioside C in the rebaudioside C product is higher than 85wt%.
[0027] Compared with the prior art, the beneficial effects of the rebaudioside C product provided by the present invention are the same as the beneficial effects of the method for separating and purifying rebaudioside C from stevia mother liquor sugar described in the above technical scheme, and will not be described in detail here.
[0028] In a fourth aspect, the present invention provides a rebaudioside F product, which is separated and obtained by using the above-mentioned method for separating and purifying rebaudioside F from stevia mother liquor sugar, and the rebaudioside F content in the rebaudioside F product is higher than 90wt%.
[0029] Compared with the prior art, the beneficial effects of the rebaudioside F product provided by the present invention are the same as the beneficial effects of the method for separating and purifying rebaudioside C from stevia mother liquor sugar described in the above technical scheme, and will not be described in detail here. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:
[0031] Figure 1 The present invention provides a schematic flow chart of the method for separating and purifying rebaudioside C from stevia mother liquor sugar provided in Example 1 of the present invention. DETAILED DESCRIPTION
[0032] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
[0033] It should be noted that when an element is referred to as being "fixed to" or "disposed on" another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element.
[0034] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined. The meaning of "several" is one or more, unless otherwise clearly and specifically defined.
[0035] In the description of the present invention, it is necessary to understand that the directions or positional relationships indicated by the terms "up", "down", "front", "back", "left", "right", etc. are based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on the present invention.
[0036] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.
[0037] Since the structures of rebaudioside F and rebaudioside C are extremely similar, the separation of the two is more difficult. The existing technology cannot effectively separate rebaudioside F and rebaudioside C. Currently, the separation research of glycosides in stevia mother liquor sugar mainly focuses on the separation of stevioside STV, rebaudioside A and rebaudioside B.
[0038] In order to solve the above technical problems, in a first aspect, the present invention provides a method for separating and purifying rebaudioside C from stevia mother liquor sugar, comprising:
[0039] Diluting stevia mother liquid sugar with water to prepare a mother liquid sugar dilution solution;
[0040] The mother sugar dilution liquid is adsorbed by a macroporous adsorption resin, and a first gradient analysis is performed using an ethanol aqueous solution with a mass concentration of 30 to 70 wt % to obtain a first analysis solution;
[0041] The first analytical solution is concentrated and dried, the dried mixture is dissolved in high-concentration methanol, stirred, crystallized, and filtered to obtain a first filter cake and a first filtrate, and the first filtrate is concentrated to obtain a concentrated solution with a solid content of 10 to 50 wt%;
[0042] The concentrated solution is diluted with 80-95 wt % methanol, the diluted solution is passed through a normal phase medium and low pressure liquid chromatography column, and a second gradient analysis is performed using an ethanol aqueous solution with a mass concentration of 70-95 wt % to obtain a second analysis solution;
[0043] The second analytical solution is concentrated and dried, and the dried mixture is dissolved in 10-50wt% methanol, stirred, crystallized, and filtered to obtain a second filter cake and a second filtrate. The content of rebaudioside C in the second filter cake is higher than 85wt%, and the content of rebaudioside F is about 30wt%.
[0044] The method for separating and purifying rebaudioside C from stevia mother liquor sugar of the present invention adopts the above technical scheme, wherein the stevia mother liquor sugar contains rebaudioside F, rebaudioside C, rebaudioside A and stevioside, and the mother liquor sugar dilution is passed through a macroporous adsorption resin column, and the rebaudioside F, rebaudioside C, rebaudioside A and stevioside in the mother liquor sugar dilution are adsorbed by the macroporous adsorption resin, and an ethanol aqueous solution with a mass concentration of 30 to 70 wt% is used for gradient analysis, so that the rebaudioside F, rebaudioside C, rebaudioside A and stevioside adsorbed by the macroporous adsorption resin can be separated and purified. Rebaudioside A and stevioside are resolved to obtain a first resolved solution; the first resolved solution is concentrated and dried to remove the ethanol aqueous solution in the first resolved solution, and the dried mixture is dissolved in high-concentration methanol, and the high-concentration methanol is used as a crystallization solvent for rebaudioside A and stevioside. During the stirring process, part of rebaudioside A and stevioside crystallize out, and after filtering, a first filter cake containing rebaudioside A and stevioside and a first filtrate containing rebaudioside F and rebaudioside C can be obtained. This step can remove most of rebaudioside A and stevioside in stevia mother liquor sugar. After the above-mentioned first filtrate is concentrated to a solid content of 10 to 50wt%, it is diluted with 80 to 95wt% of methanol, and the dilution is passed through a normal phase medium and low pressure liquid chromatography column. Rebaudioside F and rebaudioside C in the dilution are adsorbed by the stationary phase, and a second gradient analysis is performed using an ethanol aqueous solution with a mass concentration of 70 to 95wt% to obtain a second analysis solution containing most of rebaudioside F and rebaudioside C. In this step, rebaudioside F and rebaudioside C can be further enriched by analyzing the dilution after passing it through a normal phase medium and low pressure liquid chromatography column. The second analytical solution is concentrated and dried to remove the ethanol aqueous solution. The dried mixture is dissolved in 10-50 wt% methanol. Rebaudioside C crystallizes during stirring. After filtration, a second filter cake containing rebaudioside C and a second filtrate containing rebaudioside F are obtained. The content of rebaudioside C in the second filter cake is higher than 85 wt%, and can be directly used as a rebaudioside C product. At this time, the content of rebaudioside F is 30%.
[0045] It should be understood that before the mother liquor sugar dilution is adsorbed by the macroporous adsorption resin, in order to avoid clogging of the resin column due to excessive solid content of the mother liquor sugar dilution and to improve the adsorption efficiency of rebaudioside F and rebaudioside C by the macroporous adsorption resin, the mass ratio of the mother liquor sugar to water in the mother liquor sugar dilution should also be controlled. By way of example, the mass ratio of the mother liquor sugar to water in the mother liquor sugar dilution can be 1:(5-10). By way of another example, the mass ratio of the mother liquor sugar to water in the mother liquor sugar dilution can be 1:5, 1:7 or 1:10.
[0046] It should be understood that the mother liquor sugar dilution is adsorbed by the macroporous adsorption resin, and in the first gradient analysis process using an ethanol aqueous solution with a mass concentration of 30 to 70 wt%, the ethanol aqueous solution with different concentration gradients can be selected within the range according to the concentration range of the ethanol aqueous solution to analyze the macroporous adsorption resin, so as to obtain first analysis solutions of multiple concentration sections, and the first analysis solutions with high rebaudioside C content are mixed and dried, so as to further increase the content of rebaudioside C in the second filter cake; for example, the mass concentration of the ethanol aqueous solution can be 30 to 70 wt%, and the macroporous adsorption resin can be analyzed using 30 wt%, 40 wt%, 50 wt%, 60 wt% or 70 wt% ethanol aqueous solution, respectively, and the first analysis solutions corresponding to each concentration of the ethanol aqueous solution are collected, and the content of rebaudioside C in each first analysis solution is detected, and the first analysis solutions with a rebaudioside C content higher than 40 wt% are mixed and dried, so that the rebaudioside C in the second filter cake can be higher than 88 wt% through subsequent steps.
[0047] It should also be understood that in the process of concentrating and drying the first analytical solution and dissolving the dried mixture in high-concentration methanol, in order to increase the crystallization rate of rebaudioside A and stevioside in the mixture, the concentration of the high-concentration methanol and the mixing ratio of the mixture to the high-concentration methanol should also be controlled. For example, the concentration of the high-concentration methanol is not less than 90wt%, for example, it can be 90wt% to 98wt%, and the ratio of the mass of the mixture to the volume of the high-concentration methanol is 1:(1.5 to 5); specifically, the concentration of the high-concentration methanol can be 90wt%, 95wt% or 98wt%, and the ratio of the mass of the mixture to the volume of the high-concentration methanol can be 1:1.5, 1:3 or 1:5. Furthermore, the dried mixture is dissolved in high-concentration methanol. During the stirring, crystallization and filtration processes, the stirring rate is controlled to be 45-70 rpm at room temperature. Crystals gradually precipitate out of the methanol during the stirring process. The stirring is continued until the crystal content no longer increases, then the stirring is stopped and the mixture is filtered using a filtration device to obtain a first filtrate containing most of rebaudioside C and rebaudioside F, and a first filter cake containing rebaudioside A and stevioside. In the above process, in order to improve the filtering and separation effect, a Buchner funnel can be used as the filtration device, and the Buchner funnel is used for filtration by suction filtration.
[0048] It should also be understood that in the process of diluting the concentrate with 80-95wt% methanol, in order to control the column pressure of the normal phase low-pressure liquid chromatography within a reasonable range, the mixing ratio of the concentrate and the methanol should also be controlled. For example, the volume ratio of the concentrate to the methanol can be 1:(5-10), and for another example, the volume ratio of the concentrate to the methanol can be 1:5, 1:8 or 1:10. Further, the normal phase low-pressure liquid chromatography filler can be one or more of Silica, NH2 (amino), APS, CN or CPS.
[0049] Furthermore, in the process of performing the second gradient analysis on the normal phase low-pressure liquid chromatography column using an ethanol solution with a mass concentration of 70 to 95wt%, the chromatographic column can be analyzed using ethanol aqueous solutions with a mass concentration of 70wt%, 75wt%, 80wt%, 85wt% or 90wt%, respectively, and the second analysis liquid corresponding to each concentration of ethanol aqueous solution is collected, the content of rebaudioside C in each second analysis liquid is detected, the second analysis liquids in which the rebaudioside C content is higher than 40wt% are mixed, concentrated and dried, and the rebaudioside C in the second filter cake can be higher than 90wt% through subsequent steps.
[0050] It should also be understood that the dried mixture is dissolved in 10-50wt% methanol, and the 10-50wt% methanol is the crystallization solvent of rebaudioside C. In order to control the crystallization rate of rebaudioside C in the dried product, the mixing ratio of the mixture and 10-50wt% methanol should also be controlled. For example, the dried mixture is dissolved in 10-50wt% methanol, and the ratio of the mass of the mixture to the volume of methanol can be 1: (1-4). For another example, the ratio of the mass of the mixture to the volume of methanol can be 1: 1, 1: 2 or 1: 4. Further, the dried mixture is dissolved in high-concentration methanol. During stirring, crystallization and filtration, due to the different structures of rebaudioside C and rebaudioside F, rebaudioside F does not have crystallization characteristics in 10-50wt% methanol, and rebaudioside C and rebaudioside F can be separated. The stirring rate can be 45 to 70 rpm. During the stirring process, crystals gradually precipitate out of the methanol. Stirring is continued until the crystal content no longer increases, then stirring is stopped and filtered using a filtering device to obtain a second filter cake containing rebaudioside C and a second filtrate containing rebaudioside F. At this time, the content of rebaudioside F in the filtrate is increased to about 30%. The rebaudioside C content in the second filter cake is 85%, which can be directly used or sold as a rebaudioside C product. In the above process, in order to improve the filtering and separation effect, the filtering device can use a Buchner funnel, and the Buchner funnel is used for filtering by suction filtration.
[0051] In a second aspect, the present invention provides a method for separating and purifying rebaudioside F from stevia mother liquor sugar, using the second filtrate, comprising:
[0052] The second filtrate is concentrated and dried, the dried mixture is dissolved in a 60-70 wt % methanol solution and stirred evenly, after being passed through a reverse phase high pressure liquid chromatography column, a third gradient analysis is performed using a 50-70 wt % methanol solution to obtain a third analysis solution;
[0053] After the third analytical solution is concentrated and dried, a rebaudioside F product is obtained, wherein the rebaudioside F content in the rebaudioside F product is higher than 85 wt %.
[0054] According to the above technical solution, the second filtrate contains rebaudioside F and other glycoside impurities. The second filtrate is concentrated and dried, and the dried mixture is dissolved in a 60-70wt% methanol solution and stirred evenly. After the mixture is passed through a reversed-phase high-pressure liquid chromatography column, rebaudioside F is adsorbed by the stationary phase. The reversed-phase high-pressure liquid chromatography column filler can be one or more of YMCC18 filler (ODS), C8 (MOS), C4 (B) or C6H5 (Phenyl). The third analytical solution containing rebaudioside F can be obtained by performing a third gradient analysis on the reversed-phase high-pressure liquid chromatography column using a 50-70wt% methanol solution. After the third analytical solution is concentrated and dried, a rebaudioside F product having a rebaudioside F content higher than 85wt% can be obtained.
[0055] As a possible implementation, in the method for separating and purifying rebaudioside F from stevia mother liquor sugar of the present invention, the third gradient analysis using 50-70 wt % methanol solution to obtain the third analysis solution comprises:
[0056] A third gradient analysis is performed using a 50-70wt% methanol solution to obtain a plurality of third analysis solutions of different concentration segments, and the content of rebaudioside F in each segment of the third analysis solution is detected. The third analysis solutions with a rebaudioside F content higher than 90wt% are combined, concentrated, and dried to obtain a rebaudioside F product with a rebaudioside F content higher than 90wt%.
[0057] By adopting the above technical scheme, a third analytical solution with multiple different concentration ranges can be obtained by using a 50-70wt% methanol solution for a third gradient analysis; for example, a methanol solution with a mass concentration of 50wt%, 60wt% or 70wt% is used to perform a third gradient analysis on a reversed-phase high pressure liquid chromatography to obtain a third analytical solution corresponding to different methanol concentration ranges, and the content of rebaudioside F in the third analytical solution corresponding to the methanol in each concentration range is different. By combining the third analytical solutions with a rebaudioside F content higher than 90wt%, concentrating and drying them, a rebaudioside F product with a rebaudioside F content higher than 90wt% can be obtained.
[0058] As a possible implementation, in the method for separating and purifying rebaudioside F from stevia mother liquor sugar of the present invention, after the third gradient analysis using 50-70wt% methanol solution to obtain a third analysis solution of multiple different concentration ranges, the method further comprises:
[0059] The third analytical solutions with a rebaudioside F content lower than 90 wt % are combined, concentrated and dried;
[0060] The dried mixture is dissolved in 10-50 wt% methanol, stirred, crystallized, and filtered to obtain a third filter cake and a third filtrate, wherein the content of rebaudioside C in the third filter cake is higher than 90 wt%; the ratio of the volume of the 10-50 wt% methanol to the mass of the dried mixture is (1-4): 1. At this time, the content of rebaudioside in the filtrate is about 50%.
[0061] By adopting the above technical scheme, after the reversed phase high pressure liquid chromatography column is subjected to segmented analysis, the third analytical solution with a rebaudioside F content higher than 90wt% is mixed and dried to obtain a rebaudioside F product with a content higher than 90wt%, and the third analytical solution with a rebaudioside F content lower than 90wt% has a large proportion of rebaudioside C. The third analytical solution with a rebaudioside F content lower than 90wt% is combined, concentrated, and dried, and the dried mixture is dissolved in 10-50wt% methanol, and 10-50wt% methanol is used as a crystallization solvent for rebaudioside C. Rebaudioside C crystallizes and precipitates during the stirring process, and the third filter cake with a rebaudioside C content higher than 90wt% can be obtained after filtering. In order to improve the recovery rate of rebaudioside F, the filtrate can be further concentrated and dried, and as a raw material of rebaudioside F, it flows through reversed phase high pressure liquid chromatography to prepare high-purity rebaudioside F. At the same time, in order to improve the crystallization efficiency of rebaudioside C, the ratio of the volume of the 10-50wt% methanol to the mass of the dried mixture is controlled to be (1-4):1. For example, the ratio of the volume of the 10-50wt% methanol to the mass of the dried mixture can be 1:1, 2:1 or 4:1.
[0062] In a third aspect, the present invention provides a rebaudioside C product, which is separated and obtained by using the above-mentioned method for separating and purifying rebaudioside C from stevia mother liquor sugar, and the content of rebaudioside C in the rebaudioside C product is higher than 85wt%.
[0063] Compared with the prior art, the beneficial effects of the rebaudioside C product provided by the present invention are the same as the beneficial effects of the method for separating and purifying rebaudioside C from stevia mother liquor sugar described in the above technical scheme, and will not be described in detail here.
[0064] In a fourth aspect, the present invention provides a rebaudioside F product, which is separated and obtained by using the above-mentioned method for separating and purifying rebaudioside F from stevia mother liquor sugar, and the rebaudioside F content in the rebaudioside F product is higher than 90wt%.
[0065] Compared with the prior art, the beneficial effects of the rebaudioside F product provided by the present invention are the same as the beneficial effects of the method for separating and purifying rebaudioside C from stevia mother liquor sugar described in the above technical scheme, and will not be described in detail here.
[0066] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific embodiments, but the content of the present invention is not limited to the following embodiments.
[0067] Unless otherwise specified, the raw materials used in the following examples are commercially available.
[0068] Example 1
[0069] This embodiment provides a method for separating and purifying rebaudioside C from stevia mother liquor sugar, comprising:
[0070] S100, dissolving 1000 g of stevia mother liquid sugar in pure water to obtain a mother liquid sugar dilution solution, wherein the mass ratio of the stevia mother liquid sugar to the pure water is 1:5, and the components of the stevia mother liquid sugar include 4.8 wt% of rebaudioside F, 29.9 wt% of rebaudioside C, 19.4 wt% of rebaudioside A, and the remainder is stevioside and impurities;
[0071] S200, the mother liquid sugar dilution is adsorbed by a macroporous adsorption resin column; the macroporous adsorption resin is subjected to a first gradient analysis using 30wt%, 50wt% and 70wt% ethanol aqueous solutions respectively, the mother liquid sugar dilution column flow rate is 20ml / min, the ethanol aqueous solution column flow rate is 40ml / min, and the first analysis solutions of the three concentration sections are collected and mixed;
[0072] S300, concentrating and drying the first analytical solution using a rotary evaporator, wherein the temperature during the concentration and drying is 80° C. and the rotation speed is 55 rpm; dissolving the dried mixture in 92 wt % methanol, and stirring for 24 h at room temperature and a stirring speed of 45 rpm until crystals are precipitated, and filtering with a Buchner funnel to obtain a first filter cake and a first filtrate; wherein the ratio of the mass of the dried mixture to the volume of the methanol is 1:1.5;
[0073] The first filtrate was concentrated using a rotary evaporator at a temperature of 80° C. and a rotation rate of 50 rpm to obtain a concentrated solution with a solid content of 10 wt %;
[0074] S400, diluting the concentrated solution with 80wt% methanol, passing the diluted solution through a normal phase medium and low pressure liquid chromatography column, and performing a second gradient analysis on the chromatographic column using 70wt%, 80wt% and 95wt% ethanol aqueous solutions respectively, collecting the analysis solution once every 0.5BV at each ethanol aqueous solution concentration, and mixing the analysis solution at the 80wt% gradient, wherein the flow rate of the diluted solution through the column is 20ml / min, and the flow rate of the ethanol aqueous solution through the column is 30ml / min; the volume ratio of the concentrated solution to methanol is 1:5, and the amount of the ethanol aqueous solution used in each concentration section is 3 times the volume of the diluted solution;
[0075] S500, concentrating and drying the second analytical solution using a rotary evaporator, the temperature during concentration and drying is 80°C and the rotation rate is 55 rpm, the dried mixture is dissolved in 10 wt% methanol, stirred for 24 hours at room temperature and a stirring rate of 50 rpm until crystals are precipitated, and filtered through a Buchner funnel to obtain a second filtrate and 30 g of a second filter cake; wherein the ratio of the mass of the dried mixture to the volume of the methanol is 1:1.
[0076] The rebaudioside C content of the second filter cake obtained by separation and purification in this example was tested, and the rebaudioside C content in the second filter cake was 87 wt %.
[0077] Example 2
[0078] This embodiment provides a method for separating and purifying rebaudioside C and rebaudioside F from stevia mother liquor sugar, comprising:
[0079] S100, dissolving 1000 g of stevia mother liquid sugar in pure water to obtain a mother liquid sugar dilution solution, wherein the mass ratio of the stevia mother liquid sugar to the pure water is 1:10, and the components of the stevia mother liquid sugar include 4.8 wt% of rebaudioside F, 29.9 wt% of rebaudioside C, 19.4 wt% of rebaudioside A, and the remainder is stevioside and impurities;
[0080] S200, passing the mother sugar dilution through a macroporous adsorption resin column, and rebaudioside C and other glycosides in the mother sugar dilution are adsorbed by the macroporous adsorption resin; using 30wt%, 50wt% and 70wt% ethanol aqueous solutions to perform a first gradient analysis on the macroporous adsorption resin, respectively, collecting the analysis solution according to the resin column volume 2BV at each ethanol aqueous solution concentration during the analysis, detecting the analysis solution concentration, collecting the analysis solution with a rebaudioside C content higher than 40wt%, and mixing to obtain the first analysis solution; wherein the mother sugar dilution column flow rate is 20ml / min, and the ethanol aqueous solution column flow rate is 40ml / min;
[0081] S300, concentrating and drying the first analytical solution using a rotary evaporator, wherein the temperature during the concentration and drying is 80° C. and the rotation speed is 55 rpm; dissolving the dried mixture in 95 wt % methanol, stirring for 24 h at room temperature and a stirring speed of 45 rpm until crystals are precipitated, and filtering with a Buchner funnel to obtain a first filter cake and a first filtrate; wherein the ratio of the mass of the dried mixture to the volume of the methanol is 1:5;
[0082] The first filtrate was concentrated using a rotary evaporator at a temperature of 80° C. and a rotation rate of 50 rpm to obtain a concentrated solution with a solid content of 50 wt %;
[0083] S400, diluting the concentrated solution with 95wt% methanol, passing the diluted solution through a normal phase medium and low pressure liquid chromatography column, and performing a second gradient analysis on the chromatographic column using 70wt%, 85wt% and 95wt% ethanol aqueous solutions respectively, collecting the analysis solution once every 0.5BV at each ethanol aqueous solution concentration, detecting the concentration of the analysis solution, and mixing the analysis solution with a rebaudioside C content higher than 40wt% to obtain a second analysis solution; wherein, the flow rate of the diluted solution through the column is 20ml / min, the flow rate of the ethanol aqueous solution through the column is 30ml / min, the volume ratio of the concentrated solution to methanol is 1:10, and the amount of the ethanol aqueous solution used in each concentration section is 3 times the volume of the diluted solution;
[0084] S500, concentrating and drying the second analytical solution using a rotary evaporator, wherein the temperature during the concentration and drying is 80° C. and the rotation speed is 55 rpm, the dried mixture is dissolved in 50 wt % methanol, stirred for 24 h at room temperature and a stirring speed of 50 rpm until crystals are precipitated, and filtered through a Buchner funnel to obtain a second filtrate and 25 g of a second filter cake; wherein the ratio of the mass of the dried mixture to the volume of the methanol is 1:4;
[0085] S600, concentrating and drying the second filtrate, dissolving the dried mixture in a 60wt% methanol solution and stirring evenly, passing through a reversed-phase high-pressure liquid chromatography column under the conditions of a column pressure of 6MPa and a flow rate of 50ml / min, performing a third gradient analysis using 50wt%, 60wt% and 70wt% methanol solutions respectively, detecting the concentration of the analysis solution, collecting the third analysis solution with a rebaudioside F concentration higher than 90%, concentrating and drying using a rotary evaporator at a temperature of 80°C and a rotation speed of 55rpm to obtain a rebaudioside F product; the ratio of the mass of the dried mixture to the volume of the methanol is 1:3.
[0086] The rebaudioside C content of the second filter cake prepared in this example was detected, and the rebaudioside F content of the rebaudioside F product was detected. The rebaudioside C content in the second filter cake was 91wt%, and the rebaudioside F content in the rebaudioside F product was 91.5wt%.
[0087] Example 3
[0088] This embodiment provides a method for separating and purifying rebaudioside C and rebaudioside F from stevia mother liquor sugar, comprising:
[0089] S100, dissolving 1000 g of stevia mother liquid sugar in pure water to obtain a mother liquid sugar dilution solution, wherein the mass ratio of the stevia mother liquid sugar to the pure water is 1:6, and the stevia mother liquid sugar comprises 4.8 wt% of rebaudioside F, 29.9 wt% of rebaudioside C, 19.4 wt% of rebaudioside A, and the remainder is stevioside and impurities;
[0090] S200, the mother liquid sugar dilution is adsorbed by a macroporous adsorption resin column; the macroporous adsorption resin is subjected to a first gradient analysis using 30wt%, 50wt% and 70wt% ethanol aqueous solutions respectively, and the analysis solution is collected according to the resin column volume 2BV at each ethanol aqueous solution concentration during the analysis, and the concentration of the analysis solution is detected, and the analysis solution with a rebaudioside C content higher than 40wt% is collected, and the first analysis solution is obtained by mixing; wherein the mother liquid sugar dilution column flow rate is 20ml / min, and the ethanol aqueous solution column flow rate is 40ml / min;
[0091] S300, concentrating and drying the first analytical solution using a rotary evaporator, wherein the temperature during the concentration and drying is 80° C. and the rotation speed is 55 rpm; dissolving the dried mixture in 95 wt % methanol, stirring for 24 h at room temperature and a stirring speed of 45 rpm until crystals are precipitated, and filtering with a Buchner funnel to obtain a first filter cake and a first filtrate; wherein the ratio of the mass of the dried mixture to the volume of the methanol is 1:4;
[0092] The first filtrate was concentrated using a rotary evaporator at a temperature of 80° C. and a rotation rate of 50 rpm to obtain a concentrated solution with a solid content of 35 wt %;
[0093] S400, diluting the concentrated solution with 85wt% methanol, passing the diluted solution through a normal phase medium and low pressure liquid chromatography column, and performing a second gradient analysis on the chromatographic column using 70wt%, 80wt% and 95wt% ethanol aqueous solutions respectively, collecting the analysis solution once every 0.5BV at each ethanol aqueous solution concentration, detecting the concentration of the analysis solution, and mixing the analysis solution with a rebaudioside C content higher than 40wt% to obtain a second analysis solution; wherein, the flow rate of the diluted solution through the column is 20ml / min, the flow rate of the ethanol aqueous solution through the column is 30ml / min, the volume ratio of the concentrated solution to methanol is 1:8, and the amount of the ethanol aqueous solution used in each concentration section is 3 times the volume of the diluted solution;
[0094] S500, concentrating and drying the second analytical solution using a rotary evaporator, wherein the temperature during the concentration and drying is 80° C. and the rotation speed is 55 rpm, the dried mixture is dissolved in 35 wt % methanol, stirred at a stirring speed of 50 rpm for 24 h until crystals are precipitated, and filtered through a Buchner funnel to obtain a second filtrate and 27 g of a second filter cake; wherein the ratio of the mass of the dried mixture to the volume of the methanol is 1:3;
[0095] S600, concentrating and drying the second filtrate, dissolving the dried mixture in a 70wt% methanol solution and stirring evenly, passing through a reversed-phase high-pressure liquid chromatography column under the conditions of a column pressure of 6MPa and a flow rate of 50ml / min, respectively performing a third gradient analysis using 50wt%, 60wt% and 70wt% methanol solutions, detecting the concentration of the analysis solution, collecting the third analysis solution with a rebaudioside F concentration higher than 90%, concentrating and drying using a rotary evaporator at a temperature of 80°C and a rotation speed of 55rpm to obtain a rebaudioside F product, wherein the ratio of the mass of the dried mixture to the volume of the methanol is 1:4;
[0096] S700, combining the third analytical solution with a rebaudioside F content of less than 90 wt % in step S600, concentrating and drying at a temperature of 45° C. and a stirring rate of 50 rpm;
[0097] S800, dissolving the dried mixture in step S700 in 30wt% methanol, stirring for 24h at room temperature and a stirring rate of 55rpm until crystals are precipitated, filtering with a Buchner funnel to obtain a third filtrate and 15g of a third filter cake, wherein the ratio of the mass of the dried mixture to the volume of the methanol is 1:4; the third filtrate contains rebaudioside F, which can be collected as a raw material for recovering rebaudioside F.
[0098] The rebaudioside C content of the second filter cake and the third filter cake prepared in this embodiment was measured respectively, and the rebaudioside F content of the rebaudioside F product was tested. The rebaudioside C content in the second filter cake was 90wt%, the rebaudioside C content in the third filter cake was 95wt%, the rebaudioside F content in the rebaudioside F product was 93wt%, and the rebaudioside F content in the third filtrate was 50%.
[0099] Through the above-mentioned embodiments of the present invention and the test data, it can be known that under the conditions of the embodiments of the present invention, rebaudioside C and rebaudioside F with relatively high purity can be separated from stevia mother liquor sugar, and the separated rebaudioside C and rebaudioside F can be directly used or sold as products. The above-mentioned technical scheme of the present invention solves the technical problems of high difficulty in separation and purification of rebaudioside F and rebaudioside C in stevia mother liquor sugar, low separation efficiency, and low purity of the separated product.
[0100] In the description of the above embodiments, specific features, structures, materials or characteristics may be combined in a suitable manner in any one or more embodiments or examples.
[0101] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.
Claims
1. A method for separating and purifying rebaudioside C from stevia mother liquor sugar, characterized in that: include: Diluting stevia mother liquid sugar with water to prepare a mother liquid sugar dilution solution; The mother sugar dilution liquid is adsorbed by a macroporous adsorption resin, and a first gradient analysis is performed using an ethanol aqueous solution with a mass concentration of 30 to 70 wt % to obtain a first analysis solution; The first analytical solution is concentrated and dried, the dried mixture is dissolved in high-concentration methanol, stirred, crystallized, and filtered to obtain a first filter cake and a first filtrate, and the first filtrate is concentrated to obtain a concentrated solution with a solid content of 10 to 50 wt%; The concentrated solution is diluted with 80-95 wt % methanol, the diluted solution is passed through a normal phase medium and low pressure liquid chromatography column, and a second gradient analysis is performed using an ethanol aqueous solution with a mass concentration of 70-95 wt % to obtain a second analysis solution; The second analytical solution is concentrated and dried, and the dried mixture is dissolved in 10-50 wt % methanol, stirred, crystallized, and filtered to obtain a second filter cake and a second filtrate, wherein the content of rebaudioside C in the second filter cake is higher than 85 wt %.
2. The method for separating and purifying rebaudioside C from stevia mother liquor sugar according to claim 1, characterized in that: The mass ratio of mother liquid sugar to water in the mother liquid sugar dilution solution is 1:(5-10); and / or, The concentrated solution is diluted with 80-95 wt % methanol, and the volume ratio of the concentrated solution to the methanol is 1:(5-10).
3. The method for separating and purifying rebaudioside C from stevia mother liquor sugar according to claim 1, characterized in that: The concentration of the high-concentration methanol is not less than 90 wt %; and / or, The dried mixture is dissolved in high-concentration methanol, and the ratio of the mass of the mixture to the volume of the high-concentration methanol is 1:(1.5-5).
4. The method for separating and purifying rebaudioside C from stevia mother liquor sugar according to claim 1, characterized in that: The dried mixture is dissolved in 10-50 wt% methanol, and the ratio of the mass of the mixture to the volume of methanol is 1:(1-4).
5. A method for separating and purifying rebaudioside F from stevia mother liquor sugar, characterized in that: Utilizing the second filtrate according to claim 1, comprising: The second filtrate is concentrated and dried, the dried mixture is dissolved in a 60-70 wt % methanol solution and stirred evenly, after being passed through a reverse phase high pressure liquid chromatography column, a third gradient analysis is performed using a 50-70 wt % methanol solution to obtain a third analysis solution; After the third analytical solution is concentrated and dried, a rebaudioside F product is obtained, wherein the rebaudioside F content in the rebaudioside F product is higher than 85 wt %.
6. The method for separating and purifying rebaudioside F from stevia mother liquor sugar according to claim 5, characterized in that: The third analytical solution is obtained by performing a third gradient analysis using a 50-70 wt % methanol solution, comprising: A third gradient analysis is performed using a 50-70wt% methanol solution to obtain a plurality of third analysis solutions of different concentration segments, and the content of rebaudioside F in each segment of the third analysis solution is detected. The third analysis solutions with a rebaudioside F content higher than 90wt% are combined, concentrated, and dried to obtain a rebaudioside F product with a rebaudioside F content higher than 90wt%.
7. The method for separating and purifying rebaudioside F from stevia mother liquor sugar according to claim 6, characterized in that: After the third gradient analysis using 50-70 wt % methanol solution to obtain a plurality of third analysis solutions of different concentration ranges, the method further comprises: The third analytical solutions with a rebaudioside F content lower than 90 wt % are combined, concentrated and dried; The dried mixture is dissolved in 10-50wt% methanol, stirred, crystallized, and filtered to obtain a third filter cake and a third filtrate, wherein the rebaudioside C content in the third filter cake is higher than 90wt%; the ratio of the volume of the 10-50wt% methanol to the mass of the dried mixture is (1-4):
1.
8. A rebaudioside C product, characterized in that: The rebaudioside C product is separated and obtained by the method for separating and purifying rebaudioside C from stevia mother liquor sugar according to any one of claims 1 to 4, wherein the content of rebaudioside C in the rebaudioside C product is higher than 85wt%.
9. A rebaudioside F product, characterized in that: The rebaudioside F is separated and obtained by the method for separating and purifying rebaudioside F from stevia mother liquor sugar according to any one of claims 5 to 7, wherein the rebaudioside F content in the rebaudioside F product is higher than 90wt%.
Citation Information
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