Method for separating and purifying rebaudioside f and rebaudioside c from stevia mother liquor sugar

Abstract

The application discloses a method for separating and purifying rebaudioside F and rebaudioside C from stevia mother liquor sugar, and relates to the technical field of glycoside separation, aiming at solving the technical problem of high difficulty in separating and purifying rebaudioside F and rebaudioside C from stevia mother liquor sugar. The method for separating and purifying rebaudioside C from stevia mother liquor sugar comprises the following steps: diluting stevia mother liquor sugar, adsorbing the dilute solution on a macroporous adsorption resin, and obtaining a first eluate by elution; concentrating and drying the first eluate, dissolving the dried mixture in high-concentration methanol, and filtering to obtain a first filtrate; concentrating the first filtrate, diluting the concentrated solution with methanol, passing the dilute solution through a normal-phase low-pressure liquid chromatography column, and obtaining a second eluate by elution; and concentrating and drying the second eluate, dissolving the dried mixture in methanol, and filtering to obtain a second filter cake containing rebaudioside C. The technical scheme provided by the application is used for separating and purifying rebaudioside F and rebaudioside C from stevia mother liquor sugar.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of glycoside separation, and particularly relates to a method for separating and purifying rebaudioside F and rebaudioside C from stevia mother liquor sugar. BACKGROUND

[0002] Steviol glycoside is a mixture composed of various terpene glycosides, which has the same steviol glycoside core and various sugar groups connected to C13 and C19. At present, the structures of more than ten kinds of 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%). Rebaudioside B, rebaudioside D, rebaudioside E and rebaudioside F, steviolbioside and rubusoside and other glycosides exist in stevia leaves in low content (less than 1%). Among them, rebaudioside F and rebaudioside C have very similar structures and properties, and the only difference is that rebaudioside C is connected with a glucose group at C13, and rebaudioside F is connected with a xylose group at C13.

[0003] Due to the extremely similar structures of rebaudioside F and rebaudioside C, the separation of the two is difficult, and the effective separation of rebaudioside F and rebaudioside C cannot be achieved by using the prior art. At present, the separation of glycosides in stevia mother liquor sugar mainly focuses on the separation of stevioside STV, rebaudioside A and rebaudioside B. Therefore, there is an urgent need for a new separation and purification method to solve the above technical problems. SUMMARY

[0004] In order to solve the technical problems of high difficulty, low separation efficiency and low purity of the separated product in the separation and purification of rebaudioside F and rebaudioside C in the existing stevia mother liquor sugar, the present application provides a method for separating and purifying rebaudioside F and rebaudioside C from stevia mother liquor sugar. In view of this, the present application is realized by the following technical scheme.

[0005] In a first aspect, the present application provides a method for separating and purifying rebaudioside C from stevia mother liquor sugar, comprising:

[0006] The stevia mother liquor sugar is diluted with water to obtain a mother liquor sugar diluent;

[0007] The mother liquor sugar diluent is adsorbed by a macroporous adsorption resin, and a first gradient elution is performed using an ethanol aqueous solution with a mass concentration of 30 to 70 wt%, to obtain a first eluate;

[0008] The first eluate is concentrated and dried, and the dried mixture is dissolved in high-concentration methanol, stirred, crystallized, 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 80-95wt% methanol, and the diluted solution is passed through a normal-phase medium-low pressure liquid chromatography column, and then a second gradient resolution is performed using 70-95wt% ethanol aqueous solution to obtain a second resolution solution;

[0010] The second resolution solution is concentrated and dried, and the dried mixture is dissolved in 10-50wt% methanol, stirred, crystallized, filtered to obtain a second filter cake and a second filtrate, and the content of rebaudioside C in the second filter cake is higher than 85wt%.

[0011] Compared with the prior art, in the method for separating and purifying rebaudioside C from stevia mother liquor sugar, the stevia mother liquor sugar contains rebaudioside F, rebaudioside C, rebaudioside A and stevioside, the rebaudioside F, rebaudioside C, rebaudioside A and stevioside in the diluted mother liquor sugar are adsorbed by the macroporous adsorption resin after the diluted mother liquor sugar is passed through a macroporous adsorption resin column, and the rebaudioside F, rebaudioside C, rebaudioside A and stevioside adsorbed by the macroporous adsorption resin can be resolved using 30-70wt% ethanol aqueous solution to obtain a first resolution solution; the first resolution solution is concentrated and dried to remove the ethanol aqueous solution in the first resolution solution, and the dried mixture is dissolved in high-concentration methanol, and the high-concentration methanol is used as a crystallization solvent of rebaudioside A and stevioside, and the rebaudioside A and stevioside are crystallized and separated out during the stirring process, and then filtered to obtain a first filter cake containing rebaudioside A and stevioside, and a first filtrate containing rebaudioside F and rebaudioside C, and most of the rebaudioside A and stevioside in the stevia mother liquor sugar can be removed in this step; the first filtrate is concentrated to a solid content of 10-50wt%, diluted with 80-95wt% methanol, and then passed through a normal-phase medium-low pressure liquid chromatography column, and the rebaudioside F and rebaudioside C in the diluted solution are adsorbed by the stationary phase, and a second gradient resolution is performed using 70-95wt% ethanol aqueous solution to obtain a second resolution solution containing rebaudioside F and rebaudioside C, and in this step, the impurities other than rebaudioside F and rebaudioside C can be further removed by passing the diluted solution through a normal-phase medium-low pressure liquid chromatography column for resolution; the second resolution solution is concentrated and dried to remove the ethanol aqueous solution, and the dried mixture is dissolved in 10-50wt% methanol, and the rebaudioside C is crystallized and separated out during the stirring process, and then filtered to obtain a second filter cake containing rebaudioside C and a second filtrate containing rebaudioside F, and the content of rebaudioside C in the second filter cake is higher than 85wt%, and the second filter cake can be directly used as a rebaudioside C product. Through the above technical scheme, the rebaudioside C and rebaudioside F in the stevia mother liquor sugar are efficiently separated, and the technical problems of high separation and purification difficulty, low separation efficiency and low purity of the separated product of rebaudioside F and rebaudioside C in the existing stevia mother liquor sugar are solved.

[0012] Further, in the method for separating and purifying rebaudioside C from the mother liquor of stevia sugar according to the present application, the mass ratio of the mother liquor to water in the mother liquor dilution is 1:(5-10); and / or,

[0013] During the dilution of the concentrated solution with 80-95wt% methanol, the volume ratio of the concentrated solution to the methanol is 1:(5-10).

[0014] Further, in the method for separating and purifying rebaudioside C from the mother liquor of stevia sugar according to the present application, the concentration of the high-concentration methanol is not less than 90wt%; and / or,

[0015] After drying, the mixture is dissolved in high-concentration methanol, and the mass of the mixture to the volume of the high-concentration methanol is 1:(1.5-5).

[0016] Further, in the method for separating and purifying rebaudioside C from the mother liquor of stevia sugar according to the present application, after drying, the mixture is dissolved in 10-50wt% methanol, and the mass of the mixture to the volume of the methanol is 1:(1-4).

[0017] In the second aspect, the present application provides a method for separating and purifying rebaudioside F from the mother liquor of stevia sugar, which utilizes the second filtrate described above, and comprises:

[0018] The second filtrate is concentrated and dried, and the dried mixture is dissolved in 60-70wt% methanol solution and stirred uniformly. After passing through a reversed-phase high-pressure liquid chromatography column, a third gradient elution is performed using 50-70wt% methanol solution to obtain a third elution solution.

[0019] After the third elution solution is concentrated and dried, a rebaudioside F product is obtained, and the content of rebaudioside F in the rebaudioside F product is higher than 85wt%.

[0020] Compared with the prior art, the method for separating and purifying rebaudioside F from the mother liquor of stevia sugar according to the present application has the same beneficial effects as the method for separating and purifying rebaudioside C from the mother liquor of stevia sugar according to the above technical solution, and details are not repeated here.

[0021] Further, in the method for separating and purifying rebaudioside F from the mother liquor of stevia sugar according to the present application, the third gradient elution using 50-70wt% methanol solution to obtain a third elution solution comprises:

[0022] The third gradient elution using 50-70wt% methanol solution obtains a plurality of third elution solutions with different concentration sections, and the content of rebaudioside F in each section is detected. The third elution 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] Further, in the method for separating and purifying rebaudioside F from the stevia mother liquor sugar of the present application, after the third gradient resolution using 50-70wt% methanol solution to obtain multiple third resolution liquids with different concentrations, the method further comprises:

[0024] concentrating and drying the third resolution liquids with rebaudioside F content less than 90wt%;

[0025] dissolving the dried mixture in 10-50wt% methanol, stirring, crystallizing, filtering to obtain a third filter cake and a third filtrate, 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 the third aspect, the present application provides a rebaudioside C product, which is separated by the method for separating and purifying rebaudioside C from the stevia mother liquor sugar described above, and the rebaudioside C content in the rebaudioside C product is higher than 85wt%.

[0027] Compared with the prior art, the rebaudioside C product provided by the present application has the same beneficial effects as the method for separating and purifying rebaudioside C from the stevia mother liquor sugar described above, which will not be repeated here.

[0028] In the fourth aspect, the present application provides a rebaudioside F product, which is separated by the method for separating and purifying rebaudioside F from the stevia mother liquor sugar described above, and the rebaudioside F content in the rebaudioside F product is higher than 90wt%.

[0029] Compared with the prior art, the rebaudioside F product provided by the present application has the same beneficial effects as the method for separating and purifying rebaudioside C from the stevia mother liquor sugar described above, which will not be repeated here. BRIEF DESCRIPTION OF DRAWINGS

[0030] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0031] Figure 1 The flowchart of the method for separating and purifying rebaudioside C from the stevia mother liquor sugar of the present application is provided. DETAILED DESCRIPTION

[0032] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clear, the present application will be further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and do not limit the present application.

[0033] It should be noted that when an element is referred to as being "fixed" or "set" 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", "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined as "first", "second" can explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise explicitly specified and limited. The meaning of "several" is one or more, unless otherwise explicitly specified and limited.

[0035] In the description of the present application, it should be understood that the terms "upper", "lower", "front", "back", "left", "right" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the purpose of facilitating the description of the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0036] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be broadly understood, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the internal communication of two elements or the interaction relationship between two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0037] Due to the extremely similar structure of rebaudioside F and rebaudioside C, the separation of the two is difficult, and the effective separation of rebaudioside F and rebaudioside C cannot be achieved by using the prior art. At present, the separation of glycosides in the mother liquor sugar of stevia 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 application provides a method for separating and purifying rebaudioside C in a stevia mother liquor sugar, comprising:

[0039] The stevia mother liquor sugar is diluted with water to obtain a mother liquor sugar dilution;

[0040] The mother liquor sugar diluent is adsorbed by macroporous adsorption resin, and a first gradient elution is performed using an ethanol aqueous solution with a mass concentration of 30-70 wt% to obtain a first eluate;

[0041] The first eluate is concentrated and dried, and the dried mixture is dissolved in high-concentration methanol, stirred, crystallized, and filtered to obtain a first filter cake and a first filtrate; the first filtrate is concentrated to obtain a concentrated solution with a solid content of 10-50 wt%;

[0042] The concentrated solution is diluted with 80-95 wt% methanol, and the diluent is passed through a normal-phase low-pressure liquid chromatography column, and a second gradient elution is performed using an ethanol aqueous solution with a mass concentration of 70-95 wt% to obtain a second eluate;

[0043] The second eluate 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; the content of rebaudioside C in the second filter cake is higher than 85 wt%, and the content of rebaudioside F is about 30 wt%.

[0044] The method for separating and purifying rebaudioside C from stevia mother liquor sugar has the following technical solutions: the stevia mother liquor sugar contains rebaudioside F, rebaudioside C, rebaudioside A and stevioside; the rebaudioside F, rebaudioside C, rebaudioside A and stevioside in the mother liquor sugar diluent are adsorbed by the macroporous adsorption resin after the macroporous adsorption resin column is passed through; the rebaudioside F, rebaudioside C, rebaudioside A and stevioside adsorbed by the macroporous adsorption resin are resolved by using a 30-70 wt% ethanol aqueous solution for gradient resolution, so that the first resolution liquid is obtained; the first resolution liquid is concentrated and dried to remove the ethanol aqueous solution in the first resolution liquid; the mixture after drying is dissolved in high-concentration methanol, the high-concentration methanol is used as a crystallization solvent of rebaudioside A and stevioside, part of the rebaudioside A and stevioside is crystallized and precipitated during stirring, and the first filter cake containing rebaudioside A and stevioside and the first filtrate containing rebaudioside F and rebaudioside C are obtained after filtration, so that most of the rebaudioside A and stevioside in the stevia mother liquor sugar can be removed; the first filtrate is concentrated to a solid content of 10-50 wt%, then diluted by 80-95 wt% methanol, and the diluted liquid is passed through a normal-phase low-pressure liquid chromatography column, so that the rebaudioside F and rebaudioside C in the diluted liquid are adsorbed by the stationary phase; the second gradient resolution is performed by using a 70-95 wt% ethanol aqueous solution, so that the second resolution liquid containing most of the rebaudioside F and rebaudioside C is obtained; in this step, the rebaudioside F and rebaudioside C are further enriched by passing the diluted liquid through the normal-phase low-pressure liquid chromatography column and then resolving; the second resolution liquid is concentrated and dried to remove the ethanol aqueous solution, the mixture after drying is dissolved in 10-50 wt% methanol, the rebaudioside C is crystallized and precipitated during stirring, and the second filter cake containing rebaudioside C and the second filtrate containing rebaudioside F are obtained after filtration, so that the content of rebaudioside C in the second filter cake is higher than 85 wt%, and the second filter cake can be directly used as a rebaudioside C product; and the content of rebaudioside F is 30% at this time.

[0045] It should be understood that, before the macroporous adsorption resin is used to adsorb the mother liquor sugar diluent, in order to avoid clogging of the resin column due to too high solid content of the mother liquor sugar diluent and to improve the adsorption efficiency of the macroporous adsorption resin on the rebaudioside F and rebaudioside C, the mass ratio of the mother liquor sugar to water in the mother liquor sugar diluent should also be controlled; for example, the mass ratio of the mother liquor sugar to water in the mother liquor sugar diluent can be 1:(5-10), or the mass ratio of the mother liquor sugar to water in the mother liquor sugar diluent can be 1:5, 1:7 or 1:10.

[0046] It should be understood that, in the first gradient elution process using the macroporous adsorption resin adsorbed with the mother liquor sugar diluent and the aqueous ethanol solution with a mass concentration of 30-70 wt%, the aqueous ethanol solution with different concentration gradients can be selected in the concentration range to elute the macroporous adsorption resin, to obtain a plurality of first elution liquids with different concentration segments, and the first elution liquid with a high content of rebaudioside C is mixed and dried to further increase the content of rebaudioside C in the second filter cake. For example, the aqueous ethanol solution can have a mass concentration of 30-70 wt%, and the macroporous adsorption resin can be eluted with 30 wt%, 40 wt%, 50 wt%, 60 wt% or 70 wt% aqueous ethanol solution, respectively, and the first elution liquid corresponding to each concentration of aqueous ethanol solution can be collected, the content of rebaudioside C in each first elution liquid can be detected, and the first elution liquid with a rebaudioside C content higher than 40 wt% can be mixed and dried, so that the content of rebaudioside C in the second filter cake is higher than 88 wt% after subsequent steps.

[0047] It should also be understood that, in the process of concentrating and drying the first elution liquid and dissolving the dried mixture in high-concentration methanol, in order to improve 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 90 wt%, for example, it can be 90 wt%-98 wt%, and the ratio of the mass of the mixture to the volume of the high-concentration methanol is 1:(1.5-5). Specifically, the concentration of the high-concentration methanol can be 90 wt%, 95 wt% or 98 wt%, 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. Further, in the process of dissolving the dried mixture in high-concentration methanol, stirring, crystallization and filtration, the stirring rate is controlled at 45-70 rpm at room temperature. During stirring, crystals gradually precipitate from the methanol. When the crystallization content no longer increases, stirring is stopped, and a filter device is used for filtration, 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 filtration separation effect, the filter device can be a Buchner funnel, and the Buchner funnel can be used for filtration by suction filtration.

[0048] It should also be understood that, in the process of diluting the concentrated solution with 80-95wt% methanol, in order to control the pressure of the normal-phase low-pressure liquid chromatography column within a reasonable range, the mixing ratio of the concentrated solution and methanol should also be controlled. For example, the volume ratio of the concentrated solution to the methanol can be 1:(5-10), and in another example, the volume ratio of the concentrated solution 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, APS, CN or CPS.

[0049] Further, in the second gradient resolution process of the normal-phase low-pressure liquid chromatography column using an ethanol solution with a mass concentration of 70-95wt%, an ethanol aqueous solution with a mass concentration of 70wt%, 75wt%, 80wt%, 85wt% or 90wt% can be used respectively to resolve the chromatography column, and the second resolution liquid corresponding to each concentration of ethanol aqueous solution can be collected to detect the content of rebaudioside C in each second resolution liquid. The second resolution liquid with a rebaudioside C content higher than 40wt% is mixed and concentrated, dried, and subjected to subsequent steps to make the rebaudioside C content in the second filter cake higher than 90wt%.

[0050] It should also be understood that, in the process of dissolving the dried mixture in 10-50wt% methanol, the 10-50wt% methanol is a crystallization solvent of rebaudioside C, and in order to control the crystallization rate of rebaudioside C in the dried mixture, the mixing ratio of the mixture and 10-50wt% methanol should also be controlled. For example, the 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), and in 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, in the process of stirring, crystallizing and filtering the mixture dissolved in high-concentration methanol, rebaudioside C and rebaudioside F can be separated due to the structural difference between them, and rebaudioside F does not have crystallization characteristics in 10-50wt% methanol. The stirring rate can be 45-70rpm, and during the stirring process, crystallization gradually occurs in the methanol. When the crystallization content no longer increases, the stirring is stopped, and a filtering device is used for filtering 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 increases to about 30%. The content of rebaudioside C in the second filter cake reaches 85%, which can be directly used or sold as a rebaudioside C product. In the above process, in order to improve the filtering and separating effect, a Buchner funnel can be used as the filtering device, and the Buchner funnel is used for filtration by suction filtration.

[0051] In a second aspect, the present application provides a method for separating and purifying rebaudioside F from stevia mother liquor sugar, which utilizes the above-mentioned second filtrate, comprising:

[0052] concentrating and drying the third elution liquid to obtain the rebaudioside F product, wherein the content of rebaudioside F in the rebaudioside F product is higher than 85wt%.

[0053] concentrating and drying the third elution liquid to obtain the rebaudioside F product, wherein the content of rebaudioside F in the rebaudioside F product is higher than 85wt%.

[0054] 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 uniformly. After passing 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). Third gradient elution of the reversed-phase high-pressure liquid chromatography column using a 50-70wt% methanol solution can obtain a third elution liquid containing rebaudioside F. Concentrating and drying the third elution liquid can obtain a rebaudioside F product with a rebaudioside F content higher than 85wt%.

[0055] As a possible implementation, in the method for separating and purifying rebaudioside F from stevia mother liquor sugar, the third gradient elution using a 50-70wt% methanol solution to obtain a third elution liquid includes:

[0056] Third gradient elution using a 50-70wt% methanol solution can obtain third elution liquids of multiple different concentration sections. The content of rebaudioside F in each section of the third elution liquid is detected. Third elution liquids 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] The above technical solution can obtain third elution liquids of multiple different concentration sections by using a 50-70wt% methanol solution for third gradient elution. For example, a reversed-phase high-pressure liquid chromatography column is subjected to third gradient elution using a 50wt%, 60wt%, or 70wt% methanol solution to obtain third elution liquids corresponding to different methanol concentration sections. The content of rebaudioside F in each concentration section of the third elution liquid is different. Third elution liquids 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%.

[0058] As a possible implementation, in the method for separating and purifying rebaudioside F from stevia mother liquor sugar, after the third gradient resolution using 50-70 wt% methanol solution to obtain a plurality of third resolution liquids with different concentrations, the method further comprises:

[0059] The third resolution liquids with rebaudioside F content less 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 rebaudioside C content in the third filter cake is higher than 90 wt%; and 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 using the above technical solution, the third resolution liquids with rebaudioside F content higher than 90 wt% are mixed and dried to obtain a rebaudioside F product with content higher than 90 wt%; the rebaudioside C content in the third resolution liquids with rebaudioside F content less than 90 wt% is relatively large, the third resolution liquids with rebaudioside F content less than 90 wt% are combined, concentrated, and dried, and the dried mixture is dissolved in 10-50 wt% methanol, 10-50 wt% methanol is used as a crystallization solvent of rebaudioside C, rebaudioside C is crystallized and separated out during stirring, and the third filter cake with rebaudioside C content higher than 90 wt% is obtained after filtration. In order to improve the recovery rate of rebaudioside F, the filtrate can be continuously concentrated and dried, and used as a raw material of rebaudioside F to pass through the reverse-phase high-pressure liquid chromatograph to prepare high-purity rebaudioside F. In order to improve the crystallization efficiency of rebaudioside C, the ratio of the volume of the 10-50 wt% methanol to the mass of the dried mixture is controlled to be (1-4):1 in the present application, for example, the ratio of the volume of the 10-50 wt% methanol to the mass of the dried mixture can be 1:1, 2:1, or 4:1.

[0062] In a third aspect, the present application provides a rebaudioside C product, which is separated by using the above method for separating and purifying rebaudioside C from stevia mother liquor sugar, and the rebaudioside C content in the rebaudioside C product is higher than 85 wt%.

[0063] Compared with the prior art, the rebaudioside C product provided by the present application has the same beneficial effects as the method for separating and purifying rebaudioside C from stevia mother liquor sugar described in the above technical solution, and details are not repeated here.

[0064] In a fourth aspect, the present application provides a rebaudioside F product, which is obtained by the method for separating and purifying rebaudioside F from the stevia mother liquor sugar as described above, and the content of rebaudioside F in the rebaudioside F product is higher than 90 wt%.

[0065] Compared with the prior art, the rebaudioside F product provided by the present application has the same beneficial effects as the method for separating and purifying rebaudioside C from the stevia mother liquor sugar as described above, and details are not repeated here.

[0066] In order to better understand the present application, the content of the present application is further illustrated below in combination with specific examples, but the content of the present application is not limited to the following examples only.

[0067] In the following examples, the raw materials used are commercially available raw materials unless otherwise specified.

[0068] Example 1

[0069] The present example provides a method for separating and purifying rebaudioside C from stevia mother liquor sugar, comprising:

[0070] S100, 1000g of stevia mother liquor sugar is dissolved in pure water to obtain a mother liquor sugar diluent, the mass ratio of the stevia mother liquor sugar to the pure water is 1:5, the components of the stevia mother liquor sugar include rebaudioside F with a content of 4.8 wt%, rebaudioside C with a content of 29.9 wt%, rebaudioside A with a content of 19.4 wt%, and the balance is stevioside and impurities;

[0071] S200, the mother liquor sugar diluent is subjected to column adsorption by macroporous adsorption resin; 30 wt%, 50 wt% and 70 wt% ethanol aqueous solutions are used to perform first gradient elution on the macroporous adsorption resin, respectively, the column flow rate of the mother liquor sugar diluent is 20 ml / min, the column flow rate of the ethanol aqueous solution is 40 ml / min, and the first elution liquids of the three concentration sections are collected and mixed;

[0072] S300, the first elution liquid is concentrated and dried using a rotary evaporator, the temperature during the concentration and drying is 80℃, and the rotation rate is 55 rpm; the dried mixture is dissolved in 92 wt% methanol, and is stirred at room temperature for 24 h under the condition of a stirring rate of 45 rpm until crystallization occurs, and is filtered by a Buchner funnel to obtain a first filter cake and a first filtrate; wherein, the mass of the dried mixture to the volume of the methanol is 1:1.5;

[0073] The first filtrate is concentrated using a rotary evaporator under the condition of a temperature of 80℃ and a rotation rate of 50 rpm to obtain a concentrated liquid with a solid content of 10 wt%;

[0074] S400, the concentrated solution is diluted with 80wt% methanol, the diluted solution is passed through a normal phase low-pressure liquid chromatography column, and then the column is subjected to second gradient resolution using 70wt%, 80wt% and 95wt% ethanol aqueous solutions respectively, at each ethanol aqueous solution concentration, the resolution liquid is collected every 0.5BV, the resolution liquid at 80wt% gradient is mixed, wherein the flow rate of the diluted solution passing through the column is 20ml / min, and the flow rate of the ethanol aqueous solution passing 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 at each concentration section is 3 times the volume of the diluted solution;

[0075] S500, the second resolution liquid is concentrated and dried using a rotary evaporator, the temperature during the concentration and drying is 80℃, the rotation rate is 55rpm, the mixture after drying is dissolved in 10wt% methanol, and the mixture is stirred at room temperature for 24h at a stirring rate of 50rpm until crystallization occurs, and then filtered through a Buchner funnel to obtain a second filtrate and a second filter cake of 30g; wherein the mass ratio of the mixture after drying to the volume of the methanol is 1:1.

[0076] The second filter cake obtained by the separation and purification in the embodiment is subjected to rebaudioside C content detection, and the rebaudioside C content in the second filter cake is 87wt%.

[0077] Embodiment 2

[0078] The embodiment provides a method for separating and purifying rebaudioside C and rebaudioside F from stevia mother liquor sugar, comprising:

[0079] S100, 1000g of stevia mother liquor sugar is dissolved in pure water to obtain a mother liquor sugar diluent, the mass ratio of the stevia mother liquor sugar to pure water is 1:10, and the components of the stevia mother liquor sugar include 4.8wt% rebaudioside F, 29.9wt% rebaudioside C and 19.4wt% rebaudioside A, and the balance is stevioside and impurities;

[0080] S200, the mother liquor sugar diluent is passed through a macroporous adsorption resin column, and the rebaudioside C and other glycosides in the mother liquor sugar diluent are adsorbed by the macroporous adsorption resin; the macroporous adsorption resin is subjected to first gradient resolution using 30wt%, 50wt% and 70wt% ethanol aqueous solutions respectively, at each ethanol aqueous solution concentration, the resolution liquid is collected according to the resin column volume of 2BV, the resolution liquid concentration is detected, and the resolution liquid with a rebaudioside C content higher than 40wt% is collected to obtain a first resolution liquid; wherein the flow rate of the mother liquor sugar diluent passing through the column is 20ml / min, and the flow rate of the ethanol aqueous solution passing through the column is 40ml / min;

[0081] S300, the first resolving liquid is concentrated and dried using a rotary evaporator, the temperature during the concentration and drying is 80℃, and the rotation rate is 55 rpm; the dried mixture is dissolved in 95wt% methanol, and is stirred at room temperature for 24 hours under the condition that the stirring rate is 45 rpm until crystallization occurs, and is filtered through 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 is concentrated using a rotary evaporator under the condition that the temperature is 80℃ and the rotation rate is 50 rpm to obtain a concentrated liquid with a solid content of 50wt%;

[0083] S400, the concentrated liquid is diluted with 95wt% methanol, the diluted liquid is passed through a column of a normal-phase low-pressure liquid chromatograph, and then the column is subjected to second gradient resolution using 70wt%, 85wt% and 95wt% ethanol aqueous solutions respectively, the resolving liquid is collected every 0.5BV at each ethanol aqueous solution concentration, the concentration of the resolving liquid is detected, and the resolving liquids in which the content of rebaudioside C is higher than 40wt% are mixed to obtain a second resolving liquid; wherein the flow rate of the diluted liquid passing through the column is 20ml / min, the flow rate of the ethanol aqueous solution passing through the column is 30ml / min, the volume ratio of the concentrated liquid to methanol is 1:10, and the amount of the ethanol aqueous solution at each concentration is 3 times the volume of the diluted liquid;

[0084] S500, the second resolving liquid is concentrated and dried using a rotary evaporator, the temperature during the concentration and drying is 80℃, and the rotation rate is 55 rpm, the dried mixture is dissolved in 50wt% methanol, and is stirred at room temperature for 24 hours under the condition that the stirring rate is 50 rpm until crystallization occurs, and then is filtered through a Buchner funnel to obtain a second filtrate and 25g 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, the second filtrate is concentrated and dried, the dried mixture is dissolved in 60wt% methanol solution and stirred uniformly, and then is passed through a column of a reverse-phase high-pressure liquid chromatograph under the condition that the column pressure is 6MPa and the flow rate is 50ml / min, and then is subjected to third gradient resolution using 50wt%, 60wt% and 70wt% methanol solutions respectively, the concentration of the resolving liquid is detected, the third resolving liquid in which the concentration of rebaudioside F is higher than 90% is collected, and is concentrated and dried using a rotary evaporator under the condition that the temperature is 80℃ and the rotation rate is 55 rpm to obtain a rebaudioside F product; wherein the ratio of the mass of the dried mixture to the volume of the methanol is 1:3.

[0086] The second filter cake prepared in the embodiment is subjected to rebaudioside C content detection, and the rebaudioside F product is subjected to rebaudioside F content detection, the rebaudioside C content in the second filter cake is 91 wt%, and the rebaudioside F content in the rebaudioside F product is 91.5 wt%.

[0087] Example 3

[0088] The embodiment provides a method for separating and purifying rebaudioside C and rebaudioside F from stevia mother liquor sugar, comprising:

[0089] S100, 1000g of stevia mother liquor sugar is dissolved in pure water to obtain a mother liquor sugar diluent, the mass ratio of the stevia mother liquor sugar to pure water is 1:6, the stevia mother liquor sugar comprises 4.8 wt% of rebaudioside F, 29.9 wt% of rebaudioside C, and 19.4 wt% of rebaudioside A, and the balance is stevioside and impurities;

[0090] S200, the mother liquor sugar diluent is subjected to column adsorption through a macroporous adsorption resin; 30 wt%, 50 wt% and 70 wt% ethanol aqueous solutions are used for first gradient elution of the macroporous adsorption resin, and during elution, the eluate is collected at a resin column volume of 2BV for each ethanol aqueous solution concentration, the eluate concentration is detected, and the eluate with a rebaudioside C content higher than 40 wt% is collected to obtain a first eluate; wherein, the column flow rate of the mother liquor sugar diluent is 20 ml / min, and the column flow rate of the ethanol aqueous solution is 40 ml / min;

[0091] S300, the first eluate is concentrated and dried using a rotary evaporator, the concentration and drying temperature is 80℃, and the rotation rate is 55rpm; the dried mixture is dissolved in 95 wt% methanol, and is stirred at room temperature for 24h under the condition of a stirring rate of 45rpm to crystallize and precipitate, and is filtered through 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 is concentrated using a rotary evaporator at a temperature of 80℃ and a rotation rate of 50rpm to obtain a concentrated solution with a solid content of 35 wt%;

[0093] S400, the concentrated solution is diluted with 85wt% methanol, the diluted solution is passed through a normal phase low-pressure liquid chromatography column, and then a second gradient elution is performed on the chromatography column using 70wt%, 80wt% and 95wt% ethanol aqueous solutions respectively, at each concentration of the ethanol aqueous solution, the elution solution is collected every 0.5BV, the concentration of the elution solution is detected, and the elution solutions with a rebaudioside C content higher than 40wt% are mixed to obtain a second elution solution; wherein, the flow rate of the diluted solution passing through the column is 20ml / min, the flow rate of the ethanol aqueous solution passing 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 at each concentration is 3 times the volume of the diluted solution;

[0094] S500, the second elution solution is concentrated and dried using a rotary evaporator, the temperature during the concentration and drying is 80℃, and the rotation rate is 55rpm, the mixture after drying is dissolved in 35wt% methanol, and is stirred at a stirring rate of 50rpm for 24h until crystallization occurs, and then is filtered through a Buchner funnel to obtain a second filtrate and a second filter cake of 27g; wherein, the ratio of the mass of the mixture after drying to the volume of the methanol is 1:3;

[0095] S600, the second filtrate is concentrated and dried, the mixture after drying is dissolved in 70wt% methanol solution and stirred uniformly, and then is passed through a reverse phase high-pressure liquid chromatography column under the conditions of a column pressure of 6MPa and a flow rate of 50ml / min, and then a third gradient elution is performed using 50wt%, 60wt% and 70wt% methanol solutions respectively, the concentration of the elution solution is detected, a third elution solution with a rebaudioside F concentration higher than 90% is collected, and the third elution solution is concentrated and dried using a rotary evaporator under the conditions of a temperature of 80℃ and a rotation rate of 55rpm to obtain a rebaudioside F product, and the ratio of the mass of the mixture after drying to the volume of the methanol is 1:4;

[0096] S700, the third elution solutions with a rebaudioside F content lower than 90wt% in step S600 are combined, and are concentrated and dried under the conditions of a temperature of 45℃ and a stirring rate of 50rpm;

[0097] S800, the mixture after drying in step S700 is dissolved in 30wt% methanol, and is stirred at room temperature and a stirring rate of 55rpm for 24h until crystallization occurs, and then is filtered through a Buchner funnel to obtain a third filtrate and a third filter cake of 15g, wherein, the ratio of the mass of the mixture after drying to the volume of the methanol is 1:4; the third filtrate contains rebaudioside F, and can be collected uniformly as a raw material for recovering rebaudioside F.

[0098] The second filter cake and the third filter cake prepared in the embodiment are respectively subjected to rebaudioside C content determination, and the rebaudioside F product is subjected to rebaudioside F content detection, the rebaudioside C content in the second filter cake is 90 wt%, the rebaudioside C content in the third filter cake is 95 wt%, the rebaudioside F content in the rebaudioside F product is 93 wt%, and the rebaudioside F content in the third filtrate is 50%.

[0099] It can be known from the above embodiment and detection data that high-purity rebaudioside C and rebaudioside F can be separated from stevia mother liquor sugar under the condition of the embodiment, and the separated rebaudioside C and rebaudioside F can be directly used or sold as products. The above technical solution solves the technical problems of high separation and purification difficulty, low separation efficiency and low purity of the separated product of rebaudioside F and rebaudioside C in the existing stevia mother liquor sugar.

[0100] In the description of the above-described embodiments, specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner.

[0101] The above description is merely specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for separating and purifying rebaudioside C and rebaudioside F from a steviol feedstock, characterized in that, The method comprises the following steps: diluting the mother liquor sugar with water to obtain a mother liquor sugar dilution; adsorbing the mother liquor sugar dilution by using a macroporous adsorption resin, and performing first gradient elution by using an ethanol aqueous solution with a mass concentration of 30-70 wt% to obtain a first elution solution; concentrating and drying the first elution solution, dissolving the dried mixture in methanol with a concentration of not less than 90 wt%, and the ratio of the mass of the mixture to the volume of the methanol with a concentration of not less than 90 wt% is 1: (1.5-5), stirring, crystallization, and filtration to obtain a first filter cake and a first filtrate, and concentrating the first filtrate to obtain a concentrated solution with a solid content of 10-50 wt%; diluting the concentrated solution with 80-95 wt% methanol, and performing second gradient elution by using an ethanol aqueous solution with a mass concentration of 70-95 wt% after the dilution solution passes through a normal phase low-pressure liquid chromatography column to obtain a second elution solution; concentrating and drying the second elution solution, dissolving the dried mixture in 10-50 wt% methanol, stirring, crystallization, and filtration to obtain a second filter cake and a second filtrate, and the content of rebaudioside C in the second filter cake is higher than 85 wt%; concentrating and drying the second filtrate, uniformly stirring the dried mixture in 60-70 wt% methanol solution, passing the mixture through a reversed phase high-pressure liquid chromatography column, and performing third gradient elution by using a 50-70 wt% methanol solution to obtain a third elution solution; concentrating and drying the third elution solution to obtain a rebaudioside F product, and the content of rebaudioside F in the rebaudioside F product is higher than 85 wt%.

2. The method of claim 1 for separating and purifying rebaudioside C and rebaudioside F from a steviol feedstock, characterized in that, the mass ratio of the mother liquor sugar to water in the mother liquor sugar dilution 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 of claim 1, wherein the separation and purification of rebaudioside C and rebaudioside F from the steviol feedstock is characterized by, the mixture is dissolved in 10-50 wt% methanol, and the ratio of the mass of the mixture to the volume of the methanol is 1: (1-4).

4. The method of claim 1, wherein the separation and purification of rebaudioside C and rebaudioside F from the steviol feedstock is characterized by, the third elution solution is obtained by using a 50-70 wt% methanol solution to perform third gradient elution, which comprises the following steps: a plurality of third elution solutions with different concentrations are obtained by using a 50-70 wt% methanol solution to perform third gradient elution, the content of rebaudioside F in each third elution solution is detected, and the third elution solutions with a rebaudioside F content higher than 90 wt% are combined, concentrated, and dried to obtain a rebaudioside F product with a rebaudioside F content higher than 90 wt%.

5. The method of claim 4 for separating and purifying rebaudioside C and rebaudioside F from a steviol feedstock, characterized in that, after the third elution solution with a rebaudioside F content lower than 90 wt% is combined, concentrated, and dried, the mixture is dissolved in 10-50 wt% methanol, stirred, crystallized, and filtered to obtain a third filter cake and a third filtrate, and the content of rebaudioside C in the third filter cake is higher than 90 wt%; and the ratio of the volume of the 10-50 wt% methanol to the mass of the dried mixture is (1-4):

1. ​ ​

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