A method for separating rebaudioside f in a mother liquor sugar

Abstract

This invention discloses a method for separating rebaudioside F from mother liquor sugar, comprising the following steps: dissolving the mother liquor sugar and then adsorbing it with an ion exchange resin to remove impurities; drying the effluent and then adding it to a crystallization solution to obtain a crystallization mother liquor; removing the crystallization solvent from the crystallization mother liquor and redissolving it with water; then introducing it into a medium-low pressure preparative chromatography with a stationary phase of normal-phase silica gel containing amino-like polar groups; and selecting high-purity rebaudioside F product based on the peak shape of the chromatogram. This invention, through steps such as ion exchange resin adsorption to remove impurities, crystallization enrichment, and medium-low pressure preparative chromatography adsorption separation, separates high-purity rebaudioside F product with a purity of over 95% from the mother liquor sugar solution, filling a technological gap in the field of rebaudioside F preparation and providing a new approach for the reuse of mother liquor sugar.

Description

Technical Field

[0001] This invention relates to the field of food additive preparation technology, and in particular to a method for separating rebaudioside F from mother liquor sugar. Background Technology

[0002] Stevia glycosides are natural sweeteners extracted from the stevia plant, a member of the Asteraceae family. They are characterized by high sweetness and low calories; their sweetness is 100-300 times that of sucrose, while their calorie content is only 1 / 300th that of sucrose. Stevia contains various monomers. The 2022 national standard for steviol glycosides lists 13 glycosides, including RA, STV, RC, RD, DA, Rub, RB, Sbio, RF, RE, RM, RN, and RO.

[0003] Solvent extraction is currently the main process for commercially extracting stevia. After crystallization to recover high-purity rebaudioside A (RA) and steviol glycosides (ST), the remaining mother liquor is usually spray-dried into a solid powder called mother liquor sugar (MLS), which is generally sold as a low-priced sweetener. Mother liquor sugar contains 50-60% steviol glycosides and some residual polyphenols and flavonoids. RA, STV, and RC are the most abundant monoglycosides in mother liquor sugar and are relatively common and easy to extract. Other steviol glycosides have lower content and are more difficult to extract.

[0004] Rebaudioside-F (RF) is a monomer found in low concentrations in stevia. Its flavor is 200-300 times stronger than sucrose, making it an excellent sweetener. However, its content in the mother liquor is only about 5%, making extraction difficult. Furthermore, the mother liquor contains various steviol glycosides with similar properties. Therefore, existing technologies do not disclose methods for extracting high-purity RF from the mother liquor.

[0005] In summary, there is an urgent need to establish a technical solution for separating high-purity RF from mother liquor sugar. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method for separating rebaudioside F from mother liquor sugar, which fills the technical gap in the prior art for separating high-purity rebaudioside F (RF) products from mother liquor sugar, and enriches the utilization pathways of steviol glycoside mother liquor sugar.

[0007] To achieve the above objectives, the present invention provides a method for separating rebaudioside F from mother liquor saccharide, comprising the following steps:

[0008] Dissolve the mother liquor sugar in water to obtain a mother liquor sugar solution.

[0009] The mother liquor sugar solution was purified by adsorption using an ion exchange resin to obtain the effluent.

[0010] After drying the effluent, it was added to a crystallization solution and crystallized at room temperature. The crystals were then filtered to remove some RA and STV crystals, yielding the mother liquor.

[0011] The crystallization solvent was removed from the mother liquor, and the solution was redissolved in water to obtain a steviol glycoside mixture.

[0012] The steviol glycoside mixture was subjected to preparative chromatography at medium and low pressure, wherein the stationary phase was a normal-phase silica gel packing material with amino-like polar groups. After elution, the lebodiin F solution was collected according to the peak shape of the chromatogram.

[0013] This invention discloses a method for separating rebaudioside F from mother liquor sugar. The method includes dissolving the mother liquor sugar; firstly, removing impurities from the solution using an ion exchange resin to avoid affecting subsequent crystallization and chromatographic separation; then, crystallizing the purified solution to remove RA and STV, which are abundant glycosides in steviol glycosides and constitute a large proportion of the mother liquor sugar. The purpose of removing some RA and STV through crystallization is to reduce the total glycoside content and increase the proportion of RF in the total glycosides, i.e., enrichment; after removing the crystallization solvent, the crystallized mother liquor is then subjected to a medium-low pressure preparative chromatography for separation. The medium-low pressure preparative chromatography column is packed with a normal-phase silica gel packing material containing amino-like polar groups. This packing material is an adsorbent with numerous micropores. When the crystallized mother liquor enters the resin column packed with the packing material, it is adsorbed into the resin column due to the unique adsorption force of the packing material. Since the adsorption capacity of each monoglycoside in steviol glycoside is different, a chromatographic phenomenon occurs in the stationary phase, i.e., substances with weak adsorption force remain in the lower layer, and substances with strong adsorption force remain in the upper layer. In this invention, the packing material has a strong adsorption capacity for rebaudioside F. The adsorption capacity of inulin is as follows: RA > RC ≈ RF > DA > STV. Since the adsorption capacity of the packing for RC and RF is similar, adsorption by the packing alone cannot separate high-purity RF products. Therefore, in the subsequent analysis process, this invention uses the molecular sieving effect of positive silica packing, that is, substances with smaller molecular weights pass through the sieve faster and preferentially elute from the stationary phase, while substances with larger molecular weights pass through the sieve slower and elute later. Since the molecular weight of RF is slightly smaller than that of RC, RF elutes before RC during the elution process, thus achieving effective separation of RC and RF. In addition, since the RF effluent is located in the middle of the elution process, it is necessary to accurately define the start and end points of collection in order to effectively collect high-purity RF. Based on this, this invention uses chromatographic peak shape to determine the start and end points. That is, during the elution process, the peak shape of the eluent is monitored in real time by preparative chromatography at medium and low pressure (displayed by a detector). Collection begins when the peak shape of RF begins to appear and stops when the RF peak shape disappears, finally collecting the rebaudioside F solution. Experimental results show that the purity of the rebaudioside F product collected by the method described in this invention can reach over 95%. Attached Figure Description

[0014] Figure 1 The peak shape of the eluent as shown by the detector in the low-to-medium pressure preparative chromatography of Example 1;

[0015] Figure 2 The peak shape of the eluent as shown by the detector in the low-to-medium pressure preparative chromatography of Example 2;

[0016] Figure 3 The peak shape of the eluent as shown by the detector in the low-to-medium pressure preparative chromatography of Example 3. Detailed Implementation

[0017] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0018] This invention provides a method for separating rebaudioside F from mother liquor saccharide, comprising the following steps:

[0019] Dissolve the mother liquor sugar in water to obtain a mother liquor sugar solution.

[0020] The mother liquor sugar solution was purified by adsorption using an ion exchange resin to obtain the effluent.

[0021] After drying the effluent, it was added to a crystallization solution and crystallized at room temperature. The crystals were then filtered to remove some RA and STV crystals, yielding the mother liquor.

[0022] The crystallization solvent was removed from the mother liquor, and the solution was redissolved in water to obtain a steviol glycoside mixture.

[0023] The steviol glycoside mixture was subjected to preparative chromatography at medium and low pressure, wherein the stationary phase was a normal-phase silica gel packing material with amino-like polar groups. After elution, the lebodiin F solution was collected according to the peak shape of the chromatogram.

[0024] This invention first involves dissolving the mother liquor sugar in water to obtain a mother liquor sugar solution. In this invention, the solid content of the obtained mother liquor sugar solution is preferably 5-15 wt%. This invention does not have any particular limitation on the water used; for example, purified water, distilled water, or pure water can be used. In this invention, after adding the mother liquor sugar to the water, it is preferable to stir, with a stirring rate preferably of 20-100 rpm; the purpose of stirring is to promote the dissolution rate of the mother liquor sugar in the water.

[0025] After obtaining the mother liquor sugar solution, the present invention removes impurities by adsorption with an ion exchange resin to obtain the effluent. In the present invention, the ion exchange resin is preferably a cation exchange resin and / or anion exchange resin; that is, the ion exchange resin can be a cation exchange resin, anion exchange resin, or a combination of cation and anion exchange resins; when both cation and anion exchange resins are used simultaneously, the cation and anion exchange resins are preferably used in series column adsorption, and the volume ratio of the cation and anion exchange resins is preferably 1:1. In the present invention, the feed rate of the mother liquor sugar solution is preferably 1-4 BV of the exchange volume of the cation or anion exchange resin, and the flow rate is preferably 0.5-1 BV / h. This invention does not specifically limit the cation exchange resin used. For example, the cation exchange resin used in this invention is a polymer obtained by polymerizing styrene and divinylbenzene and then sulfonating it with sulfuric acid. The purpose of using cation exchange resin for adsorption in this invention is to remove impurities such as salts, saponins, and alkaloids present in the mother liquor sugar solution. This invention also does not specifically limit the anion exchange resin used. For example, the anion exchange resin used in this invention refers to an ion exchange resin containing basic groups in its molecules, which is alkaline in solution and can exchange anions in solution with its hydroxyl ions. The purpose of using anion exchange resin for adsorption in this invention is to remove impurities such as pigments, coumarins, and flavonoids from the mother liquor sugar solution. The purpose of using cation exchange resin and / or anion exchange resin for adsorption in this invention is to remove impurities from the mother liquor sugar solution and to obtain a more pure steviol glycoside solution through preliminary purification.

[0026] After obtaining the effluent, the present invention dries the effluent and then adds it to a crystallization solution for crystallization at room temperature. Filtering removes some RA and STV crystals, yielding a crystallization mother liquor. In this invention, the effluent is preferably dried using a rotary evaporator, a constant temperature chamber, or spray drying. Specifically, when using a rotary evaporator or a constant temperature chamber, the drying temperature is preferably 85-105℃; when using spray drying, the inlet air temperature is preferably 160-180℃, and the outlet air temperature is preferably 80-100℃. It should be understood that the purpose of drying the effluent in this invention is to remove moisture to facilitate subsequent crystallization. The resulting solid mixture is primarily a mixture of steviol glycosides.

[0027] In this invention, the crystallization solution is preferably an aqueous solution of methanol, ethanol, acetone, or n-butanol; the concentration of the crystallization solution is preferably 5-35% (v / v). In this invention, the ratio of the solid mixture after drying the effluent to the crystallization solution is preferably 1:(1-3)(w / v). In this invention, the crystallization temperature is preferably room temperature, such as 25°C, and the crystallization time is preferably 10-50 hours. In the mother liquor sugar solution, RA and STV are high-content steviol glycosides, while RF is a low-content steviol glycoside. The purpose of crystallization in this invention is to partially remove the high-content RA and STV from the solution, increase the proportion of RF in the steviol glycosides, and avoid interference from RA and STV on the separation effect.

[0028] After crystallization, the present invention obtains the crystallization mother liquor by filtration. In the present invention, the filtration is carried out by plate and frame filtration, more preferably by medium-speed or high-speed plate and frame filtration; the pore size of the filter cloth is preferably 30-120 μm.

[0029] After obtaining the mother liquor for crystallization, this invention removes the crystallization solvent from the mother liquor and redissolves it with water to obtain a steviol glycoside mixture. In this invention, removing the crystallization solvent from the mother liquor includes evaporating the mother liquor to dryness, preferably using a rotary evaporator and a constant temperature oven, at a temperature preferably 85-105°C. The purpose of evaporating the mother liquor to dryness is to remove the organic solvent used in the previous crystallization process, thus avoiding its impact on subsequent RF separation. In this invention, the solid content of the steviol glycoside mixture is preferably 5-20%. Controlling the solid content of the redissolved steviol glycoside solution within this range facilitates subsequent medium- and low-pressure chromatographic separation.

[0030] After obtaining the steviol glycoside mixture, the present invention uses preparative chromatography at medium and low pressure to elute the mixture and collect rebaudioside F based on the peak shape of the chromatogram. In the present invention, the preparative chromatography at medium and low pressure preferably includes a resin column, a detector, and a peristaltic pump; wherein, the resin column is packed with packing material for adsorbing and separating steviol glycosides; the detector is used to display different peak shapes in the form of chromatograms according to different components; and the peristaltic pump is used to feed the steviol glycoside mixture into the resin column. In the present invention, the rebaudioside F solution is obtained by preparative chromatography at medium and low pressure and collection based on the peak shape of the chromatogram, preferably specifically including: the peristaltic pump feeding the steviol glycoside mixture into a resin column packed with normal-phase silica gel for adsorption; eluting the adsorbed normal-phase silica gel with eluent; and collecting the rebaudioside F solution based on the peak shape displayed by the detector. In this invention, the stationary phase for the medium-low pressure preparative chromatography is preferably a normal-phase silica gel packing material with amino-like polar groups. More preferably, the normal-phase silica gel packing material with amino-like polar groups is a normal-phase silica gel packing material with cyano (such as cyanopropyl) or 1,2-dihydroxypropyl groups. More specifically, the resin column packed with normal-phase silica gel packing material can be a cyano(CN) column or a (Diol) glycol column. It should be understood that the cyano groups on the surface of the cyano column will generate a strong dipole-dipole interaction with the amino-like polar groups in the packing material, while the propyl chains contained in the amino-like polar groups in the packing material will provide hydrophobic effects, giving it unique selectivity and broadening the range of chromatographic applications. The stationary phase of the glycol column is based on ultrapure, fully porous spherical silica gel and is bonded with organosilanes containing 1,2-dihydroxypropyl functional groups. Compared with the cyano column, it has stronger polarity, and the two hydroxyl groups provide moderate normal-phase retention capacity. Combined with the normal-phase silica gel packing material with amino-like polar groups, it has superior selectivity.

[0031] The feed rate of the steviol glycoside mixture is preferably 0.1-0.5 BV of the normal-phase silica gel packing, and the flow rate is preferably 0.5-1 BV / h; the eluent is an ethanol solution with a volume fraction of 80-95% (v / v), the amount of ethanol solution used is 4-8 BV of the normal-phase silica gel packing, and the flow rate is 1-2 BV / h; when the detector displays an RF peak shape, collection begins and stops when the RF peak shape disappears, yielding a rebaudioside F solution.

[0032] It should be understood that the amino-like polar group-containing normal-phase silica gel packing material in this invention is an adsorbent with multiple micropores. After the crystallization mother liquor enters the stationary phase (resin column) packed with the packing material, it will be adsorbed into the stationary phase due to the unique adsorption force of the packing material. Since each monoglycoside in steviol glycoside has a different adsorption capacity, a chromatographic phenomenon will occur in the stationary phase, that is, substances with weak adsorption force are in the lower layer, and substances with strong adsorption force remain in the upper layer. In the subsequent desorption process, the molecular sieving effect of the normal-phase silica gel packing material is used, that is, substances with smaller molecular weights pass through the sieve faster and are preferentially removed from the stationary phase. The eluent is eluted in a fixed phase; conversely, substances with larger molecular weights elute more slowly and later. Based on this, the chromatographic separation of this invention is employed, utilizing low-to-medium pressure preparative chromatography to monitor the peak shape of the eluent in real time (via a detector) during the elution process. In terms of molecular weight, DA < STV < RF < RC < RA. Due to the effect of molecular sieves, steviol glycosides elute sequentially from smallest to largest molecular weight, and the elution order is also in ascending order of molecular weight. Collection begins when the RF peak shape begins to appear, ultimately yielding a rebaudioside F solution. This invention utilizes the principles of molecular sieves and like dissolves like to separate high-purity RF products from the mother liquor sugar. Figure 1-3 The peak shapes of the eluents displayed by the detector in the low-to-medium pressure preparative chromatography of Examples 1-3 are listed below. The coordinate axis represents time (min), and the vertical axis represents the intensity (mV) of the detector output signal. This signal intensity reflects the properties of different substances; that is, different substances absorb different response values ​​when passing through the ultraviolet detector, thus converting the light signal into an electrical signal, which is displayed in the form of a spectrum. It can be understood that one peak corresponds to one substance. RF is the third peak; we only need to collect it at the beginning of the peak and stop collecting it at the end to obtain a high-purity RF product.

[0033] After obtaining the rebaudioside F solution, the present invention further includes: evaporating the rebaudioside F solution to dryness to obtain rebaudioside F crystals. In the present invention, the drying is preferably performed using a rotary evaporator or spray drying; when using a rotary evaporator, the drying temperature is preferably 85-120°C; when using spray drying, the inlet air temperature is preferably 160-180°C, and the outlet air temperature is preferably 80-100°C.

[0034] To better illustrate the technical solution of the present invention, the following specific embodiments are also provided. It should be understood that, unless otherwise specified, all raw materials used in the following embodiments are commercially available. Specifically, Table 1 below lists the main components of three batches of steviol glycoside mother liquor, all of which were provided by Zhucheng Haotian Pharmaceutical Co., Ltd.

[0035] Table 11-3: Stevioside Components of Mother Liquor in Batch 3

[0036]

[0037] Example 1

[0038] S1. Take 100g of the mother liquor sugar from batch 1 and dissolve it in water to obtain 2L of mother liquor sugar solution with a solid content of 5wt%;

[0039] S2. 2L of mother liquor sugar solution was passed through 0.5L of cation exchange resin and 0.5L of anion exchange resin for adsorption at a flow rate of 0.8 BV / h to obtain 1.9L of effluent;

[0040] S3. The effluent is dried using a rotary evaporator at 105°C to obtain 90g of a solid mixture;

[0041] S4. Add 90g of solid mixture to 0.2L of 35% (v / v) methanol aqueous solution, crystallize at room temperature for 30h, and obtain 0.19L of crystallization mother liquor by medium-speed plate and frame filtration (filter cloth pore size 80um);

[0042] S5. The crystallization mother liquor is evaporated to dryness using a rotary evaporator at 85°C, and then redissolved in water to obtain 0.34L of a steviol glycoside mixture with a solid content of 15%;

[0043] S6. Feed 0.34L of steviol glycoside mixture into a cyano(CN) resin column packed with 2.5L of normal-phase silica gel with cyanopropyl groups for adsorption at a flow rate of 0.8 BV / h.

[0044] S7. The adsorbed normal-phase silica gel packing was eluted with 15 L of 80% (v / v) ethanol solution at a flow rate of 1.5 BV / h.

[0045] S8. When the detector displays an RF peak shape (e.g.) Figure 1 The collection process begins, and stops when the RF peak disappears, yielding 5 L of lebodiin F solution.

[0046] Example 2

[0047] S1. Take 120g of the mother liquor sugar from batch 2 and dissolve it in water to obtain 1.2L of mother liquor sugar solution with a solid content of 10wt%;

[0048] S2. 1.2 L of mother liquor sugar solution was adsorbed through 0.8 L of cation exchange resin at a flow rate of 0.5 BV / h to obtain 1.15 L of effluent;

[0049] S3. The effluent is dried in a constant temperature oven at 85°C to obtain 100g of solid mixture;

[0050] S4. Add 100g of solid mixture to 0.3L of 20% (v / v) ethanol aqueous solution, crystallize at room temperature for 10h, and obtain 0.25L of crystallization mother liquor by medium-speed plate and frame filtration (filter cloth pore size 120um);

[0051] S5. The crystallization mother liquor is evaporated to dryness using a rotary evaporator at 90°C, and then redissolved in water to obtain 1L of steviol glycoside mixture with a solid content of 5%;

[0052] S6. Feed 1L of steviol glycoside mixture into a glycol-based resin column packed with 2.5L of normal-phase silica gel containing 1,2-dihydroxypropyl groups for adsorption at a flow rate of 0.5 BV / h.

[0053] S7. The adsorbed normal-phase silica gel packing was eluted with 10 L of 85% (v / v) ethanol solution at a flow rate of 1 BV / h.

[0054] S8. When the detector displays an RF peak shape (e.g.) Figure 2 The collection was started and stopped when the RF peak disappeared, yielding 4.7 L of lebodiin F solution.

[0055] Example 3

[0056] S1. Take 100g of the mother liquor sugar from batch 3 and add it to water. Dissolve it under stirring at 50rpm to obtain 0.67L of mother liquor sugar solution with a solid content of 15wt%.

[0057] S2. Pass 0.67 L of the mother liquor sugar solution through 0.5 L of anion exchange resin for adsorption at a flow rate of 1 BV / h to obtain 0.6 L of effluent.

[0058] S3. Spray-dry the effluent at an inlet air temperature of 180°C and an outlet air temperature of 90°C to obtain 85g of solid mixture;

[0059] S4. Add 85g of solid mixture to 1.7L of 5% (v / v) acetone aqueous solution, crystallize at room temperature for 50h, and obtain 1.63L of crystallization mother liquor by medium-speed plate and frame filtration (filter cloth pore size 30um);

[0060] S5. The crystallization mother liquor is evaporated to dryness in a constant temperature oven at 105°C, and then redissolved in water to obtain 0.23L of a steviol glycoside mixture with a solid content of 20%;

[0061] S6. Feed 0.23L of steviol glycoside mixture into a cyano(CN) resin column packed with 2L of normal-phase silica gel with cyanopropyl groups for adsorption at a flow rate of 1 BV / h.

[0062] S7. The adsorbed normal-phase silica gel packing was eluted with 16 L of 95% (v / v) ethanol solution at a flow rate of 2 BV / h.

[0063] S8. When the detector displays an RF peak shape (e.g.) Figure 3 The collection process begins, and stops when the RF peak disappears, yielding 4 L of lebodiin F solution.

[0064] Example 4

[0065] The 5L solution of rebaudioside F from Example 1 was dried in a rotary evaporator at 85°C to obtain 18g of rebaudioside F crystals.

[0066] The purity of RF in the crystal was found to be 98.8%.

[0067] Example 5

[0068] The 4.7 L solution of rebaudioside F from Example 2 was dried in a rotary evaporator at 120 °C to obtain 21 g of rebaudioside F crystals.

[0069] The purity of RF in the crystal was found to be 97.6%.

[0070] Example 6

[0071] The 4L solution of rebaudioside F from Example 3 was spray-dried at an inlet air temperature of 180°C and an outlet air temperature of 90°C to obtain 16.8g of rebaudioside F crystals.

[0072] The purity of RF in the crystal was found to be 97.9%.

[0073] Comparative Example 1

[0074] In this embodiment, the "cyano(CN)-based resin column with cyanopropyl normal-phase silica gel filler" in step S6 of embodiment 1 is replaced with "NH2-based silica gel column", while other conditions remain unchanged;

[0075] 20g of rebaudioside F crystals with a purity of 34% were obtained under the same drying conditions as in Example 4.

[0076] Comparative Example 2

[0077] In this embodiment, the "35% (v / v) methanol aqueous solution" in step S4 of Example 1 is replaced with "pure water" in equal amounts, that is, no crystallization process is performed, and other conditions remain unchanged;

[0078] 15.7g of rebaudioside F crystals with a purity of 26.7% were obtained under the same drying conditions as in Example 4.

[0079] Comparative Example 3

[0080] In this embodiment, the "resin column with 1,2-dihydroxypropyl normal-phase silica gel packing" in step S6 of embodiment 2 is replaced with "C18 reverse-phase chromatography column", while other conditions remain unchanged;

[0081] 24g of rebaudioside F crystals with a purity of 27% were obtained under the same drying conditions as in Example 4.

[0082] Comparative Example 4

[0083] In this embodiment, the "80% (v / v) ethanol solution" in step S7 of embodiment 3 is replaced by an equal amount of "70% (v / v) ethanol solution", while other conditions remain unchanged;

[0084] 22g of rebaudioside F crystals with a purity of 33% were obtained under the same drying conditions as in Example 4.

[0085] Test case

[0086] The determination method of each steviol glycoside in this invention is based on the national standard GB1886.355-2022.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for separating rebaudioside F from a mother liquor sugar, characterized by, Includes the following steps: Dissolve the sugar in the mother liquor in water to obtain a sugar solution. The mother liquor sugar solution was purified by adsorption using an ion exchange resin to obtain the effluent; After drying the effluent, it was added to a crystallization solution and crystallized at room temperature. The crystals were then filtered to remove some RA and STV crystals, yielding the mother liquor. The crystallization solvent was removed from the mother liquor, and the solution was redissolved in water to obtain a steviol glycoside mixture. The steviol glycoside mixture was subjected to preparative chromatography at medium and low pressure, wherein the stationary phase was a normal-phase silica gel packing material containing cyanopropyl groups or a normal-phase silica gel packing material containing 1,2-dihydroxypropyl groups. After elution with an ethanol solution of 80-95% (v / v), the lebodiin F solution was collected according to the peak shape of the chromatogram.

2. The separation method of claim 1, wherein, The solid content of the mother liquor sugar solution is 5-15 wt%.

3. The separation method of claim 1, wherein, The ion exchange resin is a cation exchange resin and / or anion exchange resin; when both cation exchange resin and anion exchange resin are used simultaneously, the cation exchange resin and anion exchange resin are used for column adsorption; and / or, The feed rate of the mother liquor sugar solution is 1-4 BV of the volume of the cation exchange resin or anion exchange resin, and the flow rate is 0.5-1 BV / h.

4. The separation method of claim 1, wherein, The effluent is dried using a rotary evaporator, a constant temperature chamber, or a spray dryer.

5. The separation method of claim 1, wherein, The crystallization solution is an aqueous solution of methanol, ethanol, acetone or n-butanol; the concentration of the crystallization solution is 5-35% (v / v).

6. The separation method according to claim 1 or 5, characterized in that, The ratio of the solid mixture after drying the effluent to the crystallization solution is 1:(1-3)(w / v); and / or the crystallization time is 10-50 h.

7. The separation method according to claim 1, characterized in that, The removal of the crystallization solvent from the mother liquor includes: evaporating the mother liquor to dryness; The evaporation process is carried out using a rotary evaporator and a constant temperature chamber, and the evaporation temperature is 85-105℃.

8. The separation method according to claim 1, characterized in that, The solid content of the steviol glycoside mixture is 5-20%.

9. The separation method according to claim 1, characterized in that, The feed rate of the steviol glycoside mixture is 0.1-0.5 BV of the stationary phase volume, and the flow rate is 0.5-1 BV / h; and / or, The eluent is an ethanol solution with a volume fraction of 80-95% (v / v), wherein the volume of the ethanol solution is 4-8 BV of the normal-phase silica gel packing material, and the flow rate is 1-2 BV / h; and / or, When the spectral peak shape appears as an RF peak, collection begins and stops when the RF peak shape disappears, yielding a solution of lebodiin F.

10. The separation method according to claim 1, characterized in that, After obtaining the lebodiin F solution, the process further includes: drying the lebodiin F solution to obtain lebodiin F crystals.

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