A method for separating rebaudioside n, o

Abstract

This invention discloses a method for separating retinoic acid N and O, comprising the following steps: mixing steviol glycoside crystallization mother liquor with water to obtain an aqueous solution of the crystallization mother liquor; separating the aqueous solution of the crystallization mother liquor through a resin, and then eluenting it with solvent 1 to obtain steviol glycoside I; adding steviol glycoside I to solvent 2 for crystallization, then separating it through a chromatographic column, and then eluting it with solvent 3 to obtain steviol glycosides II and III from different elution segments; adding steviol glycosides II and III to solvent 4 for crystallization to obtain steviol glycoside IV, whose main component is RN, and steviol glycoside V, whose main component is RO, respectively. This invention uses specific resin adsorption and chromatographic column separation to separate the sugars in the steviol glycoside mother liquor, and then further separates them through crystallization and recrystallization methods, finally separating and purifying them to obtain high-purity RN and RO products, filling the technical gap in the treatment of steviol glycoside crystallization mother liquor, and providing a new approach for the production of RN and RO.

Description

Technical Field

[0001] This invention relates to the field of food additive preparation technology, and in particular to a method for separating rebaudioside N and O. Background Technology

[0002] Stevia glycosides are natural sweeteners extracted from the herbaceous plant Stevia repens of the Asteraceae family. They are characterized by high sweetness and low calories, with a sweetness 100-300 times that of sucrose and only 1 / 300th the calories. In the 2022 version of the new national standard for steviol glycosides, the range of glycosides has increased from 9 in the 2014 version (RA, ST, RC, RD, DA, Rub, RB, Sbio, RF) to 13, consistent with the international standard JECFA (2017). Four new compounds have been added: rebaudioside E (RE), rebaudioside M (RM), rebaudioside N (RN), and rebaudioside O (RO).

[0003] Chinese patent document CN 108712864A describes that steviosides containing low levels of rebaudioside N and O exhibit a similar taste to steviosides containing high levels of rebaudioside A, D, and M, indicating that rebaudioside N and O have a significant positive impact on the sweetness characteristics of steviosides.

[0004] Direct extraction of stevia is a common method for extracting steviol glycosides. However, the content of RN and RO in stevia leaves is low. After certain purification and enrichment, the content can be increased to a certain extent (to 10-12%), but the total glycoside content is low and the impurity content is high. It is difficult to separate and purify them using general resin and crystallization methods.

[0005] Stevioside crystallization mother liquor is a powdered solid formed by spray-drying the reaction tail liquid obtained in industrial processes after solvent extraction to recover rebaudioside A (RA) and steviol glycoside (ST). It is also known as mother liquor sugar (MLS) and is generally sold as a low-priced sweetener. Stevioside crystallization mother liquor contains approximately 60% steviol glycosides, including the aforementioned 13 glycosides. Further recovery of steviol glycosides from the crystallization mother liquor not only increases the yield of steviol glycosides but also transforms waste into valuable resources, achieving waste utilization. For example, Chinese patent CN108530503B discloses a method for separating and purifying rebaudioside C, which can recover high-purity rebaudioside C (RC) from the steviol glycoside crystallization mother liquor (mother liquor sugar) with a high yield; it can also recover rebaudioside A (RA).

[0006] The content of rebaudioside N and O in the crystallization mother liquor is 10-15%, which makes separation difficult. Therefore, there is no existing method for separating and purifying high-purity rebaudioside N and O from the mother liquor of steviol glycoside crystallization. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a method for separating rebaudioside N and O, which fills the gap in the prior art for separating and purifying high-purity rebaudioside N and O from the mother liquor of steviol glycoside crystallization.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a method for separating rebaudioside N and O, comprising the following steps:

[0009] The mother liquor for steviol glycoside crystallization was mixed with water to obtain an aqueous solution of the mother liquor for crystallization.

[0010] The aqueous solution of the crystallization mother liquor was separated by a resin. After separation, the resin was eluted with solvent 1 to obtain an eluent. The eluent was concentrated and dried to obtain steviol glycoside I.

[0011] The steviol glycoside I was added to solvent 2 for crystallization to obtain the mother liquor for steviol glycoside I crystallization;

[0012] The mother liquor of the crystallized steviol glycoside I was separated by a chromatographic column, then eluted with solvent 3, and the 2-3 BV elution was collected and concentrated and dried to obtain steviol glycoside II. The 3-5 BV elution was collected and concentrated and dried to obtain steviol glycoside III.

[0013] Steviosides II and III were added to solvent 4 and crystallized to obtain steviol glycosides IV and V, respectively.

[0014] This invention discloses a method for separating rebaudioside N and O, comprising the following steps: mixing steviol glycoside crystallization mother liquor with water to prepare an aqueous solution of the crystallization mother liquor; separating the aqueous solution of the crystallization mother liquor through a resin, wherein the resin is eluted by solvent 1 to obtain steviol glycoside I; adding the steviol glycoside I to solvent 2 for crystallization to obtain a steviol glycoside I crystallization mother liquor; separating the steviol glycoside I crystallization mother liquor by passing it through a chromatographic column, and then eluting it with solvent 3 to obtain steviol glycosides II and III from different elution segments; adding steviol glycosides II and III to solvent 4 for crystallization to obtain steviol glycoside IV, whose main component is rebaudioside N, and steviol glycoside V, whose main component is rebaudioside O, respectively. Compared with existing technologies, this invention uses the mother liquor from steviol glycoside production as raw material. It employs specific resin adsorption and chromatographic column separation of the steviol glycoside mother liquor, followed by further separation through crystallization and recrystallization. Ultimately, high-purity rebaudioside N and O products are obtained through separation and purification, filling a technological gap in the treatment of steviol glycoside crystallization feed solutions and providing a new approach for the production of rebaudioside N and O. Experimental results show that the contents of rebaudioside N and O in steviol glycosides IV and V obtained after crystallization can reach 85.3% and 79.4%, respectively; the contents of rebaudioside N and O in steviol glycosides VI and VII obtained after recrystallization can reach 97.5% and 96.2%, respectively. Attached Figure Description

[0015] Figure 1 This is the chromatogram of rebaudioside N obtained by recrystallization in Example 4;

[0016] Figure 2 This is the chromatogram of rebaudioside O obtained by recrystallization in Example 5. 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 N and O, comprising the following steps:

[0019] The mother liquor for steviol glycoside crystallization was mixed with water to obtain an aqueous solution of the mother liquor for crystallization.

[0020] The aqueous solution of the crystallization mother liquor was separated by a resin. After separation, the resin was eluted with solvent 1 to obtain an eluent. The eluent was concentrated and dried to obtain steviol glycoside I.

[0021] The steviol glycoside I was added to solvent 2 for crystallization to obtain the mother liquor for steviol glycoside I crystallization;

[0022] The mother liquor of the crystallized steviol glycoside I was separated by a chromatographic column, then eluted with solvent 3, and the 2-3 BV elution was collected and concentrated and dried to obtain steviol glycoside II. The 3-5 BV elution was collected and concentrated and dried to obtain steviol glycoside III.

[0023] Steviosides II and III were added to solvent 4 and crystallized to obtain steviol glycosides IV and V, respectively.

[0024] This invention first mixes the mother liquor for steviol glycoside crystallization with water to obtain an aqueous solution of the mother liquor. The source of the mother liquor for steviol glycoside crystallization is not particularly important in this invention; any mother liquor prepared using a steviol glycoside extraction method well-known in the art can be used. It should be noted that the mother liquor for steviol glycoside crystallization is generally a byproduct of steviol glycoside preparation using a solution extraction process, and it contains steviol glycosides including RA, ST, RC, RD, DA, Rub, RB, Sbio, RF, RE, RM, RN, and RO. In this invention, the total steviol glycoside content in the mother liquor for steviol glycoside crystallization is preferably 55-65 wt%, and the RN and RO content is preferably 6-12 wt%. This invention includes diluting the mother liquor for steviol glycoside crystallization with water to obtain an aqueous solution of the mother liquor. In this invention, the concentration of the aqueous solution of the mother liquor for crystallization is preferably 5-10 wt%, more preferably 7 wt%. In this invention, the process of diluting the steviol glycoside crystallization mother liquor with water also includes stirring. This invention does not have specific requirements for the stirring rate, as long as it achieves complete / uniform dilution. It should be understood that the steviol glycoside crystallization mother liquor itself has high viscosity (or is a powder solid formed after spray drying) and cannot be directly adsorbed and separated. The purpose of diluting with water is to improve the effect and efficiency of subsequent resin adsorption.

[0025] After obtaining the aqueous solution of the crystallization mother liquor, the present invention separates the aqueous solution of the crystallization mother liquor through a resin. After separation, the resin is eluted with solvent 1 to obtain an eluent. The eluent is then concentrated and dried to obtain steviol glycoside I. In the present invention, there is no particular limitation on the method of loading the aqueous solution of the crystallization mother liquor through the resin; for example, a pump can be used for loading. The loading volume is preferably 1-2 BV of the resin volume. In the present invention, the resin is preferably a nonpolar or weakly polar adsorption resin with a styrene structure, more preferably LX-T3, LK1300s, LX-1, LX-18, LX21SS, or LK20SS resins, and most preferably LX-18 resin. This type of resin has a strong selective adsorption effect on steviol glycosides, which can result in a higher loading capacity during the adsorption process. The present invention does not have a particular limitation on the preparation method of the resin; bonding methods well known to those skilled in the art can be used. The present invention does not impose any special restrictions on the form of the resin carrier. For example, a chromatography column can be selected, preferably a resin chromatography column or a glass chromatography column; the diameter-to-height ratio of the chromatography column in the present invention is preferably 1:(8-5).

[0026] In this invention, after resin adsorption and separation, the resin is subjected to a solvent 1 for desorption treatment to obtain a desorbed solution. In this invention, the solvent 1 is preferably an alcohol-water solution; more preferably, it is a methanol-water solution or an ethanol-water solution; most preferably, it is a methanol-water solution or an ethanol-water solution with a volume ratio of (0-30):100. In this invention, the amount of solvent 1 is preferably 1-3 BV of the resin volume, more preferably 2 BV. The feed and water washing flow rates in the desorption process are preferably 1-2 BV / h. This invention does not have special limitations on the temperature and time of the desorption process; parameters well known to those skilled in the art can be used.

[0027] After obtaining the eluent, this invention further includes concentrating and drying it to obtain steviol glycoside I. In this invention, the drying temperature is preferably 70-90°C; the concentration time is not particularly limited, as long as the weight no longer changes, for example, 4-6 hours. It should be understood that the steviol glycoside I is a composition (crystal) containing multiple steviol glycosides obtained after resin adsorption and solvent desorption of the crystallization mother liquor. Rebaudioside N and O (RN, RO) have been effectively enriched, and their content has been significantly increased compared to the original content in the crystallization mother liquor. In some embodiments, the content of RN and RO in the steviol glycoside I is 15-20% and 15-20%, respectively. Besides RN and RO, other types of steviol glycosides include RD, RM, and RE.

[0028] After obtaining steviol glycoside I, the present invention adds steviol glycoside I to solvent 2 for crystallization to obtain steviol glycoside I crystallization mother liquor. In the present invention, solvent 2 is preferably an alcohol solution, more preferably a methanol or ethanol solution, and most preferably a 90-95 wt% methanol or ethanol solution; the amount of solvent 2 added is preferably 1-3 BV, more preferably 2 BV. In the present invention, the temperature of the crystallization process is preferably 20-30℃, more preferably 15℃; the time is preferably 10-15 h, more preferably 12 h. In the present invention, the crystallization process also includes stirring. The present invention does not have a special limitation on the stirring rate, as long as it can effectively promote crystal precipitation; the preferred stirring rate is 60-120 rpm, more preferably 80-100 rpm. After crystallization, the present invention retains the crystallization mother liquor, that is, the two glycosides RN and RO do not precipitate through the crystallization process, but are retained in the crystallization mother liquor, awaiting further purification. It should be understood that the purpose of high-alcohol crystallization is to remove some other glycosides. Other glycosides crystallize out at this alcohol concentration to improve the feeding and adsorption separation effect.

[0029] After obtaining the mother liquor of steviol glycoside I crystals, the present invention separates the mother liquor of steviol glycoside I crystals by passing it through a chromatographic column; then elutes it with solvent 3, collects 2-3 BV of the elution fraction and concentrates and dries it to obtain steviol glycoside II, collects 3-5 BV of the elution fraction and concentrates and dries it to obtain steviol glycoside III. The present invention does not have special requirements for the method of loading the mother liquor of steviol glycoside I crystals through the chromatographic column; for example, a pump can be used for loading. The loading volume is preferably 2-5% of the chromatographic column volume, more preferably 3%. In the present invention, the chromatographic column is preferably a low-pressure, medium-pressure, or high-pressure chromatographic column, more preferably a low-pressure or medium-pressure chromatographic column; the diameter-to-height ratio of the chromatographic column is preferably 1:(10-12). In the present invention, the packing material of the chromatographic column is preferably a normal chromatographic packing material, more preferably one of amino silica gel packing material, aminopropyl packing material, MB70-75, or SMB70-45, and most preferably amino silica gel packing material. In this invention, the amino-bonded silica gel packing material is preferably Unisil NH2 or Amino-NH2; the aminopropyl packing material is preferably COPURE NH2. It should be understood that Unisil NH2 refers to Unisil NH2 amino-bonded silica gel packing material; Amino-NH2 refers to amino-bonded silica gel packing material; MB70-75 refers to a forward chromatography packing material manufactured in Japan, with a pore size of 7 nm and a specific surface area of ​​480 m². 2 / g, particle size 75 / 200um; SMB70-45 refers to a forward chromatography packing material produced in Japan, with a pore size of 7nm and a specific surface area of ​​280m². 2 / g, particle size 20 / 45um; COPURE NH2 refers to aminopropyl filler, with parameters of carbon content 4.5% and specific surface area 200m². 2 / g, average particle size 40-75um.

[0030] In this invention, solvent 3 is preferably one or more of water, methanol, ethanol, acetonitrile, or ethyl acetate, more preferably an ethanol-water solution or a methanol-water solution, most preferably an ethanol-water solution or methanol-water solution with a volume fraction of 60-99 v / v%, and most preferably an ethanol-water solution or methanol-water solution with a volume fraction of 85-88 v / v%. In this invention, ethyl acetate is preferably a saturated aqueous solution thereof. In this invention, the amount of solvent 3 is preferably 5 BV of the column volume; the flow rate is preferably 1-2 BV / h. In this invention, RN and RO are separated from two elution sections of 2-3 BV and 3-5 BV, respectively, and the elution sections are further concentrated and dried after separation. In this invention, the drying temperature is preferably 70-90°C; the concentration time is not particularly limited, and can be carried out until no change in mass is observed, preferably 4-6 h. It should be understood that steviol glycoside II in this invention is a steviol glycoside composition mainly containing RN, and steviol glycoside III is a steviol glycoside composition mainly containing RO. In some embodiments, the content of RN in steviol glycoside II is 40.0-55.0%, and in addition to RN, other types of steviol glycosides in steviol glycoside II include RO and RD; the content of RO in steviol glycoside III is 40-60%, and in addition to RO, other types of steviol glycosides in steviol glycoside III include RN.

[0031] After obtaining steviol glycosides II and III, the present invention adds steviol glycosides II and III to solvent 4 for crystallization to obtain steviol glycosides IV and V, respectively. In the present invention, solvent 4 is preferably one or more of water, methanol, ethanol, isopropanol, acetone, acetonitrile, or ethyl acetate, more preferably one or more of water, methanol, ethanol, isopropanol, or acetone, and most preferably methanol-isopropanol-aqueous solution or methanol-acetone-aqueous solution; when methanol-isopropanol-aqueous solution is selected, its volume ratio is preferably (5-7):(2-4):1; when methanol-acetone-aqueous solution is selected, its volume ratio is preferably (5-7):(2-4):1. In the present invention, the amount of solvent added during the crystallization process is preferably 2-10 BV of the volume of steviol glycoside II, more preferably 4-6 BV, and most preferably 5 BV. In the present invention, the amount of solvent added during the crystallization process is preferably 2-10 BV of the volume of steviol glycoside III, more preferably 4-10 BV, and most preferably 7 BV.

[0032] After adding steviol glycosides II and III to solvent 4, the present invention further adds steviol glycosides II and III to solvent 4, heats to dissolve, and then cools to crystallize. In the present invention, the heating and dissolving process is preferably carried out at 60-100°C; more preferably at 80°C. In the present invention, the temperature of the cooling and crystallization process is preferably 20-40°C, more preferably 30°C; the stirring rate is preferably 80-120 rpm. In the present invention, the crystallization reaction time is preferably 20-30 h, more preferably 24 h. It should be understood that the crystallization process of steviol glycosides II and III in the present invention is a further purification process of RN and RO; therefore, steviol glycoside IV has a higher RN content than II, and steviol glycoside V has a higher RO content than III. In some embodiments, the content of RN in steviol glycoside IV is 70-85%, and in addition to RN, other types of steviol glycosides in steviol glycoside IV include RO and RD; the content of RO in steviol glycoside V is 70-85%, and in addition to RO, other types of steviol glycosides in steviol glycoside V include RN.

[0033] Furthermore, in conjunction with the above embodiments, after crystallizing steviol glycosides II and III in solvent 4, the present invention further includes filtering, rinsing with solvent, and drying the obtained crystals to obtain steviol glycoside IV and steviol glycoside V, respectively. The present invention does not limit the filtration process, as long as it can separate the precipitated crystals from the solvent; for example, conventional filter paper can be used. The rinsing solvent in the present invention is not particularly limited, but preferably uses the same solvent as in the crystallization process. The present invention also does not limit the volume of the rinsing solvent, as long as it can remove the solvent adhering to the crystals; for example, the volume of the rinsing solvent can be 1-4 BV of the crystals. The drying temperature in the drying process after rinsing is preferably 70-90°C, and the drying time is not particularly limited; the crystals can be dried until their weight remains constant, preferably 1-2 hours.

[0034] After obtaining steviol glycosides IV and V, the present invention further includes adding steviol glycosides IV and V to solvent 5 and heating to dissolve them, followed by recrystallization to obtain steviol glycosides VI and VII. In the present invention, solvent 5 is preferably an alcohol-water solution, more preferably an alcohol-water solution with a volume ratio of (50-70):100, and most preferably a methanol-water solution or ethanol-water solution with a volume ratio of (50-70):100; the amount of solvent 5 is preferably 4-10 BV of steviol glycosides VI or VII, more preferably 4-6 BV, and most preferably 5 BV. In the present invention, the heating and dissolving temperature is preferably 80-100℃, more preferably 85℃; the recrystallization temperature is preferably 20-50℃, more preferably 40℃; and the stirring rate is preferably 80-120 rpm, more preferably 100 rpm. In the present invention, there is no particular limitation on the recrystallization time, which can be selected based on the experience of those skilled in the art; for example, the recrystallization time is preferably 10-15 h, more preferably 12 h.

[0035] Furthermore, in conjunction with the above embodiments, after obtaining steviol glycosides VI and VII through recrystallization, the present invention further includes filtering, rinsing with solvent, and drying the obtained crystals to obtain steviol glycoside VI and steviol glycoside VII, respectively. The present invention does not limit the filtration process, as long as it can separate the precipitated crystals from the solvent; for example, conventional filter paper can be used. The rinsing solvent in the present invention is not particularly limited, but is preferably the same solvent used in the crystallization process. The present invention also does not limit the volume of the rinsing solvent, as long as it can remove the solvent adhering to the crystals; for example, the volume of the rinsing solvent can be 1-4 BV of the crystals. The drying temperature in the drying process after rinsing is preferably 70-90°C, and the present invention does not limit the drying time; the crystals can be dried until their weight remains constant. It should be understood that steviol glycosides VI and VII described in this invention are the same as steviol glycosides IV and V mentioned above, and are also compositions containing multiple steviol glycosides. Furthermore, steviol glycoside VI has a further increased RN content compared to steviol glycoside IV, and steviol glycoside VII has a further increased RO content compared to steviol glycoside V. In some embodiments, the RN content in steviol glycoside VI is 95-98%, and in addition to RN, other types of steviol glycosides in steviol glycoside VI include RO; the RO content in steviol glycoside VII is 95-98%, and in addition to RO, other types of steviol glycosides in steviol glycoside VII include RN.

[0036] To better illustrate the technical solution of this 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 crystallization mother liquor, all of which were provided by Zhucheng Haotian Pharmaceutical Co., Ltd.

[0037] Table 11-3 Stevioside Components of Mother Liquor from Batch Crystallization

[0038]

[0039] Example 1

[0040] S1. Take 180g of the crystallization mother liquor from batch 1, in which the total content of steviol glycosides (13 kinds) is 56.3% and the content of RN&RO is 11.6%, and add water to prepare a crystallization mother liquor aqueous solution with a concentration of 7wt%.

[0041] S2. 1500 ml of the crystallization mother liquor aqueous solution was passed through a glass chromatography column (diameter-to-height ratio 1:6) packed with 1000 ml of LX-18 resin. After separation, the resin was eluted with 2 BV 25 v / v% methanol-water solution to obtain the eluent (flow rate 1.5 BV / h). The eluent was concentrated and dried at 80 °C for 5 h to obtain steviol glycoside I.

[0042] S3. Add 30g of steviol glycoside I to 2BV 95wt% methanol, crystallize at 15℃ and 90rpm for 12h, and then filter to obtain the mother liquor of steviol glycoside I crystallization.

[0043] S4. Separate 25g of steviol glycoside I crystallization mother liquor (10% solid content) by passing it through a medium-pressure chromatographic column packed with 1000ml COPURE NH2 packing material (column diameter to height ratio 1:11). After separation, wash with 5BV of 86% methanol at a flow rate of 2BV / h. Collect the 2-3BV eluent (RN segment) of 86% methanol and concentrate and dry it at 80℃ for 5h to obtain steviol glycoside II. Collect the 3-5BV eluent (RO segment) of 86% methanol and concentrate and dry it at 80℃ for 5h to obtain steviol glycoside III.

[0044] S5. Dissolve 4.4 g of steviol glycoside II in a 5 BV methanol:isopropanol:water mixture of 7:2:1, heat to 80 °C to dissolve, and then crystallize at 30 °C and 100 rpm for 24 h. Filter, rinse with 10 ml of crystallization solvent, and dry at 80 °C for 1 h to obtain steviol glycoside IV; Dissolve 5.2 g of steviol glycoside III in a 7 BV methanol:isopropanol:water mixture of 5:4:1, heat to 80 °C to dissolve, and then crystallize at 30 °C and 100 rpm for 24 h. Filter, rinse with 10 ml of crystallization solvent, and dry at 80 °C for 1 h to obtain steviol glycoside V.

[0045] The tests showed that the RN content in the above-obtained steviol glycoside I, II, and IV samples was 16.3%, 55.0%, and 85.3%, respectively; and the RO content in the above-obtained steviol glycoside I, III, and V samples was 20.1%, 57.1%, and 79.4%, respectively.

[0046] Example 2

[0047] S1. Take 180g of the crystallization mother liquor from batch 1, in which the total content of steviol glycosides (13 kinds) is 55% and the content of RN&RO is 11%, and add water to prepare a crystallization mother liquor aqueous solution with a concentration of 5wt%.

[0048] S2. Pass 1000 ml of the crystallization mother liquor aqueous solution through a glass chromatography column (diameter-to-height ratio 1:8) packed with 1000 ml of LX-T3 resin. After separation, the resin is eluted with 1.0 BV 30 v / v% ethanol-water solution to obtain the eluent (flow rate 1 BV / h). The eluent is concentrated and dried at 85 °C for 4 h to obtain steviol glycoside I.

[0049] S3. Add 25g of steviol glycoside I to 3BV 92wt% methanol, crystallize at 20℃ and 60rpm for 10h, and then filter to obtain the mother liquor of steviol glycoside I crystallization.

[0050] S4. Separate 20g of steviol glycoside I crystallization mother liquor (10% solid content) by passing it through a low-pressure chromatographic column packed with 1000ml Unisil NH2 packing material (column diameter to height ratio 1:10). After separation, wash with 5BV of 85% ethanol at a flow rate of 1BV / h. Collect the 2-3BV elution segment of 85% ethanol (RN segment), concentrate and dry at 70℃ for 6h to obtain steviol glycoside II; collect the 3-5BV elution segment of 85% ethanol (RO segment), concentrate and dry at 70℃ for 6h to obtain steviol glycoside III.

[0051] S5. Dissolve 4.1g of steviol glycoside II in a 4BV mixture of methanol:isopropanol:water = 6:3:1, heat to 60℃ to dissolve, and crystallize at 20℃ and 80rpm for 20h. Filter, rinse with 10ml of crystallization solvent, and dry at 80℃ for 1.5h to obtain steviol glycoside IV; Dissolve 4.7g of steviol glycoside III in a 4BV mixture of methanol:isopropanol:water = 6:3:1, heat to 60℃ to dissolve, and crystallize at 20℃ and 80rpm for 20h. Filter, rinse with 10ml of crystallization solvent, and dry at 70℃ for 1.5h to obtain steviol glycoside V.

[0052] The tests showed that the RN content in the above-obtained steviol glycoside I, II, and IV samples was 16.1%, 48.0%, and 80.9%, respectively; and the RO content in the above-obtained steviol glycoside I, III, and V samples was 18.9%, 50.0%, and 77.4%, respectively.

[0053] Example 3

[0054] S1. Take 180g of the crystallization mother liquor from batch 1, in which the total content of steviol glycosides (13 kinds) is 53.6% and the content of RN&RO is 10.4%, and add water to prepare a crystallization mother liquor aqueous solution with a concentration of 10wt%.

[0055] S2. Pass 3000 ml of crystallization mother liquor aqueous solution through a glass chromatography column (diameter-to-height ratio 1:5) packed with 1000 ml of LK20SS resin. After separation, the resin is eluted with 3.0 BV of water to obtain the eluent (flow rate 1 BV / h). The eluent is concentrated and dried at 83℃ for 6 h to obtain steviol glycoside I.

[0056] S3. Add 25g of steviol glycoside I to 1BV 90wt% methanol, crystallize at 30℃ and 120rpm for 15h, and then filter to obtain the mother liquor of steviol glycoside I crystallization.

[0057] S4. Separate 50g of steviol glycoside I crystallization mother liquor (10% solid content) by passing it through a high-pressure chromatographic column packed with 1000ml MB70-75 packing material (column diameter to height ratio 1:12). After separation, wash with 5BV of 88% methanol at a flow rate of 1.5BV / h. Collect the 2-3BV eluent (RN segment) of 88% methanol and concentrate and dry it at 90℃ for 4h to obtain steviol glycoside II. Collect the 3-5BV eluent (RO segment) of 88% methanol and concentrate and dry it at 90℃ for 4h to obtain steviol glycoside III.

[0058] S5. Dissolve 8.9g of steviol glycoside II in a 6BV methanol:acetone:water mixture of 5:4:1, heat to 100℃ to dissolve, and then crystallize at 40℃ and 120rpm for 30h. Filter, rinse with 15ml of crystallization solvent, and dry at 80℃ for 2h to obtain steviol glycoside IV; Dissolve 9.8g of steviol glycoside III in a 10BV methanol:isopropanol:water mixture of 7:2:1, heat to 100℃ to dissolve, and then crystallize at 40℃ and 120rpm for 30h. Filter, rinse with 15ml of crystallization solvent, and dry at 90℃ for 2h to obtain steviol glycoside V.

[0059] The tests showed that the RN content in the above-obtained steviol glycoside I, II, and IV samples was 17.3%, 43.0%, and 77.9%, respectively; and the RO content in the above-obtained steviol glycoside I, III, and V samples was 17.6%, 45.1%, and 70.2%, respectively.

[0060] Example 4

[0061] In Example 4, based on Example 1 above, 6BV 60v / v% methanol-water solution was added to steviol glycosides IV and V respectively, heated to 85°C to dissolve, purified and recrystallized at 40°C and 100 rpm for 12 h, filtered, washed with 10 ml of crystallization solvent, and dried at 80°C for 1.5 h to obtain steviol glycosides VI and VII respectively.

[0062] The tests showed that the RN content in the above-obtained steviol glycoside VI sample was 97.5%, and the RO content in the above-obtained steviol glycoside VII sample was 96.2%.

[0063] Example 5

[0064] Example 5: Based on Example 2, 4 BV of 60 v / v% methanol-water solution was added to steviol glycosides IV and V respectively, heated to 80°C to dissolve, purified and recrystallized at 20°C and 80 rpm for 15 h, filtered, washed with 10 ml of crystallization solvent, and dried at 70°C for 1 h to obtain steviol glycosides VI and VII respectively.

[0065] The tests showed that the RN content in the above-obtained steviol glycoside VI sample was 96.7%, and the RO content in the above-obtained steviol glycoside VII sample was 95.5%.

[0066] Example 6

[0067] In Example 6, based on Example 3 above, 10 BV 60 v / v% methanol-water solution was added to steviol glycosides IV and V respectively, heated to 100°C to dissolve, purified and recrystallized at 50°C and 120 rpm for 10 h, filtered, washed with 10 ml of crystallization solvent, and dried at 90°C for 1.2 h to obtain steviol glycosides VI and VII respectively.

[0068] The tests showed that the RN content in the obtained steviol glycoside VI sample was 95.2%, and the RO content in the obtained steviol glycoside VII sample was 95.7%.

[0069] Example 7

[0070] The resin in Example 1 was replaced with an equal amount of LK1300s resin, while other conditions remained unchanged.

[0071] The results showed that the RN content in the obtained steviol glycoside I, II, and IV samples was 16.8%, 44.5%, and 76.4%, respectively; and the RO content in the obtained steviol glycoside I, III, and V samples was 19.3%, 43.4%, and 72.4%, respectively.

[0072] Example 8

[0073] The column packing material in Example 2 was replaced with an equal amount of Amino-NH2 packing material, while other conditions remained unchanged.

[0074] The results showed that the RN content in the obtained steviol glycoside I, II, and IV samples was 16.1%, 46.6%, and 75.3%, respectively; and the RO content in the obtained steviol glycoside I, III, and V samples was 18.9%, 51.1%, and 77.7%, respectively.

[0075] Example 9

[0076] The 88 wt% methanol (solvent 3) in Example 3 was replaced by an equal amount of a mixed solvent of 86% methanol / ethyl acetate (volume ratio 8:2), while other conditions remained unchanged.

[0077] The results showed that the RN content in the obtained steviol glycoside I, II, and IV samples was 17.3%, 50.3%, and 82.8%, respectively; and the RO content in the obtained steviol glycoside I, III, and V samples was 17.6%, 58.4%, and 78.2%, respectively.

[0078] Example 10

[0079] Replace the 60 v / v% methanol-water solution (solvent 5) in Example 4 with an equal amount of 55 v / v% methanol-water solution, while keeping other conditions unchanged.

[0080] The results showed that the RN content in the obtained steviol glycoside VI sample was 93.5%, and the RO content in the obtained steviol glycoside VII sample was 96.1%.

[0081] Comparative Example 1

[0082] The resin in Example 1 was replaced with an equal amount of D101 resin, while other conditions remained unchanged.

[0083] The results showed that the RN content in the obtained steviol glycoside I sample was 7.4%, and the RO content was 8.3%.

[0084] Comparative Example 2

[0085] The column packing material in Example 2 was replaced with an equal amount of H-type SD2 packing material, while other conditions remained unchanged.

[0086] The results showed that the RN content in the obtained steviol glycoside II sample was 21.6%, and the RO content in the obtained steviol glycoside III sample was 26.1%.

[0087] Comparative Example 3

[0088] Replace 88 wt% methanol (solvent 3) in Example 3 with 50 wt% ethanol, while keeping other conditions unchanged.

[0089] The results showed that the RN content in the obtained steviol glycoside IV sample was 30.1%, and the RO content in the obtained steviol glycoside V sample was 33.5%.

[0090] Comparative Example 4

[0091] Replace the 60 v / v% methanol-water solution (solvent 5) in Example 4 with an equal amount of 70 v% ethanol-water solution, while keeping other conditions unchanged.

[0092] The results showed that the RN content in the obtained steviol glycoside VI sample was 86.4%, and the RO content in the obtained steviol glycoside VII sample was 84.2%.

[0093] Test Example 1

[0094] Furthermore, the methods for determining the content of steviol glycosides (RN, RO) in Examples 1-10 and Comparative Examples 1-4 of the present invention were performed in accordance with the national standard GB 1886.355-2022.

[0095] 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 N and O, characterized in that, Includes the following steps: The mother liquor for steviol glycoside crystallization was mixed with water to obtain an aqueous solution of the mother liquor for crystallization. The aqueous solution of the crystallization mother liquor is separated by a resin. After separation, the resin is eluted with solvent 1 to obtain an eluent. The eluent is concentrated and dried to obtain steviol glycoside I. The resin is a nonpolar or weakly polar adsorption resin with a styrene structure, and the nonpolar or weakly polar adsorption resin with a styrene structure is LX-T3, LK1300s, LX-18 or LK20SS resin. Solvent 1 is an alcohol-water solution with a volume ratio of (0-30):

100. The steviol glycoside I is added to solvent 2 for crystallization to obtain a mother liquor of steviol glycoside I crystallization; wherein, solvent 2 is a 90-95 wt% alcohol solution; The mother liquor of steviol glycoside I crystallization is separated by a chromatographic column, then eluted with solvent 3. A 2-3 BV eluent is collected, concentrated, and dried to obtain steviol glycoside II. A 3-5 BV eluent is collected, concentrated, and dried to obtain steviol glycoside III. The chromatographic column is a low-pressure, medium-pressure, or high-pressure column; the column packing material is one of the following: a normal chromatographic packing material; and solvent 3 is one or more of the following: an ethanol-water solution with a volume fraction of 60-99 v / v%, a methanol-water solution with a volume fraction of 60-99 v / v%, and ethyl acetate. Steviosides II and III were respectively added to solvent 4 for crystallization to obtain steviol glycosides IV and V respectively; wherein, solvent 4 is a methanol-isopropanol-water solution with a volume ratio of (5-7):(2-4):1 or a methanol-acetone-water solution with a volume ratio of (5-7):(2-4):1, the content of RN in steviol glycoside IV is 70-85%, and the content of RO in steviol glycoside V is 70-85%.

2. The separation method according to claim 1, characterized in that, The mass concentration of the crystallization mother liquor aqueous solution is 5-10 wt%; the sample loading amount of the crystallization mother liquor aqueous solution is 1-2 BV of the resin volume.

3. The separation method according to claim 1, characterized in that, The amount of solvent 1 used is 1-3 BV of the resin volume.

4. The separation method according to claim 1, characterized in that, The amount of solvent 2 is 1-3 BV of the volume of steviol glycoside I.

5. The separation method according to claim 1, characterized in that, The amount of the mother liquor containing the stevioside I crystals loaded is 2-5% of the volume of the chromatographic column.

6. The separation method according to claim 1, characterized in that, The amount of solvent 3 used is 5 BV of the column volume.

7. The separation method according to claim 1, characterized in that, The amount of solvent 4 is 2-10 BV of the volume of steviol glycoside II; the crystallization includes adding steviol glycosides II and III to solvent 4 respectively, dissolving them at 60-100°C, then cooling to 0-30°C, and crystallizing at 80-120 rpm for 20-30 h.

8. The separation method according to claim 1, characterized in that, After adding steviol glycosides II and III to solvent 4 for crystallization, the process further includes filtering, rinsing with solvent, and drying the obtained crystals to obtain steviol glycoside IV and steviol glycoside V, respectively.

9. The separation method according to claim 1 or 8, characterized in that, After obtaining steviol glycosides IV and V, the method further includes dissolving steviol glycosides IV and V in solvent 5 at 80-100°C, and then recrystallizing them to obtain steviol glycosides VI and VII; wherein, solvent 5 is an alcohol-water solution with a volume ratio of (50-70):100, the content of RN in steviol glycoside VI is 95-98%, and the content of RO in steviol glycoside VII is 95-98%.

10. The separation method according to claim 9, characterized in that, The amount of solvent 5 is 4-10 BV of the volume of steviol glycoside III; the recrystallization temperature is 20-50℃ and the stirring rate is 80-120 rpm.

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