CN113201034A - Obtaining high-purity stevioside from primary crystallization mother liquor of stevioside through secondary crystallization and enriching rebaudioside C - Google Patents
Obtaining high-purity stevioside from primary crystallization mother liquor of stevioside through secondary crystallization and enriching rebaudioside C Download PDFInfo
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- CANAPGLEBDTCAF-NTIPNFSCSA-N Dulcoside A Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@]23C(C[C@]4(C2)[C@H]([C@@]2(C)[C@@H]([C@](CCC2)(C)C(=O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)CC4)CC3)=C)O[C@H](CO)[C@@H](O)[C@@H]1O CANAPGLEBDTCAF-NTIPNFSCSA-N 0.000 description 2
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- QRGRAFPOLJOGRV-UHFFFAOYSA-N rebaudioside F Natural products CC12CCCC(C)(C1CCC34CC(=C)C(CCC23)(C4)OC5OC(CO)C(O)C(OC6OCC(O)C(O)C6O)C5OC7OC(CO)C(O)C(O)C7O)C(=O)OC8OC(CO)C(O)C(O)C8O QRGRAFPOLJOGRV-UHFFFAOYSA-N 0.000 description 2
- HYLAUKAHEAUVFE-AVBZULRRSA-N rebaudioside f Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)CO1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HYLAUKAHEAUVFE-AVBZULRRSA-N 0.000 description 2
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- YWPVROCHNBYFTP-UHFFFAOYSA-N Rubusoside Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC1OC(CO)C(O)C(O)C1O YWPVROCHNBYFTP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/256—Polyterpene radicals
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention relates to the field of separation of natural high-potency sweeteners, and changes the solubility of each component in stevioside through selecting a crystallization solvent so as to selectively recover stevioside in primary crystallization mother liquor. Stevioside extracted from plants is a mixture mainly containing Rebaudioside A (RA), Stevioside (ST), Rebaudioside C (RC), etc., and RA can be obtained in high purity by one-time crystallization. The remaining primary crystallization mother liquor mainly contains ST, RA and RC as well as some bitter impurities of flavone and polyphenol. The most ST content, the method utilizes the solubility difference of different components in the stevioside crystallization mother liquor in the improved crystallization solvent. And precipitating a solid when the mother liquor sugar is subjected to secondary crystallization, filtering, washing and drying to recover the ST in the mother liquor. The technical method has the advantages of no need of adding equipment, simple and short operation steps and low energy consumption. Most of ST is recovered, and RC is enriched in a liquid phase, so that the substances are further separated and purified.
Description
Technical Field
The invention relates to the field of separation of natural high-power sweeteners, in particular to a method for obtaining high-purity stevioside and enriching rebaudioside C from a primary stevioside crystallization mother liquor through secondary crystallization.
Background
Stevia Sugar (SGs) is a natural high-power sweetener extracted from a natural plant stevia rebaudiana, and is approved as a food additive in many countries, the sweetness of the stevia sugar is 200-300 times that of cane sugar, and the calorie of the stevia sugar is only 1/300 of the cane sugar. Natural STevioside is a mixture of several glycoside compounds, and all of the steviol glycosides contain the same hydrophobic steviol glycoside unit except that the glycosyl substituents at positions C13 and C18 are different, and the main components are Rebaudioside A (RA), STevioside (ST), and Rebaudioside C (RC). The total content of the three components in the natural stevia is more than 90%.
The industrial production of stevioside generally needs the procedures of water extraction, flocculation, alcohol precipitation, column chromatography, crystallization and the like. The spent mother liquor remaining after crystallization generally contains various glycosides such as RA, ST, RC, RF (rebaudioside F), Dulcoside A (DA), rubusoside and RB (rebaudioside B), wherein the ST content is highest.
The mother liquor from the primary crystallization contained a large amount of ST in addition to the non-crystallized rebaudioside a, which was slightly less palatable. However, since ST and RA only have one glucose difference in chemical structure, RA can be synthesized by grafting one glucose through enzyme, and RD with high value can also be synthesized by grafting two glucose. In addition, ST also has therapeutic effects such as lowering blood pressure. Therefore, it is important to recover and obtain ST of higher purity from the mother liquor of the stevioside crystallization. At present, the separation and purification technology of ST is difficult, which leads to high product sale price. High-purity ST can be obtained by a column chromatography (silica gel column) method, the cost is high, and the high-purity ST obtained by recrystallization is not reported at present. For example, 20mg of ST with 98% purity can be sold for 260 yuan.
RC can be used as a sweetness enhancer, so that the use amount of cane sugar can be greatly reduced, and in addition, research shows that RC also has the health-care functions of reducing blood pressure, reducing blood fat, resisting allergy and preventing cardiovascular diseases. The RC content in stevioside is very low and only accounts for 0.2 percent of the stevioside (Wangdjie, the cause mechanism of sweetness, sweetness and bitter aftertaste of stevioside is reviewed [ J ] food industry science and technology, 2010,31(05): 417-. Therefore, high purity RC cannot be obtained by the conventional method. The high-purity RC obtained at present is obtained by separation of preparative HPLC or nanofiltration membrane technology. The price of high-purity RC in the market is very high at present, for example, 20mg RC with 99% purity can be sold at a price of 2600 yuan.
The purification method of the primary crystallization mother liquor of stevioside mainly comprises two methods: crystallization and chromatography. The crystallization method has low cost and is suitable for large-scale production. The purity of the product is higher than that of the product obtained by the crystallization method, and the automatic and continuous production can be realized by using a simulated moving bed, but the cost is too high. It would be the most desirable method to obtain high purity ST by crystallization.
With respect to the purification of rebaudioside a, great progress has been made in recent years. For example, the present inventors purified crude stevia materials by crystallization using methanol water of a certain concentration as a crystallization solvent. In the product after primary crystallization, the purity of RA can reach more than 99 percent, and the yield of RA is more than 80 percent. A large amount of ST and the like remain in the primary crystallization mother liquor. At present, few effective recovery methods for stevioside crystallization mother liquor are reported at home and abroad, and the method mainly adopts a solvent crystallization method and a chromatography method. For example, qianchengcheng et al (qianchengcheng, wang yangfei, liriing. a new method for recovering stevioside mother liquor sugar [ P ].2012-10-10.) utilizes the mixed solvent of isopropanol, methanol and ethanol to carry out crystallization operation, the content of total glucoside in the obtained crystal is up to more than 90%, the recovery rate is between 45% and 75%, the main function is to improve the content of total glucoside in the mother liquor, and the proportion of RA to ST in the original mother liquor is hardly changed. Liu Yaxian et al (Liu Y, Hua X, Wang M, et al. purification of the heat sugar from induced stand specific expression production through one-steep adsorption by non-polar macroporous resin material [ J ]. Food ChemiSTry,2019,274: 337-344.) use macroporous resin material to remove flavone and polyphenol substances in stevioside mother liquor, but basically does not change the contents of RA and ST substances in the mother liquor.
In summary, it is important to recycle ST and RC of the mother solution after RA having a high purity is obtained by primary crystallization. Because the RA content in the primary stevioside crystallization mother liquor is obviously lower than the ST content (the RA content is about 1/2-2/3 of ST content), the high-purity ST can be obtained in a secondary crystallization mode, and the RC is enriched at the same time, so that an important raw material is provided for preparing the high-purity RC.
The principle of the invention is to purify stevioside and other substances according to the characteristics and solubility differences of different crystallization solvents. Stevioside ST is structurally lack of a glucosyl unit compared with RA and RC, so that the polarity is different from that of RA and RC, and after different crystallization solvents are used, the solubility balance of substances in one crystallization can be broken, so that the solubility of RA and ST is changed, but the change degrees are different. By controlling the type and the proportion of the crystallization solvent, the precipitation amount of ST is obviously more than that of RA, and RC is not precipitated, thereby realizing the selective recovery of ST in the primary crystallization mother liquor and the enrichment of RC.
Disclosure of Invention
The invention aims to obtain the high-purity Stevioside (ST) by filtering after secondary stirring crystallization of mother liquor sugar after primary crystallization of the stevioside, wherein the solubility of each component in different crystallization solvents is different, and RC is not precipitated and is remained in the secondary crystallization solvent to be enriched.
The purpose of the invention is realized by the following technical scheme:
the secondary crystallization solvent used for the stevioside mother liquor sugar is one or more than two of methanol, ethanol, isopropanol, water, chloroform and ethyl acetate, the crystallization temperature is 10-30 ℃, the crystallization standing time is 8-24 h, and the ratio v of the crystallization mixed solvent is 1: 1-1: 8.
the characteristics of the project are briefly described as follows
(1) The primary crystallization mother liquor with lower value is used as raw material. Further effectively recovering high-value Stevioside (ST), wherein the purity of the stevioside is improved from about 66 percent in the raw material mother liquor to over 95 percent, and the recovery rate can reach over 50 percent.
(2) The secondary crystallization process is simple and efficient, convenient and fast, and low in energy consumption.
(3) And most of bitter impurities of flavone and polyphenol are removed while high-purity ST is obtained by secondary crystallization, so that the recovered ST has higher economic value.
(4) High-purity ST is obtained through secondary crystallization, RC components in a secondary crystallization liquid phase are enriched, and high-value RC with higher purity is further prepared to provide raw materials.
Drawings
To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, and it should be understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope.
High performance liquid chromatography conditions: the detection wavelength is 210nm, the mobile phase is 68% acetonitrile/32% water (phosphate buffer pH is 2.60), the flow rate is 1mL/min, and the injection volume is 20 mu L; the column temperature was 40 ℃. HPLC analysis used a C18 column (Waters, Massachusetts, USA), SPD-M20A PDA detector (Shimadzu, Kyoto, Japan), column oven HCT-360LC (Hengao Tech & Dev, Tianjin, China), and workstation Class-VP (Shimadzu, Kyoto, Japan).
FIG. 1 is a flow chart of operations provided by an embodiment of the present invention.
FIG. 2 is an HPLC chart of the mother liquor of the primary crystallization provided in the example of the present invention.
FIG. 3 shows the effect of the type of crystallization solvent on the purity and yield of ST precipitates provided in the examples of the present invention.
FIG. 4 is a graph showing the effect of crystallization solvent temperature on ST precipitate purity and yield, according to an embodiment of the present invention.
FIG. 5 is a graph showing the effect of crystallization solvent time on ST precipitate purity and yield provided by an example of the present invention.
FIG. 6 shows the effect of the ratio of crystallization solvents on the purity and yield of ST precipitates provided in the examples of the present invention.
FIG. 7 is an HPLC plot of an optimized crystallization solvent versus ST precipitates provided in an example of the present invention.
FIG. 8 is an HPLC plot of mother liquor versus RC enrichment after secondary crystallization provided by an example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. The method for obtaining high-purity ST by secondary crystallization of primary crystallization mother liquor sugar provided by the embodiment of the invention is exemplified below.
Example 1
Precisely measuring 100mL of primary crystallization mother liquor by using a pipette, and drying at 100 ℃ to constant weight to obtain a solid. Taking 2g of solid for later use, preparing 90% methanol and ethanol aqueous solution as a crystallization solvent, putting the solid-liquid ratio of 1:8(g/mL) into a beaker, stirring for 30 minutes, standing for natural crystallization for 8 hours at 25 ℃, washing, drying and weighing the filtered solid by using pure methanol, and sampling according to a national standard method for HPLC analysis.
Examples 2 to 21
Examples 2-21, in which crystallization was applied to the recovery of high purity ST under other different conditions, are given in the following table. The parameter conditions not mentioned in the table are the same as in example 1.
In summary, the invention provides a method for recovering stevioside ST from industrial stevioside primary crystallization mother liquor. The influence of the crystallization solvent added with isopropanol on the solubility of various substances in the stevioside is utilized, the ST is selectively recovered, the method is simple and efficient, convenient and fast, and can remove a part of bitter impurities of flavonoids, thereby providing a raw material for the subsequent preparation of high-purity rebaudioside-C.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A method for recovering stevioside from industrial crystallization mother liquor of stevioside is characterized in that: and after the mother liquor sugar after primary crystallization of stevioside is subjected to secondary stirring crystallization after the solubility of each component in different crystallization solvents is different, and high-purity Stevioside (ST) is obtained by filtering.
2. The method of recovering ST from mother liquor of industrial crystallization of stevioside according to claim 1, wherein the solvent used for the secondary crystallization is one or a mixture of methanol, ethanol, isopropanol, water, chloroform and ethyl acetate.
3. The method for recovering ST from the crystallization mother liquor of industrial stevioside according to claim 1, characterized in that the crystallization temperature is 10 ℃ to 30 ℃, the crystallization standing time is 8h to 24h, and the ratio v of crystallization mixed solvent is 1: 1-1: 8.
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| CN113527378A (en) * | 2021-08-27 | 2021-10-22 | 江南大学 | Method for recovering stevioside from industrial stevioside mother liquor sugar through crystallization |
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