CN106967133B - A method of extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor - Google Patents
A method of extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor Download PDFInfo
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- CN106967133B CN106967133B CN201710200612.9A CN201710200612A CN106967133B CN 106967133 B CN106967133 B CN 106967133B CN 201710200612 A CN201710200612 A CN 201710200612A CN 106967133 B CN106967133 B CN 106967133B
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- mother liquor
- steviol glycoside
- silica gel
- rebaudioside
- refinement mother
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- RPYRMTHVSUWHSV-CUZJHZIBSA-N rebaudioside D 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)[C@@H](CO)O1)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]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 RPYRMTHVSUWHSV-CUZJHZIBSA-N 0.000 title claims abstract description 196
- 235000019202 steviosides Nutrition 0.000 title claims abstract description 56
- 239000012452 mother liquor Substances 0.000 title claims abstract description 47
- 239000004383 Steviol glycoside Substances 0.000 title claims abstract description 46
- 229930182488 steviol glycoside Natural products 0.000 title claims abstract description 46
- 235000019411 steviol glycoside Nutrition 0.000 title claims abstract description 46
- 150000008144 steviol glycosides Chemical class 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 44
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 60
- 239000000741 silica gel Substances 0.000 claims abstract description 60
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 60
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 54
- 238000002425 crystallisation Methods 0.000 claims abstract description 32
- 230000008025 crystallization Effects 0.000 claims abstract description 31
- 239000012043 crude product Substances 0.000 claims abstract description 19
- 239000003480 eluent Substances 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000003960 organic solvent Substances 0.000 claims abstract description 15
- 239000000706 filtrate Substances 0.000 claims abstract description 12
- 230000001476 alcoholic effect Effects 0.000 claims abstract description 10
- 238000001816 cooling Methods 0.000 claims abstract description 10
- 239000000203 mixture Substances 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 7
- 239000012535 impurity Substances 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 42
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 42
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 34
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 34
- 235000019441 ethanol Nutrition 0.000 claims description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- 238000010438 heat treatment Methods 0.000 claims description 19
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 15
- 239000012141 concentrate Substances 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 239000003292 glue Substances 0.000 claims description 3
- 229910052710 silicon Inorganic materials 0.000 claims description 3
- 239000010703 silicon Substances 0.000 claims description 3
- 230000006837 decompression Effects 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 6
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims 1
- 239000000047 product Substances 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 239000000843 powder Substances 0.000 abstract description 5
- 230000001681 protective effect Effects 0.000 abstract description 3
- 238000004090 dissolution Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 29
- 239000000243 solution Substances 0.000 description 16
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A 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)[C@@H](CO)O1)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 HELXLJCILKEWJH-NCGAPWICSA-N 0.000 description 13
- 239000001512 FEMA 4601 Substances 0.000 description 10
- HELXLJCILKEWJH-SEAGSNCFSA-N Rebaudioside A Natural products O=C(O[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@@]1(C)[C@@H]2[C@](C)([C@H]3[C@@]4(CC(=C)[C@@](O[C@H]5[C@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@H](O)[C@@H](CO)O5)(C4)CC3)CC2)CCC1 HELXLJCILKEWJH-SEAGSNCFSA-N 0.000 description 10
- UEDUENGHJMELGK-HYDKPPNVSA-N Stevioside Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[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 UEDUENGHJMELGK-HYDKPPNVSA-N 0.000 description 10
- HELXLJCILKEWJH-UHFFFAOYSA-N entered according to Sigma 01432 Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC(C1OC2C(C(O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OC(CO)C(O)C(O)C1O HELXLJCILKEWJH-UHFFFAOYSA-N 0.000 description 10
- 235000019203 rebaudioside A Nutrition 0.000 description 10
- OHHNJQXIOPOJSC-UHFFFAOYSA-N stevioside Natural products CC1(CCCC2(C)C3(C)CCC4(CC3(CCC12C)CC4=C)OC5OC(CO)C(O)C(O)C5OC6OC(CO)C(O)C(O)C6O)C(=O)OC7OC(CO)C(O)C(O)C7O OHHNJQXIOPOJSC-UHFFFAOYSA-N 0.000 description 10
- 229940013618 stevioside Drugs 0.000 description 10
- 239000002994 raw material Substances 0.000 description 6
- 239000000284 extract Substances 0.000 description 4
- 229930182470 glycoside Natural products 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- QSRAJVGDWKFOGU-WBXIDTKBSA-N rebaudioside c 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@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO)O[C@H]1O[C@]1(CC[C@H]2[C@@]3(C)[C@@H]([C@](CCC3)(C)C(=O)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)CC3)C(=C)C[C@]23C1 QSRAJVGDWKFOGU-WBXIDTKBSA-N 0.000 description 4
- 238000004809 thin layer chromatography Methods 0.000 description 4
- 241000544066 Stevia Species 0.000 description 3
- 244000228451 Stevia rebaudiana Species 0.000 description 3
- 235000006092 Stevia rebaudiana Nutrition 0.000 description 3
- 150000002338 glycosides Chemical class 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000001776 FEMA 4720 Substances 0.000 description 2
- 244000269722 Thea sinensis Species 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- 150000008131 glucosides Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 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
- 238000001953 recrystallisation Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 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
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- -1 disaccharide Glycosides Chemical class 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- YWPVROCHNBYFTP-OSHKXICASA-N rubusoside Chemical compound 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 YWPVROCHNBYFTP-OSHKXICASA-N 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000019640 taste Nutrition 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Seasonings (AREA)
- Cosmetics (AREA)
- Saccharide Compounds (AREA)
Abstract
A method of extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, comprising the following steps: (1) crystallization removal of impurities: steviol glycoside refinement mother liquor is added in alcoholic solution, dissolution, crystallization, and filtering obtains filtrate；(2) reverse phase silica gel mixes sample: no alcohol is concentrated the filtrate to, reverse phase silica gel is added, is uniformly mixed, it is dry, and sample silica gel must be mixed；(3) it fills column: in empty chromatographic column, being initially charged with reverse phase silica gel, be reloaded into and mix sample silica gel；(4) it elutes: chromatographic column being eluted with eluant, eluent, collect efflux, be concentrated, it is dry, obtain RD crude product；(5) it refines: RD crude product organic solvent or water is dissolved, cooling, crystallization filters, and it is dry, obtain RD fine work.The white crystalline powder of RD obtained by the method for the present invention, purity may be up to 99.0wt%, and yield may be up to 92.4%；The method of the present invention is environmentally protective, at low cost, and product is pure, pollution-free, and added value of product is high, is suitable for industrialized production.
Description
Technical field
The present invention relates to a kind of extracting methods of rebaudioside D, and in particular to one kind is mentioned from steviol glycoside refinement mother liquor
The method for taking high-purity rebaudioside D.
Background technique
Major glycosides in steviol glycoside have stevioside (ST) and Rebaudioside A (RA), other known glucosides include auspicious Bao
Enlightening glycosides B(RB), rebaudioside C (RC), rebaudioside D (RD), rebaudioside F (RF), Du Ke glycosides A, Rubusoside and stevia rebaudianum disaccharide
Glycosides.
Recent study discovery, the best ingredient highest RA of not instead of sugariness of mouthfeel, the sweet tea of RD, RD in steviol glycoside
Degree is about 150 times of sucrose, and in good taste, no bitter taste, still, content of the RD in STEVIA REBAUDIANA are very low, and only 1% or so, because
This, it is desirable to the RD of the extraction high-purity of high-recovery is more difficult.
CN102060892A discloses the method for purification of stevioside RD a kind of, is that mother liquor sugar material flow is crossed macroporous absorption
Resin, then with ethanol elution, Fractional Collections eluent is concentrated, dry, purification obtains RD.But due to the suction of macroreticular resin
Attached poor selectivity, by stepwise elution, the operability for collecting RD by the method for determining critical point is lower, and RD is difficult to be enriched with,
Therefore, the yield of RD is not high.
CN102406113A discloses a kind of preparation method of RA and RD edible compound stevioside, is led to using STEVIA REBAUDIANA as raw material
Water extraction is crossed, macroporous resin adsorption elutes, it decolourizes, crystallization, recrystallization and etc., obtain the product mix of RA and RD.But
This method can not obtain the RD of high-content.
CN103709215A discloses a kind of method that rebaudioside D is extracted from stevioside mother liquor sugar, is by sweet tea
Synanthrin mother liquor sugar water and methanol heat preservation, stirring, repeated crystallization obtain RD product.But this method is because crystallisation times are excessive,
Yield is lower, and the purity of RD only 50% or so.
Summary of the invention
The technical problem to be solved by the present invention is to overcome drawbacks described above of the existing technology, provide a kind of gained production
Product purity, high income, added value of product is high, environmentally protective, at low cost, and the slave steviol glycoside suitable for industrialized production refines female
The method of high-purity rebaudioside D is extracted in liquid.
The technical solution adopted by the present invention to solve the technical problems is as follows: one kind is extracted from steviol glycoside refinement mother liquor
The method of high-purity rebaudioside D, comprising the following steps:
(1) crystallization removal of impurities: steviol glycoside refinement mother liquor is added in alcoholic solution, is dissolved by heating, cooling crystallization, and filtering obtains
Filtrate；
(2) reverse phase silica gel mixes sample: filtrate decompression obtained by step (1) being concentrated into no alcohol, reverse phase silicon is added in concentrate
Glue is uniformly mixed, dry to constant weight, must mix sample silica gel；
(3) it fills column: in empty chromatographic column, being initially charged with reverse phase silica gel, then be packed on reverse phase silica gel and mix sample obtained by step (2)
Silica gel；
(4) elute: the chromatographic column after with eluant, eluent step (3) are filled with column elutes, and collects target and elutes efflux, dense
Contracting, it is dry, obtain rebaudioside D crude product；
(5) it refines: rebaudioside D crude product organic solvent obtained by step (4) or water is dissolved by heating, cooling, crystallization, mistake
Filter, it is dry, obtain rebaudioside D fine work.
Preferably, in step (1), in the steviol glycoside refinement mother liquor mass content of rebaudioside D be 5~30%(more
It is preferred that 6~10%).Steviol glycoside refinement mother liquor of the present invention is derived from using qualities of stevia extract as raw material, with organic solvent knot
Crystalline substance prepares generated mother liquor when high-purity Rebaudioside A (RA), stevioside (ST).
Preferably, in step (1), the dosage of the alcoholic solution is that 4~20 times of steviol glycoside refinement mother liquor quality are (more excellent
Select 5~10 times).
Preferably, in step (1), the alcohol in the alcoholic solution is one of methanol, ethyl alcohol, isopropanol or n-butanol etc.
Or it is several, the volumetric concentration of alcoholic solution is 50~99%.
Preferably, in step (1), the temperature of the heating for dissolving is 40~60 DEG C.
Preferably, in step (1), the temperature of the cooling crystallization is 10~25 DEG C, the time is 12~for 24 hours.
In step (1), RA and ST in the filtered filter residue of crystallization containing high-content can be with the prior arts to both originals
Material extracts.
Preferably, in step (2), the temperature of the reduced pressure is 60~90 DEG C, vacuum degree is -0.06~-
0.09MPa。
Preferably, in step (2), the temperature of the drying is 60~90 DEG C.
Preferably, in step (2), the dosage of the reverse phase silica gel is 1~2 times of concentrate quality.The purpose for mixing sample is
Material and reverse phase silica gel is set sufficiently to adsorb；If the dosage of reverse phase silica gel is very few, material can not adsorb completely；If reverse phase silica gel
Dosage it is excessive, then easily cause waste and increase the difficulty of elution.
Preferably, in step (2), the type of the reverse phase silica gel is one or more of C4, C8 or C18 etc..
Preferably, in step (3), the dosage of the reverse phase silica gel be mix sample silica gel quality 1~20 times (more preferable 5~
15 times).The purpose that reverse phase silica gel is first added is to provide a buffer area, makes in elution process various composition in reverse phase silica gel
It is middle to retain the different time.If the dosage of reverse phase silica gel is very few, the relative time that various composition retains is too close to, and is unable to reach
Isolated purpose；If the dosage of reverse phase silica gel is excessive, easily causes waste and increase the dosage of eluant, eluent.
Preferably, in step (3), the ratio of height to diameter of the chromatographic column is 5~20:1.If ratio of height to diameter is too small, isolated effect
Fruit is poor；If ratio of height to diameter is excessive, the time of the dosage and elution of eluant, eluent will be increased.
Preferably, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h.
1BV=1 bed volume.The target elution efflux is to detect the elution stream containing rebaudioside D by thin-layer chromatography
Liquid section out.
Preferably, in step (4), the eluant, eluent is the methanol of more preferable 50~80%) 40~95%(of volumetric concentration, second
One or more of alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water etc..
Preferably, in step (4), it is concentrated into solvent-free, drying to constant weight.
Preferably, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality
(more preferable 4.5~10 times).
Preferably, in step (5), the organic solvent be more preferable 60~90%) 40~95%(of volumetric concentration methanol,
One or more of ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution etc..
Preferably, in step (5), the temperature of the heating for dissolving is 40~60 DEG C.
Preferably, in step (5), the temperature of the crystallization is 10~25 DEG C, the time is 12~for 24 hours.
The principle of the method for the present invention are as follows: since the content of RD in steviol glycoside refinement mother liquor is not generally high, after being dissolved with alcohol
It is difficult to reach the concentration that RD can be crystallized, RD will not crystallize precipitation, and other impurity components, the content such as RA and ST are high, then may be used
With in alcoholic solution mass crystallization be precipitated, first by after the contaminant filter of precipitation, most RD have been left in crystalline mother solution,
The content of RD is which thereby enhanced, the difficulty of subsequent inverter silica gel column chromatography is reduced.The characteristic of reverse phase silica gel first is that eluting
In the process, the big ingredient of polarity first flows out, and flows out after the small ingredient of polarity, and RD is that polarity is maximum in all steviol glycosides, because
This, flows out at first in the elution of reverse phase silica gel column, it is possible thereby to efficiently separate RD and other glucosides, finally by
Recrystallization, obtains the RD fine work of high-purity.
The method of the present invention has the beneficial effect that:
(1) the method for the present invention can extract the RD of high-purity, gained RD essence from cheap steviol glycoside refinement mother liquor
The white crystalline powder of product, is detected through high performance liquid chromatography, and the purity of RD may be up to 99.0wt%, and the ultimate yield of RD can
Up to 92.4%, improve the utilization rate of STEVIA REBAUDIANA resource；
(2) the method for the present invention raw material is natural, and used physics extracting mode, production process is environmentally protective, at low cost, produces
Product are pure, pollution-free, and the price of RD is 10 times of raw material or more, and added value of product is high, are suitable for industrialized production.
Specific embodiment
Below with reference to embodiment, the invention will be further described.
Steviol glycoside refinement mother liquor used in the embodiment of the present invention is from the Hunan China really limited public affairs of living resources share
Department crystallizes generated mother liquor when preparing high-purity RA, ST with organic solvent, through high-efficient liquid phase color using qualities of stevia extract as raw material
External standard method detection is composed, the mass content of RD is 6.5%；C4, C8 and C18 reverse phase silica gel used in the embodiment of the present invention are purchased from
Dalian Yilite Analytical Instrument Co., Ltd；Raw material or chemical reagent used in the embodiment of the present invention, unless otherwise specified,
It is obtained by routine business approach.
Embodiment 1
(1) crystallization removal of impurities: 100g steviol glycoside refinement mother liquor being added in the ethanol solution that 500g volumetric concentration is 80%,
It at 40 DEG C, dissolves by heating, then again at 25 DEG C, cooling crystallization 16h, filtering obtains filtrate；
(2) reverse phase silica gel mixes sample: by filtrate obtained by step (1) at 65 DEG C, vacuum degree is to be concentrated under reduced pressure under -0.06MPa
Without alcohol, 60g concentrate is obtained, C4 reverse phase silica gel 60g is added in 60g concentrate, is uniformly mixed, at 70 DEG C, drying to constant weight,
It obtains 100g and mixes sample silica gel；
(3) it fills column: in the empty chromatographic column that ratio of height to diameter is 15:1, being initially charged with 1000g C4 reverse phase silica gel, then in C4 reverse phase
It is packed into 100g obtained by step (2) on silica gel and mixes sample silica gel；
(4) it elutes: using 3BV, the acetonitrile solution that volumetric concentration is 70% fills the chromatographic column after column to step (3), in 0.5BV/
It under the flow velocity of h, is eluted, is detected by thin-layer chromatography, collected the target containing rebaudioside D and elute efflux, be concentrated into
It is dry to constant weight without acetonitrile, obtain 7.6g rebaudioside D crude product；
(5) it refines: the methanol solution for being 85% with 38g volumetric concentration by 7.6g rebaudioside D crude product obtained by step (4),
At 50 DEG C, dissolves by heating, be cooled to room temperature, at 10 DEG C, crystallization for 24 hours, is filtered, and it is dry, obtain 6.1g rebaudioside D fine work.
The white crystalline powder of rebaudioside D fine work obtained by the present embodiment, is detected, institute through high-performance liquid chromatography
The purity for obtaining rebaudioside D fine work is 98.5wt%, and the ultimate yield of rebaudioside D is 92.4%.
Embodiment 2
(1) crystallization removal of impurities: 50g steviol glycoside refinement mother liquor being added in the aqueous isopropanol that 300g volumetric concentration is 55%,
It at 60 DEG C, dissolves by heating, then again at 15 DEG C, cooling crystallization 12h, filtering obtains filtrate；
(2) reverse phase silica gel mixes sample: by filtrate obtained by step (1) at 85 DEG C, vacuum degree is to be concentrated under reduced pressure under -0.08MPa
Without alcohol, 35g concentrate is obtained, C8 reverse phase silica gel 70g is added in 35g concentrate, is uniformly mixed, at 90 DEG C, drying to constant weight,
It obtains 82g and mixes sample silica gel；
(3) it fills column: in the empty chromatographic column that ratio of height to diameter is 10:1, being initially charged with 440g C8 reverse phase silica gel, then in C8 reverse phase silicon
It is packed into 82g obtained by step (2) on glue and mixes sample silica gel；
(4) it elutes: using 5BV, the methanol solution that volumetric concentration is 60% fills the chromatographic column after column to step (3), in 1BV/h
Flow velocity under, eluted, detected by thin-layer chromatography, collected target containing rebaudioside D and elute efflux, be concentrated into nothing
Methanol, it is dry to constant weight, obtain 3.9g rebaudioside D crude product；
(5) it refines: the ethanol solution for being 90% with 30g volumetric concentration by 3.9g rebaudioside D crude product obtained by step (4),
At 55 DEG C, dissolves by heating, be cooled to room temperature, at 10 DEG C, crystallization 18h is filtered, and it is dry, obtain 2.95g rebaudioside D fine work.
The white crystalline powder of rebaudioside D fine work obtained by the present embodiment, is detected, institute through high-performance liquid chromatography
The purity for obtaining rebaudioside D fine work is 99.0wt%, and the ultimate yield of rebaudioside D is 89.9%.
Embodiment 3
(1) crystallization removal of impurities: 1kg steviol glycoside refinement mother liquor being added in the methanol solution that 8kg volumetric concentration is 95%,
It at 50 DEG C, dissolves by heating, then again at 20 DEG C, for 24 hours, filtering obtains filtrate to cooling crystallization；
(2) reverse phase silica gel mixes sample: by filtrate obtained by step (1) at 60 DEG C, vacuum degree is to be concentrated under reduced pressure under -0.09MPa
Without alcohol, 650g concentrate is obtained, C18 reverse phase silica gel 650g is added in 650g concentrate, is uniformly mixed, at 75 DEG C, drying is extremely
Constant weight obtains 1.1kg and mixes sample silica gel；
(3) it fills column: in the empty chromatographic column that ratio of height to diameter is 20:1, being initially charged with 16.5kg C18 reverse phase silica gel, then anti-in C18
It is packed into 1.1kg obtained by step (2) on phase silica gel and mixes sample silica gel；
(4) it eluting: using 10BV, the ethanol solution that volumetric concentration is 50% fills the chromatographic column after column to step (3),
It under the flow velocity of 1.5BV/h, is eluted, is detected by thin-layer chromatography, collected the target containing rebaudioside D and elute efflux,
It is concentrated into no ethyl alcohol, it is dry to constant weight, obtain 77g rebaudioside D crude product；
(5) it refines: the butanol solution for being 60% with 380g volumetric concentration by 77g rebaudioside D crude product obtained by step (4),
At 55 DEG C, dissolves by heating, be cooled to room temperature, at 25 DEG C, crystallization for 24 hours, is filtered, and it is dry, obtain 60g rebaudioside D fine work.
The white crystalline powder of rebaudioside D fine work obtained by the present embodiment, is detected, institute through high-performance liquid chromatography
The purity for obtaining rebaudioside D fine work is 98.6wt%, and the ultimate yield of rebaudioside D is 91.0%.
Claims (23)
1. a kind of method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, which is characterized in that including following step
It is rapid:
(1) crystallization removal of impurities: steviol glycoside refinement mother liquor is added in alcoholic solution, is dissolved by heating, cooling crystallization, and filtering obtains filtrate；
The mass content of rebaudioside D is 5~30% in the steviol glycoside refinement mother liquor；
(2) reverse phase silica gel mixes sample: filtrate decompression obtained by step (1) being concentrated into no alcohol, reverse phase silica gel is added in concentrate, is mixed
It closes uniformly, it is dry to constant weight, sample silica gel must be mixed；
(3) it fills column: in empty chromatographic column, being initially charged with reverse phase silica gel, then be packed on reverse phase silica gel and mix sample silicon obtained by step (2)
Glue；
(4) elute: the chromatographic column after with eluant, eluent step (3) are filled with column elutes, and collects target and elutes efflux, is concentrated,
It is dry, obtain rebaudioside D crude product；
(5) it refines: rebaudioside D crude product organic solvent obtained by step (4) or water is dissolved by heating, cooling, crystallization, filtering,
It is dry, obtain rebaudioside D fine work；The organic solvent is methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution
One or more of.
2. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 1, feature exist
In in step (1), the dosage of the alcoholic solution is 4~20 times of steviol glycoside refinement mother liquor quality；Alcohol in the alcoholic solution
For one or more of methanol, ethyl alcohol, isopropanol or n-butanol, the volumetric concentration of alcoholic solution is 50~99%.
3. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special
Sign is, in step (1), the temperature of the heating for dissolving is 40~60 DEG C；The temperature of the cooling crystallization is 10~25 DEG C, when
Between for 12~for 24 hours.
4. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special
Sign is, in step (2), the temperature of the reduced pressure is 60~90 DEG C, and vacuum degree is -0.06~-0.09MPa；It is described dry
Dry temperature is 60~90 DEG C.
5. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature exist
In in step (2), the temperature of the reduced pressure is 60~90 DEG C, and vacuum degree is -0.06~-0.09MPa；The drying
Temperature is 60~90 DEG C.
6. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special
Sign is, in step (2), the dosage of the reverse phase silica gel is 1~2 times of concentrate quality；The type of the reverse phase silica gel is
One or more of C4, C8 or C18.
7. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature exist
In in step (2), the dosage of the reverse phase silica gel is 1~2 times of concentrate quality；The type of the reverse phase silica gel is C4, C8
Or one or more of C18.
8. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 4, feature exist
In in step (2), the dosage of the reverse phase silica gel is 1~2 times of concentrate quality；The type of the reverse phase silica gel is C4, C8
Or one or more of C18.
9. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special
Sign is, in step (3), the dosage of the reverse phase silica gel is 1~20 times for mixing sample silica gel quality.
10. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature
It is, in step (3), the dosage of the reverse phase silica gel is 1~20 times for mixing sample silica gel quality.
11. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 4, feature
It is, in step (3), the dosage of the reverse phase silica gel is 1~20 times for mixing sample silica gel quality.
12. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 6, feature
It is, in step (3), the dosage of the reverse phase silica gel is 1~20 times for mixing sample silica gel quality.
13. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special
Sign is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The elution
Agent is methanol, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or the water of volumetric concentration 40~95%
One or more of.
14. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature
It is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The eluant, eluent
For in the methanol of volumetric concentration 40~95%, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water
One or more.
15. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 4, feature
It is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The eluant, eluent
For in the methanol of volumetric concentration 40~95%, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water
One or more.
16. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 6, feature
It is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The eluant, eluent
For in the methanol of volumetric concentration 40~95%, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water
One or more.
17. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 9, feature
It is, in step (4), the dosage of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；The eluant, eluent
For in the methanol of volumetric concentration 40~95%, ethyl alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water
One or more.
18. the method according to claim 1 or claim 2 for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor, special
Sign is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic
Solvent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several
Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.
19. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 3, feature
It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten
Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several
Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.
20. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 4, feature
It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten
Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several
Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.
21. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 6, feature
It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten
Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several
Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.
22. the method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 9, feature
It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten
Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several
Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.
23. 3 method for extracting high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 1, feature
It is, in step (5), the dosage of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described organic molten
Agent is one of methanol, ethyl alcohol, isopropanol, n-butanol, acetone or ethyl acetate solution of volumetric concentration 40~95% or several
Kind；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization be 10~25 DEG C, the time be 12~for 24 hours.
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| CN107722070A (en) * | 2017-11-20 | 2018-02-23 | 浙江天草生物科技股份有限公司 | A kind of method for preparing high-purity steviosides RD |
| CN107955048A (en) * | 2017-12-25 | 2018-04-24 | 史迪威生物科技（苏州）有限公司 | The isolation and purification technique of rebaudioside D |
| CN108558966A (en) * | 2018-01-04 | 2018-09-21 | 常州嘉众新材料科技有限公司 | The production method of Rebaudiodside A D in a kind of enriching stevia sugar |
| BR112022008219A2 (en) * | 2019-11-01 | 2022-07-12 | Suntory Holdings Ltd | METHOD FOR PRODUCING A CRYSTALLIZED PRODUCT CONTAINING REBAUDIOSIDE D, CRYSTALLIZED PRODUCT CONTAINING REBAUDIOSIDE D, E, FOOD OR BEVERAGE PRODUCT |
| CN113201034A (en) * | 2021-04-25 | 2021-08-03 | 四川大学 | Obtaining high-purity stevioside from primary crystallization mother liquor of stevioside through secondary crystallization and enriching rebaudioside C |
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