CN106967133A - A kind of method that high-purity rebaudioside D is extracted in the refinement mother liquor from steviol glycoside - Google Patents
A kind of method that high-purity rebaudioside D is extracted in the refinement mother liquor from steviol glycoside Download PDFInfo
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- CN106967133A CN106967133A CN201710200612.9A CN201710200612A CN106967133A CN 106967133 A CN106967133 A CN 106967133A CN 201710200612 A CN201710200612 A CN 201710200612A CN 106967133 A CN106967133 A CN 106967133A
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- silica gel
- mother liquor
- steviol glycoside
- rebaudioside
- reverse phase
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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 96
- 235000019202 steviosides Nutrition 0.000 title claims abstract description 38
- 239000012452 mother liquor Substances 0.000 title claims abstract description 34
- 239000004383 Steviol glycoside Substances 0.000 title claims abstract description 33
- 229930182488 steviol glycoside Natural products 0.000 title claims abstract description 33
- 235000019411 steviol glycoside Nutrition 0.000 title claims abstract description 33
- 150000008144 steviol glycosides Chemical class 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 29
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 55
- 239000000741 silica gel Substances 0.000 claims abstract description 45
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 45
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 39
- 238000010828 elution Methods 0.000 claims abstract description 22
- 238000002425 crystallisation Methods 0.000 claims abstract description 21
- 230000008025 crystallization Effects 0.000 claims abstract description 20
- 239000012043 crude product Substances 0.000 claims abstract description 14
- 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
- 239000003480 eluent Substances 0.000 claims abstract description 10
- 238000001914 filtration Methods 0.000 claims abstract description 9
- 239000003960 organic solvent Substances 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000012535 impurity Substances 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 14
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 11
- 239000012141 concentrate Substances 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 3
- 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
- 239000000047 product Substances 0.000 abstract description 8
- 239000000843 powder Substances 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 29
- 239000000243 solution Substances 0.000 description 16
- 239000002994 raw material Substances 0.000 description 6
- 239000000499 gel Substances 0.000 description 5
- 239000000377 silicon dioxide Substances 0.000 description 5
- 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 4
- 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
- 229940013618 stevioside Drugs 0.000 description 4
- 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 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
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229930188195 rebaudioside Natural products 0.000 description 3
- 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 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 241000954177 Bangana ariza Species 0.000 description 2
- 244000269722 Thea sinensis Species 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- 150000008131 glucosides Chemical class 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 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 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 239000001776 FEMA 4720 Substances 0.000 description 1
- 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
- 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
- 238000009413 insulation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 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 1
- 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 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
- 238000000926 separation method Methods 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)
- Saccharide Compounds (AREA)
- Cosmetics (AREA)
Abstract
A kind of method that high-purity rebaudioside D is extracted in refinement mother liquor from steviol glycoside, comprises the following steps：（1）Crystallize removal of impurities：Steviol glycoside refinement mother liquor is added in alcoholic solution, dissolving, crystallization, filtering obtains filtrate；（2）Reverse phase silica gel mixes sample：No alcohol is concentrated the filtrate to, reverse phase silica gel is added, is well mixed, dries, sample silica gel must be mixed；（3）Fill post：In empty chromatographic column, reverse phase silica gel is initially charged with, is reloaded into and mixes sample silica gel；（4）Elution：Chromatographic column is eluted with eluant, eluent, efflux is collected, concentrated, dries, obtains RD crude products；（5）It is refined：RD crude products organic solvent or water are dissolved, cooling, crystallization is filtered, and is dried, is obtained RD fine work.The white crystalline powders of RD obtained by the inventive method, purity may be up to 99.0wt%, and yield may be up to 92.4%；The inventive method environmental protection, cost is low, and product is pure, pollution-free, and added value of product is high, it is adaptable to industrialized production.
Description
Technical field
The present invention relates to a kind of extracting method of rebaudioside D, and in particular to one kind is carried from steviol glycoside refinement mother liquor
The method for taking high-purity rebaudioside D.
Background technology
Major glycosides in steviol glycoside have stevioside（ST）And REBA（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 finds that the best composition of mouthfeel is not sugariness highest RA in steviol glycoside, but RD, RD sweet tea
Degree is about 150 times of sucrose, and in good taste, no bitter taste, still, contents 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 a kind of stevioside RD method of purification, is that mother liquor sugar feed liquid is flowed through into macroporous absorption
Resin, then with ethanol elution, Fractional Collections eluent is concentrated, and is dried, and is refined, is obtained RD.But, due to the suction of macroreticular resin
Attached poor selectivity, by stepwise elution, by determining that the method collection RD operability of critical point is relatively low, RD is difficult to be enriched with,
Therefore, RD yield is not high.
CN102406113A discloses a kind of preparation method of RA and RD edible compound steviosides, is, using STEVIA REBAUDIANA as raw material, to lead to
Water extraction is crossed, macroporous resin adsorption is eluted, decolourized, crystallization, the step such as recrystallization obtains RA and RD product mix.But,
This method can not obtain the RD of high content.
A kind of CN103709215A extracts rebaudioside D method in disclosing mother liquor sugar from stevioside, is by sweet tea
Synanthrin mother liquor sugar water and methanol insulation, stirring, repeated crystallization obtain RD products.But, this method is excessive because of crystallisation times,
Yield is relatively low, and RD purity only 50% or so.
The content of the invention
The technical problems to be solved by the invention are that the drawbacks described above for overcoming prior art to exist is produced there is provided one kind gained
Product purity, high income, added value of product are high, and environmental protection, cost is low, it is adaptable to industrialized production from the refined mother of steviol glycoside
The method that high-purity rebaudioside D is extracted in liquid.
The technical solution adopted for 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, comprises the following steps：
（1）Crystallize removal of impurities：Steviol glycoside refinement mother liquor is added in alcoholic solution, dissolved by heating, cooling crystallization, filtering obtains filtrate；
（2）Reverse phase silica gel mixes sample：By step（1）Gained filtrate decompression is concentrated into no alcohol, and reverse phase silica gel is added in concentrate, mixes
Close uniform, dry to constant weight, sample silica gel must be mixed；
（3）Fill post：In empty chromatographic column, reverse phase silica gel is initially charged with, then loads on reverse phase silica gel step（2）Gained mixes sample silicon
Glue；
（4）Elution：With eluant, eluent to step（3）Chromatographic column after dress post is eluted, and is collected target elution efflux, is concentrated,
Dry, obtain rebaudioside D crude product；
（5）It is refined：By step（4）Gained rebaudioside D crude product organic solvent or water are dissolved by heating, cooling, crystallization, filtering,
Dry, obtain rebaudioside D fine work.
Preferably, step（1）In, the mass content of rebaudioside D is 5~30% in the steviol glycoside refinement mother liquor（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, uses organic solvent knot
Crystalline substance prepares high-purity REBA（RA）, stevioside（ST）When produced mother liquor.
Preferably, step（1）In, the consumption of the alcoholic solution is 4~20 times of steviol glycoside refinement mother liquor quality（It is more excellent
Select 5~10 times）.
Preferably, step（1）In, the alcohol in the alcoholic solution is one kind in methanol, ethanol, isopropanol or n-butanol etc.
Or it is several, the volumetric concentration of alcoholic solution is 50~99%.
Preferably, step（1）In, the temperature of the heating for dissolving is 40~60 DEG C.
Preferably, step（1）In, the temperature of the cooling crystallization is 10~25 DEG C, and the time is 12~24h.
Step（1）In, RA and ST containing high content in the filter residue after crystallization filtering can be former to both with prior art
Material is extracted.
Preferably, step（2）In, the temperature being concentrated under reduced pressure be 60~90 DEG C, vacuum be -0.06~-
0.09MPa。
Preferably, step（2）In, the temperature of the drying is 60~90 DEG C.
Preferably, step（2）In, the consumption 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 fully to adsorb；If the consumption of reverse phase silica gel is very few, material can not be adsorbed completely；If reverse phase silica gel
Consumption it is excessive, then easily cause and waste and increase the difficulty of elution.
Preferably, step（2）In, the type of the reverse phase silica gel is the one or more in C4, C8 or C18 etc..
Preferably, step（3）In, the consumption of the reverse phase silica gel is mix sample silica gel quality 1~20 times（More preferably 5~
15 times）.The purpose for first adding reverse phase silica gel is to provide a buffer area, makes in elution process various composition in reverse phase silica gel
It is middle to retain the different times.If the consumption of reverse phase silica gel is very few, the relative time that various composition retains is too close to, it is impossible to reached
The purpose of separation；If the consumption of reverse phase silica gel is excessive, easily causes and waste and increase the consumption of eluant, eluent.
Preferably, step（3）In, the ratio of height to diameter of the chromatographic column is 5~20:1.If ratio of height to diameter is too small, the effect separated
It is really poor；If ratio of height to diameter is excessive, by the consumption for increasing eluant, eluent and the time of elution.
Preferably, step（4）In, the consumption 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
Go out liquid section.
Preferably, step（4）In, the eluant, eluent is volumetric concentration 40~95%（More preferably 50~80%）Methanol, second
One or more in alcohol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, ethyl acetate solution or water etc..
Preferably, step（4）In, it is concentrated into solvent-free, drying to constant weight.
Preferably, step（5）In, the consumption of the organic solvent or water is 4~20 times of rebaudioside D crude product quality
（More preferably 4.5~10 times）.
Preferably, step（5）In, the organic solvent is volumetric concentration 40~95%（More preferably 60~90%）Methanol,
One or more in ethanol, isopropanol, n-butanol, acetone or ethyl acetate solution etc..
Preferably, step（5）In, the temperature of the heating for dissolving is 40~60 DEG C.
Preferably, step（5）In, the temperature of the crystallization is 10~25 DEG C, and the time is 12~24h.
The principle of the inventive method is：Because the content of RD in steviol glycoside refinement mother liquor is not universal high, after being dissolved with alcohol
It is difficult to the concentration for reaching that RD can be crystallized, RD will not crystallize precipitation, and the content of other impurity components, such as RA and ST is high, then may be used
With in alcoholic solution mass crystallization separate out, first by after the contaminant filter of precipitation, most RD have been left in crystalline mother solution,
RD content is which thereby enhanced, the difficulty of subsequent inverter silica gel column chromatography is reduced.One of characteristic of reverse phase silica gel is in elution
During, the big composition of polarity first flows out, and is flowed out after the small composition of polarity, and RD is polarity maximum in all steviol glycosides, because
This, flows out at first in the elution of reverse phase silica gel post, it is possible thereby to efficient separate RD and other glucosides, finally by
Recrystallization, obtains the RD fine work of high-purity.
The inventive method has the beneficial effect that：
（1）The inventive method can extract the RD of high-purity from cheap steviol glycoside refinement mother liquor, and gained RD fine work is in
White crystalline powder, is detected through high performance liquid chromatography, and RD purity may be up to 99.0wt%, and RD ultimate yield may be up to
92.4%, improve the utilization rate of STEVIA REBAUDIANA resource；
（2）The inventive method raw material is natural, and the physics extracting mode used, production process environmental protection, cost is low, and product is pure
Only, pollution-free, RD price is more than 10 times of raw material, and added value of product is high, it is adaptable to industrialized production.
Embodiment
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 mother liquor produced 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, RD mass content is 6.5%；C4, C8 and C18 reverse phase silica gel are purchased from used in the embodiment of the present invention
Dalian Yilite Analytical Instrument Co., Ltd；Raw material used in the embodiment of the present invention or chemical reagent, unless otherwise specified,
Obtained by routine business approach.
Embodiment 1
（1）Crystallize removal of impurities：100g steviol glycosides refinement mother liquor is added in the ethanol solution that 500g volumetric concentrations are 80%, 40
At DEG C, dissolve by heating, then again at 25 DEG C, cooling crystallization 16h, filtering obtains filtrate；
（2）Reverse phase silica gel mixes sample：By step（1）Gained filtrate under vacuum is -0.06MPa, is concentrated under reduced pressure into nothing at 65 DEG C
Alcohol, obtains 60g concentrates, and C4 reverse phase silica gel 60g are added in 60g concentrates, is well mixed, at 70 DEG C, dries to constant weight, obtains
100g mixes sample silica gel；
（3）Fill post：It is 15 in ratio of height to diameter:In 1 empty chromatographic column, 1000g C4 reverse phase silica gels are initially charged with, then in C4 reverse phase silica gels
Upper loading step（2）Gained 100g mixes sample silica gel；
（4）Elution：With 3BV, the acetonitrile solution that volumetric concentration is 70% is to step（3）The chromatographic column after post is filled, 0.5BV/h's
Under flow velocity, eluted, detected by thin-layer chromatography, collected the target elution efflux containing rebaudioside D, be concentrated into no second
Nitrile, dries to constant weight, obtains 7.6g rebaudioside D crude products；
（5）It is refined：By step（4）The gained 7.6g rebaudioside Ds crude product methanol solution that 38g volumetric concentrations are 85%, at 50 DEG C
Under, dissolve by heating, be cooled to room temperature, at 10 DEG C, crystallization 24h is filtered, and is dried, is obtained 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）Crystallize removal of impurities：50g steviol glycosides refinement mother liquor is added in the aqueous isopropanol that 300g volumetric concentrations are 55%, 60
At DEG C, dissolve by heating, then again at 15 DEG C, cooling crystallization 12h, filtering obtains filtrate；
（2）Reverse phase silica gel mixes sample：By step（1）Gained filtrate under vacuum is -0.08MPa, is concentrated under reduced pressure into nothing at 85 DEG C
Alcohol, obtains 35g concentrates, and C8 reverse phase silica gel 70g are added in 35g concentrates, is well mixed, at 90 DEG C, dries to constant weight, obtains
82g mixes sample silica gel；
（3）Fill post：It is 10 in ratio of height to diameter:In 1 empty chromatographic column, 440g C8 reverse phase silica gels are initially charged with, then on C8 reverse phase silica gels
Load step（2）Gained 82g mixes sample silica gel；
（4）Elution：With 5BV, the methanol solution that volumetric concentration is 60% is to step（3）The chromatographic column after post is filled, in 1BV/h stream
Under speed, eluted, detected by thin-layer chromatography, collected the target elution efflux containing rebaudioside D, be concentrated into no first
Alcohol, dries to constant weight, obtains 3.9g rebaudioside D crude products；
（5）It is refined：By step（4）The gained 3.9g rebaudioside Ds crude product ethanol solution that 30g volumetric concentrations are 90%, at 55 DEG C
Under, dissolve by heating, be cooled to room temperature, at 10 DEG C, crystallization 18h is filtered, and is dried, is obtained 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）Crystallize removal of impurities：1kg steviol glycosides refinement mother liquor is added in the methanol solution that 8kg volumetric concentrations are 95%, at 50 DEG C
Under, dissolve by heating, then again at 20 DEG C, cooling crystallization 24h, filtering obtains filtrate；
（2）Reverse phase silica gel mixes sample：By step（1）Gained filtrate under vacuum is -0.09MPa, is concentrated under reduced pressure into nothing at 60 DEG C
Alcohol, obtains 650g concentrates, and C18 reverse phase silica gel 650g are added in 650g concentrates, is well mixed, at 75 DEG C, dries to perseverance
Weight, obtains 1.1kg and mixes sample silica gel；
（3）Fill post：It is 20 in ratio of height to diameter:In 1 empty chromatographic column, 16.5kg C18 reverse phase silica gels are initially charged with, then in the anti-phase silicon of C18
Load step on glue（2）Gained 1.1kg mixes sample silica gel；
（4）Elution：With 10BV, the ethanol solution that volumetric concentration is 50% is to step（3）The chromatographic column after post is filled, 1.5BV/h's
Under flow velocity, eluted, detected by thin-layer chromatography, collected the target elution efflux containing rebaudioside D, be concentrated into no second
Alcohol, dries to constant weight, obtains 77g rebaudioside D crude products；
（5）It is refined：By step（4）The gained 77g rebaudioside Ds crude product butanol solution that 380g volumetric concentrations are 60%, 55
At DEG C, dissolve by heating, be cooled to room temperature, at 25 DEG C, crystallization 24h is filtered, and is dried, is obtained 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 (10)
1. the method for high-purity rebaudioside D is extracted in a kind of refinement mother liquor from steviol glycoside, it is characterised in that including following step
Suddenly：
（1）Crystallize removal of impurities：Steviol glycoside refinement mother liquor is added in alcoholic solution, dissolved by heating, cooling crystallization, filtering obtains filtrate；
（2）Reverse phase silica gel mixes sample：By step（1）Gained filtrate decompression is concentrated into no alcohol, and reverse phase silica gel is added in concentrate, mixes
Close uniform, dry to constant weight, sample silica gel must be mixed；
（3）Fill post：In empty chromatographic column, reverse phase silica gel is initially charged with, then loads on reverse phase silica gel step（2）Gained mixes sample silicon
Glue；
（4）Elution：With eluant, eluent to step（3）Chromatographic column after dress post is eluted, and is collected target elution efflux, is concentrated,
Dry, obtain rebaudioside D crude product；
（5）It is refined：By step（4）Gained rebaudioside D crude product organic solvent or water are dissolved by heating, cooling, crystallization, filtering,
Dry, obtain rebaudioside D fine work.
2. extracting the method for high-purity rebaudioside D from steviol glycoside refinement mother liquor according to claim 1, its feature exists
In step（1）In, the mass content of rebaudioside D is 5~30% in the steviol glycoside refinement mother liquor.
3. extracting the method for high-purity rebaudioside D in the refinement mother liquor according to claim 1 or claim 2 from steviol glycoside, it is special
Levy and be, step（1）In, the consumption of the alcoholic solution is 4~20 times of steviol glycoside refinement mother liquor quality；In the alcoholic solution
Alcohol be one or more in methanol, ethanol, isopropanol or n-butanol, the volumetric concentration of alcoholic solution is 50~99%.
4. the method for high-purity rebaudioside D is extracted from steviol glycoside refinement mother liquor according to one of claims 1 to 3,
Characterized in that, step（1）In, the temperature of the heating for dissolving is 40~60 DEG C；The temperature of the cooling crystallization is 10~25
DEG C, the time is 12~24h.
5. the method for high-purity rebaudioside D is extracted from steviol glycoside refinement mother liquor according to one of Claims 1 to 4,
Characterized in that, step（2）In, the temperature being concentrated under reduced pressure is 60~90 DEG C, and vacuum is -0.06~-0.09MPa；Institute
Dry temperature is stated for 60~90 DEG C.
6. the method for high-purity rebaudioside D is extracted from steviol glycoside refinement mother liquor according to one of Claims 1 to 5,
Characterized in that, step（2）In, the consumption of the reverse phase silica gel is 1~2 times of concentrate quality；The class of the reverse phase silica gel
Type is the one or more in C4, C8 or C18.
7. the method for high-purity rebaudioside D is extracted from steviol glycoside refinement mother liquor according to one of claim 1~6,
Characterized in that, step（3）In, the consumption of the reverse phase silica gel is mix sample silica gel quality 1~20 times.
8. the method for high-purity rebaudioside D is extracted from steviol glycoside refinement mother liquor according to one of claim 1~7,
Characterized in that, step（3）In, the ratio of height to diameter of the chromatographic column is 5~20:1.
9. the method for high-purity rebaudioside D is extracted from steviol glycoside refinement mother liquor according to one of claim 1~8,
Characterized in that, step（4）In, the consumption of the eluant, eluent is 2~10BV, and the flow velocity of elution is 0.5~2.0BV/h；It is described
Eluant, eluent is methanol, ethanol, isopropanol, n-butanol, acetonitrile, acetic acid, acetone, the ethyl acetate solution of volumetric concentration 40~95%
Or the one or more in water.
10. the method for high-purity rebaudioside D is extracted from steviol glycoside refinement mother liquor according to one of claim 1~9,
Characterized in that, step（5）In, the consumption of the organic solvent or water is 4~20 times of rebaudioside D crude product quality；It is described
Organic solvent is one kind in methanol, ethanol, isopropanol, n-butanol, acetone or the ethyl acetate solution of volumetric concentration 40~95%
Or it is several；The temperature of the heating for dissolving is 40~60 DEG C；The temperature of the crystallization is 10~25 DEG C, and the time is 12~24h.
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