CN118047818A - Method for preparing dulcoside A and rubusoside based on resin adsorption and chromatographic separation - Google Patents
Method for preparing dulcoside A and rubusoside based on resin adsorption and chromatographic separation Download PDFInfo
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- CN118047818A CN118047818A CN202410134783.6A CN202410134783A CN118047818A CN 118047818 A CN118047818 A CN 118047818A CN 202410134783 A CN202410134783 A CN 202410134783A CN 118047818 A CN118047818 A CN 118047818A
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- rubusoside
- dulcoside
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- resin
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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 title claims abstract description 61
- 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 title claims abstract description 61
- 239000011347 resin Substances 0.000 title claims abstract description 41
- 229920005989 resin Polymers 0.000 title claims abstract description 41
- 238000001179 sorption measurement Methods 0.000 title claims abstract description 35
- 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 title claims abstract description 33
- CANAPGLEBDTCAF-QHSHOEHESA-N Dulcoside A Natural products C[C@@H]1O[C@H](O[C@@H]2[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]2O[C@]34CC[C@H]5[C@]6(C)CCC[C@](C)([C@H]6CC[C@@]5(CC3=C)C4)C(=O)O[C@@H]7O[C@H](CO)[C@@H](O)[C@H](O)[C@H]7O)[C@H](O)[C@H](O)[C@H]1O CANAPGLEBDTCAF-QHSHOEHESA-N 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000013375 chromatographic separation Methods 0.000 title claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 92
- 239000007788 liquid Substances 0.000 claims abstract description 31
- 239000003480 eluent Substances 0.000 claims abstract description 30
- 238000004458 analytical method Methods 0.000 claims abstract description 22
- 239000012452 mother liquor Substances 0.000 claims abstract description 20
- 238000010828 elution Methods 0.000 claims abstract description 13
- 239000002904 solvent Substances 0.000 claims abstract description 13
- 239000013078 crystal Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 77
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- 238000002425 crystallisation Methods 0.000 claims description 30
- 230000008025 crystallization Effects 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- CANAPGLEBDTCAF-UHFFFAOYSA-N dulcoside a Chemical compound OC1C(O)C(O)C(C)OC1OC1C(OC23C(CC4(C2)C(C2(C)C(C(CCC2)(C)C(=O)OC2C(C(O)C(O)C(CO)O2)O)CC4)CC3)=C)OC(CO)C(O)C1O CANAPGLEBDTCAF-UHFFFAOYSA-N 0.000 claims description 11
- 238000001704 evaporation Methods 0.000 claims description 11
- 239000000945 filler Substances 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 239000012065 filter cake Substances 0.000 claims description 8
- 238000005191 phase separation Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 5
- 239000012046 mixed solvent Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 235000019605 sweet taste sensations Nutrition 0.000 claims description 2
- 235000019202 steviosides Nutrition 0.000 abstract description 9
- 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 abstract description 8
- 229940013618 stevioside Drugs 0.000 abstract description 8
- 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 abstract description 8
- 239000002994 raw material Substances 0.000 abstract description 7
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 15
- 229930182470 glycoside Natural products 0.000 description 7
- 150000002338 glycosides Chemical class 0.000 description 5
- 238000012856 packing Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 4
- 238000001953 recrystallisation Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 244000228451 Stevia rebaudiana Species 0.000 description 2
- 235000006092 Stevia rebaudiana Nutrition 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method 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
- 238000011160 research Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- -1 terpene glycosides Chemical class 0.000 description 2
- 235000007586 terpenes Nutrition 0.000 description 2
- YSCJAYPKBYRXEZ-HZPINHDXSA-N (2s,3s,4s,5r,6r)-6-[[(3s,4ar,6ar,6bs,8as,12as,14ar,14br)-4,4,6a,6b,11,11,14b-heptamethyl-8a-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxycarbonyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-3-hydroxy-4-[(2s,3r,4s, Chemical compound O([C@H]1[C@H](O)[C@H](O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@]1(CCC(C[C@H]14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C(O)=O)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YSCJAYPKBYRXEZ-HZPINHDXSA-N 0.000 description 1
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 1
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 1
- 239000001512 FEMA 4601 Substances 0.000 description 1
- 239000001776 FEMA 4720 Substances 0.000 description 1
- 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 1
- QFVOYBUQQBFCRH-UHFFFAOYSA-N Steviol Natural products C1CC2(C3)CC(=C)C3(O)CCC2C2(C)C1C(C)(C(O)=O)CCC2 QFVOYBUQQBFCRH-UHFFFAOYSA-N 0.000 description 1
- 239000004383 Steviol glycoside Substances 0.000 description 1
- OMHUCGDTACNQEX-OSHKXICASA-N Steviolbioside Natural products 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]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O OMHUCGDTACNQEX-OSHKXICASA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- JLPRGBMUVNVSKP-AHUXISJXSA-M chembl2368336 Chemical compound [Na+].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]1O[C@@H](CO)[C@@H](O)[C@H](O)[C@@H]1O JLPRGBMUVNVSKP-AHUXISJXSA-M 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 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 1
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 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 1
- 235000019203 rebaudioside A Nutrition 0.000 description 1
- 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 description 1
- RLLCWNUIHGPAJY-SFUUMPFESA-N rebaudioside E 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]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 RLLCWNUIHGPAJY-SFUUMPFESA-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
- QFVOYBUQQBFCRH-VQSWZGCSSA-N steviol Chemical compound C([C@@]1(O)C(=C)C[C@@]2(C1)CC1)C[C@H]2[C@@]2(C)[C@H]1[C@](C)(C(O)=O)CCC2 QFVOYBUQQBFCRH-VQSWZGCSSA-N 0.000 description 1
- 229940032084 steviol Drugs 0.000 description 1
- 229930182488 steviol glycoside Natural products 0.000 description 1
- 235000019411 steviol glycoside Nutrition 0.000 description 1
- 150000008144 steviol glycosides Chemical class 0.000 description 1
- DRSKVOAJKLUMCL-MMUIXFKXSA-N u2n4xkx7hp 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]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O DRSKVOAJKLUMCL-MMUIXFKXSA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Saccharide Compounds (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for preparing Dukkoside A and rubusoside based on resin adsorption and chromatographic separation, which comprises the following steps: firstly, enriching stevioside mother liquor sugar feed liquid by adopting a normal pressure resin column, and then carrying out gradient analysis by adopting ethanol solutions with different concentrations to obtain analysis liquid containing dulcoside A and rubusoside; removing solvent from the solution containing Dunalioside A and rubusoside, dissolving with high alcohol solution, sequentially separating with multiple medium-low pressure chromatographic columns connected in series, respectively performing gradient elution with different concentrations of alcohol solution on single chromatographic column, and respectively collecting the eluate containing Dunalioside A and the eluate containing rubusoside; crystallizing the eluent containing Du Kegan A to obtain high-purity Dunalioside A, and extracting the eluent containing rubusoside to obtain high-purity rubusoside. The invention takes the crystal mother liquor sugar as the raw material to prepare the high-purity Dukkoside A and rubusoside, thereby greatly improving the utilization rate of the crystal mother liquor sugar and reducing the energy consumption.
Description
Technical Field
The invention relates to the technical field of stevioside preparation, in particular to a method for preparing Dukkoside A and rubusoside based on resin adsorption and chromatographic separation.
Background
Steviol glycoside is a mixture of various terpene glycosides with the same steviol aglycone core and various sugar groups attached to C13 and C19, and the structure of more than ten terpene glycosides has been identified so far, most of the glycosides in natural stevia rebaudiana are rebaudioside A (2% -10%), stevioside (2% -10%) and rebaudioside C (1% -2%), with other glycosides such as rebaudioside B, D, E and F, steviolbioside and stevioside being present in stevia rebaudiana at low levels (less than 1%). Duke's glycoside A and rubusoside belong to two components of rare glycoside in stevioside.
At present, the international research is mainly focused on the purification method of single components such as STV, RA and the like in stevioside, so that a large amount of crystallized mother liquor sugar after RA and STV are extracted needs to be treated. If stevioside crystallization mother liquor sugar can be used as a raw material, and Dunalioside A and rubusoside can be continuously separated and extracted, the raw material can be fully utilized, and a product with better taste can be obtained, but the research on separation and preparation of Dunalioside A and rubusoside is little at present.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the defects existing in the prior art, the method for preparing the dulcoside A and the rubusoside based on resin adsorption and chromatographic separation is provided, and takes the crystalline mother liquor sugar as a raw material to prepare the high-purity dulcoside A and the rubusoside, so that the utilization rate of the crystalline mother liquor sugar is greatly improved, and the consumption of energy sources is reduced.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method for preparing dulcoside A and rubusoside based on resin adsorption and chromatographic separation, comprising the following steps:
S1: enriching by using a normal pressure resin column:
Dissolving crystalline mother liquor sugar in water, then, introducing the crystalline mother liquor sugar into macroporous adsorption resin for adsorption, introducing the mixture to sweet taste, then, washing the mixture with water, then, sequentially adopting an ethanol solution with the concentration of 15-25wt%, 35-45wt% and 55-65wt% as a resolving agent for gradient resolving the macroporous adsorption resin, collecting an ethanol solution resolving section with the concentration of 55-65wt%, namely resolving liquid containing Dukkoside A and rubusoside, and collecting resolving liquid for later use when resolving the ethanol solution with the concentration of 15-25wt% and 35-45 wt%;
S2: separating by medium-low pressure chromatographic column:
Evaporating the analytical solution containing the dulcoside A and the rubusoside to remove the solvent to obtain mixed sugar containing the dulcoside A and the rubusoside, dissolving the mixed sugar by adopting a high alcohol solution, sequentially separating by adopting a plurality of medium-low pressure chromatographic columns connected in series, and respectively carrying out gradient elution on single chromatographic columns by adopting ethanol solutions with different concentrations to respectively collect eluent containing the dulcoside A and eluent containing the rubusoside;
s3: purification of dulcoside a:
Evaporating the eluent containing the dulcoside A to remove the solvent to obtain a crude product of the dulcoside A, performing primary crystallization treatment on the dulcoside A by taking a methanol solution as a crystallization solvent, filtering crystallization feed liquid, performing secondary crystallization treatment on an obtained filter cake by adopting the methanol solution, filtering the crystallization feed liquid, and drying the obtained precipitate to obtain the high-purity dulcoside A;
s4: purifying rubusoside:
and mixing the crystallization mother liquor obtained in the primary crystallization treatment with the eluent containing rubusoside, evaporating and concentrating to obtain a concentrated solution, extracting for a plurality of times by adopting a mixed solvent of ethyl acetate and n-butanol, merging raffinate phases, and evaporating to remove the solvent to obtain the high-purity rubusoside.
As an improvement of the technical scheme, in the step S1, the macroporous adsorption resin is one of LX-28, LK-1300SD, LX-T83, 69M, DM and SPD-009, the volume of the feed liquid during sample injection is 0.15-0.2BV of the volume of the resin, and the flow rate of the feed liquid is less than 1BV/h.
As an improvement of the technical scheme, in the step S1, the volume of water is 1-2BV of the volume of resin during water washing, and the flow rate of water is 1-2BV/h.
As an improvement of the technical scheme, in the step S1, the volume of the resolving agent is 1-2BV of the resin volume during gradient resolving, and the flow rate of the resolving agent is 2-3BV/h.
As an improvement of the technical scheme, in the step S2, the concentration of the high alcohol solution is 90-95wt% of ethanol solution, and the solid content of the dissolved solution is controlled to be 5-20wt% when the high alcohol solution is adopted to dissolve the mixed sugar.
As an improvement of the above technical scheme, in step S2, the packing in the middle-low pressure chromatographic column is normal phase separation packing or reverse phase separation packing, and the normal phase separation packing is silica gel and bonding phase packing with NH2 and APS, CN, CPS groups; the reversed-phase separation filler is one or more of a carbon-eight column (C18/ODS), a carbon-eight column (C8/MOS), a carbon-four column (C4/Butyl) and a Phenyl column (C6H 5/Phenyl); further, the medium-low pressure chromatographic column is a YMC-Pack CN chromatographic column.
As an improvement of the above technical scheme, in step S2, the ratio of the sample loading amount when the middle-low pressure chromatographic column is used for separation to the amount of filler in the middle-low pressure chromatographic column is 1g: (0.001-0.03 ml), and the flow rate of the feed liquid during sample loading is less than 1BV/h.
As an improvement of the technical scheme, in the step S2, the concentration of the ethanol solution adopted in gradient elution is 95-98wt%, 85-90wt%, 75-80wt% and 65-70wt%, the flow rate of the ethanol solution in elution is 1-2BV/h, and the eluent containing Du Kegan A is obtained by respectively collecting the eluent of the tail chromatographic column and the chromatographic column adjacent to the tail chromatographic column eluted by the 85-90wt% ethanol solution; and respectively collecting the eluent of the tail chromatographic column and the chromatographic columns adjacent to the tail chromatographic column when the tail chromatographic column is eluted by 75-80wt% ethanol solution, namely the eluent containing rubusoside.
As an improvement of the technical scheme, in the step S2, other chromatographic columns which are connected in series except the chromatographic column at the tail part and the chromatographic column adjacent to the chromatographic column at the tail part are respectively eluted by adopting ethanol solution with the concentration of 95-98wt% and 85-90wt%, and the eluent is combined with the analysis solution obtained in the step S1 when the ethanol solution with the concentration of 15-25wt% and the ethanol solution with the concentration of 35-45wt% is analyzed, and the combined solution is dried and reused as the crystallization mother liquid sugar.
As an improvement of the above technical scheme, in step S3, the volume ratio of methanol to water in the methanol solution during the primary crystallization treatment is 3: (1-2), the dosage ratio of the methanol solution to the crude dulcoside A product in one crystallization treatment is (2-5) ml:1g, the temperature during the primary crystallization treatment is room temperature, and the time is 20-30h.
As an improvement of the technical scheme, the volume ratio of methanol to water in the methanol solution during the secondary crystallization treatment is 3: (1-2), the dosage ratio of the methanol solution to the filter cake in the secondary crystallization treatment is (5-10) ml:1g; the temperature during the secondary crystallization treatment is room temperature and the time is 20-30h.
Due to the adoption of the technical scheme, the invention has the beneficial effects that:
According to the invention, the crystallized mother liquor sugar is used as a raw material, and is subjected to primary enrichment and middle-low pressure chromatography by a normal pressure resin column in sequence, and is treated in a mode of serial column sample injection adsorption and column separation elution during the middle-low pressure chromatography separation, so that the Dunalioside A and the rubusoside are effectively separated, and finally, the high-purity Dunalioside A and the rubusoside are obtained by a recrystallization method and an extraction purification method respectively, and the effective separation and purification of two rare glycosides are realized.
According to the invention, through optimizing process conditions, the Duchesneoside A and the rubusoside are enriched by adopting macroporous adsorption resin, and gradient analysis is carried out by adopting different concentration analysis agents, so that the analysis liquid enriched with the rubus oside and the Duchesneoside A is obtained, then, adsorption treatment is carried out by adopting a series of medium-low pressure chromatographic columns, and finally, elution is carried out on a single medium-low pressure chromatographic column by adopting different concentration ethanol solutions, so that the eluent containing the rubus oside and the eluent containing Du Kegan A are respectively obtained, and the effective separation of the Duchesneoside A and the rubus oside is realized. Other analysis waste liquid in macroporous adsorption resin analysis can be used as raw materials for continuous use, so that the recovery rate of Dukkoside A and rubusoside is greatly improved, and the energy consumption is reduced. The method of the invention is stable, the manual operation is simple, a large amount of solvent can be recovered, and the purities of the prepared dulcoside A and rubusoside are high.
Detailed Description
The invention is further illustrated below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The macroporous adsorption resin in the following examples and comparative examples was LK-1300SD, and the middle-low pressure column was YMC-Pack CN column. The crystalline mother liquor sugar used in the following examples and comparative examples had a dulcoside A content of 2wt%, rubusoside content of 5wt%, RA content of 22wt%, STV content of 18wt%, RC content of 5wt% and total glycoside content of 61%.
The raw materials in the following examples and comparative examples are outsourced unless otherwise specified, and the process conditions are conventional conditions of the prior art unless otherwise specified.
Example 1
(1) Preliminary adsorption by a normal pressure resin column:
Dissolving 10g of crystal mother liquor sugar powder in 60g of water to prepare a feed liquid, and then adsorbing the feed liquid into a resin column filled with 400ml of LK-1300SD resin at the speed of 0.5BV/h until the feed liquid is sweet; washing 800ml of water into a resin column at a speed of 1.5BV/h to remove impurities, then sequentially carrying out gradient analysis on the resin column by adopting ethanol solution with concentration of 20wt%, 40wt% and 60wt%, controlling the feeding speed at 2.5BV/h during analysis, collecting an analysis section of the 60wt% ethanol solution, namely, an analysis solution containing dulcoside A and rubusoside, and collecting the analysis solution with concentration of 20wt% ethanol solution and analysis solution with concentration of 40wt% ethanol solution for later use;
(2) Separating by medium-low pressure chromatographic column:
Removing the solvent from the solution containing Dunalioside A and rubusoside, dissolving in 95wt% ethanol solution again to obtain solution with solid content of 10wt%, separating the solution by column injection and column separation analysis, namely firstly, introducing into three medium-low pressure chromatographic columns connected in series at a speed of 0.3BV/h for adsorption, wherein the ratio of the adsorption control loading amount to the amount of filler in the medium-low pressure chromatographic columns is 1g:0.02ml, then sequentially eluting the first middle-low pressure chromatographic column by adopting an ethanol solution with the concentration of 95wt percent and 89wt percent at the speed of 1.5BV/h, sequentially eluting the middle chromatographic column by adopting an ethanol solution with the concentration of 95wt percent, 89wt percent, 80wt percent and 70wt percent, sequentially eluting the tail chromatographic column by adopting an ethanol solution with the concentration of 95wt percent, 89wt percent, 80wt percent and 70wt percent, respectively collecting effluent liquid when the middle chromatographic column and the ethanol solution with the concentration of 89wt percent of the tail chromatographic column are eluted, so as to obtain an eluent containing Du Kegan A, and respectively collecting effluent liquid when the middle chromatographic column and the ethanol solution with the concentration of 80wt percent of the tail chromatographic column are eluted, so as to obtain an eluent containing rubusoside; collecting the eluent obtained when the first chromatographic column is eluted by using 95wt% ethanol solution and 89wt% ethanol solution respectively, mixing the eluent with the analysis liquid obtained when the 20wt% ethanol solution and 40wt% ethanol solution are analyzed in the step (1), and drying the mixture to obtain powder which is used as crystal mother liquor sugar for re-separation;
(3) And (3) DA crystallization and purification:
Evaporating the eluent containing the dulcoside A to remove the solvent to obtain a crude product of the dulcoside A, and dissolving the crude product of the dulcoside A in methanol and water according to a volume ratio of 3:2, controlling the dosage ratio of the dulcoside A to the mixed solvent to be 1g:3ml, crystallization for 24h at room temperature with stirring, filtration, re-dissolution of the filter cake in methanol and water in a volume ratio of 3:1, controlling the dosage ratio of the methanol solution to the filter cake to be 7ml in the mixed solvent with the proportion composition: 1g; stirring again at room temperature for crystallization for 30 hours, filtering, collecting recrystallization mother liquor and filter cakes, and drying the filter cakes to obtain Dukkoside A;
(4) And (3) purifying rubusoside:
Mixing the recrystallization mother liquor with the eluent containing rubusoside, evaporating and concentrating to obtain feed liquid with the solid content of 50wt%, extracting the feed liquid three times by using ethyl acetate/n-butanol mixed solvent as an extracting agent, merging the raffinate phases obtained in the three extraction processes, and evaporating to remove the solvent to obtain rubusoside.
Example 2
This embodiment differs from embodiment 1 in that: in the step (1), the feed rate at the time of the analysis was controlled to 2BV/h, and the other conditions were the same as in example 1.
Example 3
This embodiment differs from embodiment 1 in that: in the step (1), the feeding rate at the time of the analysis was controlled to 3BV/h, and the other conditions were the same as in example 1.
Example 4
This embodiment differs from embodiment 1 in that: in the step (2), the feed rate at the time of elution was controlled to 1BV/h, and the other conditions were the same as in example 1.
Example 5
This embodiment differs from embodiment 1 in that: in the step (2), the feeding rate at the time of elution was controlled to 2BV/h, and the other conditions were the same as in example 1.
In order to better illustrate the isolation of highly pure dulcoside A and rubusoside according to the present invention, a number of comparative examples are given below with reference to example 1.
Comparative example 1
The difference between this comparative example and example 1 is that: in the step (1), the feed rate at the time of adsorption was controlled to 2BV/h, and the other conditions were the same as in example 1.
Comparative example 2
The difference between this comparative example and example 1 is that: in the step (1), the feed rate at the time of the analysis was controlled to 5BV/h, and the other conditions were the same as in example 1.
Comparative example 3
The difference between this comparative example and example 1 is that: in the step (2), the feed rate at the time of adsorption was controlled to 2BV/h, and the other conditions were the same as in example 1.
Comparative example 4
The difference between this comparative example and example 1 is that: in the step (2), the feeding rate at the time of elution was controlled to 3BV/h, and the other conditions were the same as in example 1.
Comparative example 5
The difference between this comparative example and example 1 is that: in the step (2), the effluent liquid obtained by eluting with 85-90wt% ethanol solution, i.e. the eluent containing Du Kegan A, is directly collected by adopting the mode of column injection and column analysis, and the effluent liquid obtained by eluting with 75-80wt% ethanol solution, i.e. the eluent of rubusoside, is collected, and other conditions are the same as those in the example 1.
The following follow-up tests were performed on the purities and recovery rates of dulcoside a and rubusoside prepared in the above examples and comparative examples, and the test results are shown in table 1, wherein the recovery rates were calculated as follows:
Recovery (%) = [ product recovery x product purity/product content in stevioside mother liquor ] ×100%.
TABLE 1
From the test results, the invention can be seen that the invention is primarily enriched by macroporous adsorption resin, then separated by a middle-low pressure chromatographic column in a mode of serial column sample injection and column separation analysis, respectively obtaining a rubusoside-containing solution and a dulcoside A-containing solution, and then obtaining a product with high purity through subsequent treatment.
Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
1. A method for preparing dulcoside A and rubusoside based on resin adsorption and chromatographic separation, which is characterized by comprising the following steps:
S1: enriching by using a normal pressure resin column:
Dissolving crystalline mother liquor sugar in water, then, introducing the crystalline mother liquor sugar into macroporous adsorption resin for adsorption, introducing the mixture to sweet taste, then, washing the mixture with water, then, sequentially adopting an ethanol solution with the concentration of 15-25wt%, 35-45wt% and 55-65wt% as a resolving agent for gradient resolving the macroporous adsorption resin, collecting an ethanol solution resolving section with the concentration of 55-65wt%, namely resolving liquid containing Dukkoside A and rubusoside, and collecting resolving liquid for later use when resolving the ethanol solution with the concentration of 15-25wt% and 35-45 wt%;
S2: separating by medium-low pressure chromatographic column:
Evaporating the analytical solution containing the dulcoside A and the rubusoside to remove the solvent to obtain mixed sugar containing the dulcoside A and the rubusoside, dissolving the mixed sugar by adopting a high alcohol solution, sequentially separating by adopting a plurality of medium-low pressure chromatographic columns connected in series, and respectively carrying out gradient elution on single chromatographic columns by adopting ethanol solutions with different concentrations to respectively collect eluent containing the dulcoside A and eluent containing the rubusoside;
s3: purification of dulcoside a:
Evaporating the eluent containing the dulcoside A to remove the solvent to obtain a crude product of the dulcoside A, performing primary crystallization treatment on the dulcoside A by taking a methanol solution as a crystallization solvent, filtering crystallization feed liquid, performing secondary crystallization treatment on an obtained filter cake by adopting the methanol solution, filtering the crystallization feed liquid, and drying the obtained precipitate to obtain the high-purity dulcoside A;
s4: purifying rubusoside:
and mixing the crystallization mother liquor obtained in the primary crystallization treatment with the eluent containing rubusoside, evaporating and concentrating to obtain a concentrated solution, extracting for a plurality of times by adopting a mixed solvent of ethyl acetate and n-butanol, merging raffinate phases, and evaporating to remove the solvent to obtain the high-purity rubusoside.
2. The method for preparing dulcoside a and rubusoside based on resin adsorption and chromatographic separation as claimed in claim 1, wherein the method comprises the following steps: in the step S1, the macroporous adsorption resin is one of LX-28, LK-1300SD, LX-T83, 69M, DM and SPD-009, the volume of the feed liquid is 0.15-0.2BV of the resin volume during sample injection, and the flow rate of the feed liquid is less than 1BV/h.
3. The method for preparing dulcoside a and rubusoside based on resin adsorption and chromatographic separation as claimed in claim 1, wherein the method comprises the following steps: in the step S1, the volume of water is 1-2BV of the volume of resin during water washing, and the flow rate of water is 1-2BV/h; and/or the volume of the resolving agent is 1-2BV of the resin volume during gradient resolving, and the flow rate of the resolving agent is 2-3BV/h.
4. The method for preparing dulcoside a and rubusoside based on resin adsorption and chromatographic separation as claimed in claim 1, wherein the method comprises the following steps: in the step S2, the concentration of the high alcohol solution is 90-95wt% of ethanol solution, and the solid content of the dissolved solution is controlled to be 5-20wt% when the high alcohol solution is adopted to dissolve the mixed sugar.
5. The method for preparing dulcoside a and rubusoside based on resin adsorption and chromatographic separation as claimed in claim 1, wherein the method comprises the following steps: in the step S2, the filler in the middle-low pressure chromatographic column is normal phase separation filler or reverse phase separation filler, wherein the normal phase separation filler is silica gel and bonding phase filler with NH 2 and APS, CN, CPS groups; the reversed-phase separation filler is one or more of a carbon-eight column, a carbon-four column and a phenyl column.
6. The method for preparing dulcoside A and rubusoside based on resin adsorption and chromatographic separation as claimed in claim 5, wherein the method comprises the following steps: the medium-low pressure chromatographic column is a YMC-Pack CN chromatographic column.
7. The method for preparing dulcoside a and rubusoside based on resin adsorption and chromatographic separation as claimed in claim 1, wherein the method comprises the following steps: in the step S2, the ratio of the loading amount of the separation by adopting the middle-low pressure chromatographic column to the amount of the filler in the middle-low pressure chromatographic column is 1g: (0.001-0.03 ml), and the flow rate of the feed liquid during sample loading is less than 1BV/h.
8. The method for preparing dulcoside a and rubusoside based on resin adsorption and chromatographic separation as claimed in claim 1, wherein the method comprises the following steps: in the step S2, the concentration of the ethanol solution adopted in gradient elution is 95-98wt%, 85-90wt%, 75-80wt% and 65-70wt%, the flow rate of the ethanol solution in elution is 1-2BV/h, and the eluent in elution of the tail chromatographic column and the chromatographic column adjacent to the tail chromatographic column by using 85-90wt% ethanol solution is collected respectively, namely the eluent containing Du Kegan A; and respectively collecting the eluent of the tail chromatographic column and the chromatographic columns adjacent to the tail chromatographic column when the tail chromatographic column is eluted by 75-80wt% ethanol solution, namely the eluent containing rubusoside.
9. The method for preparing dulcoside a and rubusoside based on resin adsorption and chromatographic separation as claimed in claim 1, wherein the method comprises the following steps: in the step S2, other chromatographic columns connected in series except the tail chromatographic column and the chromatographic columns adjacent to the tail chromatographic column are eluted by adopting ethanol solution with the concentration of 95-98wt% and 85-90wt%, and the eluent is combined with the analysis liquid obtained in the step S1 when the ethanol solution with the concentration of 15-25wt% and the ethanol solution with the concentration of 35-45wt% is analyzed, and the combined and dried solution is reused as crystal mother liquid sugar.
10. The method for preparing dulcoside a and rubusoside based on resin adsorption and chromatographic separation as claimed in claim 1, wherein the method comprises the following steps: in step S3, the volume ratio of methanol to water in the methanol solution during the primary crystallization treatment is 3: (1-2), the dosage ratio of the methanol solution to the crude dulcoside A product in one crystallization treatment is (2-5) ml:1g, the temperature during primary crystallization treatment is room temperature, and the time is 20-30h;
And/or the volume ratio of methanol to water in the methanol solution is 3: (1-2), the dosage ratio of the methanol solution to the filter cake in the secondary crystallization treatment is (5-10) ml:1g; the temperature during the secondary crystallization treatment is room temperature and the time is 20-30h.
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