CN118812608A - Method for purifying stevioside monoglycoside in mother liquor sugar - Google Patents
Method for purifying stevioside monoglycoside in mother liquor sugar Download PDFInfo
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- CN118812608A CN118812608A CN202310435243.7A CN202310435243A CN118812608A CN 118812608 A CN118812608 A CN 118812608A CN 202310435243 A CN202310435243 A CN 202310435243A CN 118812608 A CN118812608 A CN 118812608A
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- mother liquor
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- ethanol
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- 239000012452 mother liquor Substances 0.000 title claims abstract description 78
- 235000019202 steviosides Nutrition 0.000 title claims abstract description 47
- 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 title claims abstract description 44
- 229940013618 stevioside Drugs 0.000 title claims abstract description 44
- 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 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000011347 resin Substances 0.000 claims abstract description 57
- 229920005989 resin Polymers 0.000 claims abstract description 57
- 239000003480 eluent Substances 0.000 claims abstract description 51
- 239000002904 solvent Substances 0.000 claims abstract description 46
- 238000010828 elution Methods 0.000 claims abstract description 33
- 239000013078 crystal Substances 0.000 claims abstract description 26
- 238000002425 crystallisation Methods 0.000 claims abstract description 20
- 230000008025 crystallization Effects 0.000 claims abstract description 20
- 238000000746 purification Methods 0.000 claims abstract description 18
- 238000001035 drying Methods 0.000 claims abstract description 17
- 238000001179 sorption measurement Methods 0.000 claims abstract description 17
- 238000000926 separation method Methods 0.000 claims abstract description 15
- 238000004090 dissolution Methods 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 239000012264 purified product Substances 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 197
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 153
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 90
- 239000000243 solution Substances 0.000 claims description 45
- 229930182470 glycoside Natural products 0.000 claims description 26
- 150000002338 glycosides Chemical class 0.000 claims description 26
- 239000011259 mixed solution Substances 0.000 claims description 14
- -1 amino silica gel Chemical compound 0.000 claims description 12
- 239000000945 filler Substances 0.000 claims description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 6
- 229920006395 saturated elastomer Polymers 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000000047 product Substances 0.000 abstract description 19
- 239000012535 impurity Substances 0.000 abstract description 7
- 150000008442 polyphenolic compounds Chemical class 0.000 abstract description 3
- 235000013824 polyphenols Nutrition 0.000 abstract description 3
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 abstract description 2
- 229930003944 flavone Natural products 0.000 abstract description 2
- 150000002212 flavone derivatives Chemical class 0.000 abstract description 2
- 235000011949 flavones Nutrition 0.000 abstract description 2
- 235000013373 food additive Nutrition 0.000 abstract description 2
- 239000002778 food additive Substances 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 description 25
- 239000001512 FEMA 4601 Substances 0.000 description 22
- 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 22
- 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 22
- 235000019203 rebaudioside A Nutrition 0.000 description 22
- 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 22
- 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 7
- 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 7
- 244000269722 Thea sinensis Species 0.000 description 6
- 235000009508 confectionery Nutrition 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000004383 Steviol glycoside Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 235000019411 steviol glycoside Nutrition 0.000 description 3
- 229930182488 steviol glycoside Natural products 0.000 description 3
- 150000008144 steviol glycosides Chemical class 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000005191 phase separation Methods 0.000 description 2
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- 239000000843 powder Substances 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 235000019640 taste Nutrition 0.000 description 2
- 241000208838 Asteraceae Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 244000228451 Stevia rebaudiana Species 0.000 description 1
- 235000006092 Stevia rebaudiana Nutrition 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Chemical group OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 235000021096 natural sweeteners Nutrition 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Landscapes
- Saccharide Compounds (AREA)
Abstract
The invention discloses a method for purifying stevioside monoglycoside in mother liquor sugar, which relates to the technical field of food additive preparation and comprises the following steps: mixing the mother liquor sugar with a dissolution solvent to prepare a mother liquor sugar solution; carrying out adsorption separation on the mother liquor sugar solution through forward resin; sequentially carrying out gradient elution on the adsorbed resin by using a multi-stage eluting solution; respectively collecting the eluents under each level of gradient to obtain a plurality of groups of eluents after the purification of the monoglycoside; and respectively drying the plurality of groups of eluents to obtain crystals, and respectively adding the crystals into an organic crystallization solvent to crystallize to obtain a stevioside monoglycoside purified product. According to the invention, the stevioside mother liquor sugar is adsorbed by adopting a specific resin and then subjected to a multistage gradient elution mode, so that impurities such as polyphenol, flavone and the like in the mother liquor sugar are finally and effectively removed, and the stevioside monoglycoside pure product is obtained through separation and purification, thereby providing a new technical idea for the utilization of the mother liquor sugar.
Description
Technical Field
The invention relates to the technical field of food additive preparation, in particular to a method for purifying stevioside monoglycoside in mother liquor sugar.
Background
Stevioside is a natural sweetener extracted from stevia rebaudiana Bertoni of Compositae, has the functional characteristics of high sweetness and low calorie, and has the sweetness 100-300 times that of sucrose and the calorie of only 1/300 of that of sucrose. Solvent extraction is currently the primary process of commercial stevioside extraction, which eventually recovers high purity Rebaudioside A (RA) and Stevioside (ST) by crystallization, and the remaining mother liquor is usually spray dried to a powder solid, called a Mother Liquor Sugar (MLS), which is commonly sold as a low-priced sweetener.
In the current industrial production, resin separation methods, such as novel resin synthesis, mixed resin chromatography and modified resin chromatography, are mainly adopted for extracting and separating the monoglycoside in the mother liquor sugar, however, the methods cannot realize the effective separation of RA and ST in the mother liquor sugar, and have poor separation effect on other monoglycosides.
In addition, the mother liquor sugar contains about 60% stevioside and some residual polyphenol and flavonoid impurities, and the existence of the impurities not only aggravates the bitter taste of the mother liquor sugar, but also affects the further recovery of the glycoside.
Therefore, the application of the resin method for treating the mother liquor sugar in industry encounters an obstacle, and a new purification method is needed to be sought in the prior art, which can purify the monoglycoside in the mother liquor sugar and separate impurities, so that a stevioside monoglycoside product with higher purity and better taste is obtained.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for purifying stevioside monoglycoside in a mother liquor sugar, which is used for separating stevioside composition with higher purity and better taste from the mother liquor sugar.
In order to achieve the above object, the present invention provides a method for purifying stevioside monoglycoside in a mother liquor sugar, comprising the steps of:
Mixing the mother liquor sugar with a dissolution solvent to prepare a mother liquor sugar solution;
Carrying out adsorption separation on the mother liquor sugar solution through forward resin;
sequentially carrying out gradient elution on the adsorbed resin by using a multi-stage eluting solution;
Respectively collecting the eluents under each level of gradient to obtain a plurality of groups of eluents after the purification of the monoglycoside;
And respectively drying the plurality of groups of eluents to obtain crystals, and respectively adding the crystals into an organic crystallization solvent to crystallize to obtain various stevioside monoglycoside purified products.
The invention discloses a method for purifying stevioside monoglycoside in mother liquor sugar, which comprises the following steps: mixing the mother liquor sugar with a dissolution solvent to prepare a mother liquor sugar solution; carrying out adsorption separation on the mother liquor sugar solution through forward resin; sequentially carrying out gradient elution on the adsorbed resin by using a multi-stage eluting solution; respectively collecting the eluents under each level of gradient to obtain a plurality of groups of eluents after the purification of the monoglycoside; and respectively drying the plurality of groups of eluents to obtain crystals, and respectively adding the crystals into an organic crystallization solvent to crystallize to obtain a stevioside monoglycoside purified product. The forward resin skeleton selected by the invention can be bonded with various functional groups such as amino, glycol, weak anion exchange groups and the like, and can form hydrogen bonds with molecules with-OH, -NH or-SH functional groups so as to improve the reservation of polar molecules, and solvent molecules, solute molecules and solute molecules are in competitive adsorption with each other due to the fact that the interaction force is large or small, so that the difference of the reservation time in an adsorption column is caused, and different substances are separated. The adjustment of the type and proportion of the mobile phase can have a certain influence on the separation of impurities and stevioside; and (3) utilizing the difference between a normal phase separation mode and a reverse phase separation mode, selecting the polarity of the eluting solvent from low to high (the retention of polar components is stronger in forward chromatography), and separating and purifying stevioside monoglycoside respectively to obtain pure stevioside monoglycoside. According to the invention, the stevioside mother liquor sugar is adsorbed by adopting a specific resin and then subjected to a multistage gradient elution mode, so that impurities such as polyphenol, flavone and the like in the mother liquor sugar are finally and effectively removed, and the stevioside monoglycoside pure product is obtained through separation and purification, thereby providing a new technical idea for the utilization of the mother liquor sugar. Experimental results show that, by taking the mother liquor sugar with the total stevioside content of 64.8% as an example, after the purification by adopting the method disclosed by the invention, the content of rubusoside in the obtained stevioside monoglycoside pure product is increased from 1.7% to 23.8%, the DA content is increased from 1.9% to 16.5%, the RB content is increased from 1.2% to 13.5%, the STV content is increased from 14.2% to 65.3%, the RC content is increased from 18.0% to 45.8%, the RA content is increased from 21.3% to 46.7%, and the RD content is increased from 1.4% to 12.1%.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention provides a method for purifying stevioside monoglycoside in mother liquor sugar, which comprises the following steps:
Mixing the mother liquor sugar with a dissolution solvent to prepare a mother liquor sugar solution;
Carrying out adsorption separation on the mother liquor sugar solution through forward resin;
sequentially carrying out gradient elution on the adsorbed resin by using a multi-stage eluting solution;
Respectively collecting the eluents under each level of gradient to obtain a plurality of groups of eluents after the purification of the monoglycoside;
And respectively drying the plurality of groups of eluents to obtain crystals, and respectively adding the crystals into an organic crystallization solvent to crystallize to obtain various stevioside monoglycoside purified products.
The invention firstly mixes the mother liquor sugar with the dissolving solvent to prepare the mother liquor sugar solution. The source of the stevioside mother liquor sugar is not particularly required, and the mother liquor sugar prepared by the stevioside extraction method well known in the art can be used; the mother liquor sugar in the invention is solid powder prepared by spray drying a mother liquor sugar solution, wherein the content of stevioside is preferably 55-90%. In the present invention, the mass ratio of the mother liquor sugar to the dissolution solvent is preferably 1: (4-6), more preferably 1:5. In the present invention, the dissolution solvent is preferably one or more of water, methanol, ethanol and ethyl acetate saturated aqueous solution, more preferably a mixed solution of ethyl acetate/ethanol, and most preferably ethyl acetate: ethanol (volume ratio) =acetic acid of 1.5 ethyl ester/ethanol mixed solution. In the present invention, the concentration of methanol and ethanol is not limited, but the concentration of methanol and ethanol is 99% or more by mass. In the invention, the concentration of steviol glycoside composition in the mother liquor sugar solution is 3-8%. It will be appreciated that the reason for dissolving the mother liquor sugar to a suitable concentration is to enhance the effect of subsequent resin adsorption.
Further, the invention further comprises heating to 30-80 ℃ after mixing the mother liquor sugar with the dissolution solvent, stirring at a speed of 60-120rpm, and filtering after complete dissolution to obtain a mother liquor sugar solution. It should be understood that the purpose of the heating and stirring is to increase the dissolution rate and the purpose of the filtration is to remove insoluble impurities from the mother liquor sugar.
After the mother liquor sugar solution is obtained, the mother liquor sugar solution is adsorbed and separated by forward resin. In the present invention, the feed amount of the stock solution sugar solution is preferably 4 to 6% by volume of the forward resin, more preferably 5%; the feed rate is preferably 1 to 2BV/h, more preferably 1.5BV/h. In the present invention, the forward resin is preferably one of an amino silica filler, an aminopropyl filler, MB70-75 or SMB70-45, more preferably an amino silica filler; the Amino silica gel filler is preferably Unisil NH2 or Amino-NH2; the aminopropyl filler is preferably COPURE NH2. It should be understood that Unisil NH2 refers to Unisil NH amino silica gel filler; amino-NH2 refers to Amino-bonded silica gel fillers; MB70-75 refers to a forward chromatographic packing material produced in Japan, having a pore size of 7nm, a specific surface area of 480m 2/g, and a particle size of 75/200um; SMB70-45 refers to forward chromatographic packing material produced in Japan, having a pore size of 7nm, a specific surface area of 280m 2/g, and a particle size of 20/45um; COPURE NH 2A refers to aminopropyl filler, carbon content 4.5% specific surface area 200m 2/g, average particle size 40-75um. In the present invention, the carrier of the forward resin is preferably a column, more preferably a medium-low pressure column. It should be understood that, in the present invention, various functional groups, such as amino groups, glycol groups, etc., may be bonded to the positive resin skeleton, so that the retention of polar molecules may be improved, and the solvent molecules, solute molecules, and solute molecules may be competitively adsorbed to each other due to the large or small interaction force, so that the difference of retention time in the adsorption column is caused, and different substances may be separated.
Further, the invention further comprises the step of introducing a dissolving solvent into the forward resin to balance the resin after preparing the mother liquor sugar solution and before passing the mother liquor sugar solution through the forward resin. In the present invention, the feeding amount of the dissolution solvent is preferably 1-2BV of the volume of the forward resin, and the feeding speed is preferably 1-2BV/h. It will be appreciated that the use of the same solvent through the resin prior to formally performing resin adsorption is to equilibrate the resin such that adsorption of the solvent in the resin is balanced, eliminating errors that may result from direct access to the stock solution sugar solution.
After resin adsorption is finished, the resin subjected to adsorption is subjected to gradient elution sequentially through multi-stage elution solutions. In the present invention, preferably, the gradient elution sequentially through the multi-stage elution solution comprises: eluting by two or more eluting solvents in sequence, wherein the polarity of each eluting solvent is from low to high; the eluting solvent is preferably selected from one or more of methanol, ethanol and ethyl acetate saturated aqueous solution; illustratively, the eluting solvent in the present invention may be a methanol/ethanol mixed solution, a methanol/ethyl acetate mixed solution, an ethanol/ethyl acetate mixed solution, or a methanol/ethanol/ethyl acetate mixed solution.
Further preferably, the gradient elution sequentially through the multi-stage elution solution comprises: sequentially carrying out gradient elution by two or more stages of ethyl acetate/ethanol mixed solutions and two or more stages of ethanol aqueous solutions; wherein the volume ratio of ethyl acetate/ethanol in each stage is preferably gradually reduced within the range of [1.5,0.3], specifically, the volume ratio of ethyl acetate/ethanol can be selected to be any value within the range of [1.5,0.3], for example, 1.5/1.4/1.3/1.22/1.2/1.0/0.9/0.81/0.8/0.7/0.6/0.53/0.5/0.4/0.33/0.3 of a mixed solution of two or more ethyl acetate/ethanol, the volume ratio of ethyl acetate/ethanol is gradually reduced, and the polarity of the eluting solvent is gradually increased; the volume fraction of the aqueous ethanol solution of each stage is preferably gradually reduced within the range of [99%,70% ], specifically, the volume fraction of the aqueous ethanol solution may be selected to be any value within the range of [99%,70% ], for example, two or more of the aqueous ethanol solutions of 99%/98%/97%/96%/95%/94%/93%/92%/91%/90%/85%/80%/75%/70%; the volume fraction of the aqueous ethanol solution gradually decreases, and the polarity of the eluting solvent gradually increases.
In a preferred embodiment, the sequentially multistage ethyl acetate/ethanol mixed solution comprises: sequentially passing through ethyl acetate: ethanol=1.5, 1.22, 1, 0.81, 0.53, 0.33 grade 6 ethyl acetate/ethanol mixed solution; the sequential multi-stage ethanol aqueous solution comprises: the water solution of 7-grade ethanol with the volume fractions of 99%, 96%, 93%, 90%, 87%, 85% and 70% respectively is adopted in sequence.
In the present invention, the amount of the eluting solvent used in each gradient in the gradient elution is preferably 2BV of the forward resin volume, and the flow rate of the gradient elution is preferably 1-2BV/h.
After gradient elution, the invention respectively collects the eluents under each level of gradient to obtain a plurality of groups of eluents after the purification of the monoglycoside. In the invention, the eluent under each grade gradient is collected respectively, and the method for obtaining a plurality of groups of eluent after the single glycoside separation comprises the following steps: each gradient is collected for 1 BV/time, 2 groups of eluents are obtained by each gradient collection, and 2N groups of eluents are obtained by total collection, wherein N is the total stage number of gradient elution. Taking the embodiment of sequentially passing through the 6-level ethyl acetate/ethanol mixed solution and sequentially passing through the 7-level ethanol aqueous solution as an example, each level of elution adopts eluent with the volume of 2BV resin, after the elution is completed, the eluent with the volume of 2BV is collected twice (1 BV/time) in each level of elution process, so as to obtain 2 groups of eluents, and the eluent with the volume of 2BV is obtained in total by 13 levels of elution, so that 2.times.13=26 groups of eluents are obtained.
Further, after the multiple sets of eluents after the single glycoside separation are obtained, before the multiple sets of eluents are dried, the method further comprises: and combining adjacent eluents with the same single glycoside as the main component to obtain a plurality of groups of eluents with different single glycosides as the main component. It should be understood that the main components in the eluent obtained by eluting in the adjacent eluting sections have a high probability of being the same single glycoside, and the eluent with the adjacent main components being the same single glycoside is combined to obtain the eluent with a certain single glycoside, so that the subsequent treatment is convenient.
After a plurality of groups of eluents are obtained, the plurality of groups of eluents are respectively dried to obtain crystals, and the crystals are respectively added into an organic crystallization solvent for crystallization to obtain a plurality of stevioside monoglycoside purified products. In the invention, the drying conditions in the crystal obtained by drying the plurality of groups of eluents are preferably 80-85 ℃ and the time is preferably 1-2h.
In the present invention, the organic crystallization solvent is preferably one or more of methanol, ethanol, isopropanol and acetone, more preferably methanol, and in the case where the concentration of methanol, ethanol, isopropanol and acetone is not limited, the above solvents are all 99% or more by mass; the amount added is preferably 2-4BV, more preferably 3BV, of the crude steviol glycoside composition. In the present invention, the crystallization conditions are preferably: the crystallization temperature is 20-30 ℃, the stirring speed is 80-120rpm, and the crystallization time is 6-12h.
After obtaining a plurality of stevioside monoglycoside purification products, the invention also comprises the step of mixing any two or more monoglycoside products to obtain mixed monoglycoside; it is to be understood that the reason for mixing the monoglycosides is that stevioside with different flavors and sweetness can be obtained by compounding the monoglycosides of stevioside, so as to meet the diversified demands of the market.
In order to better explain the technical scheme of the invention, the invention also provides the following specific embodiments. It should be understood that the starting materials used in the examples below are commercially available unless otherwise specified. In particular, the main ingredients of 4 batches of steviol glycoside mother liquor sugar, all supplied by the pharmaceutical industry, inc., of Mihao, city, are listed in Table 1 below.
TABLE 11-4 batch of mother liquor sugar stevioside ingredients
Example 1
S1, taking 10g of mother liquor sugar of a batch 1, wherein the content of stevioside total glycoside is 55.8%, dissolving the mother liquor sugar in 40g of ethyl acetate/methanol solvent, and dissolving the ethyl acetate: methanol=1.5: 1, preparing 50g of mother liquor sugar solution;
s2, feeding 50g of a mother liquor sugar solution into a middle-low pressure chromatographic column filled with 200ml of amino silica gel resin (Unisil NH 2) for adsorption, wherein the feeding speed is 1BV/h;
S3, sequentially carrying out gradient elution on the adsorbed resin by a 6-grade ethyl acetate/methanol mixed solution (the volume ratio of ethyl acetate to methanol is 1.5, 1.22, 1, 0.81, 0.53 and 0.33) and a 7-grade methanol aqueous solution (the volume fractions of methanol are 99%, 96%, 93%, 90%, 87%, 84% and 81%) respectively; the dosage of the eluting solvent of each stage is 2BV of the resin volume, and the feeding speed is 2BV/h;
s4, collecting gradient analysis effluent liquid under each gradient, wherein the collecting mode is 1 BV/time, namely 200 ml/time, and the specific collecting mode is as follows:
① Ethyl acetate: methanol = 0.53,0-2BV segment: the sweet tea content is improved to 20.1 percent, and the yield is 81.5 percent;
② Ethyl acetate: methanol = 0.33,0-2BV segment: the DA content is improved to 15.5 percent, and the yield is 20.5 percent;
③ 99% methanol 0-1BV section: the RB content is improved to 12.5%, and the yield is 20.4%;
④ 99% methanol 1-2BV section and 96% methanol 0-1BV section: the STV content is improved to 61.2 percent, and the STV recovery rate is 62.1 percent;
⑤ 96% methanol 1-2BV section and 93% methanol 0-1BV section: the RC content is improved to 38.7 percent, and the RC recovery rate is 67.3 percent;
⑥ 93% methanol 1-2BV section and 87%, 84% methanol 0-1BV section: the RA content is improved to 47.9 percent, and the RA recovery rate is 78.2 percent;
⑦ 81% methanol 0-2BV section: the RD content is improved to 13.1 percent, and the RD recovery rate is 65.2 percent.
S5, drying the collected 7 groups of eluents at 80 ℃ for 2 hours to obtain crystals, adding the crystals into 2BV methanol respectively, crystallizing for 6 hours at the temperature of 30 ℃ and the stirring speed of 80rpm, filtering the obtained crystals, and drying at 70 ℃ to obtain the monoglycoside.
The single glycoside products are respectively crystallized according to the conditions, and the purities of the single glycoside products are 62.2 percent of rubusoside, 73.5 percent of DA, 64.1 percent of RB, 90.2 percent of STV, 55.2 percent of RC, 86.3 percent of RA and 78.6 percent of RD.
Example 2
S1, taking 10g of mother liquor sugar of a batch 2, wherein the content of stevioside total glycoside is 64.8%, and dissolving the mother liquor sugar in 50g of ethyl acetate: ethanol (volume ratio) =1.5: 1, preparing 60g of mother liquor sugar solution in the organic mixed solvent;
S2, feeding 60g of a mother liquor sugar solution into a middle-low pressure chromatographic column filled with 200ml of aminopropyl resin (COPURE NH 2) for adsorption, wherein the feeding speed is 1.5BV/h;
S3, sequentially carrying out gradient elution on the adsorbed resin by a 6-grade ethyl acetate/ethanol mixed solution (the volume ratio of ethyl acetate to ethanol is 1.5, 1.22, 1, 0.81, 0.53 and 0.33) and a 7-grade ethanol water solution (the volume fractions of ethanol are 99%, 96%, 93%, 90%, 87%, 85% and 70%) respectively; the dosage of the eluting solvent of each stage is 2BV of the volume of the resin, and the feeding speed is 1.5BV/h;
s4, collecting gradient analysis effluent liquid under each gradient, wherein the collecting mode is 1 BV/time, namely 200 ml/time, and the specific collecting mode is as follows:
① Ethyl acetate: ethanol = 0.33,0-2BV segment: the sweet tea content is improved to 23.8 percent, and the yield is 79 percent;
② 99% ethanol 0-2BV section: the DA content is improved to 16.5 percent, and the yield is 21.7 percent;
③ 96% ethanol 0-1BV section: the RB content is improved to 13.5%, and the yield is 22.4%;
④ 96% ethanol 1-2BV segment and 93% ethanol 0-1BV segment: the STV content is improved to 65.3 percent, and the STV recovery rate is 64.6 percent;
⑤ 93% ethanol 1-2BV segment and 90% ethanol 0-1BV segment: the RC content is improved to 45.8 percent, and the RC recovery rate is 65.1 percent
⑥ 90% Ethanol 1-2BV segment and 87, 85% ethanol 0-2BV segment: the RA content is improved to 46.7 percent, and the RA recovery rate is 81.2 percent;
⑦ 70% ethanol 0-2BV section: the RD content is improved to 12.1 percent, and the RD recovery rate is 74 percent.
S5, drying the collected 7 groups of eluents at 85 ℃ for 1h to obtain crystals, respectively adding the crystals into 3BV methanol, crystallizing for 8h at the temperature of 25 ℃ and the stirring speed of 100rpm, filtering the obtained crystals, and drying at 80 ℃ to obtain pure products of each monoglycoside.
The single glycoside products have the purity of 67.4 percent of rubusoside, 79.1 percent of DA, 66.3 percent of RB, 93.6 percent of STV, 73.8 percent of RC, 83.6 percent of RA and 73.5 percent of RD after crystallization respectively in each stage according to the conditions.
Example 3
S1, taking 10g of mother liquor sugar of a batch 3, wherein the content of stevioside total glycoside is 75%, and dissolving the mother liquor sugar in 60g of ethanol solvent to prepare a mother liquor sugar solution;
s2, feeding 70g of the mother liquor sugar solution into a medium-low pressure chromatographic column filled with 1200ml of MB70-75 resin for adsorption, wherein the feeding speed is 2BV/h;
s3, carrying out gradient elution on the adsorbed resin by 8 stages of 99%, 97%, 95%, 93%, 91%, 89%, 87% and 70%; the dosage of the eluting solvent of each stage is 2BV of the volume of the resin, and the feeding speed is 1BV/h;
s4, collecting gradient analysis effluent liquid under each gradient, wherein the collecting mode is 1 BV/time, namely 1200 ml/time, and the specific collecting mode is as follows:
① 97% ethanol 0-1BV section: the sweet tea content is improved to 12.2 percent, and the yield is 84.3 percent; the DA content is increased to 8.5%, and the yield is 34.7%;
② 97% ethanol 1-2BV section: the RB content is improved to 13.5%, and the yield is 22.4%;
③ 95% ethanol 1-2BV segment and 93% ethanol 0-1BV segment: the STV content is improved to 69.4 percent, and the STV recovery rate is 65.6 percent;
④ 93% ethanol 1-2BV segment and 91% ethanol 0-1BV segment: the RC content is improved to 46.8 percent, and the RC recovery rate is 67.8 percent
⑤ 91% Ethanol 1-2BV section and 89%, 87%0-2BV, RA content is improved to 56.3%, RA recovery rate is 78.5%;
⑥ 70% ethanol 0-2BV section: the RD content is improved to 12.1 percent, and the RD recovery rate is 67.3 percent.
S5, drying the collected 6 groups of eluents at 82 ℃ for 1.5 hours to obtain crystals, respectively adding the crystals into 4BV methanol, crystallizing for 12 hours at the temperature of 20 ℃ and the stirring speed of 120rpm, filtering the obtained crystals, and drying at 90 ℃ to obtain pure monoglycoside.
The single glycoside products are respectively crystallized according to the conditions, and the purities of the single glycoside products are respectively 43.8 percent of rubusoside, 36.5 percent of DA, 67.7 percent of RB, 94.3 percent of STV, 79.4 percent of RC, 95.3 percent of RA and 68.2 percent of RD.
Example 4
S1, taking 10g of mother liquor sugar of a batch 4, wherein the content of stevioside total glycoside is 90%, and dissolving the mother liquor sugar in 50g of organic mixed solvent of methanol to prepare a mother liquor sugar solution;
S2, feeding 60g of the mother liquor sugar solution into a medium-low pressure chromatographic column filled with 1000ml of SMB70-45 resin for adsorption, wherein the feeding speed is 1.5BV/h;
S3, carrying out gradient elution on the adsorbed resin by 8 stages of 99%, 97%, 95%, 93%, 91%, 89%, 87% and 70%; the dosage of the eluting solvent of each stage is 2BV of the volume of the resin, and the feeding speed is 1.5BV/h;
S4, collecting gradient analysis effluent liquid under each gradient, wherein the collecting mode is 1 BV/time, namely 1000 ml/time, and the specific collecting mode is as follows:
① 97% methanol, 0-1BV section: the sweet tea content is improved to 12.1 percent, and the yield is 82.8 percent;
② 97% methanol 1-2BV section: the RB content is increased to 6.5%, and the yield is 21.3%;
③ 95% methanol 0-2BV section and 93% methanol 0-1BV section: the STV content is improved to 86.5 percent, and the STV recovery rate is 62.1 percent;
④ 93% methanol 1-2BV,91% methanol 0-1BV section: the RC content is improved to 32.3 percent, and the RC recovery rate is 61.5 percent
⑤ 91% Methanol 1-2BV section and 89% methanol 0-1BV section: the RA content is improved to 64.3 percent, and the RA recovery rate is 73.2 percent;
⑥ 89% methanol 1-2BV section to 87% methanol 0-2BV section: the RD content is improved to 10.5 percent, and the RD recovery rate is 96.3 percent.
S5, drying the collected 7 groups of eluents at 85 ℃ for 1h to obtain crystals, respectively adding the crystals into 3BV ethanol, crystallizing for 8h at 25 ℃ and a stirring speed of 90rpm, filtering the obtained crystals, and drying at 80 ℃ to obtain pure monoglycoside.
The single glycoside products are respectively crystallized according to the conditions, and the purities of the single glycoside products are 46.4 percent of rubusoside, 36.8 percent of RB, 96.2 percent of STV, 53.9 percent of RC, 97.3 percent of RA and 46.6 percent of RD.
Comparative example 1
In this example, the forward filler amino silica gel resin (Unisil NH 2) was replaced with SKS-10 in equal amounts on the basis of example 1 above, with the other conditions unchanged.
The content and yield of each of the monoglycosides obtained were as follows:
① Ethyl acetate: methanol = 0.53,0-2BV segment: the sweet tea content is improved to 11.3 percent, and the yield is 61.4 percent;
② Ethyl acetate: methanol = 0.33,0-2BV segment: the DA content is increased to 8.5 percent, and the yield is 10.5 percent;
③ 99% methanol 0-2BV section: the RB content is increased to 7.5%, and the yield is 23.4%;
④ 96% methanol 0-2BV section: the STV content is improved to 36.2 percent, and the STV recovery rate is 72.1 percent;
⑤ 93% methanol 0-2BV section: the RC content is improved to 31.7 percent, and the RC recovery rate is 57.3 percent
⑥ 90% Methanol 0-2BV section and 87%, 84% methanol 0-1BV: the RA content is improved to 34.9 percent, and the RA recovery rate is 63.2 percent;
⑦ 84%, 81% methanol 0-2BV section: the RD content is raised to 7.1%, and the RD recovery is 54.2%.
The collected 7 sets of eluents were crystallized under the same crystallization conditions as in example 1 to obtain pure monoglycoside products.
The single glycoside products are respectively crystallized according to the conditions, and the purities of the single glycoside products are respectively 42.4 percent of rubusoside, 46.3 percent of DA, 37.4 percent of RB, 68.8 percent of STV, 57.6 percent of RC, 74.3 percent of RA and 45.3 percent of RD.
Comparative example 2
This example replaced the multistage gradient elution with an isocratic elution of 8 stages of 87% (v/v) ethanol on the basis of example 3 above, with the other conditions unchanged.
The content and yield of each of the monoglycosides obtained were as follows:
① 87% ethanol 1-2BV section: the sweet tea content is increased to 8.1 percent, and the yield is 72.3 percent; the DA content is increased to 6.5 percent, and the yield is 31.3 percent;
② 87% ethanol 2-3BV section: the RB content is increased to 6.7%, and the yield is 12.4%;
③ 87% ethanol 2-4BV: the STV content is improved to 49.2%, and the STV recovery rate is 45.6%;
④ 3-4BV section of 87% ethanol: the RC content is improved to 28.3 percent, and the RC recovery rate is 47.8 percent;
⑤ The content of the 87% ethanol in the 3-5BV section and RA is increased to 37.6%, and the RA recovery rate is 53.5%;
⑥ 5-7BV section of 87% ethanol: the RD content is increased to 5.1%, and the RD recovery rate is 97.3%.
The collected 6 sets of eluents were crystallized under the same crystallization conditions as in example 3 to obtain pure monoglycoside products.
The single glycoside products have the purity of 33.5 percent of rubusoside, 30.3 percent of DA, 35.6 percent of RB, 82.7 percent of STV, 46.3 percent of RC, 68.7 percent of RA and 23.7 percent of RD after crystallization respectively in each stage according to the conditions.
Test example 1
Further, the method for measuring the content of stevioside composition in examples 1 to 5 and comparative examples 1 to 3 of the present invention was carried out with reference to national standard GB 1886.355-2022.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The method for purifying stevioside monoglycoside in the mother liquor sugar is characterized by comprising the following steps of:
Mixing the mother liquor sugar with a dissolution solvent to prepare a mother liquor sugar solution;
Carrying out adsorption separation on the mother liquor sugar solution through forward resin;
sequentially carrying out gradient elution on the adsorbed resin by using a multi-stage eluting solution;
Respectively collecting the eluents under each level of gradient to obtain a plurality of groups of eluents after the purification of the monoglycoside;
And respectively drying the plurality of groups of eluents to obtain crystals, and respectively adding the crystals into an organic crystallization solvent to crystallize to obtain various stevioside monoglycoside purified products.
2. The purification method according to claim 1, wherein the mass ratio of the mother liquor sugar to the dissolution solvent is 1: (4-6); the dissolving solvent is one or more of water, methanol, ethanol and ethyl acetate saturated aqueous solution.
3. The purification method according to claim 1 or 2, characterized in that after preparing a mother liquor sugar solution, before passing the mother liquor sugar solution through a forward resin, further comprising introducing a dissolution solvent into the forward resin to equilibrate the resin; wherein the feeding amount of the dissolution solvent is 1-2BV of the positive resin volume, and the feeding speed is 1-2BV/h.
4. The purification method according to claim 1, wherein the carrier of the forward resin is a medium-low pressure chromatographic column; the feeding amount of the mother liquor sugar solution is 4-6% of the positive resin volume, and the feeding speed is 1-2BV/h; the forward resin is one of amino silica gel filler, aminopropyl filler, MB70-75 or SMB 70-45.
5. The purification method according to claim 1, wherein the sequentially subjecting to gradient elution with a plurality of stages of elution solutions comprises: eluting by two or more eluting solvents in sequence, wherein the polarity of each eluting solvent is from low to high; the eluting solvent is selected from one or more of methanol, ethanol and ethyl acetate saturated water solution.
6. The method of claim 5, wherein the sequentially gradient eluting with multiple eluting solutions comprises: sequentially carrying out gradient elution by two or more stages of ethyl acetate/ethanol mixed solutions and two or more stages of ethanol aqueous solutions; wherein the volume ratio of ethyl acetate/ethanol in each stage is gradually reduced within the range of [1.5,0.3], and the volume fraction of the ethanol aqueous solution in each stage is gradually reduced within the range of [99% and 70% ].
7. The purification method according to claim 1 or 5, wherein the amount of the eluting solvent used in each gradient in the gradient elution is 2BV of the forward resin volume, and the flow rate of the gradient elution is 1-2BV/h.
8. The method of claim 7, wherein the separately collecting the eluents at each stage of gradient to obtain a plurality of sets of eluents after the single glycoside separation comprises: each gradient is collected for 1 BV/time, 2 groups of eluents are obtained by each gradient collection, and 2N groups of eluents are obtained by total collection, wherein N is the total stage number of gradient elution.
9. The purification method according to claim 8, wherein after the obtaining of the plurality of sets of eluents after the single glycoside separation, before the plurality of sets of eluents are dried, further comprising: and combining adjacent eluents with the same single glycoside as the main component to obtain a plurality of groups of eluents with different single glycosides as the main component.
10. The purification method according to claim 1, wherein the drying conditions in the plurality of sets of eluents are such that the drying temperature is 80-85 ℃ and the time is 1-2 hours;
The organic crystallization solvent is one or more of methanol, ethanol, isopropanol and acetone, and the addition amount is 2-4BV of the volume of the crystal; the crystallization conditions are as follows: the crystallization temperature is 20-30 ℃, the stirring speed is 80-120rpm, and the crystallization time is 6-12h.
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