CN114031499A - Method for obtaining high-purity isochlorogenic acid from stevia rebaudiana and application - Google Patents
Method for obtaining high-purity isochlorogenic acid from stevia rebaudiana and application Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 47
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 title claims abstract description 44
- CWVRJTMFETXNAD-BMNNCGMMSA-N (1s,3r,4s,5r)-3-[(e)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-1,4,5-trihydroxycyclohexane-1-carboxylic acid Chemical compound O[C@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-BMNNCGMMSA-N 0.000 title claims abstract description 40
- KRZBCHWVBQOTNZ-DLDRDHNVSA-N isochlorogenic acid Natural products O[C@@H]1[C@H](C[C@@](O)(C[C@H]1OC(=O)C=Cc2ccc(O)c(O)c2)C(=O)O)OC(=O)C=Cc3ccc(O)c(O)c3 KRZBCHWVBQOTNZ-DLDRDHNVSA-N 0.000 title claims abstract description 40
- 244000228451 Stevia rebaudiana Species 0.000 title claims abstract description 26
- 235000006092 Stevia rebaudiana Nutrition 0.000 title claims abstract description 26
- 239000012528 membrane Substances 0.000 claims abstract description 43
- 239000003960 organic solvent Substances 0.000 claims abstract description 20
- 238000001914 filtration Methods 0.000 claims abstract description 16
- 235000019202 steviosides Nutrition 0.000 claims abstract description 10
- UEDUENGHJMELGK-HYDKPPNVSA-N Stevioside Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UEDUENGHJMELGK-HYDKPPNVSA-N 0.000 claims abstract description 9
- 229940013618 stevioside Drugs 0.000 claims abstract description 9
- 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 claims abstract description 9
- 239000000706 filtrate Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 238000003825 pressing Methods 0.000 claims description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 241000196324 Embryophyta Species 0.000 claims description 20
- 108090000790 Enzymes Proteins 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 20
- 229940088598 enzyme Drugs 0.000 claims description 20
- 238000000967 suction filtration Methods 0.000 claims description 20
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 16
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 13
- 241000544066 Stevia Species 0.000 claims description 12
- 239000013078 crystal Substances 0.000 claims description 12
- 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 claims description 12
- 239000002002 slurry Substances 0.000 claims description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 11
- 239000002244 precipitate Substances 0.000 claims description 10
- 238000002425 crystallisation Methods 0.000 claims description 9
- 230000008025 crystallization Effects 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 239000002131 composite material Substances 0.000 claims description 8
- 108010059892 Cellulase Proteins 0.000 claims description 7
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 7
- 229930003268 Vitamin C Natural products 0.000 claims description 7
- 229940106157 cellulase Drugs 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- 239000003208 petroleum Substances 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 235000019154 vitamin C Nutrition 0.000 claims description 7
- 239000011718 vitamin C Substances 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 239000004744 fabric Substances 0.000 claims description 5
- 238000000703 high-speed centrifugation Methods 0.000 claims description 5
- 238000000464 low-speed centrifugation Methods 0.000 claims description 5
- 238000001728 nano-filtration Methods 0.000 claims description 5
- 238000004537 pulping Methods 0.000 claims description 5
- 238000004042 decolorization Methods 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 22
- 239000000284 extract Substances 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- 238000012545 processing Methods 0.000 abstract description 4
- 239000011347 resin Substances 0.000 abstract description 4
- 229920005989 resin Polymers 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 3
- 239000013543 active substance Substances 0.000 abstract description 2
- 238000004587 chromatography analysis Methods 0.000 abstract description 2
- 238000004821 distillation Methods 0.000 abstract description 2
- 239000008394 flocculating agent Substances 0.000 abstract description 2
- 238000012994 industrial processing Methods 0.000 abstract description 2
- 238000003672 processing method Methods 0.000 abstract description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 abstract 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 abstract 1
- 235000001368 chlorogenic acid Nutrition 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000002994 raw material Substances 0.000 description 6
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical class O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 description 5
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 description 4
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 description 4
- 229940074393 chlorogenic acid Drugs 0.000 description 4
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 description 4
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 description 4
- 238000011049 filling Methods 0.000 description 3
- 229930182470 glycoside Natural products 0.000 description 3
- 150000002338 glycosides Chemical class 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 239000012535 impurity Substances 0.000 description 2
- 238000000874 microwave-assisted extraction Methods 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- KRZBCHWVBQOTNZ-UHFFFAOYSA-N (-) 3,5-dicaffeoyl-muco-quinic acid Natural products OC1C(OC(=O)C=CC=2C=C(O)C(O)=CC=2)CC(O)(C(O)=O)CC1OC(=O)C=CC1=CC=C(O)C(O)=C1 KRZBCHWVBQOTNZ-UHFFFAOYSA-N 0.000 description 1
- KRZBCHWVBQOTNZ-RDJMKVHDSA-M (-)-3,5-Dicaffeoyl quinic acid Natural products O([C@@H]1CC(O)(C[C@H](C1O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C([O-])=O)C(=O)\C=C\C1=CC=C(O)C(O)=C1 KRZBCHWVBQOTNZ-RDJMKVHDSA-M 0.000 description 1
- KRZBCHWVBQOTNZ-PSEXTPKNSA-N 3,5-di-O-caffeoyl quinic acid Chemical compound O([C@@H]1C[C@](O)(C[C@H]([C@@H]1O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C(O)=O)C(=O)\C=C\C1=CC=C(O)C(O)=C1 KRZBCHWVBQOTNZ-PSEXTPKNSA-N 0.000 description 1
- UFCLZKMFXSILNL-RVXRWRFUSA-N 4,5-di-O-caffeoylquinic acid Chemical compound O([C@@H]1C[C@](O)(C[C@H]([C@@H]1OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)O)C(O)=O)C(=O)\C=C\C1=CC=C(O)C(O)=C1 UFCLZKMFXSILNL-RVXRWRFUSA-N 0.000 description 1
- 241000205585 Aquilegia canadensis Species 0.000 description 1
- 241001501111 Erycibe Species 0.000 description 1
- 241000628997 Flos Species 0.000 description 1
- 241000382298 Lagotis Species 0.000 description 1
- 239000004383 Steviol glycoside Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000001179 sorption measurement Methods 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
- 238000003786 synthesis reaction Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/60—Separation; Purification; Stabilisation; Use of additives by treatment giving rise to chemical modification
-
- 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
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/256—Polyterpene radicals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/14—The ring being saturated
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
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Abstract
The invention discloses a method for obtaining high-purity isochlorogenic acid from stevia rebaudiana and application thereof. According to the method, the complex enzymatic hydrolysis system is optimized, the extraction means is improved, the extraction rate of stevioside is improved, the content of isochlorogenic acid in filter residue is improved, substances such as a flocculating agent and the like are not required to be added, the pollution risk is reduced, and the further extraction of other active substances in the later period is facilitated. The isochlorogenic acid is extracted by using the combined mode of the organic membrane, a high-purity decolored crystalline isochlorogenic acid product is obtained at one time, complex processes such as activated carbon filtration/resin chromatography/distillation and the like in the conventional processing method are not needed, the processing time is reduced, the operation is convenient, the treatment capacity is large, and the method is very suitable for industrial processing production. According to the technical scheme, under the condition that stevioside extraction is not influenced, the production process is optimized to simultaneously and efficiently extract isochlorogenic acid, the used organic solvent is few, the operation is simple, the yield is high, and the method is practical and suitable for deep processing of stevia rebaudiana.
Description
Technical Field
The invention relates to a separation and extraction method, in particular to a method for obtaining high-purity isochlorogenic acid from stevia rebaudiana and application thereof.
Background
The source of the natural isochlorogenic acid is very wide and can be obtained by extracting by a hot reflux extraction method, a macroporous resin adsorption method, an ultrasonic-assisted extraction method, a microwave-assisted extraction method, an enzymolysis method and the like; the isochlorogenic acid chemically synthesized has more byproducts, difficult separation and purification and high synthesis process cost. It mainly extracts isochlorogenic acid from natural plants, but the current research on the extraction process of isochlorogenic acid is still in the early stage.
The chlorogenic acid compounds are mainly derived from flos Lonicerae, folium Ilicis, sweet stevia, folium Artemisiae Argyi, herba Lagotis, and caulis Erycibes. Modern researches have shown that chlorogenic acid substance in dry leaves of stevia rebaudiana Bertoni is as high as 52.69mg/g, and the whole plant contains chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C. In the conventional stevia extract production process, only the steviol glycosides in stevia are often obtained, but the chlorogenic acids in stevia are regarded as impurities, and the impurities are physically or chemically changed into precipitated waste residues for removal, which is definitely a waste of resources if the chlorogenic acids in the waste residues are not recovered.
In recent years, researchers have developed extraction methods such as a thermal reflux extraction method, a macroporous resin absorption method, an ultrasonic-assisted extraction method, a microwave-assisted extraction method, an enzymolysis method and the like for chlorogenic acid from different natural plant sources. However, compared with chlorogenic acid, the extraction process of isochlorogenic acid is less studied. Most of them are studied from honeysuckle. The method adopts multiple separation means such as multi-stage countercurrent extraction, fractional extraction, chromatographic elution and the like, and has complex process. The operation is complicated, and key points are not easy to control, so that the product yield, content and quality are unstable.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for obtaining high-purity isochlorogenic acid from stevia rebaudiana.
The invention also aims to provide application of the method for obtaining high-purity isochlorogenic acid from stevia rebaudiana.
The purpose of the invention is realized by the following technical scheme:
a method for obtaining high-purity isochlorogenic acid from stevia comprises the following steps:
s1, crushing a dried stevia rebaudiana Bertoni product, adding water and pulping to obtain stevia rebaudiana Bertoni aqueous slurry, adjusting the pH value, adding enzyme and vitamin C, stirring uniformly, performing enzymolysis and filtering; centrifuging the obtained filtrate, and centrifuging the obtained precipitate for later use; filter pressing the obtained filter residue; centrifuging and filter-pressing to obtain filtrate of stevioside, and filter-pressing to obtain filter residue;
s2, combining the precipitate obtained by centrifugation in the step S1 and the filter residue obtained by filter pressing, adding a solvent, and stirring to obtain an extracting solution;
s3, carrying out suction filtration on the extracting solution obtained in the step S2 to obtain a filtrate;
and S4, purifying the suction filtration liquid obtained in the step S3 by using an organic membrane to obtain a decolorization trapping membrane liquid.
S5, concentrating the decolorized trapped membrane liquid obtained in the step S4, adding an organic solvent for crystallization, performing suction filtration after crystallization, and washing and drying the obtained crystals to obtain the high-purity isochlorogenic acid crystals.
The amount of the added water in the step S1 is 3-5 times of the mass of the dried stevia rebaudiana product.
The pulverization in step S1 is preferably performed to a particle size of 100 to 200 mesh.
The adjustment of pH described in step S1 is to adjust the pH of the stevia aqueous slurry to 4.0 using a 1 wt% citric acid solution.
The enzyme in the step S1 is at least one of plant cellulase and plant composite wall-breaking enzyme; preferably plant cellulase and plant composite wall-breaking enzyme; more preferably, the plant cellulase and the plant composite wall-breaking enzyme are mixed according to the volume ratio of 1:1, mixing and obtaining the product.
The amount of the enzyme added in step S1 is preferably selected from the group consisting of dried stevia: enzyme 1 kg: calculating the proportion of 4-6 mL; more preferably dried stevia: enzyme 1 kg: 5mL of the mixture ratio is calculated.
The addition amount of the vitamin C in the step S1 is 0.2-0.4 per mill of the mass of the stevia rebaudiana water slurry.
Performing water bath enzymolysis for 1-1.5 h at 33-37 ℃ under the enzymolysis condition in the step S1; more preferably, the enzymolysis is carried out for 1 to 1.5 hours in water bath at the temperature of 35 ℃.
The filtering in the step S1 is filtering with 200-300 mesh filter cloth.
The centrifugation in the step S1 is firstly performed for 30 minutes through a butterfly type low-speed centrifugation at 1470r/min and then performed for 1.5 hours through a tubular type high-speed centrifugation at 14000 r/min.
And the pressure of the filter pressing in the step S1 is 1-3 MPa.
The solvent in the step S2 is at least one of acetic acid, n-hexane, and n-heptane; preferably a mixed solvent composed of two or more of acetic acid, n-hexane and n-heptane; more preferably, the solvent is a mixed solvent composed of acetic acid, n-hexane and n-heptane according to a volume ratio of 1-3: 1-2: 1-4.
And in the step S2, the adding amount of the solvent is 5-8 times of the mass of the combined filter residue.
The stirring condition in the step S2 is that the mixture is stirred for 1-3 hours at the temperature of 4-9 ℃.
The number of times of stirring described in step S2 is at least one; preferably at least twice.
And in the step S3, the suction filtration is carried out by using pad filter paper, and the aperture of the filter paper is 80-120 microns.
The suction filtration in the step S3 is carried out at least once; preferably at least twice.
The organic membrane in the step S4 is an organic solvent nanofiltration membrane.
And S4, performing two-stage organic membrane purification, wherein the molecular weight cutoff of the first-stage organic membrane is 650-850, the molecular weight cutoff of the second-stage organic membrane is 200-350, and taking the second filtrate as the decoloration membrane cutoff liquid.
The first membrane feeding pressure in the purification is 290-870 psi, and the second membrane feeding pressure is 500-850 psi.
And concentrating to 15-20 Baume degrees in the step S5.
And the adding amount of the organic solvent in the step S5 is 3-4 times of the volume of the decolorization trapping solution.
The organic solvent in the step S5 is at least one of petroleum ether, methanol and ethanol; preferably an organic solvent obtained by mixing petroleum ether, methanol and ethanol; more preferably, the organic solvent is obtained by mixing petroleum ether, methanol and ethanol according to the volume ratio of 1-2: 1-3: 1-2.
The crystallization condition in the step S5 is crystallization for more than 4 hours at a temperature of-4 to 10 ℃.
And in the step S5, the suction filtration is carried out by using pad filter paper, and the aperture of the filter paper is 80-120 microns.
The washing in the step S5 is washing with pure water at 4-8 ℃.
The isochlorogenic acid content of the high-purity isochlorogenic acid crystal described in step S5 is greater than 80%.
Compared with the prior art, the application of the method for obtaining high-purity isochlorogenic acid from stevia rebaudiana in preparing isochlorogenic acid has the following advantages and effects:
1. according to the method, the extraction rate of stevioside is improved by optimizing a composite biological enzyme enzymolysis system and improving an extraction means, the content of isochlorogenic acid in filter residue is improved, substances such as a flocculating agent and the like are not required to be added, the pollution risk is reduced, and the further extraction of other active substances in the later period is facilitated.
2. The isochlorogenic acid is extracted by using the combined mode of the organic membrane, a high-purity decolored crystalline isochlorogenic acid product is obtained at one time, complex processes such as activated carbon filtration/resin chromatography/distillation and the like in the conventional processing method are not needed, the processing time is reduced, the operation is convenient, the treatment capacity is large, and the method is very suitable for industrial processing production.
3. According to the technical scheme, under the condition that stevioside extraction is not influenced, the production process is optimized to simultaneously and efficiently extract isochlorogenic acid, the used organic solvent is few, the method is safe and environment-friendly, the operation is simple, the yield is high, and the method is a practical novel method suitable for deep processing of stevia rebaudiana.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples in any way. The starting reagents employed in the examples of the present invention are, unless otherwise specified, those that are conventionally purchased.
Example 1
(1) Taking 200g of stevia rebaudiana dry product (total content of raw materials is 4%), crushing to 150 meshes, adding 3 times of pure water, and pulping to obtain stevia rebaudiana water slurry. Adjusting pH to 4.0 with 1 wt% citric acid solution, adding 0.5mL plant cellulase (500U/mL) and 0.5mL plant composite wall-breaking enzyme (also called plant extraction special enzyme from Henghuadao Biotech limited in Nanning), adding 0.2 ‰ vitamin C of stevia water slurry, stirring, performing enzymolysis in 35 deg.C water bath for 1h (h ═ h), filtering with 200 mesh filter cloth, centrifuging the obtained filtrate by stage centrifuge, performing butterfly low-speed centrifugation at 1470r/min for 30 min, and then performing tubular high-speed centrifugation at 14000r/min for 1.5h, and centrifuging the obtained precipitate for use. And (3) pressing the filter residue obtained by filtering to dry the filtrate under the pressure of 1MPa, centrifuging and pressing to obtain filtrate which is stevioside (total glycoside), and pressing and filtering to obtain filter residue for later use.
(2) And (2) combining the precipitate obtained by centrifugation in the step (1) and the filter residue obtained by filter pressing, adding acetic acid with the mass of 5 times of that of the mixture, stirring and extracting at a low temperature of 4 ℃ for 1 hour, and combining the two extractions to obtain an extracting solution.
(3) And carrying out suction filtration twice on the obtained extract pad filter paper, wherein the aperture of the filter paper is 80 microns, and combining and collecting filtrate obtained twice for later use.
(4) Purifying the filtrate by two-stage OSN (organic solvent nanofiltration) organic membrane, firstly passing through an organic membrane with the molecular weight cutoff of 650 (the membrane feeding pressure is 290psi), then passing through an organic membrane with the molecular weight cutoff of 200 (the membrane feeding pressure is 500psi), and taking the second filtrate to obtain the decoloration membrane-intercepting liquid.
(5) Concentrating the decolorized entrapment membrane solution to 15 Baume degrees, adding 3 times volume of organic solvent (AR grade petroleum ether, methanol and ethanol are mixed according to the volume ratio of 1:1:1), crystallizing for more than 4 hours at low temperature (-4 ℃), filling filter paper for suction filtration after crystallization, wherein the aperture of the filter paper is 80 microns to obtain crystals, washing with 10 times volume of pure ice water (4 ℃), drying to obtain a high-purity isochlorogenic acid product which is in a white crystal shape, measuring the content of isochlorogenic acid by HPLC (high performance liquid chromatography) to be 83.5%, and calculating the total isochlorogenic acid recovery rate to be 95.5% by using raw materials.
Example 2
(1) Taking 200g of stevia rebaudiana dry product (total content of raw materials is 4%), crushing to 150 meshes, adding 4 times of pure water, and pulping to obtain stevia rebaudiana water slurry. Adjusting pH to 4.0 with 1 wt% citric acid solution, adding 0.5mL plant cellulase (500U/mL) and 0.5mL plant composite wall-breaking enzyme (also called plant extraction special enzyme from Henghuadao Biotech limited in Nanning), adding 0.2 ‰ vitamin C of stevia water slurry, stirring, performing enzymolysis in 35 deg.C water bath for 1h (h ═ h), filtering with 250 mesh filter cloth, centrifuging the obtained filtrate by stage centrifuge, performing butterfly low-speed centrifugation at 1470r/min for 30 min, and then performing tubular high-speed centrifugation at 14000r/min for 1.5h, and centrifuging the obtained precipitate for use. And (3) pressing the filter residue obtained by filtering to dry the filtrate under the pressure of 2MPa, centrifuging and pressing to obtain filtrate which is stevioside (total glycosides), and pressing and filtering to obtain filter residue for later use.
(2) And (2) combining the precipitate obtained by centrifugation in the step (1) and filter residue obtained by filter pressing, adding 6 times of mixed organic solvent (acetic acid, normal hexane and normal heptane in a volume ratio of 1:1:1), stirring and extracting at a low temperature of 7 ℃ for 2 hours, and combining the extraction twice to obtain an extracting solution.
(3) And carrying out suction filtration twice on the obtained extract pad filter paper, wherein the aperture of the filter paper is 100 microns, and combining and collecting filtrate obtained twice for later use.
(4) Purifying the filtrate by two-stage OSN (organic solvent nanofiltration) organic membrane, firstly passing through an organic membrane with the molecular weight cutoff of 750 (the membrane inlet pressure of 590psi), then passing through an organic membrane with the molecular weight cutoff of 270 (the membrane inlet pressure of 700psi), and taking the second filtrate to obtain the decoloration membrane-trapping liquid.
(5) Concentrating the decolorized entrapment membrane liquid to 17 Baume degrees, adding 3.5 times volume of organic solvent (AR grade petroleum ether, methanol and ethanol are mixed according to the volume ratio of 1:1:1), crystallizing for more than 4 hours at low temperature (5 ℃), filling filter paper for suction filtration after crystallization, wherein the aperture of the filter paper is 100 microns to obtain crystals, washing with 10 times volume of pure ice water (6 ℃), drying to obtain a high-purity isochlorogenic acid product which is in a white crystal shape, measuring the content of isochlorogenic acid by HPLC (high performance liquid chromatography) to be 81.5%, and calculating the total isochlorogenic acid recovery rate to be 96.1% by using raw materials.
Example 3
(1) Taking 200g of stevia rebaudiana dry product (total content of raw materials is 4%), crushing to 150 meshes, adding 5 times of pure water, and pulping to obtain stevia rebaudiana water slurry. Adjusting pH to 4.0 with 1 wt% citric acid solution, adding 0.5mL plant cellulase (500U/mL) and 0.5mL plant composite wall-breaking enzyme (also called plant extraction specific enzyme from Hengchang Huadao Biotech limited Co., Ltd.), adding 0.4 ‰ vitamin C of stevia water slurry, stirring, performing enzymolysis in 35 deg.C water bath for 1.5h (h ═ h), filtering with 300 mesh filter cloth, centrifuging the obtained filtrate by stage centrifuge, performing butterfly low-speed centrifugation at 1470r/min for 30 min, and then performing tubular high-speed centrifugation at 14000r/min for 1.5h, and centrifuging the obtained precipitate for use. Pressing the filter residue obtained by filtering under the pressure of 3MPa to dry the filtrate, centrifuging and pressing to obtain filtrate which is stevioside (total glycoside), and pressing to obtain filter residue for later use;
(2) and (2) combining the precipitate obtained by centrifugation in the step (1) and filter residue obtained by filter pressing, adding 8 times of mixed organic solvent (acetic acid, n-hexane and n-heptane in a volume ratio of 3:2:4), stirring and extracting at a low temperature of 9 ℃ for 3 hours, and combining the extraction solutions to obtain an extracting solution.
(3) And (4) carrying out suction filtration on the extract pad filter paper twice, wherein the aperture of the filter paper is 120 microns, and combining and collecting filtrate obtained in two times for later use.
(4) Purifying the filtrate by two-stage OSN (organic solvent nanofiltration) organic membrane, passing through an organic membrane with the molecular weight cutoff of 850 (inlet membrane pressure of 870psi), passing through an organic membrane with the molecular weight cutoff of 350 (inlet membrane pressure of 850psi), and taking the second filtrate to obtain decolorized membrane-trapping liquid.
(5) Concentrating the decolorized entrapment membrane solution to 20 Baume degrees, adding 4 times volume of organic solvent (AR grade petroleum ether, methanol and ethanol are mixed according to a volume ratio of 2:3:2), crystallizing for more than 4 hours at low temperature (10 ℃), filling filter paper for suction filtration after crystallization, wherein the aperture of the filter paper is 120 microns to obtain crystals, washing with 10 times volume of pure ice water (8 ℃), drying to obtain a high-purity isochlorogenic acid product which is in a white crystal shape, measuring the content of isochlorogenic acid by HPLC (high performance liquid chromatography) and ensuring that the total isochlorogenic acid recovery rate is 96.2% by taking the raw materials as a reference.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for obtaining high-purity isochlorogenic acid from stevia rebaudiana is characterized by comprising the following steps:
s1, crushing a dried stevia rebaudiana Bertoni product, adding water and pulping to obtain stevia rebaudiana Bertoni aqueous slurry, adjusting the pH value, adding enzyme and vitamin C, stirring uniformly, performing enzymolysis and filtering; centrifuging the obtained filtrate, and centrifuging the obtained precipitate for later use; filter pressing the obtained filter residue; centrifuging and filter-pressing to obtain filtrate of stevioside, and filter-pressing to obtain filter residue;
s2, combining the precipitate obtained by centrifugation in the step S1 and the filter residue obtained by filter pressing, adding a solvent, and stirring and extracting to obtain an extracting solution;
s3, carrying out suction filtration on the extracting solution obtained in the step S2 to obtain a filtrate;
s4, purifying the suction filtration liquid obtained in the step S3 by using an organic membrane to obtain a decolorization trapping membrane liquid;
s5, concentrating the decolorized trapped membrane liquid obtained in the step S4, adding an organic solvent for crystallization, performing suction filtration after crystallization, and washing and drying the obtained crystals to obtain the high-purity isochlorogenic acid crystals.
2. The method of claim 1, wherein the method comprises the steps of:
the amount of the added water in the step S1 is 3-5 times of the mass of the dried stevia rebaudiana product;
the pulverization degree in the step S1 is to be pulverized to a particle size of 100-200 meshes.
3. The method of claim 1, wherein the method comprises the steps of:
the adjustment of pH described in step S1 is to adjust the pH of the stevia aqueous slurry to 4.0 using a 1 wt% citric acid solution;
the enzyme in the step S1 is at least one of plant cellulase and plant composite wall-breaking enzyme;
the addition amount of the enzyme in the step S1 is determined according to the following ratio: enzyme 1 kg: calculating the proportion of 4-6 mL;
the addition amount of the vitamin C in the step S1 is 0.2-0.4 per mill of the mass of the stevia rebaudiana water slurry;
and the enzymolysis condition in the step S1 is water bath enzymolysis for 1-1.5 h at 33-37 ℃.
4. The method of claim 1, wherein the method comprises the steps of:
the filtering in the step S1 is filtering by using filter cloth with 200-300 meshes;
the centrifugation in the step S1 is firstly performed for 30 minutes through a butterfly type low-speed centrifugation at 1470r/min and then performed for 1.5 hours through a tubular type high-speed centrifugation at 14000 r/min;
and the pressure of the filter pressing in the step S1 is 1-3 MPa.
5. The method of claim 1, wherein the method comprises the steps of:
the solvent in the step S2 is at least one of acetic acid, n-hexane, and n-heptane;
the adding amount of the solvent in the step S2 is 5-8 times of the mass of the combined filter residue;
stirring conditions in the step S2 are that stirring is carried out for 1-3 h at 4-9 ℃;
the number of times of stirring described in step S2 is at least one.
6. The method of claim 1, wherein the method comprises the steps of:
the suction filtration in the step S3 is the suction filtration of pad filter paper, and the aperture of the filter paper is 80-120 microns;
the number of suction filtration in step S3 is at least one.
7. The method of claim 1, wherein the method comprises the steps of:
the organic membrane in the step S4 is an organic solvent nanofiltration membrane;
the purification in the step S4 is two-stage organic membrane purification, the molecular weight cut-off of the first-stage organic membrane is 650-850, the molecular weight cut-off of the second-stage organic membrane is 200-350, and the second filtrate is taken as decoloration cut-off membrane liquid;
in the purification, the first membrane feeding pressure is 290-870 psi, and the second membrane feeding pressure is 500-850 psi.
8. The method of claim 1, wherein the method comprises the steps of:
concentrating to 15-20 Baume degrees in the step S5;
the adding amount of the organic solvent in the step S5 is 3-4 times of the volume of the decolorization trapping membrane liquid;
the organic solvent in step S5 is at least one of petroleum ether, methanol, and ethanol.
9. The method of claim 1, wherein the method comprises the steps of:
crystallizing at the temperature of-4-10 ℃ for more than 4 hours under the crystallizing condition in the step S5;
the suction filtration in the step S5 is the suction filtration of pad filter paper, and the aperture of the filter paper is 80-120 microns;
the washing in the step S5 is washing with pure water at 4-8 ℃;
the isochlorogenic acid content of the high-purity isochlorogenic acid crystal described in step S5 is greater than 80%.
10. Use of the method of any one of claims 1 to 9 for obtaining high purity isochlorogenic acid from stevia rebaudiana Bertoni in the preparation of isochlorogenic acid.
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