CN105616709A - Novel technology for extracting tea polyphenol in tea leaves - Google Patents
Novel technology for extracting tea polyphenol in tea leaves Download PDFInfo
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- CN105616709A CN105616709A CN201410669799.3A CN201410669799A CN105616709A CN 105616709 A CN105616709 A CN 105616709A CN 201410669799 A CN201410669799 A CN 201410669799A CN 105616709 A CN105616709 A CN 105616709A
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Abstract
The invention provides a novel technology for extracting tea polyphenol in tea leaves. The technology employs four steps of enzyme extraction, ultrasonic extraction, membrane filtration and molecular imprinting for comprehensively extracting the tea polyphenol. According to the technology, the enzyme extraction is employed for destroying tea cell walls, so that the reaction temperature is normal temperature, energy consumption is little, chemical bonds such as glycosidic bond in cellulose can be better destroyed, and then superpower penetrating capability and cavitation effect of supersonic wave are used, cell walls can be rapidly dispersed, cell membranes are destroyed, and the tea polyphenol can be rapidly released; the molecular imprinting is employed for specifically absorbing and purifying a main component catechin in the tea polyphenol, the specific adsorption capability of a synthesized imprinting polymer on catechin is very strong, extraction step is greatly reduced, and extraction purity of the tea polyphenol is greatly increased. The extraction method has the advantages of reasonable, scientific, effective performances and little energy consumption.
Description
Technical field
The invention belongs to extractive technique field, especially relate to a kind of Folium Camelliae sinensis extracts the novel process of tea polyphenols.
Background technology
Modern medicine study shows: in Folium Camelliae sinensis, wholesome main component has tea polyphenols, caffeine, polysaccharide, aminoacid, mineral etc., and wherein tea polyphenols is a kind of novel antioxidant from natural food, is approved by China in nineteen ninety. Tea polyphenols also has a series of pharmacological functions such as anticancer, defying age, radioprotective, elimination human free radical, blood fat reducing efficiently, is widely applied at industrial circles such as fatty foods, medicine, daily use chemicals. Therefore, recent domestic experts and scholars are to the extraction of tea polyphenols and applied research growing interest.
Tea polyphenols (Teapolyphenols is called for short TP) is the main component of Folium Camelliae sinensis, also known as tea tannin, tea tannic acid, tea polyphenols, is the general name of tea leaf polyphenols class material. By the difference of its chemical constitution, tea polyphenols contains Multiple components, and wherein flavanol compound material catechin is the bulk composition of tea polyphenols, accounts for 60%��80% in tea polyphenols. Catechin is divided into 6 kinds, respectively ester catechin and non-ester catechin; Flavone and flavonols; Leucocyanidin; Phenolic acids and depside. Ester catechin has L-Epicatechin gallate (L-ECG) and epigallocatechin gallate (EGCG) (L-EGCG). Non-ester catechin has epicatechin (L-EC), nutgall catechin (D, L-GC), epigallo catechin (L-EGC) and catechin (D, L-C) etc. Flavone and flavonols are mainly flavonol and glycosides thereof; Leucocyanidin and hydroxyl flavonol and salt thereof, including leucoanthocyanidin and anthocyanidin.
At present, from Folium Camelliae sinensis, extract tea polyphenols and have multiple method. Current factory mainly adopts single extracting method, such as hot water extraction method, organic solvent extraction, ultrasonic extraction, microwave loss mechanisms, Enzymatic Extraction method, super critical extraction. But every kind of method has its shortcoming and advantage. Such as hot-water soak extraction method, extraction efficiency is not high, and energy resource consumption is high, but extraction cost is low. Organic solvent extraction, seriously polluted, environment is unfriendly. Ultrasonic, microwave extract method, it is too high to be used alone the consumption energy, and extraction effect neither be ideal, although super critical extraction extraction effect is fine, but equipment cost is too high, and extractability is limited.
The advantage of the comprehensive various extracting method of the present invention, maximizes favourable factors and minimizes unfavourable ones, takes its chief, keep away its shortage. First the polyphenol substance in Folium Camelliae sinensis is present in plant cell, and what plant cell wall was made up of fine and close cellulose. If only using physical disruption methods or extracting method, such as hot water extraction, ultrasound wave, supertension and microwave loss mechanisms, it is not easy to effectively, making Folium Camelliae sinensis cell breakage rapidly, tea polyphenols is discharged in extracting solution. On the contrary, if using enzyme process to carry out destroying Folium Camelliae sinensis cell wall, reaction temperature room temperature, energy resource consumption is little, environmental friendliness, it is possible to destroy the chemical bond in cellulose very well, such as glycosidic bond. But not having the auxiliary of external force, the cell wall of destruction remains attached on cell membrane. So we want the advantage of comprehensive both approaches, utilizing enzymatic treatment Folium Camelliae sinensis cell wall thickness, recycling hyperacoustic superpower penetration capacity and cavitation effect, rapidly cell wall is broken up, destroying cell membrane, make tea leaf polyphenols discharge rapidly. And the time required for this process is than being used alone short many of ultrasonic disruption Folium Camelliae sinensis cell, so energy resource consumption is also little. Therefore, the extracting method of this tea polyphenols compared with other method more rationally, science, effectively.
Additionally in the present invention, another innovative point is in that to adopt molecular engram method that the main component catechin in tea polyphenols is carried out specific adsorption and purification. The imprinted polymer of synthesis is very strong to the specificity absorbability of catechin, so greatly reduces extraction step, reduces tea polyphenols loss and extraction cost.
Summary of the invention
The problem to be solved in the present invention is to provide the novel process extracting tea polyphenols in a kind of Folium Camelliae sinensis, and this technique greatly reduces extraction step, substantially increases the DNA purity of tea polyphenols.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
Extracting the novel process of tea polyphenols in Folium Camelliae sinensis, this technique includes step in detail below:
(1) break process of Folium Camelliae sinensis: add a certain amount of compound enzyme in Folium Camelliae sinensis and Folium Camelliae sinensis somatic cell is carried out break process;
(2) the preliminary extraction of tea polyphenols: utilize ultrasound wave to carry out assisted extraction tea polyphenols the Folium Camelliae sinensis processed in step (1);
(3) preliminary purification of tea polyphenols: adopt the mode of membrane filtration to carry out the primary purification of tea polyphenols;
(4) imprinted polymer of molecular engram method synthesis tea polyphenols main component catechin is adopted;
(5) the high performance liquid chromatography detection imprinted polymer absorbability to catechin is utilized: molecularly imprinted polymer is filled post, make and prepare post, then elution requirement is studied, it is determined that utilize molecularly imprinted polymer to carry out the optimum process condition of refining Catechin in Tea;
(6) pilot scale test run: be amplified trial production according to the lab scale craft studied well, adjusts the technological parameter of pilot scale, it is determined that the extraction and purification process of polyphenol in Folium Camelliae sinensis.
Further, compound enzyme described in step (1) includes following components by weight percent: 0.1��0.3% cellulase, 0.1��0.3% hemicellulase, 0.1��0.3% pectase, the hydrolysis temperature 50��55 DEG C of the break process of the biological Folium Camelliae sinensis of step (1), enzymolysis time 30��45min.
Further, adopting ethanol to carry out the preliminary extraction of tea polyphenols in step (2), in preliminary extraction process, ultrasonic power is 2800��3200W, time 10��15min, temperature 45��55 DEG C.
Further, what adopt in membrane filtration processes in step (3) is the molecular cut off hollow-fibre membrane at more than 1000Da, and by the protein of macromolecule, the polyphenol substance such as the catechin of the material such as nucleic acid and molecular weight is easily separated.
Further, in step (4), the preparation process of the imprinted polymer of catechin is: with ��-methacrylic acid for monomer, ethylene glycol dimethacrylate is cross-linking agent, catechin is template molecule, the imprinted polymer of lower polymerization preparation catechin is penetrated in UV illumination, the polymer prepared is pulverized, after sieving, carries out surname extraction.
Further, in step (4), in the process of surname extraction, eluting solution is: methanol: acetic acid (v/v)=9:1, elution speed 5mL/min, then removes microsphere, drying for standby.
The present invention adopts enzyme process to carry out destroying Folium Camelliae sinensis cell wall, reaction temperature room temperature, energy resource consumption is little, the chemical bond in cellulose can be destroyed very well, such as glycosidic bond, then utilize hyperacoustic superpower penetration capacity and cavitation effect, rapidly cell wall is broken up, destroy cell membrane, make tea leaf polyphenols discharge rapidly; And the present invention adopts molecular engram method that the main component catechin in tea polyphenols is carried out specific adsorption and purification, the imprinted polymer of synthesis is very strong to the specificity absorbability of catechin, greatly reduce extraction step, substantially increase tea polyphenol extract purity, this extracting method has rationally, science, effectively, the little advantage of energy resource consumption.
Detailed description of the invention
Molecular imprinting is the experiment technology of preparing for obtaining the polymer mated completely on space structure and binding sites with certain a part (being commonly referred to template molecule). its preparation process is first to use function monomer and template molecule with covalent bond or non-covalent bond (hydrogen bond, electrostatic force, metal-chelating active force, intermolecular force, the intermolecular forces of group and hydrophobic interaction etc.) form complex, add cross-linking agent, initiator and organic solvent, (heating under certain conditions, ultraviolet light or other roentgenization) polymerization, so as to generate imprinted polymer, method finally by eluting, template molecule is removed from polymer, thus leave template molecule space structure on polymer, the three-dimensional hole that binding sites mates completely, this three-dimensional hole is single-minded, under suitable conditions, this three-dimensional lumen " can remember " physical dimension and other the physico-chemical property of template satisfactorily, and can effectively and selectively go bonding template (or the like) molecule.
Catechin is the material containing multiple hydroxy functional groups, and it can be combined with hydrogen bond formation with function monomer ��-methacrylic acid (MAA). Although due to sterically hindered existence, a part of hydroxyl being made with MAA to be combined difficulty, but still has multiple binding site, can form stable complex with function monomer MAA. After polymer obtained by crosslinked polymerization removes template molecule, it is possible to stay and the hole of catechin complementary element, " making a reservation for " of becoming absorption catechin identifies position.
Embodiment 1
A kind of Folium Camelliae sinensis extracts the novel process of tea polyphenols, comprises the following steps:
(1) break process of Folium Camelliae sinensis: add a certain amount of compound enzyme in Folium Camelliae sinensis and Folium Camelliae sinensis somatic cell is carried out break process; Described compound enzyme includes following components by weight percent: 0.1��0.3% cellulase, 0.1��0.3% hemicellulase, 0.1��0.3% pectase, the hydrolysis temperature 50��55 DEG C of the break process of the biological Folium Camelliae sinensis of step (1), enzymolysis time 30��45min.
(2) the preliminary extraction of tea polyphenols: utilize ultrasound wave to carry out assisted extraction tea polyphenols the Folium Camelliae sinensis processed in step (1); Adopting ethanol to carry out the preliminary extraction of tea polyphenols, in preliminary extraction process, ultrasonic power is 2800��3200W, time 10��15min, temperature 45��55 DEG C.
(3) preliminary purification of tea polyphenols: adopt molecular cut off at the hollow-fibre membrane of more than 1000Da, by the protein of macromolecule, the polyphenol substance such as the catechin of the material such as nucleic acid and molecular weight is easily separated.
(4) imprinted polymer of molecular engram method synthesis tea polyphenols main component catechin is adopted:
1. the preparation process of the imprinted polymer of epicatechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.4584g epicatechin (EC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer one.
2. the preparation process of the imprinted polymer of epigallo catechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.4584g epigallo catechin (EGC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer two.
3. the preparation process of the imprinted polymer of L-Epicatechin gallate is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.4584g L-Epicatechin gallate (ECG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer three.
4. the preparation process of the imprinted polymer of epigallocatechin gallate (EGCG) is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.4584g epigallocatechin gallate (EGCG) (EGCG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer four.
(5) the high performance liquid chromatography detection imprinted polymer absorbability to catechin is utilized: take 100mg molecularly imprinted polymer one, imprinted polymer two, imprinted polymer three, imprinted polymer four respectively, it is suspended in 3mL methanol respectively, ultrasonic vibration 10min, then it is sequentially loaded in self-control politef solid-phase extraction column (60mm �� 8mmi.d.), the porous ceramic support in extraction column lower end. Extraction column 15mLV (methanol): V (the acetic acid)=1:8 solution loaded cleans, and flow speed control is at 0.1mL/min. Cleaned pillar vacuum drying is standby. Preparation 0.1mg/mL tea polyphenols solution adds in extraction column, flows through with the flow velocity of 0.1mL/min. Upload rear extraction column V (methanol): V (water)=1:9 eluant solution, detect with HPLC after collecting liquid micro-filtrate membrane filtration, mobile phase is V (methanol): V (acetic acid): V (water)=24.5:0.5:75, flow velocity is 0.8mL/min, sample size is 6 �� L, detects wavelength 265nm.
(6) pilot scale test run: be amplified trial production according to the lab scale craft studied well, adjusts the technological parameter of pilot scale, it is determined that the extraction and purification process of polyphenol in Folium Camelliae sinensis. If Folium Camelliae sinensis total amount is 100g, the total amount of the catechin in eluent is 13g, and obtaining the response rate is 13%, and the purity of catechin is 100%.
Embodiment 1
A kind of Folium Camelliae sinensis extracts the novel process of tea polyphenols, comprises the following steps:
(1) break process of Folium Camelliae sinensis: add a certain amount of compound enzyme in Folium Camelliae sinensis and Folium Camelliae sinensis somatic cell is carried out break process; Described compound enzyme includes following components by weight percent: 0.1��0.3% cellulase, 0.1��0.3% hemicellulase, 0.1��0.3% pectase, the hydrolysis temperature 50��55 DEG C of the break process of the biological Folium Camelliae sinensis of step (1), enzymolysis time 30��45min.
(2) the preliminary extraction of tea polyphenols: utilize ultrasound wave to carry out assisted extraction tea polyphenols the Folium Camelliae sinensis processed in step (1); Adopting ethanol to carry out the preliminary extraction of tea polyphenols, in preliminary extraction process, ultrasonic power is 2800��3200W, time 10��15min, temperature 45��55 DEG C.
(3) preliminary purification of tea polyphenols: adopt molecular cut off at the hollow-fibre membrane of more than 1000Da, by the protein of macromolecule, the polyphenol substance such as the catechin of the material such as nucleic acid and molecular weight is easily separated.
(4) imprinted polymer of molecular engram method synthesis tea polyphenols main component catechin is adopted:
1. the preparation process of the imprinted polymer of epicatechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.5501g epicatechin (EC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer one.
2. the preparation process of the imprinted polymer of epigallo catechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.5501g epigallo catechin (EGC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer two.
3. the preparation process of the imprinted polymer of L-Epicatechin gallate is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.5501g L-Epicatechin gallate (ECG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer three.
4. the preparation process of the imprinted polymer of epigallocatechin gallate (EGCG) is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.5501g epigallocatechin gallate (EGCG) (EGCG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer four.
(5) the high performance liquid chromatography detection imprinted polymer absorbability to catechin is utilized: take 100mg molecularly imprinted polymer one, imprinted polymer two, imprinted polymer three, imprinted polymer four respectively, it is suspended in 3mL methanol respectively, ultrasonic vibration 10min, then it is sequentially loaded in self-control politef solid-phase extraction column (60mm �� 8mmi.d.), the porous ceramic support in extraction column lower end. Extraction column 15mLV (methanol): V (the acetic acid)=1:8 solution loaded cleans, and flow speed control is at 0.1mL/min. Cleaned pillar vacuum drying is standby. Preparation 0.1mg/mL tea polyphenols solution adds in extraction column, flows through with the flow velocity of 0.1mL/min. Upload rear extraction column V (methanol): V (water)=1:9 eluant solution, detect with HPLC after collecting liquid micro-filtrate membrane filtration, mobile phase is V (methanol): V (acetic acid): V (water)=24.5:0.5:75, flow velocity is 0.8mL/min, sample size is 6 �� L, detects wavelength 265nm.
(6) pilot scale test run: be amplified trial production according to the lab scale craft studied well, adjusts the technological parameter of pilot scale, it is determined that the extraction and purification process of polyphenol in Folium Camelliae sinensis. If Folium Camelliae sinensis total amount is 100g, the total amount of the catechin in eluent is 14.6g, and obtaining the response rate is 14.6%, and the purity of catechin is 100%.
Embodiment 2
A kind of Folium Camelliae sinensis extracts the novel process of tea polyphenols, comprises the following steps:
(1) break process of Folium Camelliae sinensis: add a certain amount of compound enzyme in Folium Camelliae sinensis and Folium Camelliae sinensis somatic cell is carried out break process; Described compound enzyme includes following components by weight percent: 0.1��0.3% cellulase, 0.1��0.3% hemicellulase, 0.1��0.3% pectase, the hydrolysis temperature 50��55 DEG C of the break process of the biological Folium Camelliae sinensis of step (1), enzymolysis time 30��45min.
(2) the preliminary extraction of tea polyphenols: utilize ultrasound wave to carry out assisted extraction tea polyphenols the Folium Camelliae sinensis processed in step (1); Adopting ethanol to carry out the preliminary extraction of tea polyphenols, in preliminary extraction process, ultrasonic power is 2800��3200W, time 10��15min, temperature 45��55 DEG C.
(3) preliminary purification of tea polyphenols: adopt molecular cut off at the hollow-fibre membrane of more than 1000Da, by the protein of macromolecule, the polyphenol substance such as the catechin of the material such as nucleic acid and molecular weight is easily separated.
(4) imprinted polymer of molecular engram method synthesis tea polyphenols main component catechin is adopted:
1. the preparation process of the imprinted polymer of epicatechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.6876g epicatechin (EC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer one.
2. the preparation process of the imprinted polymer of epigallo catechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.6876g epigallo catechin (EGC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer two.
3. the preparation process of the imprinted polymer of L-Epicatechin gallate is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.6876g L-Epicatechin gallate (ECG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer three.
4. the preparation process of the imprinted polymer of epigallocatechin gallate (EGCG) is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.6876g epigallocatechin gallate (EGCG) (EGCG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer four.
(5) the high performance liquid chromatography detection imprinted polymer absorbability to catechin is utilized: take 100mg molecularly imprinted polymer one, imprinted polymer two, imprinted polymer three, imprinted polymer four respectively, it is suspended in 3mL methanol respectively, ultrasonic vibration 10min, then it is sequentially loaded in self-control politef solid-phase extraction column (60mm �� 8mmi.d.), the porous ceramic support in extraction column lower end. Extraction column 15mLV (methanol): V (the acetic acid)=1:8 solution loaded cleans, and flow speed control is at 0.1mL/min. Cleaned pillar vacuum drying is standby. Preparation 0.1mg/mL tea polyphenols solution adds in extraction column, flows through with the flow velocity of 0.1mL/min. Upload rear extraction column V (methanol): V (water)=6:4 solution, methanol solution eluting, detect with HPLC after collecting liquid micro-filtrate membrane filtration, mobile phase is V (methanol): V (acetic acid): V (water)=24.5:0.5:75, flow velocity is 0.8mL/min, sample size is 6 �� L, detects wavelength 270nm.
(6) pilot scale test run: be amplified trial production according to the lab scale craft studied well, adjusts the technological parameter of pilot scale, it is determined that the extraction and purification process of polyphenol in Folium Camelliae sinensis. If Folium Camelliae sinensis total amount is 100g, the total amount of the catechin in eluent is 16.7g, and the response rate is 16.7%, and the purity of catechin is 99%.
Embodiment 3
A kind of Folium Camelliae sinensis extracts the novel process of tea polyphenols, comprises the following steps:
(1) break process of Folium Camelliae sinensis: add a certain amount of compound enzyme in Folium Camelliae sinensis and Folium Camelliae sinensis somatic cell is carried out break process; Described compound enzyme includes following components by weight percent: 0.1��0.3% cellulase, 0.1��0.3% hemicellulase, 0.1��0.3% pectase, the hydrolysis temperature 50��55 DEG C of the break process of the biological Folium Camelliae sinensis of step (1), enzymolysis time 30��45min.
(2) the preliminary extraction of tea polyphenols: utilize ultrasound wave to carry out assisted extraction tea polyphenols the Folium Camelliae sinensis processed in step (1); Adopting ethanol to carry out the preliminary extraction of tea polyphenols, in preliminary extraction process, ultrasonic power is 2800��3200W, time 10��15min, temperature 45��55 DEG C.
(3) preliminary purification of tea polyphenols: adopt molecular cut off at the hollow-fibre membrane of more than 1000Da, by the protein of macromolecule, the polyphenol substance such as the catechin of the material such as nucleic acid and molecular weight is easily separated.
(4) imprinted polymer of molecular engram method synthesis tea polyphenols main component catechin is adopted:
1. the preparation process of the imprinted polymer of epicatechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.9168g epicatechin (EC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer one.
2. the preparation process of the imprinted polymer of epigallo catechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.9168g epigallo catechin (EGC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer two.
3. the preparation process of the imprinted polymer of L-Epicatechin gallate is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.9168g L-Epicatechin gallate (ECG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer three.
4. the preparation process of the imprinted polymer of epigallocatechin gallate (EGCG) is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 0.9168g epigallocatechin gallate (EGCG) (EGCG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer four.
(5) the high performance liquid chromatography detection imprinted polymer absorbability to catechin is utilized: take 100mg molecularly imprinted polymer one, imprinted polymer two, imprinted polymer three, imprinted polymer four respectively, it is suspended in 3mL methanol respectively, ultrasonic vibration 10min, then it is sequentially loaded in self-control politef solid-phase extraction column (60mm �� 8mmi.d.), the porous ceramic support in extraction column lower end. Extraction column 15mLV (methanol): V (the acetic acid)=1:8 solution loaded cleans, and flow speed control is at 0.1mL/min. Cleaned pillar vacuum drying is standby. Preparation 0.1mg/mL tea polyphenols solution adds in extraction column, flows through with the flow velocity of 0.1mL/min. Upload rear extraction column V (methanol): V (acetic acid)=9:1 eluant solution, detect with HPLC after collecting liquid micro-filtrate membrane filtration, mobile phase is V (methanol): V (acetic acid): V (water)=24.5:0.5:75, flow velocity is 0.8mL/min, sample size is 6 �� L, detects wavelength 278nm.
(6) pilot scale test run: be amplified trial production according to the lab scale craft studied well, adjusts the technological parameter of pilot scale, it is determined that the extraction and purification process of polyphenol in Folium Camelliae sinensis. If Folium Camelliae sinensis total amount is 100g, the total amount of the catechin in eluent is 17g, and the response rate is 17%, and the purity of catechin is 100%.
Embodiment 4
A kind of Folium Camelliae sinensis extracts the novel process of tea polyphenols, comprises the following steps:
(1) break process of Folium Camelliae sinensis: add a certain amount of compound enzyme in Folium Camelliae sinensis and Folium Camelliae sinensis somatic cell is carried out break process; Described compound enzyme includes following components by weight percent: 0.1��0.3% cellulase, 0.1��0.3% hemicellulase, 0.1��0.3% pectase, the hydrolysis temperature 50��55 DEG C of the break process of biological Folium Camelliae sinensis, enzymolysis time 30��45min.
(2) the preliminary extraction of tea polyphenols: utilize ultrasound wave to carry out assisted extraction tea polyphenols the Folium Camelliae sinensis processed in step (1); Adopting ethanol to carry out the preliminary extraction of tea polyphenols, in preliminary extraction process, ultrasonic power is 2800��3200W, time 10��15min, temperature 45��55 DEG C.
(3) preliminary purification of tea polyphenols: adopt molecular cut off at the hollow-fibre membrane of more than 1000Da, by the protein of macromolecule, the polyphenol substance such as the catechin of the material such as nucleic acid and molecular weight is easily separated.
(4) imprinted polymer of molecular engram method synthesis tea polyphenols main component catechin is adopted:
1. the preparation process of the imprinted polymer of epicatechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 1.146g epicatechin (EC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer one.
2. the preparation process of the imprinted polymer of epigallo catechin is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 1.146g epigallo catechin (EGC), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer two.
3. the preparation process of the imprinted polymer of L-Epicatechin gallate is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 1.146g L-Epicatechin gallate (ECG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer three.
4. the preparation process of the imprinted polymer of epigallocatechin gallate (EGCG) is: weigh 1.3774g ��-methacrylic acid, 3.9600g ethylene glycol dimethacrylate, 1.146g epigallocatechin gallate (EGCG) (EGCG), it is subsequently adding the dehydrated alcohol of 2mL and the chloroform of 2mL, in mixed solution, passes into N2Be sealed in after 20min 0 DEG C, 366nm UV illumination penetrate lower polymerization 48h, make polymer reaction complete, obtain solid imprinted polymer; The polymer prepared is pulverized, carries out surname extraction after sieving, again with methanol: the solution of acetic acid (v/v)=9:1 carries out eluting, elution speed 5mL/min, reflux 24h in apparatus,Soxhlet's, removes microsphere, drying for standby, will obtain imprinted polymer four.
(5) the high performance liquid chromatography detection imprinted polymer absorbability to catechin is utilized: take 100mg molecularly imprinted polymer one, imprinted polymer two, imprinted polymer three, imprinted polymer four respectively, it is suspended in 3mL methanol respectively, ultrasonic vibration 10min, then it is sequentially loaded in self-control politef solid-phase extraction column (60mm �� 8mmi.d.), the porous ceramic support in extraction column lower end. Extraction column 15mLV (methanol): V (the acetic acid)=1:8 solution loaded cleans, and flow speed control is at 0.1mL/min. Cleaned pillar vacuum drying is standby. Preparation 0.1mg/mL tea polyphenols solution adds in extraction column, flows through with the flow velocity of 0.1mL/min. Upload rear extraction column 20mL0.01moL/L acetic acid solution eluting, detect with HPLC after collecting liquid micro-filtrate membrane filtration, mobile phase is V (methanol): V (acetic acid): V (water)=24.5:0.5:75, flow velocity is 0.8mL/min, sample size is 6 �� L, detects wavelength 280nm.
(6) pilot scale test run: be amplified trial production according to the lab scale craft studied well, adjusts the technological parameter of pilot scale, it is determined that the extraction and purification process of polyphenol in Folium Camelliae sinensis. If Folium Camelliae sinensis total amount is 100g, the total amount of the catechin in eluent is 17g, and the response rate is 17%, and the response rate is higher, and the purity of catechin is 100%.
Above several embodiments of the present invention are described in detail, but described content has been only presently preferred embodiments of the present invention, it is impossible to be considered the practical range for limiting the present invention. All equalizations made according to the present patent application scope change and improvement etc., all should still belong within the patent covering scope of the present invention.
Claims (6)
1. Folium Camelliae sinensis extracts the novel process of tea polyphenols, it is characterised in that include step in detail below:
(1) break process of Folium Camelliae sinensis: add a certain amount of compound enzyme in Folium Camelliae sinensis and Folium Camelliae sinensis somatic cell is carried out break process;
(2) the preliminary extraction of tea polyphenols: utilize ultrasound wave to carry out assisted extraction tea polyphenols the Folium Camelliae sinensis processed in step (1);
(3) preliminary purification of tea polyphenols: adopt the mode of membrane filtration to carry out the primary purification of tea polyphenols;
(4) imprinted polymer of molecular engram method synthesis tea polyphenols main component catechin is adopted;
(5) the high performance liquid chromatography detection imprinted polymer absorbability to catechin is utilized: molecularly imprinted polymer is filled post, make and prepare post, then elution requirement is studied, it is determined that utilize molecularly imprinted polymer to carry out the optimum process condition of refining Catechin in Tea;
(6) pilot scale test run: be amplified trial production according to the lab scale craft studied well, adjusts the technological parameter of pilot scale, it is determined that the extraction and purification process of polyphenol in Folium Camelliae sinensis.
2. Folium Camelliae sinensis according to claim 1 extracts the novel process of tea polyphenols, it is characterized in that: the compound enzyme described in step (1) includes following components by weight percent: 0.1��0.3% cellulase, 0.1��0.3% hemicellulase, 0.1��0.3% pectase, the hydrolysis temperature 50��55 DEG C of the break process of the biological Folium Camelliae sinensis of step (1), enzymolysis time 30��45min.
3. Folium Camelliae sinensis according to claim 1 extracts the novel process of tea polyphenols, it is characterized in that: step (2) adopts ethanol carry out the preliminary extraction of tea polyphenols, in preliminary extraction process, ultrasonic power is 2800��3200W, time 10��15min, temperature 45��55 DEG C.
4. Folium Camelliae sinensis according to claim 1 extracts the novel process of tea polyphenols, it is characterized in that: what adopt in membrane filtration processes in step (3) is the molecular cut off hollow-fibre membrane at more than 1000Da, by the protein of macromolecule, the polyphenol substance such as the catechin of the material such as nucleic acid and molecular weight is easily separated.
5. Folium Camelliae sinensis according to claim 1 extracts the novel process of tea polyphenols, it is characterized in that: in step (4), the preparation process of the imprinted polymer of catechin is: with ��-methacrylic acid for monomer, ethylene glycol dimethacrylate is cross-linking agent, catechin is template molecule, the imprinted polymer of lower polymerization preparation catechin is penetrated in UV illumination, the polymer prepared is pulverized, after sieving, carries out surname extraction.
6. Folium Camelliae sinensis according to claim 5 extracts the novel process of tea polyphenols, it is characterized in that, in step (4), in the process of surname extraction, eluting solution is: methanol: acetic acid (v/v)=9:1, elution speed 5mL/min, then microsphere, drying for standby are removed.
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| CN105754035A (en) * | 2016-02-16 | 2016-07-13 | 湖南农业大学 | Preparation method of ECG (epigallocatechin gallate) monomer |
| CN106943768A (en) * | 2017-04-01 | 2017-07-14 | 北京国康本草物种生物科学技术研究院有限公司 | Tea Polyphenols and its extracting method and application |
| CN107912567A (en) * | 2017-12-28 | 2018-04-17 | 唐秀廷 | A kind of extracting method of golden camellia tea active material |
| CN108478663A (en) * | 2018-06-27 | 2018-09-04 | 广西克鲁尼茶叶生物科技有限公司 | A kind of technique of the extraction of the high yield pulp1 from fresh tea passes tea polyphenols |
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| CN105754035A (en) * | 2016-02-16 | 2016-07-13 | 湖南农业大学 | Preparation method of ECG (epigallocatechin gallate) monomer |
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| CN106943768A (en) * | 2017-04-01 | 2017-07-14 | 北京国康本草物种生物科学技术研究院有限公司 | Tea Polyphenols and its extracting method and application |
| CN107912567A (en) * | 2017-12-28 | 2018-04-17 | 唐秀廷 | A kind of extracting method of golden camellia tea active material |
| CN108478663A (en) * | 2018-06-27 | 2018-09-04 | 广西克鲁尼茶叶生物科技有限公司 | A kind of technique of the extraction of the high yield pulp1 from fresh tea passes tea polyphenols |
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| CN110585093A (en) * | 2019-11-01 | 2019-12-20 | 信阳学院 | Hand-washing-free disinfection gel containing natural green tea extract and preparation method thereof |
| CN110755459A (en) * | 2019-11-22 | 2020-02-07 | 广西南亚热带农业科学研究所 | Extraction process of annona squamosa leaf polyphenol |
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