CN104998271B - A kind of preparation method of fat-soluble tea polyphenol - Google Patents
A kind of preparation method of fat-soluble tea polyphenol Download PDFInfo
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- CN104998271B CN104998271B CN201510394484.7A CN201510394484A CN104998271B CN 104998271 B CN104998271 B CN 104998271B CN 201510394484 A CN201510394484 A CN 201510394484A CN 104998271 B CN104998271 B CN 104998271B
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Abstract
The present invention provides a kind of preparation method of fat-soluble tea polyphenol, comprise the following steps：(1) tea polyphenols are taken, add ethyl acetate, are uniformly mixed, lipase and protease is added, is eventually adding disodium hydrogen phosphate phosphate sodium dihydrogen buffer solution, obtains reaction solution；(2) above-mentioned reaction solution is subjected to microwave radiation, reacts 10 20min under the conditions of being 35 45 DEG C in temperature；(3) after reaction, add water and remove water-solubility impurity, stratification, takes ethyl acetate layer, vacuum distillation, vacuum freeze drying, to obtain the final product.Relative to the prior art, the present invention takes microwave radiation enzyme coupling and catalyzing technology to prepare fat-soluble tea polyphenol, avoids using the organic solvent for largely having severe toxicity, and greatly shortens the reaction time, substantially increases the fat-soluble of tea polyphenols.
Description
Technical field
The present invention relates to a kind of preparation method of fat-soluble tea polyphenol, belong to tea polyphenols technical field of modification.
Background technology
Solubility of the tea polyphenols in grease is smaller, and shows higher solubility in water, this is greatly limited
Its application in lipid product, especially as the antioxidant of table oil, is difficult to reach effective in grease
Anti-oxidant concentration, inoxidizability can get a desired effect.Also, under physiological environment, the stability of tea polyphenols is poor,
The concentration that can be efficiently used is low, and causing it, bioavilability is not high in vivo, these all greatly limit tea polyphenols as natural
Antioxidant is in food, the latent application of pharmaceuticals industry.
The research that related tea polyphenols are modified at present mainly includes physical method and utilizes biology enzyme or point of chemical synthesis
Sub- method of modifying.The essence that physical method is different from molecular modification is that physical method does not change its molecular structure, merely with
Carrier mass increases stability and dissolubility of the tea polyphenols in grease by emulsification method, to improve its inoxidizability
Deng physiological activity.The fat-soluble tea polyphenol that such a method is modified is not also very stable, will also result in the moisture of vegetable oil and can wave
The parameter indexes such as hair property content, transparency, viscosity are unqualified.Molecular modification is the main path for transforming tea polyphenols, at present synthesis
The main method of fat-soluble tea polyphenol is the carbon of oxygen atom or phenyl ring that oil-soluble aliphatic hydrocarbon is incorporated into tea polyphenols polyhydroxy
Atom gets on.This method uses fatty acid chloride or acid anhydrides as acylating agent at present.Currently used fat acyl chloride is generally by fat
The reagent reacting such as acid and phosphorus trichloride, thionyl chloride or phosphorus pentachloride, but phosphorus trichloride, phosphorus pentachloride and thionyl chloride
All it is toxic articles, is particularly phosphorus trichloride, it is inflammable, explosive, belongs to the reagent of hypertoxic level, these reagents are applied to acylating agent
Prepare, obtained fat-soluble tea polyphenol is applied to food service industry, its harm to human body is unclear.Acyl chlorides general at the same time
It is more volatile, white cigarette is easily emitted, how to ensure the security in industrial production is also a problem urgently to be resolved hurrily.This is this method
An existing big drawback.In addition, reaction be strong acid medium, although and on phenyl ring the substitution of hydrogen atom avoid phenolic hydroxyl group
Destroy, but its reagent used such as prepares fat-soluble tea polyphenol with Friedel-Crafts acylation reactions, and most-often used is molten
Agent is nitrobenzene, and nitrobenzene meets high fever and can burn or even explode, and is a kind of raw material for making explosive, is also not appropriate for being added to
In food.Its problem of solvent residual will hinder application of the fat-soluble tea polyphenol in the industries such as food.In addition, the introducing of Long carbon chain
The shielding action produced to phenolic hydroxyl group around increases its steric hindrance, while Long carbon chain can also cause to assemble, and not reach solubilising
Effect.
Enzymatic modification is occurred mainly on 3 hydroxyls of non-ester catechin molecule, and B rings and D rings in EGCG molecules
Hydroxyl on.Enzyme reaction mild condition, regioselectivity is high, is not required to radical protection, and reactions steps are few, environmental-friendly, reacts bar
Part is simply controllable, and the operation of product downstream separation is relatively easy.Enzyme process molecular modification has high selectivity at the same time.So enzyme process point
Son modification is to prepare one of most potential method of fat-soluble tea polyphenol.
But enzyme modification still suffers from some defects at present, the enzyme cost as enzymolysis efficiency is low, uses is higher, during reaction
Between it is long, up to 8-24h, to improve reaction effect organic solvent, the usage amounts such as excessive acetone, chloroform need to be used often to surpass
Original more than 500 times are crossed, and are all hypertoxic solvents.
The content of the invention
Goal of the invention：In order to overcome the deficiencies in the prior art, the present invention provides a kind of fat-soluble tea polyphenol
Preparation method.
Technical solution：To achieve the above object, the present invention provides a kind of preparation method of fat-soluble tea polyphenol, including following
Step：
(1) tea polyphenols are taken, add ethyl acetate, are uniformly mixed, lipase and protease is added, is eventually adding phosphoric acid hydrogen two
Sodium-phosphate sodium dihydrogen buffer solution, obtains reaction solution；
(2) above-mentioned reaction solution is subjected to microwave radiation, reacts 10-20min under the conditions of being 35-45 DEG C in temperature；
(3) after reaction, add water and remove water-solubility impurity, stratification, takes ethyl acetate layer, vacuum distillation, very
Vacuum freecing-dry, to obtain the final product.
Preferably, in the step (1), ethyl acetate is food level ethyl acetate, its addition is every gram of tea polyphenols
Add ethyl acetate 300-400ml.
As another preferred embodiment, in the step (1), the gross mass of the lipase and protease and the quality of tea polyphenols
Than for (0.0625-0.1875)：1, wherein lipase quality accounts for the 20-50% of the gross mass.
As another preferred embodiment, in the step (1), the addition of disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution is every
Gram tea polyphenols add buffer solution 0.25-1.25ml, its pH is 6.5-8.0.
As another preferred embodiment, in the step (2), the power of microwave is 300W-700W.
As another preferred embodiment, in the step (3), the temperature of vacuum distillation is 50 DEG C.
Fat-soluble tea polyphenol is prepared using combined-enzyme method in the present invention, the acetyl group of short chain is introduced in tea polyphenols structure,
And product is fat-soluble significantly better than using single enzyme.In preparation process, acylation is used as using excessive food level ethyl acetate
Agent also serves as solvent, is heated using microwave radiation and is applied to enzymic catalytic reaction instead of traditional mode of heating, that is, takes microwave spoke
Penetrate-enzyme coupling and catalyzing technology prepares fat-soluble tea polyphenol, use organic solvent without extra, and greatly shorten when reacting
Between.
Beneficial effect：Relative to the prior art, preparation method of the present invention has the advantage that：
(1) compared with traditional chemical method, avoid having used a large amount of poisonous and harmful, be not suitable for the chemistry of food service industry
Reagent, preferably resolves reagent residue problem.
(2) fat-soluble tea polyphenol is prepared using microwave radiation-enzyme coupling and catalyzing technology, liposoluble is prepared with traditional enzyme process
Property tea polyphenols are compared, by the reaction time by 8-24 it is small when shorten to 10-20 minutes.
(3) lipase-protease composite enzyme formula is used, product is fat-soluble to be greatly improved.
Embodiment
The present invention is further described with reference to embodiment.
Embodiment 1
(1) tea polyphenols are taken, add ethyl acetate, are uniformly mixed, lipase and protease is added, is eventually adding phosphoric acid hydrogen two
Sodium-phosphate sodium dihydrogen buffer solution, obtains reaction solution；
(2) above-mentioned reaction solution is subjected to microwave radiation, reacts 10min under the conditions of being 35 DEG C in temperature；
(3) after reaction, add water and remove water-solubility impurity, stratification, takes ethyl acetate layer, vacuum distillation, very
Vacuum freecing-dry, to obtain the final product.
In step (1), ethyl acetate is food level ethyl acetate, its addition adds ethyl acetate for every gram of tea polyphenols
300ml。
In step (1), the gross mass of the lipase and protease and the mass ratio of tea polyphenols are 0.0625：1, wherein fat
Fat enzyme quality accounts for the 20% of the gross mass.
In step (1), the addition of disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution adds buffer solution for every gram of tea polyphenols
0.25ml, its pH are 6.5.
In step (2), the power of microwave is 300W.
In step (3), the temperature of vacuum distillation is 50 DEG C.
Embodiment 2
(1) tea polyphenols are taken, add ethyl acetate, are uniformly mixed, lipase and protease is added, is eventually adding phosphoric acid hydrogen two
Sodium-phosphate sodium dihydrogen buffer solution, obtains reaction solution；
(2) above-mentioned reaction solution is subjected to microwave radiation, reacts 20min under the conditions of being 45 DEG C in temperature；
(3) after reaction, add water and remove water-solubility impurity, stratification, takes ethyl acetate layer, vacuum distillation, very
Vacuum freecing-dry, to obtain the final product.
In step (1), ethyl acetate is food level ethyl acetate, its addition adds ethyl acetate for every gram of tea polyphenols
400ml。
In step (1), the gross mass of the lipase and protease and the mass ratio of tea polyphenols are 0.1875：1, wherein fat
Fat enzyme quality accounts for the 50% of the gross mass.
In step (1), the addition of disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution adds buffer solution for every gram of tea polyphenols
1.25ml, its pH are 8.0.
In step (2), the power of microwave is 700W.
In step (3), the temperature of vacuum distillation is 50 DEG C.
Embodiment 3
(1) tea polyphenols are taken, add ethyl acetate, are uniformly mixed, lipase and protease is added, is eventually adding phosphoric acid hydrogen two
Sodium-phosphate sodium dihydrogen buffer solution, obtains reaction solution；
(2) above-mentioned reaction solution is subjected to microwave radiation, reacts 15min under the conditions of being 40 DEG C in temperature；
(3) after reaction, add water and remove water-solubility impurity, stratification, takes ethyl acetate layer, vacuum distillation, very
Vacuum freecing-dry, to obtain the final product.
In step (1), ethyl acetate is food level ethyl acetate, its addition adds ethyl acetate for every gram of tea polyphenols
350ml。
In step (1), the gross mass of the lipase and protease and the mass ratio of tea polyphenols are 0.125：1, wherein fat
Fat enzyme quality accounts for the 35% of the gross mass.
In step (1), the addition of disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution adds buffer solution for every gram of tea polyphenols
0.75ml, its pH are 7.5.
In step (2), the power of microwave is 500W.
In step (3), the temperature of vacuum distillation is 50 DEG C.
Embodiment 4
(1) tea polyphenols are taken, add ethyl acetate, are uniformly mixed, lipase and protease is added, is eventually adding phosphoric acid hydrogen two
Sodium-phosphate sodium dihydrogen buffer solution, obtains reaction solution；
(2) above-mentioned reaction solution is subjected to microwave radiation, reacts 12min under the conditions of being 38 DEG C in temperature；
(3) after reaction, add water and remove water-solubility impurity, stratification, takes ethyl acetate layer, vacuum distillation, very
Vacuum freecing-dry, to obtain the final product.
In step (1), ethyl acetate is food level ethyl acetate, its addition adds ethyl acetate for every gram of tea polyphenols
320ml。
In step (1), the gross mass of the lipase and protease and the mass ratio of tea polyphenols are 0.1：1, wherein fat
Enzyme quality accounts for the 28% of the gross mass.
In step (1), the addition of disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution adds buffer solution for every gram of tea polyphenols
0.5ml, its pH are 7.
In step (2), the power of microwave is 400W.
In step (3), the temperature of vacuum distillation is 50 DEG C.
Embodiment 5
(1) tea polyphenols are taken, add ethyl acetate, are uniformly mixed, lipase and protease is added, is eventually adding phosphoric acid hydrogen two
Sodium-phosphate sodium dihydrogen buffer solution, obtains reaction solution；
(2) above-mentioned reaction solution is subjected to microwave radiation, reacts 18min under the conditions of being 42 DEG C in temperature；
(3) after reaction, add water and remove water-solubility impurity, stratification, takes ethyl acetate layer, vacuum distillation, very
Vacuum freecing-dry, to obtain the final product.
In step (1), ethyl acetate is food level ethyl acetate, its addition adds ethyl acetate for every gram of tea polyphenols
320ml。
In step (1), the gross mass of the lipase and protease and the mass ratio of tea polyphenols are 0.15：1, wherein fat
Enzyme quality accounts for the 45% of the gross mass.
In step (1), the addition of disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution adds buffer solution for every gram of tea polyphenols
1ml, its pH are 7.5.
In step (2), the power of microwave is 600W.
In step (3), the temperature of vacuum distillation is 50 DEG C.
Experimental example tea polyphenol liposoluble measures
The dried products of 10mg are weighed, are dissolved in 10mL chloroforms, ultrasonic 10min makes its dissolving, and it is 1g/L to be made into concentration
Solution or suspension, measure solution or suspension with ultraviolet specrophotometer after concussion mixes on earthquake device again before survey and exist
Light transmittance under 800nm.
Yield=product actual production * 100%/raw material inventorys
Blank group is without modified tea polyphenols；
It is according to 3 preparation method of embodiment and raw material proportioning to compare 1 group, and lipase and protease are replaced with and its total matter
Measure identical lipase, obtained fat-soluble tea polyphenol；
It is according to 3 preparation method of embodiment and raw material proportioning to compare 2 groups, and lipase and protease are replaced with and its total matter
Measure identical protease, obtained fat-soluble tea polyphenol；
Embodiment 3,4,5 groups be respectively fat-soluble tea polyphenol obtained by the embodiment of the present invention 3,4 and 5.
Above each group products obtained therefrom is taken, it is fat-soluble according to said determination method measure product, it the results are shown in Table 1.
Fat-soluble raising multiplying power, refers to multiple of the light transmittance relative to blank group light transmittance.
The 1 fat-soluble measurement result of each set product of table
It can be obtained by 1 result of table, compare blank group, and the light transmittance of the embodiment of the present invention 3,4 and 5 gained tea polyphenols significantly carries
Height, fat-soluble raising multiplying power respectively reaches 3.4 times, 3.2 times and 3.3 times, fat-soluble to greatly improve；
For compareing 1 group and 2 groups of control group, that is, be used alone lipase or protease be catalyzed obtained by tea polyphenols,
Its fat-soluble raising multiplying power is only 1.5 times and 1.9 times, more than 3 times far below the present invention respectively, shows lipase and albumen
The complex enzyme catalytic effect of enzyme is significantly more preferably.
Claims (3)
1. a kind of preparation method of fat-soluble tea polyphenol, it is characterised in that comprise the following steps：
(1) take tea polyphenols, add ethyl acetate, be uniformly mixed, add lipase and protease, be eventually adding disodium hydrogen phosphate-
Phosphate sodium dihydrogen buffer solution, obtains reaction solution；In the step (1), ethyl acetate is food level ethyl acetate, its addition is every
Gram tea polyphenols add ethyl acetate 300-400ml；The gross mass of the lipase and protease and the mass ratio of tea polyphenols are
(0.0625-0.1875)：1, wherein lipase quality accounts for the 20-50% of the gross mass；
(2) above-mentioned reaction solution is subjected to microwave radiation, power 300W-700W, reacts 10- under the conditions of being 35-45 DEG C in temperature
20min；
(3) after reaction, add water and remove water-solubility impurity, stratification, takes ethyl acetate layer, vacuum distillation, and vacuum is cold
It is lyophilized dry, to obtain the final product.
2. the preparation method of fat-soluble tea polyphenol according to claim 1, it is characterised in that in the step (1), phosphoric acid
The addition of disodium hydrogen-phosphate sodium dihydrogen buffer solution adds buffer solution 0.25-1.25ml for every gram of tea polyphenols, its pH is 6.5-
8.0。
3. the preparation method of fat-soluble tea polyphenol according to claim 1, it is characterised in that in the step (3), decompression
The temperature of distillation is 50 DEG C.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747117A (en) * | 2012-07-28 | 2012-10-24 | 江南大学 | Method for synthetizing liposoluble tea polyphenols by enzymic method |
| CN102787146A (en) * | 2012-07-28 | 2012-11-21 | 江南大学 | Method utilizing microwave assisted lipase catalysis to synthesize esterification modified EGCG |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747117A (en) * | 2012-07-28 | 2012-10-24 | 江南大学 | Method for synthetizing liposoluble tea polyphenols by enzymic method |
| CN102787146A (en) * | 2012-07-28 | 2012-11-21 | 江南大学 | Method utilizing microwave assisted lipase catalysis to synthesize esterification modified EGCG |
Non-Patent Citations (1)
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| "茶多酚—蛋白质之间的络合及沉淀回收研究";黄惠华等;《食品科学》;20021231;第23卷(第1期);第26-30页 * |
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