CN104926680B - The method extracting bioactive substance from low-grade tea and tea making leftover bits and pieces - Google Patents
The method extracting bioactive substance from low-grade tea and tea making leftover bits and pieces Download PDFInfo
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Abstract
The present invention extracts the method for bioactive substance from low-grade tea and tea making leftover bits and pieces, comprises the following steps: 1) from raw material, extract caffeine；2) tea polyphenols is extracted；3) tea polysaccharide is extracted；4) theanine and γ mono-aminobutyric acid are extracted；5) extract and raffinate albumen feedstuff are extracted；6) extract separates, obtains high-purity lutein ester and chlorophyll.The inventive method low cost, environmental protection, it is suitable for large-scale production.
Description
Technical field:
The present invention relates to a kind of low-grade tea and the green method of tea making leftover bits and pieces high-efficiency comprehensive utilization, particularly to be
A kind of method extracting bioactive substance from low-grade tea.
Background technology:
Folium Camelliae sinensis is as the Traditional health care beverage of the Chinese nation, the history of existing 4000 or five thousand years.Since reform and opening-up, at tea
While leaf industry fast development, there is easy situation in traditional tea products, and outstanding day by day, has several every year
Measuring unsalable low-grade tea (spring tea, summer tea) and substantial amounts of tea making leftover bits and pieces goes out of use, the new application of anxious Folium Camelliae sinensis to be developed is with comprehensive
Close the green technology utilized, it is impossible to have the huge waste of resource.
Multi-disciplinary intersection carries out the result of numerous studies it has proven convenient that in low-grade tea and tea making leftover bits and pieces with infiltration,
All contain abundant tea polysaccharide, tea polyphenols, theanine, gamma aminobutyric acid, chlorophyll, lutein ester, caffeine etc. and have bright
The bioactive substance of aobvious medicine healthy sofa function, develops the green method of a kind of high-efficiency comprehensive utilization, these biological activitys
Material all extracts and separates, it is achieved the comprehensive complete utilization of raw material, not only can produce significant economic benefit, social benefit and ring
Border benefit, additionally aids the sustainable development of Tea Industry.
At present, the domestic extraction separation method about bioactive substances such as tea polyphenols in Folium Camelliae sinensis and tea making leftover bits and pieces is special
Profit application, has CN1344748A, CN1379044A, CN1431226A, CN1176748A, in these patents: one is most patents
Application is all only extracted from raw material and has been separated a kind of, two kinds, three kinds of bioactive substances, and comprehensive utilization degree is very poor；Two be
Extract in separation process and all employ the big chloroform of toxicity, all employ and be easily decomposed to acetic acid and second in operation
The ethyl acetate of alcohol, exists and pollutes environment and the big problem of solvent consumption；Three is the relevant auxiliary of use in extracting separation process
The degree of material recycled is the most very poor；Four be the most do not use can significantly energy saving, product quality is guaranteed, meet
The leading complete set of equipments that GMP requires, such as low temperature subcritical abstraction equipment, membrane separation concentration equipment, economic benefits and social benefits falling liquid film
Vaporizer, vacuum and low temperature microwave dryer etc..Separately there is CN1557841A, although extract and isolated tea polysaccharide, tea polyphenols and coffee
Coffee alkali, but the degree or poor of comprehensive utilization, and in adjuvant, still used chloroform that toxicity is big and hold labile acetic acid
Ethyl ester.
Summary of the invention:
For above-mentioned deficiency, the invention aims to provide a kind of low cost, environmental protection, be suitable for large-scale production
The method extracting bioactive substance from low-grade tea and tea making leftover bits and pieces.
The object of the present invention is achieved like this:
The present invention extracts the method for bioactive substance from low-grade tea and tea making leftover bits and pieces, comprises the following steps:
1) raw material powder being broken into 20～40 mesh coarse powder, adding weight is coarse powder weight 8～the water of 12 times, in pH value 5～6,70
～90 DEG C extracted and separate, obtain aqueous extract and wet feed slag, aqueous extract, inhale with 732 large hole cation exchanger resins
Attached with separate, obtain caffeine and by 732 large hole cation exchanger resins absorption aqueous solution；
2) not by the aqueous solution of 732 large hole cation exchanger resin absorption, after adjusting pH value to be 3～3.5, warp in step 1)
3500Da membrane ultrafiltration and centrifugal filtration, obtain filtrate and solid content, and filtrate is adsorbed with macroporous adsorbent resin and separates, and obtains
Tea polyphenols and not by the acidic aqueous solution of absorption with macroporous adsorbent resin；
3) not by the acidic aqueous solution of absorption with macroporous adsorbent resin, through vaccum cryogenic distillation, distilled water and concentrated solution are obtained,
Concentrated solution through precipitate with ethanol with separate, obtain tea polysaccharide and ethanol water；
4), after reclaiming ethanol from ethanol water, adsorb with 732 large hole cation exchanger resins and separate, obtaining tea ammonia
Acid and an ammonia tea butanoic acid；
5) the wet feed slag obtained in step 1) and step 2) in solid content merge, use subcritical abstraction equipment, use
Extractant carries out low-temperature extraction, is extracted thing and raffinate (albumen feedstuff, dry product)；
6) extract obtained in step 5), uses lead oxide column chromatography for separation, obtains high-purity lutein ester (orange/yellow solid
Powder) and chlorophyll (dark green solid divides end).
The present invention extracts the method for bioactive substance from low-grade tea and tea making leftover bits and pieces, specifically includes following step
Rapid:
1) raw material (low-grade tea and tea making leftover bits and pieces) being ground into 20 40 mesh coarse powder, adding weight is coarse powder 6～8 times of weights
Amount water (tap water, distilled water), start in extraction vessel stirring under heat temperature raising, add simultaneously concentration expressed in percentage by weight be 25～
The phosphate aqueous solution of 35% adjusts pH value 5～6, container inside holding 70～90 DEG C of stirrings to extract 1.5～2 hours, stops heating and stirs
Mixing, suction strainer goes out an aqueous extract, and separately adding weight in container is coarse powder 6～the water of 8 times of weight, repeats same operation discharging
Filter, obtains the limpid of wet feed slag and twice merging, the aqueous extract of pH5～6.
Aqueous extract, uses 732 large hole cation exchanger resins being processed into H type in advance, carries out under room temperature low rate mixing
Absorption；The consumption of 732 large hole cation exchanger resins, according to the content of caffeine in aqueous extract (CAF) and 732 macropores sun from
The exchange capacity of sub-exchange resin, after calculating, excess 10～15%, adsorbs 1～2 hour, quickly detects by non-aqueous titration
When reaching to adsorb in terminal ie in solution almost without caffeine (CAF), stopping stirring, discharging is filtered, and obtains being adsorbed with caffeine
(CAF) 732 large hole cation exchanger resins and by the aqueous solution of 732 large hole cation exchanger resins absorption；
Be adsorbed with caffeine (CAF) 732 large hole cation exchanger resins, with ethanol (such as 95% ethanol, dehydrated alcohol) in
Carrying out eluting under 60～65 DEG C of low rate mixings, ethanol consumption is 2 times of volumes of 732 large hole cation exchanger resin volumes, 1～2
After hour, stopping heating and stirring, discharging is filtered, is obtained 732 large hole cation exchanger resins and ethanol elution, ethanol elution
Liquid with dry, obtains ethanol, distillate and caffeine (CAF) in 60～70 DEG C of vacuum distillinges；
2) aqueous solution that will not adsorbed by 732 large hole cation exchanger resins in step 1), uses weight under stirring at normal temperature
Percentage concentration be 25～35% phosphate aqueous solution adjust pH3 3.5, through 3500Da electronegative membrane ultrafiltration, obtain film and pass through
Liquid and trapped fluid, trapped fluid machine by centrifugation is filtrated to get filtrate (merging with film permeate) and solid content (for albumen feedstuff
In)；
Film permeate containing filtrate, under room temperature low rate mixing, adds macroporous adsorbent resin and adsorbs, macroporous absorption tree
The consumption of fat is 2～2.5 times of weight the excess 10～15% of tea polyphenols (TP) content, adsorbs 1～2 hour, takes solution and see almost
For absorption terminal time colourless, stop drawing material and filter, appropriate distilled water wash macroporous adsorbent resin 1～2 times, obtain being adsorbed with tea
The macroporous adsorbent resin of polyphenol (TP) and not by the acidic aqueous solution containing wash water of absorption with macroporous adsorbent resin；
The macroporous adsorbent resin being adsorbed with tea polyphenols (TP) is added in ethanol, washes under 40～50 DEG C of low rate mixings
De-, ethanol consumption is advisable can mix material under low rate mixing, stops stirring after 1～1.5 hour, and suction strainer goes out an eluent,
Separately add ethanol and repeat same operation 1～2 time, until eluent is almost colourless, obtains macroporous adsorbent resin and merge several times
Ethanol elution；
Ethanol elution reverse osmosis membrane separation and filtration, obtain ethanol film permeate and trapped fluid；Trapped fluid is through low temperature
Vacuum drying, obtains ethanol distillate and tea polyphenols (TP)；
3) step 2) in not by pH3 4 acidic aqueous solution of absorption with macroporous adsorbent resin, in room temperature 60～70 DEG C of fine vacuum
Distillation and concentration, obtains distilled water distillate and concentrated solution, and concentrated solution volume is concentrated solution volume 2～the ethanol of 3 times, in room temperature
Stirring and evenly mixing, stand, filter separation, obtain filter cake and ethanol filtrate.Filter cake obtains ethanol in 60～70 DEG C of vacuum drying and distillates
Liquid and tea polysaccharide (TPS)；
4) ethanol filtrate obtained in step 3), first reclaims ethanol, divides with 732 large hole cation exchanger resin column chromatographies afterwards
From, obtain theanine (TAA) and gamma aminobutyric acid (GABA)；
5) the wet feed slag obtained in step 1), after pelletize or tabletting, with extractant in 40～50 DEG C, 0.6～
0.7MPa carries out low temperature subcritical abstraction and separates, and obtains solid content and liquid, the two respectively while row pressure to normal pressure, its
In extractant be evaporated into gas, compressed and liquefied again and apply mechanically, and then obtain albumen feedstuff raffinate and extract (pulpous state
Thing)；
6) extract obtained in step 5), separates through 40～50 DEG C of distilled water washs, obtains water-fast pastel
With wash water (add in the water extraction aqueous solution of step 1 and apply mechanically), water-fast pastel, the volume by its weight 3 times is anhydrous
Ethanol and petroleum ether mixture, after stirring at normal temperature 0.5～1 hour, separately add dehydrant (such as anhydrous sodium sulfate), continue stirring 10～
20 minutes, stop stirring, stand 1～2 hour, first sucking-off upper liquid, filters lower floor afterwards, obtains solid content and filtrate (containing upper strata
Liquid), after solid content such as then washes 1～2 time (washing liquid merges) with filtrate with a small amount of dehydrated alcohol in blue green, drying obtains anhydrous
Sodium sulfate (is applied mechanically)；
Filtrate separates with neutral alumina column: pillar Φ H=1 20；Alumina powder is 100～200 mesh；Oxygen in post
Change aluminium powder and use petroleum ether moistening in advance；On filtrate after complete post, first use petroleum ether eluting, until eluent is colourless, contained
The petroleum ether eluent of a small amount of ethanol；Again with ethanol elution, until eluent is colourless, obtain ethanol elution and oxidation
Aluminium powder chromatographic column (is applied mechanically)；
Petroleum ether eluent merges with the post liquid of crossing during upper prop, obtains distillate (containing a small amount of in 60～70 DEG C of vacuum distillinges
The petroleum ether of ethanol, applies mechanically) and concentrated solution, this concentrated solution is through being recrystallized to give lutein ester (orange/yellow solid powder)；
Ethanol elution with dry, obtains ethanol distillate and chlorophyll (dark green solid in 60～70 DEG C of vacuum distillinges
Powder).
Above-mentioned macroporous adsorbent resin uses D101 macroporous adsorbent resin or AB 8 macroporous adsorbent resin, and extractant uses
Dimethyl ether or normal butane or propane, the boiling point of petroleum ether is 60～90 DEG C, ethanol that ethanol uses concentration expressed in percentage by volume to be 95% or
Dehydrated alcohol.
The invention have the advantages that
1, technological process is short and easy, and has isolated caffeine, tea polyphenols, tea polysaccharide, theanine, γ from raw material
Aminobutyric acid, albumen feedstuff, chlorophyll, lutein ester, it is achieved that the comprehensive complete utilization of raw material and the recycled of adjuvant, success
Got rid of the big chloroform of adjuvant toxic and used during be easily decomposed into the ethyl acetate of acetic acid and ethanol.
2, the low temperature subcritical abstraction equipment of selection, membrane separation concentration machine, double-effect falling film evaporator, vacuum in technological process
Low-temperature microwave drying machine, is and advanced both at home and abroad meets the complete set of equipments that GMP requires, the most energy-conservation, product quality is guaranteed,
Production capacity is big, and the also recovery rate to DME equal solvent can rise to 99%, also can the environmental friendliness of reinforcement process flow process.Continue
After complete again scale-up.Low cost, high efficiency, environmental protection, the beneficial effect of applicable large-scale production are obtained.
Detailed description of the invention:
Embodiment 1:
The present embodiment 1 extracts the method for bioactive substance from low-grade tea, comprises the following steps:
1) weigh low-grade tea 500 grams, be ground into 20 mesh, add in 5 liters of extraction vessels, add tap water 3000ML, at water
Begin to warm up intensification under stirring in bath, adjust pH5.0, when container contents temperature reaches with concentration about 30% phosphate aqueous solution simultaneously
When 70 DEG C, being incubated 70～75 DEG C of stirrings and extract 1.5 hours, stop heating and stirring, suction strainer goes out an aqueous extract.In container
Separately adding 3 liters of tap water, repeat same operation, discharging is filtrated to get second time aqueous extract and wet feed slag (extruding through big glass plug)
731 grams.The aqueous extract merging twice has 5583 grams, and pH is 5.0；
Aqueous extract, under room temperature and low rate mixing, adds 732 large hole cation exchanger resins being processed into H type in advance
Carry out adsorbing (732 large hole cation exchanger resin consumptions 230 grams, excess 15% in amount of calculation), with non-after 0.5 hour
Water titration (method) quickly detects, and also has CAF in solution, continues stirring, crosses again and within 0.5 hour, arrive terminal after testing, stops stirring discharging
Sucking filtration, obtains being adsorbed with 732 large hole cation exchanger resins of CAF and not by the water of 732 large hole cation exchanger resin absorption
Solution 5480 grams.
It is adsorbed with 732 large hole cation exchanger resins of CAF, is added in 95% ethanol under stirring (95% ethanol 300 milli
Rise, material can be made to mix), it is incubated 60 DEG C of low rate mixing eluting 1 hour, suction strainer goes out an eluent.Separately add 95% ethanol 300 milli
Rising, repeat same operation, reach eluting terminal after 0.5 hour, stop heating and filter with stirring discharging, tree washed by a small amount of 95% ethanol
Fat once, obtains 732 large hole cation exchanger resins (applying mechanically) and merges the ethanol elution (containing cleaning mixture) of twice.Ethanol is washed
De-liquid, in 60 DEG C of vacuum distillinges with dry, obtains distillate (ethanol is applied mechanically) and caffeine (CAF) 5.1 grams, and yellowish white is solid
Body powder；Purity 88%, GB8312 87 method)；
2) not by the aqueous solution 5480 grams of 732 large hole cation exchanger resin absorption in step 1), use under stirring at normal temperature
The phosphate aqueous solution of concentration expressed in percentage by weight about 30% adjusts pH value 3～3.5, with membrane ultrafiltration machine electronegative for 3500Da separate with
Concentrate, obtain film permeate and trapped fluid.Trapped fluid obtains filter cake and filtrate (merging with film permeate) through sucking filtration.Filter cake contains
Vegetable protein, plant gum, tannin, cellulose etc., merge with albumen feedstuff after drying；
Film permeate (containing filtrate) 5380 grams, under room temperature and low rate mixing, adds D101 macroporous adsorbent resin 150 grams and carries out
Absorption: D101 large hole cation exchanger resin consumption is 2～2.5 times of weight the excess 15% of TP content；TP content is used
GB8313 2002 method measures；Take solution after 1 hour and be seen as faint yellow；After 0.5 hour, take solution the most colourless, stop stirring
Mix discharging sucking filtration, and with distilled water wash 2 times, obtain being adsorbed with the D101 resin of TP and not by the acid water of D101 resin absorption
Solution (containing wash water, pH3.5) 4630 grams；
Be adsorbed with the D101 resin of TP, with 95% ethanol in 40～～45 DEG C of low rate mixings under carry out eluting (95% ethanol use
Measure and be advisable so that material can be mixed under low rate mixing), be incubated 40～45 DEG C after 0.5 hour and stop stirring, suction filter liquor after 15 minutes
Body, separately adds 95% ethanol and repeats same operation 1～2 time, until eluent is almost colourless, stops heating discharging and filters, obtain
D101 resin (applying mechanically) and the ethanol elution merged several times；
Ethanol elution, with reverse osmosis membrane separation and concentration, obtains film permeate (ethanol is applied mechanically) and trapped fluid.Retain
Liquid with dry, obtains distillate (ethanol is applied mechanically) and tea polyphenols (TP) 56 grams, orange/yellow solid powder in 60 DEG C of 0.095mPa distillations
End, ECG.EGCG.GCG, 3 kinds of catechin contents are 93.26%, type HPLC method)；
3) step 2) in not by the acidic aqueous solution (pH3.5) 4630 grams of D101 resin absorption, use glass rotary evaporation
Device, in 70 DEG C of 0.095mPa distillation and concentrations, obtains distillate (distilled water is applied mechanically) and concentrated solution (608 grams)；
Concentrated solution is added in 1200 milliliters of dehydrated alcohol under stirring, stops stirring, stand 1.5 in room temperature little after 10 minutes
Time, first sucking-off upper liquid, lower floor's sucking filtration, obtain ethanol filtrate (containing the supernatant) and filter cake.Filter cake dehydrated alcohol proceeds to steam
Evaporate in bottle, in 60 DEG C of 0.095mPa rotary distillations with dry, obtain distillate (ethanol is applied mechanically) and tea polysaccharide (TPS) 1.6 grams,
Pale solid powder, total sugar content 60.1%, Phenol sulfuric acid procedure；
4) ethanol filtrate obtained in step 3), the concentrated solution obtained after 60 DEG C of 0.095mPa Distillation recovery ethanol, directly
Connect the 732 resin chromatography posts being processed into H type in advance, with PH8～9 phosphate buffer gradient elutions, be collected in multiple container
In, TLC method detect, merge TAA one-component, GABA one-component, the eluent of TAA and GABA intersection component respectively；
TAA one-component, distills through 60～70 DEG C of 0.095mPa and is concentrated to give distillate (distilled water is applied mechanically) and paste
Thing, the latter is recrystallized to give theanine (TAA) sterling 3.8 grams, white crystalline powder, purity 96.3%, HPLC through dehydrated alcohol
Method；
GABA one-component, by TAA one-component same method, obtains gamma aminobutyric acid (GABA) sterling 0.9 gram, in vain
Color crystalline powder, purity 95.6%, HPLC method；
TAA with GABA intersects component, under apply mechanically when pulling on post.Post liquid (pH3.5 water liquid) of crossing during upper prop enters for step 1)
Apply mechanically during row water extraction；
5) the 731 grams of wet feed slags obtained in step 1), are carried out in 40～50 DEG C of 0.7mPa with DME extractant after pelletize
Extraction with isolated solid content and liquid, the two respectively while row pressure to normal pressure, DME therein be evaporated into gas,
Compressed and liquefied again and applied mechanically, obtain raffinate (i.e. albumen feedstuff, ivory buff particulate, 346 grams) and extract (yellow green,
380 grams)；
6) in the extract obtained in step 5), adding 200 grams of 50 DEG C of distilled water, low rate mixing mixes 5 minutes, in 45～50
DEG C insulation stratification, about 1 hour release lower layer of water (add in the aqueous extract of lower batch and apply mechanically)；
The water-fast pastel in upper strata has 63 grams to be added to 180 milliliters of intermixtures (containing the petroleum ether of a small amount of dehydrated alcohol)
In, dissolve 0.5 hour under stirring at normal temperature, separately add dehydrant (anhydrous sodium sulfate) (addition becomes limpid being advisable with solution), continue
Continuous stirring stopped stirring after 0.5 hour, stood about 2 hours, sucking-off upper strata, and lower floor filters, obtains solid content and filtrate.Solid
Thing is if any color, and after washing 1～2 time (washing liquid merges) with filtrate with dehydrated alcohol, drying obtains anhydrous sodium sulfate (white solid
Powder, applies mechanically)；
Upper liquid, filtrate, washing liquid merge, separate with neutral alumina powder chromatographic column: neutral alumina aluminium powder 100～
200 mesh, consumption be 200～300 grams (being 50 times of chlorophyll content), in advance uses petroleum ether moistening, glass column Φ: H is 1:20；
First with BP60～90 DEG C of petroleum ether eluting after upper prop is complete, until eluent is almost colourless；Then dehydrated alcohol eluting is used, directly
To eluent almost colourless.Obtain alumina powder chromatographic column (applying mechanically), ethanol elution, cross post liquid and petroleum ether eluent
Amalgamation liquid；
Ethanol elution, concentrates by reverse osmosis membrane separation, obtains film permeate (ethanol is applied mechanically) and trapped fluid.Trapped fluid
Distillate (ethanol is applied mechanically) and 4.2 grams of chlorophylls (dark green solid powder, purity 98%, ultraviolets is obtained in 60 DEG C of vacuum drying
Visible spectrophotometry)；
Amalgamation liquid obtains distillate (containing the petroleum ether of a small amount of ethanol, apply mechanically) and concentrated solution in 60 DEG C of 0.095mPa distillations.
Concentrated solution is through being recrystallized to give 0.8 gram of lutein ester (orange/yellow solid powder, purity 81.3%, UV-VIS spectrophotometry).
Embodiment 2:
The present embodiment 2 extracts the method for bioactive substance from low-grade green tea, except low-grade green tea 500 grams changes green tea dust into
Outside 500 grams, other conditions are same as in Example 1.The caffeine of extracted isolated have 3.9 grams, tea polyphenols 35.2 grams, tea many
Sugar have 0.9 gram, theanine have 2.3 grams, gamma aminobutyric acid 0.36 gram, chlorophyll 3.1 grams, lutein ester 0.3 gram, albumen feedstuff
330 grams.
Embodiment 3:
The present embodiment 3 extracts the method for bioactive substance from low-grade green tea, comprises the following steps:
1, weigh low-grade green tea 50 kilograms, be ground into 20 mesh, add in 500 liters of enamel reactors, separately add tap water 300
Rise, start to stir lower heat temperature raising, adjust PH5.0 with concentration about 30% phosphate aqueous solution simultaneously.As tank Nei Wenduda 70 DEG C,
Being incubated 70～75 DEG C of stirrings to extract 1.5 hours, stop heating and stirring, the pipe suction strainer having filtering layer head in being arranged on tank goes out
Aqueous extract for the first time.In tank, separately add 300 liters of tap waters, repeat same operation, from the material mouth discharging of tank bottoms center, through row automatically
Slag filter filters, and obtains the aqueous extract 560 kilograms (limpid, pH5.0) of 72 kilograms of wet feed slags and twice merging；
560 kg of water extracting solution, at 1M3Enamel reactor in, under room temperature and low rate mixing, 23 kilograms of prior places
Manage into 732 large hole cation exchanger resins of H type to add and adsorb, within 1.5 hours, reach terminal, stop stirring, discharging through from
Scheming is filtered, and obtains being adsorbed with 732 large hole cation exchanger resins of caffeine (CAF) and not exchanged by 732 macroporous cations
The aqueous solution of resin absorption 542 kilograms；
It is adsorbed with 732 large hole cation exchanger resins of CAF, is added in the enamel reactor of 100 liters, separately adds 95% ethanol
30 liters, start heat temperature raising under low rate mixing, as tank Nei Wenduda 60 DEG C, protect 60～65 DEG C of low rate mixing eluting 1.5 hours,
Stopping heated and stirred, discharging adds in centrifuge filters (first natural filtration, rear centrifuge dripping；60～65 DEG C are put into again in tank
95% washing with alcohol with dry once), obtain ethanol elution and 732 resins (applying mechanically)；
Ethanol elution, by 5 liters of glass rotary evaporators in 60 DEG C, 0.09mPa distillation and concentration, obtain distillate (ethanol,
Apply mechanically) and concentrated solution.In concentrator loading dish, in 60 DEG C of vacuum microwave dryings, obtain distillate (Diluted Alcohol is applied mechanically) and coffee
0.6 kilogram of alkali (CAF) (yellowish white pressed powder, purity 90.5%, GB8312 87 method)；
2) not by the aqueous solution 542 kilograms of 732 large hole cation exchanger resin absorption in step 1), at 1M3Enamel reacts
In tank, under stirring at normal temperature, adjust pH3.0～3.5 with the phosphate aqueous solution of concentration about 30%, with the electronegative film of 3500Da
Ultrafilter separates and concentrates, and obtains film permeate and trapped fluid.Trapped fluid machine the most by centrifugation is filtrated to get filtrate and filter cake (has and plants
Thing albumen, plant gum, tannin, cellulose etc.).Filter cake after drying, merges with albumen feedstuff；
Filtrate merges with film permeate and has 538 kilograms, at 1 M3In enamel reactor, under room temperature and low rate mixing,
Adding D101 macroporous adsorbent resin 15 kilograms to adsorb, within 1.5 hours, detection solution is almost colourless, stops stirring discharging and was centrifuged
Filter, and with appropriate distilled water wash D101 resin 1～2 times, obtain being adsorbed with the D101 resin of TP and not by D101 resin absorption
Acidic aqueous solution (containing wash water) 483 kilograms, pH3.5；
It is adsorbed with the D101 resin of TP, at 1M3In enamel reactor, add 95% ethanol 420 liters, start under low rate mixing
Heat temperature raising, as tank Nei Wenduda 40 DEG C, is incubated 40 45 DEG C 0.5 hour, stops heating and stirring, after 15 minutes, suction strainer
Go out eluent for the first time.In tank, separately add 400 liter of 95% ethanol, repeat same operation 12 times, until eluent is almost colourless be
Only, discharging centrifugal filtration (first natural filtration, then get rid of in), obtain D101 resin (applying mechanically) and the ethanol elution merged several times；
Ethanol elution, with reverse osmosis membrane separation and concentration, obtains film permeate (ethanol is applied mechanically) and trapped fluid.Retain
In liquid loading dish, in 60 DEG C of vacuum microwave dryings, obtain distillate (ethanol is applied mechanically) and tea polyphenols (TP) 6 kilograms, orange-yellow solid
Body powder, ECG, EGCG, GCG, 3 kinds of several catechin content 93.8%, HPLCI method；
3) step 2) in not by the acidic aqueous solution (PH35) 483 kilograms of D101 resin absorption, use economic benefits and social benefits falling film evaporation
Device, in 70 DEG C of 0.09MP distillations and concentration, obtains distillate (distilled water is applied mechanically) and concentrated solution 66 kilograms；
In 500 liters of enamel reactors, add 95% ethanol 200 liters, under stirring at normal temperature, 66 kilograms of concentrated solutions added,
Stopping stirring after 10 15 minutes, room temperature stands 1.5 hours, sucking-off upper liquid, and lower floor's feed liquid puts into the most natural mistake of centrifuge
Filter, rear centrifuge dripping, obtain ethanol water (containing upper liquid and filtrate) and wet cake.Wet cake enters in dish, in 60 DEG C of vacuum
Microwave drying, obtains distillate (Diluted Alcohol is applied mechanically) and tea polysaccharide (TPS) 0.2 kilogram, pale solid powder, total sugar content
60%, Phenol sulfuric acid procedure；
4) ethanol water obtained in step 3), is distillated in 60 DEG C of 0.09mPa with double-effect falling film evaporator
Liquid (ethanol is applied mechanically) and concentrated solution (pH3.5) 53 kilograms.This concentrated solution, directly on be processed into 732 resin chromatography of H type in advance
Post, with the phosphate buffer gradient elution of PH8 9, is detected by TLC method, merges TAA one-component, single group of GABA respectively
Point, TAA with GABA intersect the eluent of component；
TAA one-component eluent, uses double-effect falling film evaporator distillation and concentration, obtains distillate (ethanol is applied mechanically) and dense
Contracting liquid.Concentrated solution phosphoric acid,diluted aqueous solution is adjusted after pH5 6, with dehydrated alcohol recrystallization 2 times, obtains theanine (TAA) sterling
0.36 kilogram (white crystalline powder, purity 93.2%, HPCC method)；
GABA one-component eluent, operational approach is identical with TAA one-component, obtains gamma aminobutyric acid (GABA) pure
Product 0.08 kilogram, white crystalline powder, purity 92.8%, HPLC method；
TAA with GABA intersects and applies mechanically when pulling on post under component.Cross post liquid to go to apply mechanically during step 1 water extraction；
5) the wet feed slag obtained in step 1) 72 kilograms, after pelletize, with DME extractant in 40～60 DEG C, under 0.7mPa
Carry out subcritical abstraction and separate, obtain raffinate (i.e. albumen feedstuff 33 kilograms, ivory buff particulate) and extract 39 kilograms；
6) extract obtained in step 5), proceeds in 100 liters of containers with 50～60 DEG C of distilled water, is incubated 50～60 DEG C and stirs
Mix about 10 minutes stop stirring, be incubated 50～60 DEG C stand 1 hour, with filter centrifugal separation (first put lower layer of water, after put
Layer thing), obtain filtrate (add in the aqueous extract that step 1 obtains and apply mechanically) and pastel 6.3 kilograms；
6.3 kilograms of pastel, proceed in 50 liters of containers with intermixture 18 liters (petroleum ether and ethanol), dissolve under stirring at normal temperature
0.5 hour, separately add dehydrant (anhydrous sodium sulfate powder) and be stirred for 15～30 minutes, as the most limpid in solution should add anhydrous slufuric acid
Sodium powder, until solution is limpid, stopping is stirred and is stood 2 hours in room temperature, first sucking-off upper liquid, rear centrifugal filtration lower floor material
(first natural filtration, then start centrifuge dry), obtains solid content (washing 1～2 time with a small amount of dehydrated alcohol) and lysate (containing upper
Layer liquid, filtrate, washing liquid).Solid content drying obtains anhydrous sodium sulfate (applying mechanically)；
Lysate separates with 100～200 mesh neutral alumina powder chromatographic columns: alumina powder consumption is chlorophyll content
50 times of weight；Chromatography pillar Φ H=1 20；In post, alumina powder uses petroleum ether moistening in advance；After material upper prop is complete, first use stone
Oil ether eluting, until eluent is colourless；Then dehydrated alcohol eluting is used, until eluent is colourless.Alumina powder post
Son (applying mechanically)；
Crossing post liquid and petroleum ether eluent merges, 60 DEG C of 0.095mPa distillations obtain the distillate (oil containing a small amount of ethanol
Ether, applies mechanically) and concentrated solution.The blended solvent recrystallization of concentrated solution, obtains lutein ester 0.08 kilogram, orange/yellow solid powder,
Purity 82%, UV-VIS spectrophotometry；
Ethanol elution, concentrates by reverse osmosis membrane separation, obtains film permeate (ethanol is applied mechanically) and trapped fluid.Trapped fluid
In loading dish, 60 DEG C of vacuum microwave dryings obtain distillate (ethanol is applied mechanically) and chlorophyll 0.45 kilogram, dark green solid powder
End, purity 98%, UV-VIS spectrophotometry.
Embodiment 4:
The present embodiment 4 extracts the method for bioactive substance from low-grade green tea, except low-grade green tea 50 kilograms changes green tea into
Outside 50 kilograms of end, other conditions are same as in Example 3.The caffeine of extracted isolated has 0.41 kilogram, tea polyphenols 3.5 public affairs
Jin, tea polysaccharide have 0.09 kilogram, theanine have 0.23 kilogram, gamma aminobutyric acid 0.04 kilogram, chlorophyll 0.32 kilogram, leaf yellow
Element ester 0.03 kilogram, albumen feedstuff 31 kilograms.
Above-described embodiment is that the foregoing to the present invention is described further, but this should not being interpreted as, the present invention is above-mentioned
The scope of theme is only limitted to above-described embodiment.All technology realized based on foregoing belong to the volume scope of the present invention.
Claims (2)
1. the method extracting bioactive substance from low-grade tea and tea making leftover bits and pieces, comprises the following steps:
1) raw material powder being broken into 20-40 mesh coarse powder, adding weight is coarse powder 6～the water of 8 times of weight, starts stirring in extraction vessel
Lower heat temperature raising, adds the phosphoric acid solution tune pH value 5～6 that concentration expressed in percentage by weight is 25～35%, container inside holding 70～90 simultaneously
DEG C, stirring extract 1.5～2 hours, stop heating with stirring, suction strainer goes out an aqueous extract, separately adds weight in extraction vessel
For coarse powder 6～the water of 8 times of weight, repeating same operation, discharging is filtered, and obtains the pH value 5～6 of wet feed slag and twice merging, clear
Bright aqueous extract；
Aqueous extract, uses 732 large hole cation exchanger resins being processed into H type in advance, adsorbs under room temperature low rate mixing:
The consumption of 732 large hole cation exchanger resins is according to the content of caffeine in aqueous extract and 732 large hole cation exchanger resins
Exchange capacity, after calculating excess 10～15%, adsorb 1～2 hour, quickly detect by non-aqueous titration reach absorption end
Point, time in ie in solution almost without caffeine, stops stirring, and discharging is filtered, and 732 macroporous cations obtaining being adsorbed with caffeine are handed over
Change resin and not by the aqueous solution of 732 large hole cation exchanger resin absorption；
It is adsorbed with 732 large hole cation exchanger resins of caffeine, under 60～65 DEG C of low rate mixings, carries out eluting, second with ethanol
Alcohol consumption is 2 times of volumes of 732 large hole cation exchanger resin volumes, after 1～2 hour, stops heating and filters with stirring discharging
Obtain 732 large hole cation exchanger resins and ethanol elution；Ethanol elution, obtains with dry in 60～70 DEG C of vacuum distillinges
Distillate ethanol and caffeine；
2) not by the aqueous solution of 732 macroporous cation exchange adsorption resin absorptioies in step 1), by weight hundred under stirring at normal temperature
Point concentration be 25～35% phosphate aqueous solution adjust pH3～3.5, through the membrane ultrafiltration that 3500Da is electronegative, obtain film permeate and cut
Staying liquid, trapped fluid machine by centrifugation is filtrated to get filtrate and albumen feedstuff solid content, and filtrate merges with film permeate；In room temperature at a slow speed
Under stirring, add macroporous adsorbent resin and adsorb, the consumption of macroporous adsorbent resin be 2～2.5 times of weight of polyphenol content also
Excess 10～15%, adsorbs 1～2 hour, takes and stops stirring discharging filtration when solution is seen almost colourless, and with distilling washing in right amount
Wash macroporous resin 1～2 times, obtain being adsorbed with the macroporous adsorbent resin of tea polyphenols and not by absorption with macroporous adsorbent resin containing wash water
The acidic aqueous solution of pH3～4；
The macroporous adsorbent resin being adsorbed with tea polyphenols is added in ethanol, carries out eluting under 40～50 DEG C of low rate mixings, and ethanol is used
Measuring and be advisable can mix material under low rate mixing, stop stirring after 0.5～1.5 hour, suction strainer goes out an eluent, separately adds ethanol
Repeat same operation 1～2 time, until eluent is almost colourless, obtains macroporous adsorbent resin and the ethanol merged several times is washed
De-liquid；
Ethanol elution reverse osmosis membrane separation and concentration, obtain film permeate and trapped fluid, trapped fluid through low-temperature vacuum drying,
Obtain the distillate containing ethanol and tea polyphenols；
3) step 2) in not dense in 60～70 DEG C of high vacuum distillations by the pH3 of absorption with macroporous adsorbent resin～the acidic aqueous solution of 4
Contracting, obtain distillate and concentrated solution, concentrated solution volume is concentrated solution volume 2～the ethanol of 3 times, in stirring at normal temperature mixing, quiet
Putting, filter separation, obtain filter cake and ethanol filtrate, filter cake obtains distillate and tea polysaccharide in 60～70 DEG C of vacuum drying；
4) ethanol filtrate from step 3) first reclaims ethanol, afterwards by 732 large hole cation exchanger resin column chromatography for separation,
Obtain theanine and γ-aminobutyric acid；
5) the wet feed slag obtained in step 1), after pelletize or tabletting, enters in 40～50 DEG C, 0.6～0.7MPa with extractant
Row low temperature subcritical abstraction with separate, obtain solid content and liquid, the two is respectively during being decompressed to normal pressure, extraction therein
Take agent to be evaporated into gas, compressed and liquefied again, and then obtain raffinate albumen feedstuff and extract；
6) extract obtained in step 5) separates through 40～50 DEG C of distilled water washs, obtains water-fast pastel and washes
Water, water-fast pastel, with 3 times of volume dehydrated alcohol of its weight and petroleum ether mixture, in stirring at normal temperature 0.5～1 hour
After, separately adding dehydrant and continue to stir 10～20 minutes, stop stirring, stand 1～2 hour, first sucking-off upper liquid, under rear filtration
Layer, obtains solid content and the filtrate containing upper liquid, after solid content then washes 1～2 time with a small amount of dehydrated alcohol if any color, drying
Obtain anhydrous sodium sulfate；
Filtrate separates with neutral alumina chromatographic column: pillar Φ H=1 20；Alumina powder is 100～200 mesh；Oxygen in post
Change aluminium powder and use petroleum ether moistening in advance；On filtrate after complete post, first use petroleum ether eluting, until eluent is colourless, contained
The petroleum ether eluent of a small amount of dehydrated alcohol；Again with ethanol elution, until eluent is colourless, obtain ethanol elution and oxygen
Change aluminum chromatographic column；
Petroleum ether eluent merges with the post liquid of crossing during upper prop, obtains the petroleum ether containing a small amount of ethanol in 60～70 DEG C of vacuum distillinges
Distillate and concentrated solution: concentrated solution is through being recrystallized to give the lutein ester of orange/yellow solid powder；
Ethanol elution, in 60～70 DEG C of vacuum distillinges with dry, obtains ethanol distillate and dark green solid powder chlorophyll.
The method extracting bioactive substance from low-grade tea and tea making leftover bits and pieces the most according to claim 1, it is special
Levying and be that macroporous adsorbent resin uses D101 macroporous adsorbent resin or AB-8 macroporous adsorbent resin, extractant uses dimethyl ether or just
Butane or propane, the boiling point of petroleum ether is 60～90 DEG C, ethanol or the dehydrated alcohol that ethanol uses concentration expressed in percentage by volume to be 95%.
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| CN105166199A (en) * | 2015-10-10 | 2015-12-23 | 浙江长兴随易茶叶科技有限公司 | Resourceful treatment technology for tea waste |
| CN106035760A (en) * | 2016-06-06 | 2016-10-26 | 大新县生产力促进中心 | Processing technology of green tea |
| CN106434059A (en) * | 2016-09-15 | 2017-02-22 | 湖南古洞春茶业有限公司 | Method for producing health-care soap with camellia sinensis var assamica, and forming machine thereof |
| CN107455521A (en) * | 2017-08-16 | 2017-12-12 | 广西克鲁尼茶叶生物科技有限公司 | A kind of complex art for being used for the high efficiency extraction Tea Polyphenols from discarded tealeaves |
| CN107439739A (en) * | 2017-09-07 | 2017-12-08 | 广西昭平县天成生态农业有限公司 | A kind of high-quality slender joss stick green tea of scenting and preparation method thereof |
| CN110117231B (en) * | 2018-02-07 | 2022-05-10 | 内蒙古昶辉生物科技股份有限公司 | Method for extracting 4-hydroxyisoleucine and total saponins from fenugreek seeds |
| CN108634311A (en) * | 2018-05-08 | 2018-10-12 | 紫阳县硒资源开发研究所 | A kind of process of extraction tealeaves active material |
| CN111393539A (en) * | 2020-05-22 | 2020-07-10 | 陕西理工大学 | Preparation method for co-producing tea polysaccharide, theanine and caffeine in summer and autumn tea |
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| CN1557841A (en) * | 2004-01-14 | 2004-12-29 | 中国农业科学院茶叶研究所 | Process for synthetic extraction of polysaccharides, tea-polyphenol, theanine, caffeine from tea |
| CN101798355A (en) * | 2010-04-08 | 2010-08-11 | 晋江市恒源科技开发有限公司 | Method for integrated separation and extraction of tea polysaccharide, theanine, polyphenol and caffeine |
| CN103910705A (en) * | 2014-03-20 | 2014-07-09 | 苏州市职业大学 | Method for rapidly extracting, separating and purifying epigallocatechin gallate(EGCG) from leftover of green tea |
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| CN1557841A (en) * | 2004-01-14 | 2004-12-29 | 中国农业科学院茶叶研究所 | Process for synthetic extraction of polysaccharides, tea-polyphenol, theanine, caffeine from tea |
| CN101798355A (en) * | 2010-04-08 | 2010-08-11 | 晋江市恒源科技开发有限公司 | Method for integrated separation and extraction of tea polysaccharide, theanine, polyphenol and caffeine |
| CN103910705A (en) * | 2014-03-20 | 2014-07-09 | 苏州市职业大学 | Method for rapidly extracting, separating and purifying epigallocatechin gallate(EGCG) from leftover of green tea |
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