CN102336841B - Method for extracting tea polysaccharide - Google Patents
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Abstract
The invention discloses a method for extracting tea polysaccharide, which comprises the following steps of: (1) performing ultralow temperature freeze drying on coarse and old tea at -80DEG C for 12 to 24 hours, grinding, and sieving to obtain tea powder; (2) performing refluxing extraction on the tea powder by using an organic solvent for 2 to 4 hours, soaking in 70 to 90 percent aqueous solution of ethanol for 24 to 36 hours, filtering, drying a filter cake, and thus obtaining the pretreated tea powder; and (3) adding water into the pretreated tea powder in the step (2), performing ultrasonic extraction at the temperature of between 0 and 40DEG C and the frequency of between 20 and 40KHz for 10 to 60 minutes to obtain a primary extracting solution, centrifuging the primary extracting solution to obtain primary supernate and primary residue, and separating and purifying the primary supernate to obtain the tea polysaccharide. The obtained product has high purity and high antioxidant activity; and process conditions are mild, the structure of the polysaccharide is hardly damaged, and the method is environment-friendly, meets the production requirement of health foods, is suitable for keeping the activity of thermosensitive polysaccharide, and is suitable for industrial production.
Description
(1) technical field
The present invention relates to a kind of extracting method of tea polysaccharide, particularly a kind of method of from the coarse tea leaf, extracting tea polysaccharide.
(2) background technology
China is one of main Tea Production state, and the tea tree planting area accounts for 50% of tea place, world area, ranks first in the world, and the tealeaves ultimate production surpasses 1,000,000 tons, accounts for 31% of world's tea yield.Yet only tender leaf partly is fully utilized for a long time, and coarse tea is overstock in the warehouse in a large number or discards in the tea place, and overstocked coarse tea finally can only be used as fertilizer or fuel more than 200,000 tons, causes the significant wastage of resource.Thereby the processing industry of urgent need development coarse tea, seek the breach in the coarse tea deep processing, widen the applicable surface of product, improve added value of product.
The content of tea polysaccharide is far above tender tealeaves in the coarse tea, the experience of often drinking coarse tea treatment diabetes arranged in that China and Japan are among the people, weak tea or the strong tea made from age of tree tea tree ' s old leaf more than 30 years as Japan, through clinical observation, glucose in urine is reduced, and sx is until getting well.Relevant tea polysaccharide and Pharmacological action study report thereof increase gradually in recent years, and tea polysaccharide is the another important biological polysaccharide that tealeaves relaying tea-polyphenol is found afterwards.Since early 1990s, research mainly concentrates on the pharmacological action aspect of thick tea polysaccharide, and studies show that: tea polysaccharide has antitumor (Richard B é liveau, 2004; Fang Y.Z., 1995), anti-oxidant (Yen G.C., 1997; Nie Shaoping, 2008), antithrombotic (Wang Shuru, 1992), hypoglycemic (Takeo, T, 1998; Zhou Jie, 1997; Wang Dongfeng, 1994), raise immunity (Wang Dongfeng, 2000), hypotensive (Wang; Wang, 1991b), coronary blood flow increasing (Wang Dinggang, 1991) and reducing blood-fat (Zhu; Effect such as Wang, 1992).From low and middle-grade coarse tea leaves, extract active tea polysaccharide, not only be conducive to taking full advantage of of tea resources, increase economic efficiency, promote the Tea Industry development, solve the agriculture, rural areas and farmers problem, and preventing disease is promoted that human health is also significant.
Problems such as although the extracting method of at present relevant tea polysaccharide has more report, and it is not high that ubiquity polysaccharide extract rate, and purity is low.Because same tea raw material, adopt the resulting tea polysaccharide proterties of different extracting method, purity difference, biological activity is (Zhang Huiling inequality also, 1995), adopt which kind of technology can keep the tea polysaccharide biological activity better actually, how they influence the tea polysaccharide structure, are problems that is worth research all the time.Extraction is the first step of tea polysaccharide separation and purification, directly influences tea polysaccharide yield and biological activity.Japan once did research with regard to the different temperature gained tea polysaccharide of extracting for hypoglycemic test, and domestic research all concentrates on follow-up separation and purification aspect.How the extraction process of inquiring into tea polysaccharide influences its biological activity, and adopts suitable separation and Extraction tea polysaccharide method, to improve tea polysaccharide purity and activity, will have great application prospect.
Temperature is to influence its biological activity important factor in the tea polysaccharide extraction process, though tea polysaccharide solvability in hot water is better, hot water lixiviate yield is higher than the cold water lixiviate, and activity can be affected simultaneously.Patent record as Japanese doctor alpine rush or palm-bark rain cape and doctor Tian with hot water lixiviate more than 85 ℃, can destroy hypoglycemic effective constituent; Wang Dongfeng etc. (1996) studies confirm that the tea polysaccharide thermostability is relatively poor, and degraded is accelerated more than 60 ℃; And clear water Cen husband's (1990) the extraction temperature that studies show that the best hypoglycemic activity of maintenance tealeaves is relevant with hypoglycemic effect, and for green tea, frozen water (0 ℃)＞warm water (40～50 ℃)＞hot water (100 ℃) should be considered Temperature Influence when extracting tea polysaccharide.Therefore, will take into account yield and active during the extraction tea polysaccharide, pulverizing at ultralow temperature technology and ultrasonic technology are one of important means that guarantees the effective broken wall of low temperature, raising yield and activity.
The pulverizing at ultralow temperature technology is to combine with pulverizing 2 kinds of unit operations freezing, makes material under frozen state, utilizes the fragility of very low temperature to realize pulverizing.It can be pulverized and be difficult to the material pulverized at normal temperatures, make material particles mobile better, size-grade distribution is better, can phenomenons such as oxidation, decomposition, variable color not occur because pulverizing makes the material heating, is particularly suitable for material grinding such as functional component.
Ultrasonic wave is the above high frequency mechanical wave of a kind of 20KHz, the acoustic cavitation process that it produces in solution system make the liquid hollow chamber formation, vibration, grow, be contracted to collapse, be the process of concentrating sound field energy and abrupt release.During the cavitation bubble collapse, in the extremely short time with in the little space around the cavitation bubble, can produce the high pressure of about 50Mpa, and with strong impact.The ultrasonic wave radiation is radiated time length no matter, and the depolymerization molecular weight has individual lower bound; And, depolymerizing substance has the molecular weight distribution that is rather narrow, distribution has certain rules through the polysaccharide molecular weight behind the ultrasonic degradation, namely no matter the molecular weight distribution of polysaccharide how before handling, the molecular weight total distributed of product is in narrower scope, and ultrasonic degradation can not cause that the polysaccharide space conformation changes, and can keep original activity.Behind usefulness ultrasonic degradation black fungus acid heteroglycans such as Li Xiang, its relative molecular mass and relative molecular mass dispersity index all increase in time and reduce, and ultrasonic wave does not influence the glucuronic acid content of this polysaccharide.Think and to adopt ultrasonic method to prepare the acid heteroglycan of serial different relative molecular masses.And think, the easier bigger chain of relative molecular mass that interrupts of ultrasonic wave, the possibility that is interrupted with the reduction of relative molecular mass diminishes; The reduction of relative molecular mass is progressively carried out, and can not have the fragment of too high or too low in a large number relative molecular mass.
Because tea polysaccharide is a kind of acid heteroglycan (SP Nie ﹠amp; MY Xie, 2011; Lee's cloth green grass or young crops 1996), acidic polysaccharose contains more electronegative uronic acid, and namely the O atom has more negative charge and the complexing steric hindrance is little, so it is higher to remove the OH activity, contains the big and O of positive charge of the polysaccharide molecule C atom of uronic acid simultaneously
2It is less that he of attack hinders, so remove O
2Active higher; In the acid assorted poly-polysaccharide, glucuronic acid content and anti-oxidant activity positive correlation (Wang Yuanfeng, 2005).Traditional tea polysaccharide is extracted and is adopted cold water or warm water extraction, and polysaccharide is difficult to come out from born of the same parents' internal diffusion; High temperature easily makes the tea polysaccharide inactivation; And structure sour, that alkaline process destroys polysaccharide easily reduces its bioavailability.The present invention adopt the pulverizing at ultralow temperature technology in conjunction with ultrasonic wave synergistic reaction in the coarse tea leaf, and glucuronic acid content extracts the index of yield and anti-oxidant activity as optimizing tea polysaccharide simultaneously in the extract to follow the trail of in leaching process, and in conjunction with ultrafiltration membrane treatment, further purification polysaccharide, a new operational path and method are extracted in the exploitation that is the tea polysaccharide protective foods, and this is for the comprehensive exploitation of coarse tea leaf and improve its economic worth and have practical significance.
(3) summary of the invention
The object of the invention provides a kind of extracting method of tea polysaccharide, and described method is raw material with the coarse tea leaf, utilizes the pulverizing at ultralow temperature technology in conjunction with ultrasonic extracting process, adopt ethanol sedimentation and ultrafiltration membrane treatment, gained tea polysaccharide purity height, anti-oxidant activity height, the processing condition gentleness, environmental protection.
The technical solution used in the present invention is:
A kind of extracting method of tea polysaccharide, described method is: (1) is got the coarse tea leaf and is pulverized behind-80 ℃ of cryogenic temperature freezing drying 12～24h, crosses 60～100 mesh sieves, obtains the tea powder; Described coarse tea leaf is to pluck in belonging to Theaceae (Theaceae), and Camellia (Camellia) plant is often referred to the thick old blade of plucking from tea tree summer, and the mouthfeel bitterness mostly is low-grade tea, and is undressed; (2) the tea powder is used organic solvent refluxing extraction 2～4h, soaked 24～36h with 70～90% aqueous ethanolic solutions again, remove small molecular weight impurity, filter, filtration cakes torrefaction obtains pretreated tea powder; Described organic solvent is dehydrated alcohol, ether or sherwood oil; 3) the pretreated tea powder of step (2) is added water, 0～40 ℃, ultrasonic extraction 10～60min under 20～40KHz, obtain No. one time extracting solution, an extracting solution is centrifugal, obtain supernatant liquor and a defective material slag, a supernatant liquor separation and purification is obtained tea polysaccharide, the mass ratio of described pretreated tea powder and water is 1: 20～80.
Described step (1) sublimation drying is 18h.
Described step (2) organic solvent is preferably dehydrated alcohol.
The mass ratio of described step (2) tea powder and organic solvent is 1: 3～5, and the volumetric usage of described 80% ethanolic soln can get final product by the submergence raw material usually to not influence of the present invention.
The mass ratio of the pretreated tea powder of described step (3) and water is preferably 1: 30～and 50.
Further, the pretreated tea powder of preferred described step (3) mixes with water, and 10～20 ℃, ultrasonic extraction 40min under 20～30KHz obtains No. one time extracting solution.
A supernatant liquor separation purification method is in the described step (3): with supernatant liquor with 30～90% ethanol sedimentations, centrifugal, obtain supernatant A and precipitate A, precipitate A is water-soluble, concentrate the mixture that the back adds diatomite and gac, stir, filter, filtrate is carried out ultrafiltration membrance filter with the ultra-filtration membrane of 50～200kD, collects the not material of filtration membrane, concentrates, vacuum-drying, obtain described tea polysaccharide, the mixture add-on of described diatomite and gac is counted 2～8wt% with a supernatant liquor quality, and diatomite is 1: 3 with the quality of activated carbon ratio in the described mixture.
Described ultra-filtration membrane is preferably poly (ether sulfone) film, and described poly (ether sulfone) film is 50～200kD poly (ether sulfone) film, preferred 100kD poly (ether sulfone) film.
Described diatomite and Mixture of Activated Carbon add-on are preferably 5wt% in the supernatant A quality.
Described vacuum-drying is microwave vacuum drying, microwave frequency 2450MHz, and microwave power 1kW, vacuum tightness is 35～45mmHg, dry 10～30min.
Further, the extracting method of described tea polysaccharide, supersound extraction is carried out N time in the described step (3), described N is 2～5 integer, centrifugal, a defective material slag is added water, 10～20 ℃, ultrasonic extraction 20～40min under 10～30KHz, obtain secondary raffinate, secondary raffinate is centrifugal, obtain secondary supernatant liquor and two defective material slags, again two defective material slags are carried out supersound extraction next time, by that analogy, finish extraction after, obtain N defective material slag and 1～N time supernatant liquor, merge all supernatant liquors, all supernatant liquors with 30～90% ethanol sedimentations, are obtained supernatant liquor B and precipitate B, the water-soluble concentrated back of precipitate B is added the mixture of diatomite and gac, stir, filter, filtrate is carried out membrane filtration with the film of 100kD, collects the not material of filtration membrane, concentrate, vacuum-drying obtains described tea polysaccharide, and the mixture add-on of described diatomite and gac is counted 2～8wt% with a supernatant liquor B quality, preferred 5wt%, diatomite is 1: 3 with the quality of activated carbon ratio in the described mixture.
The described employing flame filter press that is filtered into of step (3) uses 6～8 metafiltration cloth to filter.
Step of the present invention (2) refluxes with dehydrated alcohol and carries the fat that 2h removes the encirclement of tealeaves extracellular lipid, adopts volume fraction 80% ethanol soaking at room temperature 24h to remove materials such as small molecular weight impurity, monose, disaccharide, oligose, glucoside, alkaloid, tea-polyphenol, amino acid and protein,alcohol-soluble in the tealeaves again.
The poly (ether sulfone) film of usefulness 50～200kD of the present invention carries out membrane filtration, the preferred tangential flow filtration technology that adopts, be liquid flow direction and filtering direction filtered version in vertical direction, filtering with respect to the vertical current of routine has the filtration velocity that can keep stable, the characteristics that can circulate continuously and filter and do not form gel coat on the film surface.
The coarse tea leaf that the present invention is adopted with the Dragon Well tea tea tree that Hangzhou Mei Jiawu is produced is raw material, adopt pulverizing at ultralow temperature in conjunction with ultrasonic wave synergistic reaction in the coarse tea leaf, guarantee the anti-oxidant activity of polysaccharide, and in conjunction with ethanol sedimentation and ultrafiltration membrane treatment, the polysaccharide of further purifying.
The present invention adopts the pulverizing at ultralow temperature technology in conjunction with ultrasonic wave synergistic reaction, guarantees the anti-oxidant activity of polysaccharide when impelling the effective stripping of polysaccharide, and in conjunction with ethanol sedimentation and ultrafiltration membrane treatment, the purity of polysaccharide height that obtains, activity are destroyed little.
Supernatant A of the present invention and supernatant liquor B are supernatant liquor, and for distinguishing different name of supernatant liquor that different step obtains, described precipitate A and precipitate B are precipitation, for distinguishing different name of precipitation that different step obtains.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
1) products obtained therefrom purity height of the present invention, wherein polysaccharide content can be up to more than 55%, the anti-oxidant activity height;
2) be to extract the important factor in order of tea polysaccharide activity at temperature, adopt pulverizing at ultralow temperature and ultrasonic synergistic treatment process, guarantee the effective broken wall of low temperature, improve yield and anti-oxidant activity; The yield of tea Crude polysaccharides can reach 5.94%, and the Crude polysaccharides that obtains than traditional hot water lixiviate mechanical type pulverizing meal has improved 3.09 times, and anti-oxidant activity has improved 2.47 times than traditional technology; The processing condition gentleness, not fragile polysaccharide structures, environmental protection meets the health care food production requirement;
3) the superfreeze preconditioning technique that adopts can make that material particles is mobile better, size-grade distribution is better, can phenomenons such as oxidation, decomposition, variable color not appear because pulverizing makes the material heating, be particularly suitable for when pulverizing, oxidation, variable color occurring owing to material generates heat such as tea-polyphenol constituents in the tealeaves, influence the refining of follow-up tea polysaccharide, this technology is very suitable to the maintenance of thermo-sensitivity active polysaccharide simultaneously;
4) adopt ethanol sedimentation in conjunction with the ultrafiltration membrane treatment tea polysaccharide of purifying, can guarantee the product purity height, treatment capacity is big, especially the cross-flow ultrafiltration technology is applied in the preparation of tea polysaccharide, also to the poly (ether sulfone) film surface erosion, makes the film surface can not form impurity when filtering, guarantee stable ultrasiltrated rate fast, shorten the process time greatly, also sample liquid is concentrated and decolouring in the time of in the purification process, be convenient to suitability for industrialized production.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1
1 material and reagent
Fresh coarse tea leaf (Xihu Longjing Tea, dragon hat Industrial Co., Ltd. provides by Hangzhou); 1,1-phenylbenzene picryl phenylhydrazine, dehydrated alcohol, toluene, carbazole, galacturonic acid, phenol, the vitriol oil are analytical pure.
2 laboratory apparatuss
The UV-2000 ultraviolet spectrophotometer; The DFY-400 pulverizer; The SANYO Ultralow Temperature Freezer; The RE-52A rotatory evaporator; WVD-01 microwave vacuum drying case.
3 experimental techniques
1) coarse tea leaf pre-treatment
Get the fresh coarse tea leaf of 1000g freezing and pulverizing behind-80 ℃ of superfreeze 18h, cross 60 mesh sieves, obtain the tea powder, carry 3h with the dehydrated alcohol backflow and remove the fat that the tealeaves extracellular lipid is surrounded, adopt volume fraction 80% ethanol soaking at room temperature 24h again, filter, remove materials such as small molecular weight impurity, monose, disaccharide, oligose, glucoside, alkaloid, tea-polyphenol, amino acid and protein,alcohol-soluble in the tealeaves, then filter cake is dried standbyly, obtain pretreated tea powder 186g.
2) extraction process of tea polysaccharide
Get the pretreated tea powder 30g that step 1) obtains and mix (mass ratio of pretreated tea powder and water is 1: 20～80) with water, adopt ultrasonic technology to extract the tea Crude polysaccharides, 0～40 ℃, ultrasonic extraction 10～60min under 20～40KHz, ultrasonic extracting process sees Table 1 orthogonal design, obtain extracting solution, extracting solution is centrifugal, obtain supernatant liquor and precipitation.
Table 1 tea polysaccharide extraction process orthogonal design
3) preparation of tea polysaccharide
Get step 2) supernatant liquor add the aqueous ethanolic solution precipitation of volume fraction 70%, the water-soluble rotary evaporation of throw out is removed residual ethanol, get the liquid that evaporation removes ethanol and carry out membrane filtration with 100kD poly (ether sulfone) film (Co., Ltd among the Mi Libo), adopt the cross-flow ultrafiltration technology, pressure is no more than 0.4MPa, collect the not macromole of filtration membrane, concentrate, macromole concentrated solution microwave vacuum drying (microwave frequency 2450MHz, microwave power 1kW, vacuum tightness is 35～45mmHg, and drying 10～30min) obtains tea polysaccharide.
4) the tea polysaccharide stripping quantity calculates
Tea polysaccharide adopts the phenolsulfuric acid method of improvement that it is carried out assay:
A) typical curve: get glucose 20mg in 500mL volumetric flask distilled water constant volume, be the glucose standardized solution, get 2mL distilled water as blank, get 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL, 1.4mL, 1.6mL glucose standardized solution successively, supply 2mL with distilled water again, the phenol 1mL of adding 5% in 9 standard models, 95% vitriol oil 5mL, shake up, room temperature leaves standstill 30min, the light absorption value of measurement standard sample under 490nm then obtains glucose typical curve regression equation and is: Y=0.0095X-0.018, R
2=0.9975, Y is absorbancy, and X is polysaccharide content (μ g/mL).
B) determination of polysaccharide: the tea polysaccharide of getting the preparation of 0.6mg step 3) method is joined solution 1mL with distilled water, be settled to 100mL, polysaccharide soln 0.5mL behind the absorption constant volume, react by method in a), be blank then with distilled water, test 490nm place light absorption value, according to glucose typical curve regression equation calculation polysaccharide content, by formula (1) calculates the tea polysaccharide stripping quantity:
Formula (1)
In the formula (1), C is that the polysaccharide content of mensuration (μ g), V are the supernatant volume (mL) that obtains after extracting through after diluting, and N is extension rate, and v is the supernatant volume (mL) that is used for mensuration, and M is for the weight (mg) of trying tealeaves.
5) uronic acid detects
The galacturonic aqueous acid of accurate preparation 0.4mg/mL respectively with its dilution, obtains galacturonic acid standardized solution series concentration 0.4mg/mL, 0.2mg/mL, 0.1mg/mL, 0.05mg/mL, 0.025mg/mL according to multiple dilution principle.Precision is measured galacturonic acid standard solution series concentration 1mL in 10mL tool plug test tube respectively, adds concentrated sulfuric acid solution 6mL then, and jolting mixes, and boils 5min in boiling water bath.Be cooled to room temperature with ice-water bath, add 0.1mg/mL carbazole ethanol solution 0.2mL respectively.After shaking up, boiling water with in boil 10min.After cooling shakes up, be blank with the double distilled water of same processing, carry out colorimetric, locate sequentially determining OD value in the measured maximum absorption wavelength of spectrophotometer (530nm).Be ordinate zou with the OD value, corresponding galacturonic acid concentration value (mg/mL) is X-coordinate production standard curve, is set up the equation of linear regression of galacturonic acid concentration and absorbance by the gained result.Regression equation is: Y=0.1611X+0.1586, R
2=0.9927, Y is absorbancy, and X is acid sugar concentration (mg/mL), according to acid sugar content in the tea polysaccharide of regression equation calculation step 3) method extraction, namely get the 0.6mg tea polysaccharide and join solution 1mL with distilled water, measure acid sugar content in the tea polysaccharide sample as stated above.
6) anti-oxidant detection
Tea polysaccharide is to the clearance test of DPPH: 1,1-phenylbenzene picryl phenylhydrazine (DPPH) is stable organic free radical.After DPPH is dissolved in a spot of toluene (the toluene consumption can dissolve DPPH and get final product not influence of the present invention), be made into the DPPH mixing solutions of 0.1g/L with 50% aqueous ethanolic solution.The tea polysaccharide water of step 3) method preparation is mixed with the tea polysaccharide solution of 52mg/L, the 52mg/L tea polysaccharide solution of getting 0.1mL adds in the 5mL DPPH mixing solutions, rapid mixing, in 10min, measuring solution absorbance at the 525nm place every 1min, calculate absorbancy rate over time, is control sample with BHT (butylated hydroxytoluene) methanol solution of 50mg/L, measure absorbancy rate over time under the similarity condition, calculate the tea polysaccharide resistance of oxidation according to formula (2).
Resistance of oxidation (AA, %)=[the velocity of variation Δ A of velocity of variation Δ A/ control sample (50mg/LBHT methanol solution) absorbancy of sample absorbancy] * 100 formula (2)
4 experimental results
The orthogonal experiments of ultrasonic extraction tea polysaccharide technology sees Table 2.
Table 2 quadrature result and analysis
By experiment as can be known, press table 1 design ultrasonic extraction tea polysaccharide, in the table 2 in the tea polysaccharide content detection result of uronic acid wherein extract temperature to the content influence maximum of uronic acid in the tea polysaccharide as can be known, next is extraction time, ultrasonic power, material-water ratio, i.e. A＞B＞D＞C.The optimised process of ultrasonic extraction tea polysaccharide is A
2B
2C
2D
2, namely extract 20 ℃ of temperature, extraction time 30min, ultrasonic power 30KHz, material-water ratio 1: 40 o'clock, the content of uronic acid is the highest in the prepared tea polysaccharide.With this understanding, obtain tea polysaccharide 1.78g, polysaccharide content 58.33%, the stripping quantity of tea polysaccharide are 5.94%.
Comparative example 1
Experimental implementation is with embodiment 1, and difference is, in step 1), gets 100g coarse tea leaf directly after the standard machinery coarse reduction, sieves with 60 mesh standard sieves, obtains the green tea meal; Follow-up step and operation are all with embodiment 1.Adopt this technology to obtain tea polysaccharide 1.04g, polysaccharide content 56.16%.The stripping quantity of tea polysaccharide and anti-oxidant activity see the following form 3 respectively, table 4.
The stripping quantity of the different disintegrating process tea polysaccharide of table 3
| Tea machinery is pulverized | The tealeaves superfreeze is pulverized | |
| Polysaccharide stripping quantity (%) | 3.47 | 5.94 |
As shown in Table 3, the disintegrating process of tealeaves has certain influence to the stripping quantity of polysaccharide, and raw material can make the polysaccharide stripping quantity increase after superfreeze is handled, and its stripping quantity is 1.71 times that standard machinery is pulverized.
The oxidation-resistance of the different disintegrating process tea polysaccharide of table 4
| Tea machinery is pulverized | The tealeaves superfreeze is pulverized | |
| The polysaccharide resistance of oxidation (AA, %) | 46.6 | 85.3 |
As shown in Table 4, the disintegrating process of tealeaves is bigger to the resistance of oxidation influence of polysaccharide, and raw material can make the resistance of oxidation of polysaccharide keep higher level after superfreeze is handled, and its resistance of oxidation is 1.83 times that standard machinery is pulverized.
Comparative example 2
Experimental implementation is with embodiment 1, difference is, in step 1), get 100g coarse tea leaf directly after the standard machinery coarse reduction, sieve with 60 mesh standard sieves, obtain the green tea meal, carry 2h except degrease with the dehydrated alcohol backflow, adopt volume fraction 80% ethanol soaking at room temperature 24h to remove small molecular weight impurity in the tealeaves again, raw material is dried standby, obtain pretreated tea powder 67g.Get pretreated tea powder 30g, press solid-liquid ratio and add deionized water at 1: 60, under 40 ℃ of conditions, extract 180min, after centrifugal, precipitation is pressed solid-liquid ratio and added deionized water at 1: 30, extracts 180min under 40 ℃ of conditions, and is centrifugal, concentrate after merging twice supernatant liquor, follow-up step and operation are all with embodiment 1.Adopt this technology to obtain tea polysaccharide 0.576g, polysaccharide content 57.05%.The stripping quantity of tea polysaccharide and anti-oxidant activity see the following form 5 respectively, table 6.
The stripping quantity of table 5 different process tea polysaccharide
| Tea machinery is pulverized, hot water extraction | The tealeaves superfreeze is pulverized, supersound extraction | |
| Polysaccharide stripping quantity (%) | 1.92 | 5.94 |
As shown in Table 5, the extraction process of tealeaves is very big to the stripping quantity influence of polysaccharide, and raw material can make the big leap ahead of polysaccharide stripping quantity after superfreeze processing, ultrasonic extraction, and its stripping quantity is standard machinery pulverizing, hot water extraction 3.09 times.
The oxidation-resistance of the different disintegrating process tea polysaccharide of table 6
| Tea machinery is pulverized | The tealeaves superfreeze is pulverized | |
| The polysaccharide resistance of oxidation (AA, %) | 34.5 | 85.3 |
As shown in Table 6, the extraction process of tealeaves is very big to the resistance of oxidation influence of polysaccharide, raw material can make the resistance of oxidation of polysaccharide keep higher level after superfreeze, ultrasonic extraction, and its resistance of oxidation is standard machinery pulverizing, hot water extraction 2.47 times.
Embodiment 2
Experimental implementation is with embodiment 1, and difference is to get the coarse tea leaf at-80 ℃ of superfreeze 12h, and other operations and condition obtain tea polysaccharide 1.36g with embodiment 1, and polysaccharide content 56.84%, the stripping quantity of tea polysaccharide are 4.53%, and resistance of oxidation is 81.6%.
Embodiment 3
Experimental implementation is with embodiment 1, and difference is to get the coarse tea leaf at-80 ℃ of superfreeze 24h, and other operations and condition obtain tea polysaccharide 1.73g with embodiment 1, and polysaccharide content 57.36%, the stripping quantity of tea polysaccharide are 5.76%, and resistance of oxidation is 84.7%.
Embodiment 4
Experimental implementation is with embodiment 1, difference is to get coarse tea leaf freezing and pulverizing behind-80 ℃ of superfreeze 18h, cross 60 mesh sieves, obtain green tea powder, carry 3h with aether backflow and remove the fat that the tealeaves extracellular lipid is surrounded, other operations and condition are with embodiment 1, obtain tea polysaccharide 1.45g, polysaccharide content 56.24%, the stripping quantity of tea polysaccharide are 4.83%, and resistance of oxidation is 82.1%.
Embodiment 5
Get coarse tea leaf freezing and pulverizing behind-80 ℃ of superfreeze 15h, cross 60 mesh sieves, in degreasing, removal tealeaves, behind the small molecular weight impurity, obtain pretreated tea powder, get pretreated tea powder and mix at 1: 30 by mass ratio with water, under 20 ℃ of conditions of temperature, with the ultrasonic extraction 20min of 40KHz, extract 2 times, centrifugal, merge 2 times and extract the supernatant liquor that obtains, following step and operation are all with embodiment 1.Preferred microwave vacuum drying when dry, described microwave frequency 2450MHz, microwave power 1kW, vacuum tightness is 40mmHg, and dry 12 minutes, other operations and condition were with embodiment 1, obtain tea polysaccharide 1.51g, polysaccharide content 56.31%, the stripping quantity of tea polysaccharide are 5.03%, and resistance of oxidation is 78.8%.
Embodiment 6
Get coarse tea leaf freezing and pulverizing behind-80 ℃ of superfreeze 20h, cross 60 mesh sieves, in degreasing, removal tealeaves, behind the small molecular weight impurity, obtain pretreated tea powder, get pretreated tea powder and mix at 1: 60 by mass ratio with water, under 40 ℃ of conditions of temperature, with the ultrasonic extraction 60min of 20KHz, extract 2 times, centrifugal, merge 2 times and extract the supernatant liquor that obtains, following step and operation are all with embodiment 1.Preferred microwave vacuum drying when dry, described microwave frequency 2450MHz, microwave power 1kW, vacuum tightness is 45mmHg, dry 8 minutes, other operations and condition obtained tea polysaccharide 1.72g with embodiment 1, polysaccharide content 55.87%, the stripping quantity of tea polysaccharide are 5.74%, and resistance of oxidation is 80.7%.
Embodiment 7
Get freezing and pulverizing after the coarse tea leaf superfreeze, cross 60 mesh sieves, through degreasing, remove in the tealeaves behind the small molecular weight impurity, obtain pretreated tea powder, following step and operation are all with embodiment 1, wherein, concentrate the mixture that the back adds diatomite and gac, stir, filter, filtrate is carried out membrane filtration with the poly (ether sulfone) film of 50kD, other operations and condition obtain tea polysaccharide 1.85g, polysaccharide content 56.91% with embodiment 1, the stripping quantity of tea polysaccharide is 6.16%, and resistance of oxidation is 67.4%.
Embodiment 8
Experimental implementation is with embodiment 1, and difference is to get freezing and pulverizing after the coarse tea leaf superfreeze, crosses 60 mesh sieves, through degreasing, remove in the tealeaves behind the small molecular weight impurity, obtains pretreated tea powder, and following step and operation are all with embodiment 1.Wherein, concentrate the mixture that the back adds diatomite and gac, stir, filter, filtrate is carried out membrane filtration with the poly (ether sulfone) film of 200kD, and other operations and condition are with embodiment 1, obtain tea polysaccharide 0.71g, polysaccharide content 61.25%, the stripping quantity of tea polysaccharide are 2.37%, and resistance of oxidation is 40.24%.
Claims (9)
1. the extracting method of a tea polysaccharide, described method is: (1) is got the coarse tea leaf and is being pulverized behind lyophilize 12～24h under-80 ℃ of conditions, crosses 60～100 mesh sieves, obtains the tea powder; Described coarse tea leaf is the thick old blade of plucking from tea tree summer, and is undressed; (2) described tea powder is used organic solvent refluxing extraction 2～4h, soaked 24～36h with 70～90% aqueous ethanolic solutions again, filter, filtration cakes torrefaction obtains pretreated tea powder; Described organic solvent is dehydrated alcohol, ether or sherwood oil; (3) the pretreated tea powder of step (2) is added water, 0～40 ℃, ultrasonic extraction 10～60min under 20～40KHz, obtain No. one time extracting solution, an extracting solution is centrifugal, obtain supernatant liquor and a defective material slag, a supernatant liquor separation and purification is obtained tea polysaccharide, the mass ratio of described pretreated tea powder and water is 1:20～80.
2. the extracting method of tea polysaccharide as claimed in claim 1 is characterized in that described step (1) sublimation drying is 18h.
3. the extracting method of tea polysaccharide as claimed in claim 1 is characterized in that described step (2) organic solvent is dehydrated alcohol.
4. the extracting method of tea polysaccharide as claimed in claim 1, the mass ratio that it is characterized in that described step (2) tea powder and organic solvent is 1:3～5.
5. the extracting method of tea polysaccharide as claimed in claim 1 is characterized in that the mass ratio of pretreated tea powder and water is 1:30～50 in the described step (3).
6. the extracting method of tea polysaccharide as claimed in claim 1 is characterized in that the pretreated tea powder of described step (3) mixes with water, and 10～20 ℃, ultrasonic extraction 40min under 20～30KHz obtains No. one time extracting solution.
7. the extracting method of tea polysaccharide as claimed in claim 1, it is characterized in that a supernatant liquor separation purification method is in the described step (3): with supernatant liquor with 30～90% ethanol sedimentations, centrifugal, obtain supernatant A and precipitate A, precipitate A is water-soluble, concentrate the mixture that the back adds diatomite and gac, stir, filter, filtrate is carried out membrane filtration with the ultra-filtration membrane of 50～200kD, collect the not material of filtration membrane, concentrate, vacuum-drying obtains described tea polysaccharide, the mixture add-on of described diatomite and gac is counted 2～8wt% with a supernatant liquor quality, and diatomite is 1:3 with the quality of activated carbon ratio in the described mixture.
8. the extracting method of tea polysaccharide as claimed in claim 7 is characterized in that described ultra-filtration membrane is 50～200kD poly (ether sulfone) film.
9. the extracting method of tea polysaccharide as claimed in claim 7 is characterized in that described vacuum-drying is microwave vacuum drying, microwave power 1kW, and vacuum tightness is 35～45mmHg, dry 10～30min.
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